Identification Of Novel Cell Surface Markers For Pancreatic Progenitor Cells And Definite Endodermal Cells

Abstract

Methods of identifying, isolating and qualifying pancreatic progenitor cells and definite endodermal cells. An isolated population of pancreatic progenitor cells, including at least 75% of cells having a TROP-2+ and/or TROP-2+/GPR50+ expression pattern and an isolated population of definite endodermal cells, including at least 50% of cells having a SOX17+/SOX7+/GSC+/CER+/FOXA2+/CXCR4+/NANOG expression pattern. Nucleic acid constructs including a reporter protein under the transcriptional regulation of SOX17 regulatory sequence or of PDX1 regulatory sequence, and cells comprising same, and methods and kits using same.

Description

FIELD OF THE INVENTION

The present invention, in some embodiments thereof, relates to methods of identifying, isolating and qualifying pancreatic progenitor cells and definite endodermal cells, and, more particularly, but not exclusively, to isolated cell populations generated thereby.

BACKGROUND OF THE INVENTION

Type 1 Diabetes Mellitus is an autoimmune disease affecting the life of millions world wide with enormous financial costs. It is caused by the destruction and loss of function of beta cells in the pancreatic Islets of Langerhans. The lack of insulin production causes deregulation of blood glucose levels, and leads to a large number of symptoms that can eventually be fatal. Daily insulin injections are the most prevalent treatment for type I Diabetes Mellitus and for insulin-dependent type II Diabetes Mellitus. However, insulin injections are expensive, cumbersome and do not enable the patient to attain a real steady state in blood glucose levels, but instead lead to fluctuations above and below the optimal base line, which do not ultimately prevent complications of diabetes. A potential cure for these diseases is transplantation therapy whereby islets are transplanted into the patient. However, the limited number of donor organs presently restricts the use of this procedure.

New sources of beta cells are needed in order to develop cell therapies for patients with insulin-dependent diabetes. An alternative to forced expansion of post-mitotic beta cells is the induction of differentiation of pluripotent stem/progenitor cells (which have a natural self-expansion capacity) of different origins, into insulin-producing cells. Various publications describe protocols for differentiation of human embryonic stem cells (hESCs) (5-9), induced pluripotent stem cells (iPSCs) (10, 11), or cord blood mesenchymal stem cells (Chao C K et al., PLoS ONE. 2008; 3(1): e1451; Santos T M et al., Transplantation Proceedings, 42: 563-565, 2010; Bhandari D R et al., Differentiation. 2011 Jul. 20. [Epub ahead of print]) into pancreatic endocrine cells which might provide a source of insulin producing cells for diabetics.

The in vitro differentiation of cells to pancreatic beta cells, like in vivo embryonic development, is a stepwise process by which the initially pluripotent/multipotent cells, such as human ESCs or iPSCs, progressively commit towards a more specialized cell fate ultimately resulting in insulin producing cells. Though insulin is a classic marker for pancreatic endocrine cells it is only a useful marker for cells that have reached the endpoint of differentiation and are fully functional beta islet cells.

Two significant points along the differentiation process are the endodermal progenitor stage and the pancreatic progenitor stage. These stages are characterized by the expression of stage specific transcription factors. Two examples of these are SRY box 17 (SOX17) and pancreatic and duodenal homeobox 1 (PDX1), which are expressed relatively early in pancreatic differentiation. SOX17 is expressed at the earliest stage of hESC differentiation towards definitive endoderm and PDX1 is expressed at onset of the earliest commitment stages towards pancreas (12).

Lavon et al. (25) transfected hESCs with a reporter construct which included the enhanced green fluorescent protein (EGFP) under the albumin promoter (ALB-eGFP).

Additional background art includes Wang P., et al. Cell Stem Cell, 8: 335-346, 2011; WO 2005116073; Zwaka T P and J A Thomson, 2003 [Homologous recombination in human embryonic stem cells. Nat Biotechnol 21:319-321]; Micallef S J., 2005 (Diabetes 54: 301-305); Nikakan K K., 2010 (Genes Dev. 24:312-326); D'Amour K A, et al., 2005 (Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat Biotechnol 23:1534-1541); Yasunaga M, et al., 2005 (Induction and monitoring of definitive and visceral endoderm differentiation of mouse ES cells. Nat Biotechnol 23:1542-1550); US 2011/0070645 (Chen et al.); WO 2005/116073 A2 (D'Amour K A et al.); Borowiak M, 2009 (Cell Stem Cell 4, 348-358); Jiang W., 2007 (Cell Res 17:333-344); D'amour 2006 (Nat Biotechnol 24:1392-1401); and Kroon 2008 (Nat Biotechnol 26:443-452).

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present invention there is provided a method of identifying pancreatic progenitor cells, comprising determining in a population of cells which comprises pancreatic progenitor cells at least one marker that is: (i) positively associated with pancreatic differentiation, the marker being selected from the group consisting of: TACSTD2 (TROP-2), GPR50, BST2, NTRK2, ITGA4, KDR, PTPRN, LGI1, VIPR2, SLC2A1, MUC15, MUC12, LPHN3, MUC16, VTCN1, MMP16, FZD3, ITGB6, GFRA3, ROBO1, NLGN1, BST2, MUC12, CNTFR, LPHN1, SULF1, ADAM23, SCUBE3, PLAU, CDON, SLIT2, C7orf68, PLXDC2, CD74, MUC15, GPR56, VTCN1, ITGB6, NTRK2, AREG, BOC, ITGA4, KLRK1, PRTG, PTPRZ1, GABRP, SILV, KIAA1772, PLP1, OVOS, HAPLN1, EPHA7, ENO2, PCDHB5, OVOS, SYT1, DCT, GPAM, SLITRK6, DCC, FREM2, SDK2, CGA, ATP1B2, SEMA3D, PCDHB15, CDH1, WNT8B, LPAR4, NPIPL3, FAM171B, PTN, ABCC2, ADAMTS3, RASA4, CPT1C, SLC6A6, PCDHB3, LRRC37B, RNFT2, KCNG3, TRPC1, ALPPL2, OR4F21, CCL2, KIF5A, OLFM2, CACNG7, MPHOSPH9, SLC13A4, MOXD1, C6orf186, SLC4A8, STX16, AMY2A, SPARCL1, MGP, A2M, DCN, ATP8B1, MMRN1, EMP1, PLA2G2A, PDE3A, TLR3, CYP1B1, PTGIS, RFTN2, PLEKHA2, SMOC1, STOM, JAM2, CHL1, SCG5, IGFBP7, NPR3, IFI6, CR1L, OR2A4, OR2A7, KCNG3, CACNG7, GRID2, CDH1, LPAR3, SEMA6A, PTPRZ1, ATP1A3, CAMKV, SCNN1G, SYT6, SLC18A2, PCDHB5, ABCG2, HLA-DRA, CR1L, HTR2C, EDNRB, PCDH11X, SLC17A7, SCNN1A, CD9, CXCL16, FXYD5, GABRQ, GFRA3, CACNA2D2, CLDN4, PTPRN, PLP1, PDPN, MMP24, SDK2, GPR176, GPR64, GPR160, PCDH11Y, NKAIN4, ATP1B2, SCN8A, THBS4, CR2, HLA-DQA1, HLA-DRA, HTR7, SLC2A1, HLA-DRA, KCNS3, SLC7A3, HLA-DPB2, CACNA1B, and GPR143, wherein an upregulation above a predetermined threshold of an expression level of the marker positively associated with the pancreatic differentiation as compared to the expression level of the marker in a reference cell, wherein said reference cell is a definite endodermal cell which expresses SOX17, is indicative of a pancreatic progenitor cell, and alternatively or additionally (ii) negatively associated with the pancreatic differentiation, the marker being selected from the group consisting of: LGR5, CXCR4, FLRT3, TRPA1, SLC40A1, LRIG3, COLEC12, EPSTI1, GPR128, CDH2, SLC5A9, FSHR, SLC30A10, IL13RA1, SLC7A7, PCDH10, SLC1A1, GPR141, LIFR, TMEM27, GPC4, LYPD6B, FOLH1, TRPC4, PCDH7, KEL, KCNJ3, OR2T4, VIPR2, FLRT2, CD34, SLC39A8, CLDN11, CXCR7, ITGA5, ITGAV, CALCR, CLDN18, CCKBR, SLC7A5, TMBIM4, SLCO2A1, CDH10, AMHR2, ASAM, CLDN1, DSCAM, TMEM88, PLXNA2, CD177, TMEM144, GPR37, GJA5, SEMA6D, NIPAL2, GPR151, MCC, TMEM136, KCNG1, LHFPL2, MOSPD1, SLC37A1, LRIT3, EPHA4, GPR177, and IL1R1, wherein a downregulation above a predetermined threshold of an expression level of the marker negatively associated with the pancreatic differentiation as compared to the expression level of the marker in a reference cell, wherein said reference cell is a definite endodermal cell which expresses SOX17, is indicative of a pancreatic progenitor cell, thereby identifying the pancreatic progenitor cells.

According to an aspect of some embodiments of the present invention there is provided a method of identifying pancreatic progenitor cells, comprising determining in a population of cells which comprises pancreatic progenitor cells at least one marker that is: (i) negatively associated with the pancreatic differentiation, the marker being selected from the group consisting of: LGR5, CCKBR, CXCR4, FLRT3, TRPA1, SLC40A1, LRIG3, COLEC12, EPSTI1, GPR128, CDH2, SLC5A9, FSHR, SLC30A10, IL13RA1, SLC7A7, PCDH10, SLC1A1, GPR141, LIFR, TMEM27, GPC4, LYPD6B, FOLH1, TRPC4, PCDH7, KEL, KCNJ3, OR2T4, VIPR2, FLRT2, CD34, SLC39A8, CLDN11, CXCR7, ITGA5, ITGAV, CALCR, CLDN18, SLC7A5, TMBIM4, SLCO2A1, CDH10, AMHR2, ASAM, CLDN1, DSCAM, TMEM88, PLXNA2, CD177, TMEM144, GPR37, GJA5, SEMA6D, NIPAL2, GPR151, MCC, TMEM136, KCNG1, LHFPL2, MOSPD1, SLC37A1, LRIT3, EPHA4, GPR177, IL1R1, CST1, CER1, ANKRD1, TRY6, HAS2, DKK1, PRSS2, HP, APOA2, RHOBTB3, BMP2, ACE2, STC1, PDZK1, HHEX, VIL1, PRDM1, EOMES, DNAJC15, TNIK, IGFBP5, RLBP1L2, ADAMTS9, EPSTI1, C5, ARHGAP24, TRY6, ANGPT2, TTR, MYL7, FST, KITLG, GATA3, ST8SIA4, CCDC141, TSPYL5, EGFLAM, TTN, LEFTY2, FOXA2, FAM184A, STMN2, DIO3, FN1, PRSS1, NPPB, and OTX2, wherein a downregulation above a predetermined threshold of an expression level of the marker negatively associated with the pancreatic differentiation as compared to the expression level of the marker in a reference cell, wherein said reference cell is a definite endodermal cell which expresses SOX17, is indicative of a pancreatic progenitor cell and alternatively or additionally; (ii) positively associated with the pancreatic differentiation, the marker being selected from the group consisting of: TACSTD2 (TROP-2), GPR50, BST2, NTRK2, ITGA4, KDR, PTPRN, LGI1, VIPR2, SLC2A1, MUC15, MUC12, LPHN3, MUC16, VTCN1, MMP16, FZD3, ITGB6, GFRA3, ROBO1, NLGN1, MUC12, CNTFR, LPHN1, SULF1, ADAM23, SCUBE3, PLAU, CDON, SLIT2, C7orf68, PLXDC2, CD74, MUC15, GPR56, VTCN1, ITGB6, AREG, BOC, KLRK1, PRTG, PTPRZ1, GABRP, SILV, KIAA1772, PLP1, OVOS, HAPLN1, EPHA7, ENO2, PCDHB5, OVOS, SYT1, DCT, GPAM, SLITRK6, DCC, FREM2, SDK2, CGA, ATP1B2, SEMA3D, PCDHB15, CDH1, WNT8B, LPAR4, NPIPL3, FAM171B, PTN, ABCC2, ADAMTS3, RASA4, CPT1C, SLC6A6, PCDHB3, LRRC37B, RNFT2, KCNG3, TRPC1, ALPPL2, OR4F21, CCL2, KIF5A, OLFM2, CACNG7, MPHOSPH9, SLC13A4, MOXD1, C6orf186, SLC4A8, STX16, AMY2A, SPARCL1, MGP, A2M, DCN, ATP8B1, MMRN1, EMP1, PLA2G2A, PDE3A, TLR3, CYP1B1, PTGIS, RFTN2, PLEKHA2, SMOC1, STOM, JAM2, CHL1, SCG5, IGFBP7, NPR3, IFI6, CR1L, OR2A4, OR2A7, KCNG3, CACNG7, GRID2, CDH1, LPAR3, SEMA6A, PTPRZ1, ATP1A3, CAMKV, SCNN1G, SYT6, SLC18A2, PCDHB5, ABCG2, HLA-DRA, CR1L, HTR2C, EDNRB, PCDH11X, SLC17A7, SCNN1A, CD9, CXCL16, FXYD5, GABRQ, GFRA3, CACNA2D2, CLDN4, PDPN, MMP24, SDK2, GPR176, GPR64, GPR160, PCDH11Y, NKAIN4, ATP1B2, SCN8A, THBS4, CR2, HLA-DQA1, HLA-DRA, HTR7, SLC2A1, HLA-DRA, KCNS3, SLC7A3, HLA-DPB2, CACNA1B, GPR143, ALPPL2, DPPA5, H19, CRYZ, CXCL12, TYW3, ZYG11A, CRABP1, IDO1, POU5F1, HEY2, HIST1H1A, TFAP2C, DPPA2, ZFP42, LECT1, NECAB1, CKMT1A, SAMHD1, FGF2, PLA2G2A, PRDM14, POU5F1, GLI3, GSTT2, OLFML3, DAZL, GALNT3, SOX2, POU5F1B, ACTN3, CPT1A, DCLK1, EDIL3, NANOG, THUMPD3, VASH2, ATCAY, USP44, HIST1H4F, NANOG, PIM2, DNMT3B, ZNF483, FEZF1, SCARNA9L, and SILV, wherein an upregulation above a predetermined threshold of an expression level of the marker positively associated with the pancreatic differentiation as compared to the expression level of the marker in a reference cell, wherein said reference cell is a definite endodermal cell which expresses SOX17, is indicative of a pancreatic progenitor cell.

According to an aspect of some embodiments of the present invention there is provided a method of identifying pancreatic progenitor cells, comprising determining in a population of cells which comprises pancreatic progenitor cells at least one marker that is: (i) negatively associated with pancreatic differentiation, the marker being selected from the group consisting of: LGR5, CCKBR, CXCR4, FLRT3, TRPA1, SLC40A1, LRIG3, COLEC12, EPSTI1, GPR128, CDH2, SLC5A9, FSHR, SLC30A10, IL13RA1, SLC7A7, PCDH10, SLC1A1, GPR141, LIFR, TMEM27, GPC4, LYPD6B, FOLH1, TRPC4, PCDH7, KEL, KCNJ3, OR2T4, VIPR2, FLRT2, CD34, SLC39A8, CLDN11, CXCR7, ITGA5, ITGAV, CALCR, CLDN18, SLC7A5, TMBIM4, SLCO2A1, CDH10, AMHR2, ASAM, CLDN1, DSCAM, TMEM88, PLXNA2, CD177, TMEM144, GPR37, GJA5, SEMA6D, NIPAL2, GPR151, MCC, TMEM136, KCNG1, LHFPL2, MOSPD1, SLC37A1, LRIT3, EPHA4, GPR177, and IL1R1, wherein downregulation above a predetermined threshold of an expression level of the marker negatively associated with the pancreatic differentiation as compared to the expression level of the marker in a reference cell, wherein said reference cell is a definite endodermal cell which expresses SOX17, is indicative of a pancreatic progenitor cell, and alternatively or additionally; (ii) positively associated with pancreatic differentiation, the marker being selected from the group consisting of: TACSTD2 (TROP-2), GPR50, BST2, NTRK2, ITGA4, KDR, PTPRN, LGI1, VIPR2, SLC2A1, MUC15, MUC12, LPHN3, MUC16, VTCN1, MMP16, FZD3, ITGB6, GFRA3, ROBO1, NLGN1, MUC12, CNTFR, LPHN1, SULF1, ADAM23, SCUBE3, PLAU, CDON, SLIT2, C7orf68, PLXDC2, CD74, MUC15, GPR56, VTCN1, ITGB6, AREG, BOC, KLRK1, PRTG, PTPRZ1, GABRP, SILV, KIAA1772, PLP1, OVOS, HAPLN1, EPHA7, ENO2, PCDHB5, OVOS, SYT1, DCT, GPAM, SLITRK6, DCC, FREM2, SDK2, CGA, ATP1B2, SEMA3D, PCDHB15, CDH1, WNT8B, LPAR4, NPIPL3, FAM171B, PTN, ABCC2, ADAMTS3, RASA4, CPT1C, SLC6A6, PCDHB3, LRRC37B, RNFT2, KCNG3, TRPC1, ALPPL2, OR4F21, CCL2, KIF5A, OLFM2, CACNG7, MPHOSPH9, SLC13A4, MOXD1, C6orf186, SLC4A8, STX16, AMY2A, SPARCL1, MGP, A2M, DCN, ATP8B1, MMRN1, EMP1, PLA2G2A, PDE3A, TLR3, CYP1B1, PTGIS, RFTN2, PLEKHA2, SMOC1, STOM, JAM2, CHL1, SCG5, IGFBP7, NPR3, IFI6, CR1L, OR2A4, OR2A7, KCNG3, CACNG7, GRID2, CDH1, LPAR3, SEMA6A, PTPRZ1, ATP1A3, CAMKV, SCNN1G, SYT6, SLC18A2, PCDHB5, ABCG2, HLA-DRA, CR1L, HTR2C, EDNRB, PCDH11X, SLC17A7, SCNN1A, CD9, CXCL16, FXYD5, GABRQ, GFRA3, CACNA2D2, CLDN4, PTPRN, PLP1, PDPN, MMP24, SDK2, GPR176, GPR64, GPR160, PCDH11Y, NKAIN4, ATP1B2, SCN8A, THBS4, CR2, HLA-DQA1, HLA-DRA, HTR7, SLC2A1, HLA-DRA, KCNS3, SLC7A3, HLA-DPB2, CACNA1B, and GPR143, wherein upregulation above a predetermined threshold of an expression level of the marker positively associated with the pancreatic differentiation as compared to the expression level of the marker in a reference cell, wherein said reference cell is a definite endodermal cell which expresses SOX17, is indicative of a pancreatic progenitor cells, and alternatively or additionally; (iii) negatively associated with pancreatic differentiation, the marker being selected from the group consisting of: TMPRSS11E, LGR5, SLC39A8, TM4SF18, CUZD1, GPC4, SLC22A3, CXCR4, NRN1, TMBIM4, THBS2, SLC7A5, and TMEM47, wherein downregulation above a predetermined threshold of an expression level of the marker negatively associated with pancreatic differentiation as compared to the expression level of the marker in a reference cell, wherein said reference cell is an undifferentiated human embryonic stem cell (hESC), is indicative of a pancreatic progenitor cell, and alternatively or additionally; (iv) positively associated with pancreatic differentiation, the marker being selected from the group consisting of: TACSTD2 (TROP-2), LRP2, LAMA1, GPC3, LGI1, FN1, LPHN3, FGB, KIT, DPP4, TRO, SPA17, ROBO2, DLK1, BST2, PRTG, LRP2, CACNG7, DLK1, CACNA2D2, CR1L, SCNN1G, HTR2C, LPAR3, THBS3, KCNG3, SDK2, HLA-DRA, SLC18A2, CXCL16, TMEM63C, SLC17A7, GFRA3, PRTG, DKK1, APOA2, AFP, APOB, FLRT3, KIAA1772, FBN2, FRZB, KLK6, FREM2, RSPO3, APOA1, APOA1, SEMA3D, KEL, BMP5, HEPH, STMN2, CACNB3, CHST9, TMC7, FAM171B, ATP5G1, FOLR1, ABCC2, EFNA5, CPT1C, MAGED1, RASA4, KDELR3, ST6GALNAC2, EDNRA, LRRC37B, ABCC4, CLCN5, MBOAT2, SLC44A5, NPC2, ELFN1, MMD, SLC5A3, IMPAD1, OSTC, DSC2, SLC31A1, SLC5A12, LRRN3, NPIPL3, GDPD1, NPIPL3, NPIPL3, LINGO2, and LYPD6B, wherein upregulation above a predetermined threshold of an expression level of the marker positively associated with pancreatic differentiation as compared to the expression level of the marker in a reference cell, wherein said reference cell is an undifferentiated human embryonic stem cell (hESC), is indicative of a pancreatic progenitor cell; thereby identifying the pancreatic progenitor cells.

According to an aspect of some embodiments of the present invention there is provided a method of isolating pancreatic progenitor cells, comprising: (a) identifying the pancreatic progenitor cells according to the method of some embodiments of the invention, and (b) isolating the pancreatic progenitor cells identified according to step (a) to thereby obtain isolated pancreatic progenitor cells, thereby isolating the pancreatic progenitor cells.

According to an aspect of some embodiments of the present invention there is provided a method of qualifying a pancreatic progenitor cell population, comprising: determining in a sample of the cell population a percentage of the pancreatic progenitor cells which are identified according to the method of some embodiments of the invention out of the total cells in the sample, thereby qualifying the pancreatic progenitor cell population.

According to an aspect of some embodiments of the present invention there is provided a method of isolating endocrine progenitors or insulin producing cells, comprising culturing the pancreatic progenitor cells isolated by the method of some embodiments of the invention or the pancreatic progenitor cell population qualified according to the method of some embodiments of the invention under conditions suitable for maturation of the pancreatic progenitor cells into endocrine progenitors or beta cells, thereby generating insulin producing cells.

According to an aspect of some embodiments of the present invention there is provided a method of transplanting pancreatic progenitor cells or cells derived therefrom in a subject, comprising (a) qualifying the pancreatic progenitor cells according to the method of some embodiments of the invention, wherein presence of at least a predetermined percentage of the pancreatic progenitor cells in the cell sample indicates the suitability of the pancreatic progenitor cells for transplantation in a subject, to thereby obtain a pancreatic progenitor cell population being suitable for transplantation in a subject, (b) transplanting in the subject the pancreatic progenitor cell population being suitable for transplantation in a subject or cells derived therefrom, thereby transplanting the pancreatic progenitor cells or cells derived therefrom in the subject.

According to an aspect of some embodiments of the present invention there is provided a method of identifying definite endodermal cells, comprising determining in a population of cells which comprises definite endodermal cells at least one marker that is negatively associated with definite endodermal cells, the marker being selected from the group consisting of: KDR, PCDHB5, FAT4, FLT1, NRN1, THBS2, PTPRZ1, SLC6A15, GPR176, SEMA6A, THBS1, CDH11, GRID2, SLC7A11, CDH1, LRFN5, EDNRB, THY1, NETO1, KCND2, TMPRSS11E, CD44, PDPN, SLC7A1, KAL1, KCNG3, GPM6B, FXYD5, PCDH18, ICAM3, MCTP1, TACR3, and TMEM155, wherein downregulation above a predetermined threshold of an expression level of the marker negatively associated with the definite endodermal cells as compared to the expression level of the marker in a reference cell, wherein said reference cell is an undifferentiated human embryonic stem cell (hESC), is indicative of a definite endodermal cell.

According to an aspect of some embodiments of the present invention there is provided a method of identifying definite endodermal cells, comprising determining in a population of cells which comprises definite endodermal cells at least one marker that is positively associated with definite endodermal cells, the marker being selected from the group consisting of: FSHR, COLEC12, ROR2 ITGA5, LRP2, CD177, CCKBR, TRPA1, KEL, FOLR1, FOLH1, APOA1, APOA1, FZD5, FN1, BMP2, ADAMTS9, DPP4, FGA, GPR128, IGFBP5, LIFR, FZD4, PRTG, STC1, TNFSF4, CD177, IHH, LAMA1, GPC3, LGI1, FN1, LPHN3, FGB, KIT, DPP4, TRO, SPA17, ROBO2, DLK1, CST1, DKK1, HAS2, APOA2, CDH2, LGR5, AFP, FLRT3, APOB, DGKK, HP, SYTL5, SLC7A7, TTR, FRZB, RSPO3, GCNT1, DIO3, TMEM27, APOC1, EPHA4, C4orf18, GLIPR2, PCDH10, F10, BMP5, FMO5, STMN2, SLC5A9, PORCN, EGFLAM, RAB17, CST2, PNPLA3, MOSPD1, ELMO1, CHST9, SLC30A10, TMC7, C8orf49, CDH12, ST8SIA4, SLCO2A1, MANEA, LRIG3, HCN1, ADAMTS12, TMEM144, VTN, CAMK2N1, ABCC4, PCDH7, OR2T4, UNC93A, COL4A6, PAMR1, SLC1A1, PROS1, APOM, APOM, APOM, LYPD6B, TMEM88, ITLN2, BMPER, GPR141, VEGFA, DKK1, APOA2, AFP, APOB, FLRT3, KIAA1772, FBN2, FRZB, KLK6, FREM2, RSPO3, APOA1, APOA1, SEMA3D, KEL, BMP5, HEPH, STMN2, CACNB3, CHST9, TMC7, FAM171B, ATP5G1, FOLR1, ABCC2, EFNA5, CPT1C, MAGED1, RASA4, KDELR3, ST6GALNAC2, EDNRA, LRRC37B, ABCC4, CLCN5, MBOAT2, SLC44A5, NPC2, ELFN1, MMD, SLC5A3, IMPAD1, OSTC, DSC2, SLC31A1, SLC5A12, LRRN3, NPIPL3, GDPD1, NPIPL3, NPIPL3, LINGO2, LYPD6B, TRPA1, SLC40A1, SLC30A10, CCKBR, VIPR2, COLEC12, FLRT3, LGR5, GPR141, BST2, SLC5A9, GPR128, KEL, LRIG3, LYPD6B, FSHR, LIFR, FOLH1, CXCR4, ITGA5, AMOT, LY6E, SEMA6D, GJA5, PRTG, CD34, TMEM144, ROR2, GPR177, OR2T4, SLC7A7, KCNJ3, CLDN18, GPR151, SLC44A5, CDH10, TMEM27, SLC1A1, TMEM56, CD177, PLXNA2, SLC26A2, DSCAM, TMEM133, IL13RA1, ATP2B1, CD302, MEGF9, EDNRA, CDH2, GPR161, TYRO3, FLRT2, LRIT3, PCDH7, NRCAM, SMAGP, AMHR2, ELTD1, GRPR, EPHA4, and CD99, wherein upregulation above a predetermined threshold of an expression level of the marker positively associated with the definite endodermal cells as compared to the expression level of the marker in a reference cell, wherein said reference cell is an undifferentiated human embryonic stem cell (hESC), is indicative of a definite endodermal cell.

According to an aspect of some embodiments of the present invention there is provided a method of isolating definite endodermal cells, comprising: (a) identifying the definite endodermal cells according to the method of some embodiments of the invention, (b) isolating the definite endodermal cells identified according to step (a) to thereby obtain an isolated population of the definite endodermal cells, thereby isolating the definite endodermal cells.

According to an aspect of some embodiments of the present invention there is provided a method of qualifying a definite endodermal cell population, comprising: determining in a sample of the cell population a percentage of the definite endodermal cells which are identified according to the method of some embodiments of the invention out of the total cells in the sample, thereby qualifying the definite endodermal cell population.

According to an aspect of some embodiments of the present invention there is provided an isolated population of pancreatic progenitor cells obtained according to the method of some embodiments of the invention.

According to an aspect of some embodiments of the present invention there is provided an isolated population of pancreatic progenitor cells, comprising at least 75% of cells having a TROP-2+ and/or TROP-2+/GPR50+ expression pattern.

According to an aspect of some embodiments of the present invention there is provided an isolated population of definite endodermal cells obtained according to the method of some embodiments of the invention.

According to an aspect of some embodiments of the present invention there is provided an isolated population of definite endodermal cells, comprising at least 50% of cells having a SOX17+/SOX7+ expression pattern.

According to an aspect of some embodiments of the present invention there is provided a nucleic acid construct comprising a first polynucleotide encoding a reporter protein and a second polynucleotide which comprises a human endogenous SOX17 regulatory sequence, wherein the first polynucleotide being under transcriptional regulation of the SOX17 regulatory sequence, wherein the SOX17 regulatory sequence comprises an upstream sequence and a downstream sequence, wherein the upstream sequence comprises the nucleotide sequence set forth in SEQ ID NO:38; and wherein the downstream sequence comprises the nucleotide sequence set forth in SEQ ID NO:39.

According to an aspect of some embodiments of the invention there is provided a nucleic acid construct comprising a first polynucleotide encoding a reporter protein and a second polynucleotide which comprises a human endogenous PDX1 regulatory sequence, wherein the first polynucleotide being under transcriptional regulation of the PDX1 regulatory sequence, wherein the PDX1 regulatory sequence comprises an upstream sequence and a downstream sequence, wherein the upstream sequence comprises the nucleotide sequence set forth in SEQ ID NO:16; and wherein the downstream sequence comprises the nucleotide sequence set forth in SEQ ID NO:17.

According to an aspect of some embodiments of the present invention there is provided a cell comprising the nucleic acid construct of some embodiments of the invention.

According to an aspect of some embodiments of the present invention there is provided a method of screening for markers which differentiate a definite endodermal cell from an undifferentiated pluripotent stem cell, comprising comparing the expression level of markers between the undifferentiated pluripotent stem cell and the cell of some embodiments of the invention, wherein upregulation or downregulation in the expression level above a predetermined threshold indicates that the markers differentiate the definite endodermal cell from the undifferentiated pluripotent stem cell, thereby screening for markers which differentiate the definite endodermal cell from the undifferentiated pluripotent stem cell.

According to an aspect of some embodiments of the present invention there is provided a method of screening for compounds capable of inducing differentiation of undifferentiated pluripotent stem cells to definite endodermal cells, comprising: (a) contacting undifferentiated pluripotent stem cells which comprise the nucleic acid construct of some embodiments of the invention with at least one compound of a plurality of candidate compounds, and; (b) monitoring an expression level of the reporter protein in the cells following the contacting, wherein an increase above a predetermined level in the expression level of the reporter protein following the contacting as compared to the expression level prior to the contacting is indicative that the at least one compound is capable of inducing differentiation of the undifferentiated pluripotent stem cells to the definite endodermal cells, thereby screening for the compounds capable of inducing differentiation of undifferentiated pluripotent stem cells to definite endodermal cells.

According to an aspect of some embodiments of the present invention there is provided a cell comprising the nucleic acid construct of some embodiments of the invention.

According to an aspect of some embodiments of the present invention there is provided a method of screening for markers which differentiate a pancreatic progenitor cell from a definite endodermal cell, comprising comparing the expression level of markers between the cell of some embodiments of the invention and the cell of some embodiments of the invention, wherein upregulation or downregulation in the expression level above a predetermined threshold indicates that the markers differentiate the pancreatic progenitor cell from the definite endodermal cell, thereby screening for markers which differentiate the pancreatic progenitor cell from the definite endodermal cell.

According to an aspect of some embodiments of the present invention there is provided a method of screening for compounds capable of inducing differentiation of definite endodermal cells or undifferentiated pluripotent stem cells to pancreatic progenitor cells, comprising: (a) contacting definite endodermal cells or undifferentiated pluripotent stem cells which comprise the nucleic acid construct of some embodiments of the invention with at least one compound of a plurality of candidate compounds, and; (b) monitoring an expression level of the reporter protein in the cells following the contacting, wherein an increase above a predetermined level in the expression level of the reporter protein following the contacting as compared to the expression level prior to the contacting is indicative that the at least one compound is capable of inducing differentiation of the definite endodermal cells or undifferentiated pluripotent stem cells to the pancreatic progenitor cells, thereby screening for the compounds capable of inducing differentiation of definite endodermal cells or undifferentiated pluripotent stem cells to the pancreatic progenitor cells.

According to an aspect of some embodiments of the present invention there is provided a kit for screening for markers which differentiate a definite endodermal cell from a pluripotent stem cell, comprising the cell of some embodiments of the invention.

According to an aspect of some embodiments of the present invention there is provided a kit for screening for markers which differentiate a pancreatic progenitor cell from a definite endodermal cell, comprising the cell of some embodiments of the invention and the cell of some embodiments of the invention.

According to some embodiments of the invention, the method further comprising determining in a population of cells which comprises pancreatic progenitor cells at least one marker that is: (i) negatively associated with the pancreatic differentiation, the marker being selected from the group consisting of: CST1, CER1, ANKRD1, TRY6, HAS2, DKK1, PRSS2, HP, APOA2, RHOBTB3, BMP2, ACE2, STC1, PDZK1, HHEX, VIL1, PRDM1, EOMES, DNAJC15, TNIK, IGFBP5, RLBP1L2, ADAMTS9, EPSTI1, C5, ARHGAP24, TRY6, ANGPT2, TTR, MYL7, FST, KITLG, GATA3, ST8SIA4, CCDC141, TSPYL5, EGFLAM, TTN, LEFTY2, FOXA2, FAM184A, STMN2, DIO3, FN1, PRSS1, NPPB, and OTX2, wherein a downregulation above a predetermined threshold of an expression level of the marker negatively associated with the pancreatic differentiation as compared to the expression level of the marker in a reference cell, wherein said reference cell is a definite endodermal cell which expresses SOX17, is indicative of a pancreatic progenitor cell and alternatively or additionally; (ii) positively associated with the pancreatic differentiation, the marker being selected from the group consisting of: ALPPL2, DPPA5, H19, CRYZ, CXCL12, TYW3, ZYG11A, CRABP1, IDO1, POU5F1, HEY2, HIST1H1A, TFAP2C, DPPA2, ZFP42, LECT1, NECAB1, CKMT1A, SAMHD1, FGF2, PLA2G2A, PRDM14, POU5F1, GLI3, GSTT2, OLFML3, DAZL, GALNT3, SOX2, POU5F1B, ACTN3, CPT1A, DCLK1, EDIL3, NANOG, THUMPD3, VASH2, ATCAY, USP44, HIST1H4F, NANOG, PIM2, DNMT3B, ZNF483, FEZF1, SCARNA9L, and SILV, wherein an upregulation above a predetermined threshold of an expression level of the marker positively associated with the pancreatic differentiation as compared to the expression level of the marker in a reference cell, wherein said reference cell is a definite endodermal cell which expresses SOX17, is indicative of a pancreatic progenitor cell.

According to some embodiments of the invention, the method further comprising determining in a population of cells which comprises pancreatic progenitor cells at least one marker that is: (i) negatively associated with the pancreatic differentiation, the marker being selected from the group consisting of: TMPRSS11E, LGR5, SLC39A8, TM4SF18, CUZD1, GPC4, SLC22A3, CXCR4, NRN1, TMBIM4, THBS2, SLC7A5, TMEM47, NTS, CER1, CST1, NODAL, PRRX1, KGFLP1, NFIB, GCNT4, MIXL1, CAV1, LUM, RASGRF2, OXCT1, GLIPR1, VSNL1, FST, POSTN, GNA14, CBR1, TNIK, RGS5, KGFLP1, ETS1, MPPED2, ACTA2, SEMA3A, DACT1, ANXA1, COL12A1, KITLG, MMP2, DLEU2, ACE2, ACTG2, PUS7L, RNU5B-1, COL3A1, LEFTY2, NPPB, S1AE, AFP, ZFP42, HAS2, TRY6, NR5A2, and EBF1, wherein a downregulation above a predetermined threshold of an expression level of the marker negatively associated with the pancreatic differentiation as compared to the expression level of the marker in a reference cell, wherein said reference cell is an undifferentiated human embryonic stem cell (hESC), is indicative of a pancreatic progenitor cell, and alternatively or additionally; (ii) positively associated with the pancreatic differentiation, the marker being selected from the group consisting of: TACSTD2 (TROP-2), BST2 LRP2, LAMA1, GPC3, LGI1, FN1, LPHN3, FGB, KIT, DPP4, TRO, SPA17, ROBO2, DLK1, PRTG, DKK1, APOA2, AFP, APOB, FLRT3, KIAA1772, FBN2, FRZB, KLK6, FREM2, RSPO3, APOA1, APOA1, SEMA3D, KEL, BMP5, HEPH, STMN2, CACNB3, CHST9, TMC7, FAM171B, ATP5G1, FOLR1, ABCC2, EFNA5, CPT1C, MAGED1, RASA4, KDELR3, ST6GALNAC2, EDNRA, LRRC37B, ABCC4, CLCN5, MBOAT2, SLC44A5, NPC2, ELFN1, MMD, SLC5A3, IMPAD1, OSTC, DSC2, SLC31A1, SLC5A12, LRRN3, NPIPL3, GDPD1, NPIPL3, NPIPL3, LINGO2, LYPD6B, PRTG, LRP2, CACNG7, DLK1, CACNA2D2, CR1L, SCNN1G, HTR2C, LPAR3, THBS3, KCNG3, SDK2, HLA-DRA, SLC18A2, CXCL16, TMEM63C, SLC17A7, GFRA3, DPPA5, ALPPL2, CRYZ, H19, ZYG11A, TYW3, ARRDC4, TXNIP, FOS, NCRNA00173, GSTT2, C3, DDX43, B2M, PYGM, CYP4F22, ZNF578, ENO2, ZNF248, NLRP4, SNORD59B, SNORD113-4, CASZ1, MIR21, IFI16, ZNF560, TDRD1, ZNF680, HSPA1B, HSPA1A, EGR1, RASGRP2, ASMTL, CPT1C, ATCAY, ACSF2, PAMR1, SILV, ACTN3, HORMAD1, ACSM3, RNF157, SAMHD1, and RCN1, wherein an upregulation above a predetermined threshold of an expression level of the marker positively associated with the pancreatic differentiation as compared to the expression level of the marker in a reference cell, wherein said reference cell is an undifferentiated human embryonic stem cell (hESC), is indicative of a pancreatic progenitor cell.

According to some embodiments of the invention, the population of cells which comprises pancreatic progenitor cells express a transcription factor selected from the group consisting of PDX1, ngn3, pax4, hlxb9, nkx6.1, Hnf6, and sox9.

According to some embodiments of the invention, the definite endodermal cells are identified by a method comprising determining in a population of cells which comprises definite endodermal cells at least one marker that is: (i) negatively associated with definite endodermal cells, the marker being selected from the group consisting of: KDR, PCDHB5, FAT4, FLT1, NRN1, THBS2, PTPRZ1, SLC6A15, GPR176, SEMA6A, THBS1, CDH11, GRID2, SLC7A11, CDH1, LRFN5, EDNRB, THY1, NETO1, KCND2, TMPRSS11E, CD44, PDPN, SLC7A1, KAL1, KCNG3, GPM6B, FXYD5, PCDH18, ICAM3, MCTP1, TACR3, TMEM155, ZFP42, THUMPD3, ANXA1, SPP1, PRDM14, GNA14, EDIL3, CXCL12, PSMD5, PRRX1, NANOG, TRIM22, NANOG, RASGRF2, POU5F1B, POLR3G, HHLA1, POU5F1, VSNL1, SCG3, B3GALT1, LECT1, NTS, MBNL1, CKMT1A, NECAB1, FGF2, SFRP2, DCLK1, DACT1, CRABP1, TFAP2C, SCGB3A2, LRAT, CUZD1, GLB1L3, METTL7A, VAT1L, COL12A1, OLFML3, SOX2, USP44, HIST1H4F, KGFLP1, CPT1A, and DBC1, wherein downregulation above a predetermined threshold of an expression level of the marker negatively associated with definite endodermal cells as compared to the expression level of the marker in a reference cell, wherein said reference cell is an undifferentiated human embryonic stem cell (hESC), is indicative of a definite endodermal cell, and alternatively or additionally; (ii) positively associated with definite endodermal cells, the marker being selected from the group consisting of: FLRT3, FSHR, LIFR, ROR2, KEL, TRPA1, CD177, CCKBR, APOA1, APOA1, FZD5, FN1, BMP2, ADAMTS9, DPP4, FGA, COLEC12, GPR128, IGFBP5, FZD4, ITGA5, STC1, TNFSF4, CD177, IHH, LRP2, LAMA1, GPC3, LGI1, FN1, LPHN3, FGB, KIT, DPP4, TRO, SPA17, ROBO2, DLK1, CST1, DKK1, HAS2, APOA2, CDH2, LGR5, AFP, APOB, DGKK, HP, SYTL5, SLC7A7, TTR, FRZB, RSPO3, GCNT1, DIO3, TMEM27, APOC1, EPHA4, C4orf18, GLIPR2, PCDH10, F10, BMP5, FMO5, FOLR1, STMN2, SLC5A9, PORCN, EGFLAM, RAB17, CST2, PNPLA3, MOSPD1, ELMO1, CHST9, SLC30A10, TMC7, C8orf49, CDH12, ST8SIA4, SLCO2A1, MANEA, LRIG3, HCN1, ADAMTS12, FOLH1, TMEM144, VTN, CAMK2N1, ABCC4, PCDH7, OR2T4, UNC93A, COL4A6, PAMR1, SLC1A1, PROS1, APOM, APOM, APOM, LYPD6B, TMEM88, ITLN2, BMPER, GPR141, VEGFA, PRTG, DKK1, APOA2, AFP, APOB, FLRT3, KIAA1772, FBN2, FRZB, KLK6, FREM2, RSPO3, APOA1, APOA1, SEMA3D, KEL, BMP5, HEPH, STMN2, CACNB3, CHST9, TMC7, FAM171B, ATP5G1, FOLR1, ABCC2, EFNA5, CPT1C, MAGED1, RASA4, KDELR3, ST6GALNAC2, EDNRA, LRRC37B, ABCC4, CLCN5, MBOAT2, SLC44A5, NPC2, ELFN1, MMD, SLC5A3, IMPAD1, OSTC, DSC2, SLC31A1, SLC5A12, LRRN3, NPIPL3, GDPD1, NPIPL3, NPIPL3, LINGO2, SLC40A1, SLC30A10, CCKBR, VIPR2, COLEC12, FLRT3, LGR5, GPR141, BST2, SLC5A9, GPR128, KEL, LRIG3, LYPD6B, FSHR, LIFR, FOLH1, CXCR4, ITGA5, AMOT, LY6E, SEMA6D, GJA5, PRTG, CD34, TMEM144, ROR2, GPR177, OR2T4, SLC7A7, KCNJ3, CLDN18, GPR151, SLC44A5, CDH10, TMEM27, SLC1A1, TMEM56, CD177, PLXNA2, SLC26A2, DSCAM, TMEM133, IL13RA1, ATP2B1, CD302, MEGF9, EDNRA, CDH2, GPR161, TYRO3, FLRT2, LRIT3, PCDH7, NRCAM, SMAGP, AMHR2, ELTD1, GRPR, EPHA4, CD99, GATA3, SEMA3E, HHEX, ZNF280A, FAM184A, WDR72, PDZK1, RLBP1L2, SHISA2, VIL1, STMN2, APOA2, SERHL, PPFIBP2, DKK1, MUM1L1, IGFBP5, ST6GALNAC2, TSPYL5, STC1, SYTL5, EPSTI1, ANKRD1, ARHGAP24, KRT18P49, PRSS2, RHOBTB3, FRZB, RARB, ADAMTS9, ARL4D, PRDM1, HP, FZD5, TRY6, ATP6V0D2, ANGPT2, DENND2C, BMP5, FOXA2, HAS2, BMP2, S100A16, FOLH1B, FAM122C, and FZD4, wherein upregulation above a predetermined threshold of an expression level of the marker positively associated with definite endodermal cells as compared to the expression level of the marker in a reference cell, wherein said reference cell is an undifferentiated human embryonic stem cell (hESC), is indicative of a definite endodermal cell, thereby identifying the definite endodermal cells.

According to some embodiments of the invention, the at least one marker positively associated with pancreatic differentiation is selected from the group consisting of: TACSTD2 (TROP-2), GPR50, BST2, LGI1, VIPR2, SLC2A1, MUC15, MUC12, LPHN3, MUC16, VTCN1, MMP16, FZD3, ITGB6, GFRA3, ROBO1, NLGN1, MUC12, CNTFR, LPHN1, SULF1, ADAM23, SCUBE3, PLAU, CDON, SLIT2, C7orf68, PLXDC2, CD74, MUC15, GPR56, VTCN1, ITGB6, NTRK2, AREG, BOC, ITGA4, KLRK1, PRTG, PTPRZ1, GABRP, SILV, KIAA1772, PLP1, OVOS, HAPLN1, EPHA7, ENO2, PCDHB5, OVOS, SYT1, DCT, GPAM, SLITRK6, DCC, FREM2, SDK2, CGA, ATP1B2, SEMA3D, PCDHB15, CDH1, WNT8B, LPAR4, NPIPL3, FAM171B, PTN, ABCC2, ADAMTS3, RASA4, CPT1C, SLC6A6, PCDHB3, LRRC37B, RNFT2, KCNG3, TRPC1, ALPPL2, OR4F21, CCL2, KIF5A, OLFM2, CACNG7, MPHOSPH9, SLC13A4, MOXD1, C6orf186, SLC4A8, STX16, AMY2A, SPARCL1, MGP, A2M, DCN, ATP8B1, MMRN1, EMP1, PLA2G2A, PDE3A, TLR3, CYP1B1, PTGIS, RFTN2, PLEKHA2, SMOC1, STOM, JAM2, CHL1, SCG5, IGFBP7, NPR3, IFI6, CR1L, OR2A4, and OR2A7.

According to some embodiments of the invention, the at least one marker positively associated with pancreatic differentiation is selected from the group consisting of: TACSTD2 (TROP-2), BST2, GPR50, ROBO1, NTRK2, ITGA4, LGI1, VIPR2, SLC2A1, MUC15, MUC12, LPHN3, MUC16, VTCN1, MMP16, FZD3, ITGB6, GFRA3, NLGN1, MUC12, CNTFR, LPHN1, SULF1, ADAM23, SCUBE3, PLAU, CDON, SLIT2, C7orf68, PLXDC2, CD74, MUC15, GPR56, VTCN1, ITGB6, AREG, BOC, and KLRK1.

According to some embodiments of the invention, the at least one marker positively associated with pancreatic differentiation is TROP-2.

According to some embodiments of the invention, the at least one marker positively associated with pancreatic differentiation is GPR50.

According to some embodiments of the invention, the at least one marker positively associated with pancreatic differentiation comprises at least two markers, said at least two markers are TROP-2 and GPR50.

According to some embodiments of the invention, the at least one marker positively associated with pancreatic differentiation comprises at least three markers, said at least three markers comprise TROP-2, GPR50 and a marker selected from the group consisting of BST2, NTRK2, ITGA4, KDR, PTPRN, LGI1, VIPR2, SLC2A1, MUC15, MUC12, LPHN3, MUC16, VTCN1, MMP16, FZD3, ITGB6, GFRA3, ROBO1, NLGN1, MUC12, CNTFR, LPHN1, SULF1, ADAM23, SCUBE3, PLAU, CDON, SLIT2, C7orf68, PLXDC2, CD74, MUC15, GPR56, VTCN1, ITGB6, AREG, BOC, KLRK1, PRTG, PTPRZ1, GABRP, SILV, KIAA1772, PLP1, OVOS, HAPLN1, EPHA7, ENO2, PCDHB5, OVOS, SYT1, DCT, GPAM, SLITRK6, DCC, FREM2, SDK2, CGA, ATP1B2, SEMA3D, PCDHB15, CDH1, WNT8B, LPAR4, NPIPL3, FAM171B, PTN, ABCC2, ADAMTS3, RASA4, CPT1C, SLC6A6, PCDHB3, LRRC37B, RNFT2, KCNG3, TRPC1, ALPPL2, OR4F21, CCL2, KIF5A, OLFM2, CACNG7, MPHOSPH9, SLC13A4, MOXD1, C6orf186, SLC4A8, STX16, AMY2A, SPARCL1, MGP, A2M, DCN, ATP8B1, MMRN1, EMP1, PLA2G2A, PDE3A, TLR3, CYP1B1, PTGIS, RFTN2, PLEKHA2, SMOC1, STOM, JAM2, CHL1, SCG5, IGFBP7, NPR3, IFI6, CR1L, OR2A4, OR2A7, KCNG3, CACNG7, GRID2, CDH1, LPAR3, SEMA6A, PTPRZ1, ATP1A3, CAMKV, SCNN1G, SYT6, SLC18A2, PCDHB5, ABCG2, HLA-DRA, CR1L, HTR2C, EDNRB, PCDH11X, SLC17A7, SCNN1A, CD9, CXCL16, FXYD5, GABRQ, GFRA3, CACNA2D2, CLDN4, PLP1, PDPN, MMP24, SDK2, GPR176, GPR64, GPR160, PCDH11Y, NKAIN4, ATP1B2, SCN8A, THBS4, CR2, HLA-DQA1, HLA-DRA, HTR7, SLC2A1, HLA-DRA, KCNS3, SLC7A3, HLA-DPB2, CACNA1B, and GPR143

According to some embodiments of the invention, the at least one marker positively associated with definite endodermal cells is selected from the group consisting of: COLEC12, ROR2, FLRT3, LGR5, LIFR, KEL, FSHR, TRPA1, FOLR1, LRP2, FOLH1, CD177, CCKBR, ITGA5, FZD5, FN1, BMP2, ADAMTS9, DPP4, FGA, GPR128, IGFBP5, FZD4, STC1, TNFSF4, CD177, IHH, APOA1, LAMA1, GPC3, LGI1, FN1, LPHN3, FGB, KIT, DPP4, TRO, SPA17, ROBO2, DLK1, CST1, DKK1, HAS2, APOA2, CDH2, AFP, APOB, DGKK, HP, SYTL5, SLC7A7, TTR, FRZB, RSPO3, GCNT1, DIO3, TMEM27, APOC1, EPHA4, C4orf18, GLIPR2, PCDH10, F10, BMP5, FMO5, STMN2, SLC5A9, PORCN, EGFLAM, RAB17, CST2, PNPLA3, MOSPD1, ELMO1, CHST9, SLC30A10, TMC7, C8orf49, CDH12, ST8SIA4, SLCO2A1, MANEA, LRIG3, HCN1, ADAMTS12, TMEM144, VTN, CAMK2N1, ABCC4, PCDH7, OR2T4, UNC93A, COL4A6, PAMR1, SLC1A1, PROS1, APOM, APOM, APOM, LYPD6B, TMEM88, ITLN2, BMPER, GPR141, VEGFA, PRTG, DKK1, APOA2, AFP, APOB, FLRT3, KIAA1772, FBN2, FRZB, KLK6, FREM2, RSPO3, APOA1, APOA1, SEMA3D, KEL, BMP5, HEPH, STMN2, CACNB3, CHST9, TMC7, FAM171B, ATP5G1, FOLR1, ABCC2, EFNA5, CPT1C, MAGED1, RASA4, KDELR3, ST6GALNAC2, EDNRA, LRRC37B, ABCC4, CLCN5, MBOAT2, SLC44A5, NPC2, ELFN1, MMD, SLC5A3, IMPAD1, OSTC, DSC2, SLC31A1, SLC5A12, LRRN3, NPIPL3, GDPD1, NPIPL3, NPIPL3, LINGO2, and LYPD6B.

According to some embodiments of the invention, the at least one marker positively associated with definite endodermal cells is selected from the group consisting of: FSHR, LIFR, COLEC12, ROR2, ITGA5, CD177, CCKBR, APOA1, APOA1, FZD5, FN1, BMP2, ADAMTS9, DPP4, FGA, GPR128, IGFBP5, FZD4, STC1, TNFSF4, CD177, IHH, LRP2, LAMA1, GPC3, LGI1, FN1, LPHN3, FGB, KIT, DPP4, TRO, SPA17, ROBO2, and DLK1.

According to some embodiments of the invention, the cell sample comprises the isolated pancreatic progenitor cells of some embodiments of the invention.

According to some embodiments of the invention, presence of at least a predetermined percentage of the pancreatic progenitor cells in the cell sample indicates the suitability of the pancreatic progenitor cells for transplantation in a subject.

According to some embodiments of the invention, the method further comprising determining in the population of cells which comprises definite endodermal cells at least one marker that is positively associated with definite endodermal cells, the marker being selected from the group consisting of: FSHR, COLEC12, ROR2, LIFR, LIFR, FLRT3, KEL, LGR5, FOLR1, CD177, CCKBR, APOA1, APOA1, FZD5, FN1, BMP2, ADAMTS9, DPP4, FGA, GPR128, IGFBP5, FZD4, ITGA5, STC1, TNFSF4, CD177, IHH, LRP2, LAMA1, GPC3, LGI1, FN1, LPHN3, FGB, KIT, DPP4, TRO, SPA17, ROBO2, DLK1, CST1, DKK1, HAS2, APOA2, CDH2, AFP, APOB, DGKK, HP, SYTL5, SLC7A7, TTR, FRZB, RSPO3, GCNT1, TRPA1, DIO3, TMEM27, APOC1, EPHA4, C4orf18, GLIPR2, PCDH10, F10, BMP5, FMO5, STMN2, SLC5A9, PORCN, EGFLAM, RAB17, CST2, PNPLA3, MOSPD1, ELMO1, CHST9, SLC30A10, TMC7, C8orf49, CDH12, ST8SIA4, SLCO2A1, MANEA, LRIG3, HCN1, ADAMTS12, FOLH1, TMEM144, VTN, CAMK2N1, ABCC4, PCDH7, OR2T4, UNC93A, COL4A6, PAMR1, SLC1A1, PROS1, APOM, APOM, APOM, LYPD6B, TMEM88, ITLN2, BMPER, GPR141, VEGFA, PRTG, DKK1, APOA2, AFP, APOB, FLRT3, KIAA1772, FBN2, FRZB, KLK6, FREM2, RSPO3, APOA1, APOA1, SEMA3D, KEL, BMP5, HEPH, STMN2, CACNB3, CHST9, TMC7, FAM171B, ATP5G1, FOLR1, ABCC2, EFNA5, CPT1C, MAGED1, RASA4, KDELR3, ST6GALNAC2, EDNRA, LRRC37B, ABCC4, CLCN5, MBOAT2, SLC44A5, NPC2, ELFN1, MMD, SLC5A3, IMPAD1, OSTC, DSC2, SLC31A1, SLC5A12, LRRN3, NPIPL3, GDPD1, NPIPL3, NPIPL3, LINGO2, LYPD6B, TRPA1, SLC40A1, SLC30A10, CCKBR, VIPR2, COLEC12, FLRT3, LGR5, GPR141, BST2, SLC5A9, GPR128, KEL, LRIG3, LYPD6B, FSHR, LIFR, FOLH1, CXCR4, ITGA5, AMOT, LY6E, SEMA6D, GJA5, PRTG, CD34, TMEM144, ROR2, GPR177, OR2T4, SLC7A7, KCNJ3, CLDN18, GPR151, SLC44A5, CDH10, TMEM27, SLC1A1, TMEM56, CD177, PLXNA2, SLC26A2, DSCAM, TMEM133, IL13RA1, ATP2B1, CD302, MEGF9, EDNRA, CDH2, GPR161, TYRO3, FLRT2, LRIT3, PCDH7, NRCAM, SMAGP, AMHR2, ELTD1, GRPR, EPHA4, CD99, GATA3, SEMA3E, HHEX, ZNF280A, FAM184A, WDR72, PDZK1, RLBP1L2, SHISA2, VIL1, STMN2, APOA2, SERHL, PPFIBP2, DKK1, MUM1L1, IGFBP5, ST6GALNAC2, TSPYL5, STC1, SYTL5, EPSTI1, ANKRD1, ARHGAP24, KRT18P49, PRSS2, RHOBTB3, FRZB, RARB, ADAMTS9, ARL4D, PRDM1, HP, FZD5, TRY6, ATP6V0D2, ANGPT2, DENND2C, BMP5, FOXA2, HAS2, BMP2, S100A16, FOLH1B, FAM122C, and FZD4, wherein upregulation above a predetermined threshold of an expression level of the marker positively associated with definite endodermal cells as compared to the expression level of the marker in a reference cell, wherein said reference cell is an undifferentiated human embryonic stem cell (hESC), is indicative of a definite endodermal cell.

According to some embodiments of the invention, the method further comprising determining in the population of cells which comprises definite endodermal cells at least one marker that is positively associated with definite endodermal cells, the marker being selected from the group consisting of: GATA3, SEMA3E, HHEX, ZNF280A, FAM184A, WDR72, PDZK1, RLBP1L2, SHISA2, VIL1, STMN2, APOA2, SERHL, PPFIBP2, DKK1, MUM1L1, IGFBP5, ST6GALNAC2, TSPYL5, STC1, SYTL5, EPSTI1, ANKRD1, ARHGAP24, KRT18P49, PRSS2, RHOBTB3, FRZB, RARB, ADAMTS9, ARL4D, PRDM1, HP, FZD5, TRY6, ATP6V0D2, ANGPT2, DENND2C, BMP5, FOXA2, HAS2, BMP2, S100A16, FOLH1B, FAM122C, and FZD4, wherein upregulation above a predetermined threshold of an expression level of the definite endodermal cells as compared to the expression level of the marker in a reference cell, wherein said reference cell is an undifferentiated human embryonic stem cell (hESC), is indicative of a definite endodermal cell.

According to some embodiments of the invention, the method further comprising determining in the population of cells which comprises definite endodermal cells at least one marker that is negatively associated with definite endodermal cells, the marker being selected from the group consisting of: KDR, PCDHB5, FAT4, FLT1, NRN1, THBS2, PTPRZ1, SLC6A15, GPR176, SEMA6A, THBS1, CDH11, GRID2, SLC7A11, CDH1, LRFN5, EDNRB, THY1, NETO1, KCND2, TMPRSS11E, CD44, PDPN, SLC7A1, KAL1, KCNG3, GPM6B, FXYD5, PCDH18, ICAM3, MCTP1, TACR3, TMEM155, ZFP42, THUMPD3, ANXA1, SPP1, PRDM14, GNA14, EDIL3, CXCL12, PSMD5, PRRX1, NANOG, TRIM22, NANOG, RASGRF2, POU5F1B, POLR3G, HHLA1, POU5F1, VSNL1, SCG3, B3GALT1, LECT1, NTS, MBNL1, CKMT1A, NECAB1, FGF2, SFRP2, DCLK1, DACT1, CRABP1, TFAP2C, SCGB3A2, LRAT, CUZD1, GLB1L3, METTL7A, VAT1L, COL12A1, OLFML3, SOX2, USP44, HIST1H4F, KGFLP1, CPT1A, and DBC1, wherein downregulation above a predetermined threshold of an expression level of the marker negatively associated with definite endodermal cells as compared to the expression level of the marker in a reference cell, wherein said reference cell is an undifferentiated human embryonic stem cell (hESC), is indicative of a definite endodermal cell.

According to some embodiments of the invention, the at least one marker positively associated with definite endodermal cells is selected from the group consisting of: FSHR, COLEC12, ROR2 ITGA5, LRP2, CD177, CCKBR, TRPA1, KEL, FOLR1, FOLH1, APOA1, APOA1, FZD5, FN1, BMP2, ADAMTS9, DPP4, FGA, GPR128, IGFBP5, LIFR, FZD4, PRTG, STC1, TNFSF4, CD177, IHH, LAMA1, GPC3, LGI1, FN1, LPHN3, FGB, KIT, DPP4, TRO, SPA17, ROBO2, DLK1, CST1, DKK1, HAS2, APOA2, CDH2, LGR5, AFP, FLRT3, APOB, DGKK, HP, SYTL5, SLC7A7, TTR, FRZB, RSPO3, GCNT1, DIO3, TMEM27, APOC1, EPHA4, C4orf18, GLIPR2, PCDH10, F10, BMP5, FMO5, STMN2, SLC5A9, PORCN, EGFLAM, RAB17, CST2, PNPLA3, MOSPD1, ELMO1, CHST9, SLC30A10, TMC7, C8orf49, CDH12, ST8SIA4, SLCO2A1, MANEA, LRIG3, HCN1, ADAMTS12, TMEM144, VTN, CAMK2N1, ABCC4, PCDH7, OR2T4, UNC93A, COL4A6, PAMR1, SLC1A1, PROS1, APOM, APOM, APOM, LYPD6B, TMEM88, ITLN2, BMPER, GPR141, VEGFA, DKK1, APOA2, AFP, APOB, FLRT3, KIAA1772, FBN2, FRZB, KLK6, FREM2, RSPO3, APOA1, APOA1, SEMA3D, KEL, BMP5, HEPH, STMN2, CACNB3, CHST9, TMC7, FAM171B, ATP5G1, FOLR1, ABCC2, EFNA5, CPT1C, MAGED1, RASA4, KDELR3, ST6GALNAC2, EDNRA, LRRC37B, ABCC4, CLCN5, MBOAT2, SLC44A5, NPC2, ELFN1, MMD, SLC5A3, IMPAD1, OSTC, DSC2, SLC31A1, SLC5A12, LRRN3, NPIPL3, GDPD1, NPIPL3, NPIPL3, LINGO2, and LYPD6B.

According to some embodiments of the invention, the at least one marker positively associated with definite endodermal cells is selected from the group consisting of: FSHR, COLEC12, ROR2 ITGA5, LRP2, CD177, CCKBR, TRPA1, KEL, FOLR1, FOLH1, APOA1, APOA1, FZD5, FN1, BMP2, ADAMTS9, DPP4, FGA, GPR128, IGFBP5, LIFR, FZD4, PRTG, STC1, TNFSF4, CD177, IHH, LAMA1, GPC3, LGI1, FN1, LPHN3, FGB, KIT, DPP4, TRO, SPA17, ROBO2, and DLK1.

According to some embodiments of the invention, the at least one marker positively associated with definite endodermal cells is selected from the group consisting of: CD177, CCKBR, APOA1, APOA1, FSHR, FZD5, FN1, BMP2, ADAMTS9, DPP4, FGA, COLEC12, ROR2, GPR128, IGFBP5, LIFR, FZD4, ITGA5, STC1, TNFSF4, CD177 and IHH.

According to some embodiments of the invention, the isolated population of pancreatic progenitor cells are further characterized by an expression signature of PDX1+/ngn3+/pax4+/hlxb9+/nkx6.1+/Hnf6+/sox9+.

According to some embodiments of the invention, the pancreatic progenitor cells are genetically unmodified.

According to some embodiments of the invention, the definite endodermal cells are genetically unmodified.

According to some embodiments of the invention, the definite endodermal cells are characterized by a SOX17+/SOX7+ expression signature.

According to some embodiments of the invention, the definite endodermal cells are characterized by a SOX17+/SOX7+/GSC+/CER+/FOXA2+/CD34+/CXCR4+/NANOG− expression signature.

According to some embodiments of the invention, the definite endodermal cells are characterized by a SOX17+/SOX7+/GSC+/CER+/FOXA2+/CXCR4+/NANOG− expression signature.

According to some embodiments of the invention, the undifferentiated embryonic stem cells (ESCs) are characterized by Oct4+ expression pattern.

According to some embodiments of the invention, the pancreatic progenitor cells are obtained by differentiating stem cells into pancreatic progenitor cells.

According to some embodiments of the invention, the definite endodermal cells are obtained by differentiating stem cells into the definite endodermal cells.

According to some embodiments of the invention, the stem cells are undifferentiated pluripotent stem cells.

According to some embodiments of the invention, the stem cells are adult stem cells.

According to some embodiments of the invention, the stem cells are fetal stem cells.

According to some embodiments of the invention, the undifferentiated pluripotent stem cells are embryonic stem cells (ESCs).

According to some embodiments of the invention, the undifferentiated pluripotent stem cells are induced pluripotent stem cells (iPSCs).

According to some embodiments of the invention, the cells are human cells.

According to some embodiments of the invention, differentiating the undifferentiated pluripotent stem cells into the pancreatic progenitor cells is performed by: (a) differentiating the pluripotent stem cells into definite endodermal cells to thereby obtain a population of cells which comprises definite endodermal cells, and (b) differentiating the population of cells which comprises the definite endodermal cells into the pancreatic progenitor cells, thereby inducing the differentiation of the pluripotent stem cells into the pancreatic progenitor cells.

According to some embodiments of the invention, differentiating the undifferentiated pluripotent stem cells into the pancreatic progenitor cells is performed by differentiation of the pluripotent stem cells into embryoid bodies.

According to some embodiments of the invention, the embryoid bodies are differentiated until about day 7-21 of human EBs differentiation.

According to some embodiments of the invention, differentiating the undifferentiated pluripotent stem cells into the definite endodermal cells is performed by culturing the pluripotent stem cells in the presence of activin A, Wnt3A, a small molecule Induce Definitive Endoderm 1 (IDE1) and/or a small molecule Induce Definitive Endoderm 2 (IDE2).

According to some embodiments of the invention, differentiating the definite endodermal cells into the pancreatic progenitor cells is performed by culturing the definite endodermal cells in the presence of retinoic acid.

According to some embodiments of the invention, differentiating the definite endodermal cells into the pancreatic progenitor cells is performed by culturing the definite endodermal cells in the presence of bFGF, KGF, FGF10, noggin, cyclopamine, KAAD cyclopamine, B27, Indolactam V, nicotinamide and/or epidermal growth factor.

According to some embodiments of the invention, step (b) of the method of some embodiments of the invention is effected by an immunological isolation assay selected from the group consisting of fluorescent activated cell sorter (FACS), Magnetic-activated cell sorting (MACS) or immunopanning.

According to some embodiments of the invention, the nucleic acid construct is a bacterial artificial chromosome (BAC).

According to some embodiments of the invention, the cell is a stem cell.

According to some embodiments of the invention, the stem cell is an embryonic stem cell or an induced pluripotent stem cell.

According to some embodiments of the invention, the cell is a human cell.

According to some embodiments of the invention, the method further comprising synthesizing the compound capable of inducing differentiation of the undifferentiated pluripotent stem cells to the definite endodermal cells.

According to some embodiments of the invention, the undifferentiated pluripotent stem cell is an embryonic stem cell, an adult stem cell or an adult-derived stem cell such as induced pluripotent stem cells (iPSC).

According to some embodiments of the invention, the pluripotent stem cell is characterized by an Oct4+/SSEA4+/SSEA3+/TRA1-60+ expression signature.

According to some embodiments of the invention, the method further comprising synthesizing the compound capable of inducing differentiation of the definite endodermal cells or undifferentiated pluripotent stem cells to the pancreatic progenitor cells.

According to some embodiments of the invention, the kit further comprising a pluripotent stem cell.

According to some embodiments of the invention, the kit further comprising at least one agent suitable for detecting an expression level of a marker of interest.

According to some embodiments of the invention, the expression level is detected by an RNA detection method.

According to some embodiments of the invention, the expression level is detected by a protein detection method.

According to some embodiments of the invention, the kit further comprising a genetic micro array chip.

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

In the drawings:

FIGS. 1A-C depict generation and expression of the SOX17-GFP reporter constructs. EGFP and floxed neomycin resistance gene was knocked into the SOX17 gene locus in a human genomic BAC by replacing the coding sequence of SOX17 (ATG to TGA) with the coding sequence of GFP and also the floxed neomycin resistance gene. FIG. 1A schematically illustrates the structure of a recombinant SOX17-GFP BAC construct (using BAC RP11-53M11) according to some embodiments of the invention. FIG. 1B schematically illustrates the structure of a recombinant SOX17-GFP plasmid according to some embodiments of the invention. The positions of the 5′-arm, the 3′-arm in the plasmid construct are shown relative to nucleotide number 1 of the recombinant BAC RP11-53M11 SOX17-GFP (SEQ ID NO:1). In addition the positions of two non repetitive 3′ external screening probes are also shown with positions relative to nucleotide number 1 of the recombinant BAC RP11-53M11 SOX17-GFP (SEQ ID NO:1). “Ext.”=external. FIG. 1C schematically illustrates the structure of the recombinant RP11-47H10 SOX17-GFP BAC construct according to some embodiments of the invention.

FIGS. 2A-H depict expression of the SOX17-GFP reporter construct in human embryonic stem cells and cells differentiated therefrom. FIG. 2A-FACS (fluorescence-activated cell sorting) of GFP expressing cells in two day old embryoid bodies (EBs) of a SOX17-GFP clone grown in the presence of activin A (66 ng/ml). A hESC clone containing the SOX17 reporter BAC was allowed to differentiate to form EBs. At day 2, the cells were dissociated and subjected to sorting by FACS into GFP⁺ (GFP-positive cells; green cells in gate R2) and GFP⁻ (GFP-negative cells; red cells) cell populations. The GFP⁺ population represents a population of SOX17⁺ (SOX17-positive) endoderm progenitor cells. FIGS. 2B-2E are microscopic images depicting SOX17 GFP reporter cells observed in phase contrast (FIGS. 2B-C) or using fluorescent microscopy (FIGS. 2D-E). A SOX17 GFP reporter clone was grown in the presence of activin A (66 ng/ml) for three days, and GFP expression was observed using a fluorescent microscope. FIG. 2F—A microscopic image depicting SOX17 expression in a SOX17 reporter clone. An 80% confluent monolayer of the SOX17-GFP reporter ES cell line was treated for three days with activin A (66 ng/ml), following which immunohistochemistry was performed with anti-SOX17 (red staining) and anti-GFP (green staining) antibodies. Note the clear correlation between SOX17 and GFP expression. FIG. 2G—A microscopic image depicting double staining of the cells described in FIG. 2F with both OCT4 (green staining), which marks undifferentiated cells, and SOX17 (red staining), which marks the definitive endoderm population. Note that OCT4 positive cells are negative for SOX17. FIG. 2H—A histogram depicting relative gene expression levels in SOX17-GFP+ cells as compared to SOX17-GFP-cells. SOX17-GFP reporter cells were grown as a monolayer in the presence of activin A (66 ng/ml), were harvested and FACS-sorted into GFP⁺ and GFP⁻ cells. RNA was extracted from the sorted populations which was reverse transcribed and was analyzed by real-time qPCR. The relative expression of various genes in the SOX17-GFP⁻ population as compared to the SOX17-GFP⁻ population is presented. Note that while SOX17, SOX7, GSC, CER, FOXA2, CD34 and CXCR4 are highly expressed in SOX17-GFP⁺ cells as compared to the SOX17-GFP⁻ cells, the expression of NANOG, a marker of pluripotency, is higher in SOX17-GFP⁻ cells as compared to SOX17-GFP⁺ cells.

FIGS. 3A-E depict generation of PDX1-GFP reporter constructs according to some embodiments of the invention. FIG. 3A—a schematic illustration of the PDX1 ATG-GFP-knock in recombinant BAC construct of some embodiments of the invention. FIG. 3B—a schematic illustration with a nucleotide positions of the 5′-arm, GFP gene, floxed neomycin resistance gene and 3′-arm of the PDX1 ATG-GFP-knock in BAC construct of some embodiments of the invention. FIG. 3C—a schematic illustration of the PDX1 ATG-GFP-knock in plasmid construct (which includes the 5′-arm, the GFP gene, the floxed neomycin resistance gene and the 3′-arm) along with the relative position of the 3′-external probe (not included in the plasmid construct) in the recombinant BAC which was used to generate the recombinant plasmid construct; FIG. 3D—a schematic illustration of the PDX1-IRES-GFP plasmid construct according to some embodiments of the invention. FIG. 3E—a schematic illustration of the PDX1-mCherry BAC construct according to some embodiments of the invention.

FIGS. 4A-D depicts expression of the PDX1-GFP reporter BAC. FIGS. 4A-B—Dot plots depicting FACS analyses of 15 day-old EBs from PDX1 GFP reporter BAC clones. The PDX1 BAC reporter clone was allowed to form EBs for fifteen days. The EBs were then dissociated into single cells which were analysed by FACS. FIG. 4A—represents control cells from wild type hESCs that do not contain a reporter construct. FIG. 4B—represents GFP expression (R2) in EBs from PDX1-GFP reporter clone. Note that while in the control cells there are no cells which express GFP (R2), cells harbouring the PDX1-GFP construct express GFP (R2). FIG. 4C—A confocal microscopy image depicting 15 day-old EBs developed from a hESC harbouring the PDX1-GFP reporter clone that were subjected to immunostaining. Note that the EBs contain cells which express PDX1 in the nucleus (red staining) and GFP in the cytoplasm (green staining). The nuclei were counterstained with TO-PRO-3 (blue stain). FIG. 4D—A histogram depicting gene expression levels in PDX1-GFP⁺ cells and PDX1-GFP⁻ cells as compared to unsorted EBs. The PDX1 BAC reporter clone was allowed to form EBs for fifteen days after which they were dissociated into single cells and the PDX1-GFP⁺ cells and PDX1-GFP⁻ cells were sorted by FACS, and RNA was extracted from the GFP⁺ and GFP⁻ populations. qRT-PCR (quantitative RT-PCR) was performed to compare expression of various markers in the two different cell populations. Red bars=GFP⁺ cells; Blue bars=GFP− cells. Note that PDX1 is enriched in the GFP⁺ population. Also note the upregulation of markers of pancreatic differentiation HLXB9, HNF6, NGN3 and PAX4 in PDX1-GFP+ cells as compared to PDX1-GFP⁻ cells.

FIGS. 5A-E depict gene profiling of the SOX17⁺ and PDX1⁺ precursor cells. FIG. 5A—Schematic description of the strategy for identification of unique, stage specific expression markers. Each colored ellipse represents a different differentiation stage in pancreatic development from undifferentiated embryonic cells (OCT4 positive cells), through endodermal progenitor cells (SOX17 positive cells); pancreatic progenitor cells (PDX1 positive cells) to mature pancreatic cells (insulin positive cells). RNA from each cell group was extracted and expression profiling was performed. Comparison between each group enables stage specific identification. FIGS. 5B-E—graphs depicting clustering of gene expression profiles obtained by Affymetrix analysis of mRNA extracted from GFP positive cells that were isolated by FACS from SOX17 reporter clones or from PDX1 reporter clones, and compared with the gene expression profile of undifferentiated hESC and of mature pancreas (as reported in the Affymetrix site). FIG. 5B—“SOX17 only genes” (definite endodermal-associated genes, represented by the newly identified markers: TMEM45A, RELL2 and PPFIA4; FIG. 5C—PDX1 only genes, represented by SILV (Official gene symbol PMEL) and GPR50; FIG. 5D—SOX17 and PDX1 genes, represented by the STX16, DLK1, CPT1C, LRP2 and BST2; and FIG. 5E—PDX1 and Pancreases genes, represented by TROP-2 (also known as TROP2; official gene symbol TACSTD2).

FIGS. 6A-B depict cell sorting using antibodies which specifically bind GPR50 and TROP-2 in twenty five day old genetically-unmodified EBs and gene expression analysis of the various subgroups. FIG. 6A—Twenty five day old dissociated EBs were immunostained with GPR50 (FITC-labeled) and TROP-2 (APC, Allophycocyanin-labeled) antibodies. Four populations were separated by FACS: GPR50−/TROP-2−, GPR50−/TROP-2⁺, GPR50⁺/TROP-2− and GPR50⁺/TROP-2⁺. FIG. 6B—RT-qPCR was performed to compare expression of various pancreatic markers in the four different cell populations isolated by FACS as described in FIG. 6B.

FIG. 7 is a graph depicting gene clustering by Partek analysis. The x axis: cell types; The y axis: normalized expression means value. Purple, red, green and blue curves represent the specific genes for the SOX17⁺, SOX17⁺/PDX1⁺, PDX1⁺ and PDX1⁺/Pancreas cell populations, respectively.

FIG. 8 is a histogram depicting SOX17⁺ stage specific genes. The x axis: genes; The y axis: expression ±SE. Blue, red, green and purple bars represent HESC, SOX17, PDX1 and Pancreas expression, respectively.

FIG. 9 is a histogram depicting PDX1⁺ stage-specific genes. The x axis: genes; The y axis: expression ±SE. Blue, red, green and purple bars represent HESC, SOX17, PDX1 and Pancreas expression, respectively.

FIG. 10 is a histogram depicting SOX17⁺/PDX1⁺ stage specific genes. The x axis: genes; The y axis: expression ±SE. Blue, red, green and purple bars represent HESC, SOX17, PDX1 and Pancreas expression, respectively.

FIG. 11 is a histogram depicting PDX1⁺-Pancreas specific genes. The x axis: genes; The y axis: expression ±SE. Blue, red, green and purple bars represent HESC, SOX17, PDX1 and Pancreas expression, respectively.

FIGS. 12A-E are FACS analyses depicting the expression of the new endodermal surface markers BST (FIG. 12A), FLRT3 (FIG. 12B), COLEC12 (FIG. 12C), GPR49 (FIG. 12D) and LIFR (FIG. 12E) in cells of day-7 embryoid bodies. Embryoid bodies were prepared from H9.2 hESCs grown for 7 days and then separated to single cells with TrypLE Select. Next, the cells were incubated for 30 minutes at room temperature with one of the following antibodies: Rabbit anti BST-2 1:200; Rabbit anti FLRT-3 1:50; Goat anti CL-P1/COLEC12 1:40; Rabbit anti GPR-49/LGR5 1:100; Anti human LIF-R-PE 1:10 or control non treated cells, followed by secondary antibodies anti rabbit Cy3 (for BST2, FLRT-3 and GPR-49) or anti goat Cy3 (COLEC12) for 15 minutes at room temperature. The expression of these markers in the EBs (green) were compared to undifferentiated hESCs cells (red) by FACS calibrator (BD) using Flow-Jo analysis. Note that 43.3% of the EBs cells are BST+ (FIG. 12A), 70.19% of the EBs cells are COLEC12+ (FIG. 12C), 38.32% of the EBs cells are PGR49+ (FIG. 12D), and 16.13% of the EBs cells are LIFR+ (FIG. 12E).

FIGS. 13A-D are FACS analyses depicting BST2, KDR and CXCR4 expression in IDE treated cells. Undifferentiated H9.2 cells were treated with IDE1/2 and then stained with an anti-BST2 antibody (1:200), an anti-KDR antibody Fitc conjugated (1:10) or with a combination of both, followed by a cy3-conjugated secondary antibody; or stained with 1:10 anti-human CXCR4-PerCP and 1:10 anti-human VEGF R2/KDR-Fitc, and taken for FACS analysis. Note that incubation with an anti-BST2 antibody resulted in isolation of 11.9% BST2+ cells from the IDE-treated cells (FIG. 13A); incubation with an anti-KDR antibody resulted in 4.32% KDR+ cells from IDE-treated cells (FIG. 13B); incubation with an anti-BST2 and anti-KDR antibodies resulted in 10.7% Bst2⁺/KDR⁻ (BST2 positive and KDR negative) cells of the IDE-treated cells and 0.39% of KDR+/BST2− (KDR positive and BST2 negative) cells of the IDE-treated cells (FIG. 13C); and incubation with the anti-CXCR4 and anti-KDR antibodies resulted in 1.05% CXCR4+/KDR− (CXCR4 positive and KDR negative) cells of the IDE-treated cells (FIG. 13D).

FIGS. 14A-C are representative images of confocal microscopy depicting expression of PDX1 in day-28 of human embryoid bodies which were sorted with an anti-TROP-2 antibody, demonstrating enrichment of a pancreatic progenitor cells. Blue—DAPI, nuclear staining; Red—PDX1. Note that most of the cells presented in each of the microscopic fields express PDX1 in their cytoplasm.

DETAILED DESCRIPTION OF THE INVENTION

The present invention, in some embodiments thereof, relates to methods of isolating pancreatic progenitor cells, definite endodermal cells and isolated cell population obtained thereby and, to nucleic acid constructs, cells and kit for identifying markers which characterize pancreatic progenitor cells or definite endodermal cells, and to methods and kits using same for identifying compounds involved in the differentiation of same.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

In the quest for developing cutting edge therapeutics, a significant effort is put on the production of stem cells (hESC and/or iPS) derived treatments. A key step in a successful production of new insulin secreting cells is the identification, isolation and characterization of stage-specific progenitor cells. The present inventors used genetic labeling for screening stage specific genes, and examined the expression profiling of cells in specific stages of the pancreatic differentiation process.

FIG. 5A depicts the general strategy used to identify markers presenting differential expression across different cell types. To achieve that goal, pluripotent stem cells were used for two major differentiation stages: (1) Definitive endoderm stage represented by SOX17 positive population; (2) Pancreatic progenitor cells represented by PDX1 positive population. Undifferentiated pluripotent stem cells (e.g., ESCs) were used as control for the experiment. In addition, DNA microarray data from pancreas cells was used for further comparisons. To enhance the reliability of the analysis, three replicas from each cell type were included in the experiment.

Thus, the present inventors have uncovered various markers which characterize the endoderm progenitor population and the pancreatic progenitor population, two intermediate stages along the differentiation pathway to insulin producing cells. As described in the Examples section which follows, the present inventors have generated human embryonic stem cells clones harboring SOX17 (definitive endoderm marker) regulatory sequences fused to a reporter gene (e.g., GFP) (FIGS. 1A-C, Example 1 of the Examples section which follows), and the PDX1 (pancreatic progenitor marker) regulatory sequences fused to a reporter gene (e.g., GFP or mCherry) (FIGS. 3A-E, Example 2 of the Examples section which follows), introduced the constructs into embryonic stem cells and identified subpopulations of GFP⁺ cells [FIG. 2A (SOX17+ cells) and FIG. 4B (PDX1+ cells); Example 1 and 2 of the Examples section which follows]. Using FACS-based purification, the present inventors identified and isolated stage-specific human progenitor cell populations. As is further shown in FIGS. 2H and 4D, Tables 1-12 and described in Examples 1-3 of the Examples section which follows, the present inventors have determined the expression level of various genes in the isolated SOX17+ or PDX1+ stage-specific populations and identified genes that are upregulated or downregulated at specific stages of differentiation and hence define those stages. The identified markers (genes) are involved in the differentiation process and can be used for optimizing conditions for ultimately producing beta cells for transplantation therapy, for selecting pancreatic progenitors from mixed populations of differentiating cells and for carrying out quality control of candidate islet replacement cells. In addition, as is shown in Tables 13-20 and described in Example 4 of the Examples section which follows, by employing the PARTEK cluster analysis the present inventors have identified additional genes/markers which can be used to identify pancreatic progenitor cells, definite endodermal cells, SOX17 and PDX1 expressing cells, and PDX1+ and pancreatic cells. Further experiments using some of the novel markers identified herein were used to isolate pancreatic progenitor cells. Thus, as shown in FIGS. 14A-C and described in Example 5 of the Examples section which follows, cells of embryoid bodies (day 28), which were sorted using an anti-TROP-2 antibody, were found to include at least 84% of PDX1-positive cells. In addition, as is shown in FIGS. 12A-E and described in Table 21 (Example 6 of the Examples section which follows), cells of 7-day-old EBs were found to express high levels of BST2, FLRT3, COLEC12, GPR-49/LGR5 and LIF-R as compared to undifferentiated hESCs. Moreover, induction of undifferentiated hESCs towards the definite endoderm stage by treatment with IDE1/IDE2 resulted in cells which are BST2+/KDR−, and cells which are CXCR4+/KDR− (FIGS. 13A-D, Example 7 of the Examples section which follows).

Thus, according to an aspect of some embodiments of the invention there is provided a method of identifying pancreatic progenitor cells. The method is effected by determining in a population of cells which comprises pancreatic progenitor cells at least one marker that is:

(i) positively associated with pancreatic differentiation, the marker being selected from the group consisting of: TACSTD2 (TROP-2), GPR50, BST2, NTRK2, ITGA4, KDR, PTPRN, LGI1, VIPR2, SLC2A1, MUC15, MUC12, LPHN3, MUC16, VTCN1, MMP16, FZD3, ITGB6, GFRA3, ROBO1, NLGN1, MUC12, CNTFR, LPHN1, SULF1, ADAM23, SCUBE3, PLAU, CDON, SLIT2, C7orf68, PLXDC2, CD74, MUC15, GPR56, VTCN1, ITGB6, AREG, BOC, KLRK1, PRTG, PTPRZ1, GABRP, SILV, KIAA1772, PLP1, OVOS, HAPLN1, EPHA7, ENO2, PCDHB5, OVOS, SYT1, DCT, GPAM, SLITRK6, DCC, FREM2, SDK2, CGA, ATP1B2, SEMA3D, PCDHB15, CDH1, WNT8B, LPAR4, NPIPL3, FAM171B, PTN, ABCC2, ADAMTS3, RASA4, CPT1C, SLC6A6, PCDHB3, LRRC37B, RNFT2, KCNG3, TRPC1, ALPPL2, OR4F21, CCL2, KIF5A, OLFM2, CACNG7, MPHOSPH9, SLC13A4, MOXD1, C6orf186, SLC4A8, STX16, AMY2A, SPARCL1, MGP, A2M, DCN, ATP8B1, MMRN1, EMP1, PLA2G2A, PDE3A, TLR3, CYP1B1, PTGIS, RFTN2, PLEKHA2, SMOC1, STOM, JAM2, CHL1, SCG5, IGFBP7, NPR3, IFI6, CR1L, OR2A4, OR2A7, KCNG3, CACNG7, GRID2, CDH1, LPAR3, SEMA6A, PTPRZ1, ATP1A3, CAMKV, SCNN1G, SYT6, SLC18A2, PCDHB5, ABCG2, HLA-DRA, CR1L, HTR2C, EDNRB, PCDH11X, SLC17A7, SCNN1A, CD9, CXCL16, FXYD5, GABRQ, GFRA3, CACNA2D2, CLDN4, PLP1, PDPN, MMP24, SDK2, GPR176, GPR64, GPR160, PCDH11Y, NKAIN4, ATP1B2, SCN8A, THBS4, CR2, HLA-DQA1, HLA-DRA, HTR7, SLC2A1, HLA-DRA, KCNS3, SLC7A3, HLA-DPB2, CACNA1B, and GPR143, wherein an upregulation above a predetermined threshold of an expression level of the marker positively associated with the pancreatic differentiation as compared to the expression level of the marker in a reference cell, wherein the reference cell is a definite endodermal cell which expresses SOX17, is indicative of a positive identification of a pancreatic progenitor cell, and alternatively or additionally

(ii) negatively associated with the pancreatic differentiation, the marker being selected from the group consisting of: LGR5, CCKBR, CXCR4, FLRT3, TRPA1, SLC40A1, LRIG3, COLEC12, EPSTI1, GPR128, CDH2, SLC5A9, FSHR, SLC30A10, IL13RA1, SLC7A7, PCDH10, SLC1A1, GPR141, LIFR, TMEM27, GPC4, LYPD6B, FOLH1, TRPC4, PCDH7, KEL, KCNJ3, OR2T4, VIPR2, FLRT2, CD34, SLC39A8, CLDN11, CXCR7, ITGA5, ITGAV, CALCR, CLDN18, SLC7A5, TMBIM4, SLCO2A1, CDH10, AMHR2, ASAM, CLDN1, DSCAM, TMEM88, PLXNA2, CD177, TMEM144, GPR37, GJA5, SEMA6D, NIPAL2, GPR151, MCC, TMEM136, KCNG1, LHFPL2, MOSPD1, SLC37A1, LRIT3, EPHA4, GPR177, and IL1R1, wherein a downregulation above a predetermined threshold of an expression level of the marker negatively associated with the pancreatic differentiation as compared to the expression level of the marker in a reference cell, wherein the reference cell is a definite endodermal cell which expresses SOX17, is indicative of a positive identification of a pancreatic progenitor cell,

thereby identifying the pancreatic progenitor cells.

As used herein the phrase “pancreatic progenitor cell” relates to a cell which express the duodenal homoebox factor-1 (PDX1) and which is not terminally differentiated but has the potential to differentiate to pancreatic endocrine, exocrine and/or duct cells.

As used herein the phrase “terminally differentiated” refers to a cell which cannot further differentiate in the lineage.

According to some embodiments of the invention, the pancreatic progenitor cell is capable of differentiating into at least two types of cells present in the pancreas.

It should be noted that the pancreas includes endocrine, exocrine and duct cells.

Pancreatic endocrine cells are present in the pancreatic islets of Langerhans, and produce hormones that underlie the endocrine functions of the pancreas, such as insulin, glucagon, and somatostatin. Pancreatic exocrine cells are present in the pancreas and secrete the pancreatic juice containing digestive enzymes that assist the absorption of nutrients and the digestion in the small intestine (e.g., proteases and peptidases, lipases, carbohydrases, and nucleases). Pancreatic duct cells transport bile and pancreatic enzymes from the pancreas to the hepatopancreatic ampulla.

According to some embodiments of the invention, the pancreatic progenitor cells are capable of differentiating into insulin-producing cells.

As used herein the term “identifying” refers to classifying a cell according the expression level of a marker characterizing the cell.

For example, a cell can be classified as being a pancreatic progenitor cell or as being a non-pancreatic progenitor cell based on the expression level of a marker characterizing the pancreatic progenitor cell.

As used herein the phrase “associated with pancreatic differentiation” refers to a marker which is upregulated (i.e., positively associated) or downregulated (i.e., negatively associated) when a cell is induced towards the pancreatic cell lineage, e.g., in a pancreatic progenitor cell.

As used herein the phrase “expression level” refers to the degree of gene expression and/or gene product activity in a specific cell. For example, up-regulation or down-regulation of various genes can affect the level of the gene product (i.e., RNA and/or protein) in a specific cell.

It should be noted that the level of expression can be determined in arbitrary absolute units, or in normalized units (relative to known expression levels of a control reference). For example, when using DNA chips, the expression levels are normalized according to the chips' internal controls or by using quantile normalization such as RMA (Robust Multichip Average).

As used herein the term “marker” refers to a gene or a gene product described using an accepted gene symbol.

According to some embodiments of the invention, the level of the marker is determined by the expression level of any of the polynucleotide transcripts which comprise the target nucleotide sequence identified by the Affymetrix probe set ID and/or any of the proteins which comprise the amino acid sequence encoded by the target nucleotide sequence identified by the Affymetrix probe set ID. The target nucleotide sequences identified by each of the Affymetrix probe set IDs are provided in Tables 1-20 in the Examples section which follows. The polynucleotides and polypeptides (which are identified by the Affynetrix probe set ID) are presented in Tables 1-20 by their sequence identifiers (SEQ ID NO:) and in some cases also by their GenBank Accession numbers. All polypeptides and polynucleotides are provided in the sequence listing of the application.

According to some embodiments of the invention, each of the polynucleotides or polypeptides, for which the expression level is determined, comprises the entire target sequence (with 100% sequence identity) or the amino acid sequence encoded by the target sequence, respectively.

The cells used by the method of some embodiments of the invention can be isolated cells (at least partially removed from a subject), cultured cells and/or non-cultured cells (e.g., primary cells obtained from a subject). The cells can be obtained from the subject by any known method including, but not limited to, tissue biopsy (e.g., pancreas biopsy) obtained using a surgical tool or a needle), fine needle aspiration, and the like. It should be noted that the cells may be isolated from the subject (e.g., for in vitro detection) or may optionally comprise a cell that has not been physically removed from the subject (e.g., in vivo detection).

According to some embodiments of the invention, the expression level of the marker in the test cell (e.g., the cell of the population of cells which is subject to the method of identifying pancreatic progenitor cells according to some embodiments of the invention) and in the reference cell (e.g., the definite endodermal cell which expresses SOX17) is determined under identical assay conditions.

According to some embodiments of the invention, the level of expression of the marker (gene) of the invention is determined using an RNA or a protein detection method.

According to some embodiments of the invention, the RNA or protein molecules are extracted from the cell of the subject.

Methods of extracting RNA or protein molecules from cells of a subject are well known in the art. Once obtained, the RNA or protein molecules can be characterized for the expression and/or activity level of various RNA and/or protein molecules using methods known in the arts.

Non-limiting examples of assays for detecting the expression level of RNA molecules in a cell sample include Northern blot analysis, RT-PCR, RNA in situ hybridization (using e.g., DNA or RNA probes to hybridize RNA molecules present in the cells or tissue sections), in situ RT-PCR (e.g., as described in Nuovo G J, et al. Am J Surg Pathol. 1993, 17: 683-90; Komminoth P, et al. Pathol Res Pract. 1994, 190: 1017-25), and oligonucleotide microarray (e.g., by hybridization of polynucleotide sequences derived from a sample to oligonucleotides attached to a solid surface [e.g., a glass wafer) with addressable location, such as Affymetrix microarray (Affymetrix®, Santa Clara, Calif.)].

Non-limiting examples of assays for detecting the expression level and/or activity of specific protein molecules in a cell sample include Enzyme linked immunosorbent assay (ELISA), Western blot analysis, radio-immunoassay (RIA), Fluorescence activated cell sorting (FACS), immunohistochemical analysis, in situ activity assay (using e.g., a chromogenic substrate applied on the cells containing an active enzyme), in vitro activity assays (in which the activity of a particular enzyme is measured in a protein mixture extracted from the cells). For example, in case the detection of the expression level of a secreted protein is desired, ELISA assay may be performed on a sample of fluid obtained from the subject (e.g., serum), which contains cell-secreted content.

According to some embodiments of the invention, the definite endodermal cell, which express SOX17, is characterized by a SOX17+/SOX7+ expression signature.

According to some embodiments of the invention, the definite endodermal cell is characterized by the SOX17+/SOX7+/GSC+/CER+/FOXA2+/CXCR4+/NANOG− expression signature.

According to some embodiments of the invention, the definite endodermal cell is characterized by the SOX17+/SOX7+/GSC+/CER+/FOXA2+/CXCR4+/NANOG− expression signature.

According to some embodiments of the invention, the definite endodermal cell is characterized by the SOX17+/SOX7+/GSC+/CER+/FOXA2+/CXCR4+/CD34+/NANOG− expression signature.

According to some embodiments of the invention, the expression level of the marker in the definite endodermal cell is determined from at least one definite endodermal cells, e.g., from at least 2, from at least 3, from at least 4, from at least 5, from at least 6, from at least 7, from at least 8, from at least 9, from at least 10, from at least 20, from at least 100, from at least 1000, from at least 1×10⁴, from at least 1×10⁵, e.g., from at least 1×10⁶ definite endodermal cells. It should be noted that when more than one definite endodermal cell is used, the expression level of the marker may comprise an average of the expression level of several or all cells, and those of skills in the art are capable of averaging expression levels from 2 or more cells, using e.g., normalized expression values.

As used herein the phrase “an upregulation above a predetermined threshold” refers to an increase in the level of expression in the cell relative to a reference cell (e.g., the definite endodermal cell) which is higher than a predetermined threshold such as a about 10%, e.g., higher than about 20%, e.g., higher than about 30%, e.g., higher than about 40%, e.g., higher than about 50%, e.g., higher than about 60%, higher than about 70%, higher than about 80%, higher than about 90%, higher than about 2 times, higher than about three times, higher than about four time, higher than about five times, higher than about six times, higher than about seven times, higher than about eight times, higher than about nine times, higher than about 20 times, higher than about 50 times, higher than about 100 times, higher than about 200 times of at least one reference cell (e.g., definite endodermal cell). The upregulation in the expression level can be also determined using logarithmic fold changes as shown in the Examples section which follows.

According to some embodiments of the invention, the method further comprising determining in a population of cells which comprises pancreatic progenitor cells at least one marker that is: (i) negatively associated with the pancreatic differentiation, the marker being selected from the group consisting of: CST1, CER1, ANKRD1, TRY6, HAS2, DKK1, PRSS2, HP, APOA2, RHOBTB3, BMP2, ACE2, STC1, PDZK1, HHEX, VIL1, PRDM1, EOMES, DNAJC15, TNIK, IGFBP5, RLBP1L2, ADAMTS9, EPSTI1, C5, ARHGAP24, TRY6, ANGPT2, TTR, MYL7, FST, KITLG, GATA3, ST8SIA4, CCDC141, TSPYL5, EGFLAM, TTN, LEFTY2, FOXA2, FAM184A, STMN2, DIO3, FN1, PRSS1, NPPB, RGSS, MANEA, OTX2, CFLAR, FZD5, LOC151009, IFLTD1, PPFIBP2, SYTL5, ARHGAP28, NTS, APOB, CST2, FOLH1B, TFF1, GPAM, RNF152, SEMA3E, OXCT1, DUSP4, RSPO3, EHHADH, DGKK, VEGFA, APOA1, RAB17, LYPD6B, TNNC1, ZNF280A, FRZB, APOA1, SOX17, SAMD3, BMP5, GATA4, MATN3, FAM122C, SPOCK3, PLOD2, NPL, ATP6V0D2, SERHL, SERPINE2, TGFB2, ELMO1, C8orf49, ENC1, COL5A2, FMO5, COL4A1, MGST2, GLUD2, CLIP4, UNC93A, GLIPR2, GSTA2, H2AFY2, ST6GALNAC2, DUSP6, NTN4, LHX1, PUS7L, TNC, BARB, HNF1B, SHISA2, NEXN, MSL3L2, IRAK4, S100A16, CDK6, FAM159B, ELL2, B3GALNT1, ARL4D, ETS2, MUM1L1, VTN, SEMA3A, ZNF611, VWA5A, ZNF518B, NUDT4P1, RASGEF1B, TPK1, ANXA3, MYL4, BMI1, ODC1, ARSE, PTPN13, ZNF321, NODAL, ANKMY2, LPGAT1, PROS1, KLF8, PAX6, GCNT1, S100Z, ZNF702P, CCDC129, LRRFIP1, TBC1D9, YPEL2, GCNT4, F10, ZNF585B, ELMOD2, GLT8D3, ETS1, SALL1, IAH1, RFC1, ZNF214, DPPA3, FGA, B3GNT5, COL4A6, DENND2C, FZD4, MYOCD, DDIT4L, PLCXD3, ELL2, SPOCK1, IGFBP6, SERHL2, SERPINI1, FAM26E, RNF128, RBM24, GALNT4, CTSL2, ALPK2, ANKRD20B, PLCE1, TAGLN, STAT4, SOAT1, MIXL1, HNF4A, IQCA1, KLHL14, MAML3, EGF, LEFTY1, ANKRD20B, RGS8, TAL2, ADI1, SPA17, KHDRBS2, and MCC, wherein a downregulation above a predetermined threshold of an expression level of the marker negatively associated with the pancreatic differentiation as compared to the expression level of the marker in a reference cell, wherein the reference cell is a definite endodermal cell which expresses SOX17 is indicative of a positive identification of a pancreatic progenitor cell and alternatively or additionally; (ii) positively associated with the pancreatic differentiation, the marker being selected from the group consisting of: ALPPL2, DPPA5, H19, CRYZ, CXCL12, TYW3, ZYG11A, CRABP1, IDO1, POU5F1, HEY2, HIST1H1A, TFAP2C, DPPA2, ZFP42, LECT1, NECAB1, CKMT1A, SAMHD1, FGF2, PLA2G2A, PRDM14, POU5F1, GLI3, GSTT2, OLFML3, DAZL, GALNT3, SOX2, POU5F1B, ACTN3, CPT1A, DCLK1, EDIL3, NANOG, THUMPD3, VASH2, ATCAY, USP44, HIST1H4F, NANOG, PIM2, DNMT3B, ZNF483, FEZF1, SCARNA9L, SCGB3A2, SILV, ENPP1, MYC, NLRP7, TDRD1, HHLA1, MT1G, RASL11B, PYGM, APOBEC3B, COL14A1, PTRF, HOXA1, CYP2S1, NRK, RASGRP2, FBXO2, KIF5A, PLEKHA2, HERC5, TRIML2, ARRDC4, HSPA2, ZNF248, NPTX2, MT1X, SPP1, PHC1, LRAT, NLRP4, GFPT2, ZNF680, MYO1E, B2M, DDX43, FABP3, GRHL2, ACOXL, CDCA7L, LDB2, KIAA1772, SFRP2, TRIM71, PLAU, AIM1, MT1E, STAT3, ZSCAN10, SCG3, VAT1L, HPDL, ZFP57, BNC2, CYP4F22, NOTCH1, LIX1, QPRT, ZNF398, RGS10, MT1G, GAA, PAX3, RHBDL3, POLR3G, FAM46B, MT1F, GLB1L3, RBM46, PRODH, ZNF300, STOM, PLA2G16, GAL, TOX, B3GALT1, MDGA2, MAD2L2, ATP6V0A4, LAMA2, HORMAD1, TRIM22, RNF157, ADD2, UPRT, PSMB8, ZNF562, FAM9C, UGT8, KIF5A, SNRPN, UNC13A, DOC2A, ZNF560, VENTX, RARRES2, CCDC109B, MT2A, ALDOC, STARD9, and AKAP1, wherein an upregulation above a predetermined threshold of an expression level of the marker positively associated with the pancreatic differentiation as compared to the expression level of the marker in a reference cell, wherein the reference cell is a definite endodermal cell which expresses SOX17, is indicative of a positive identification of a pancreatic progenitor cell.

According to an aspect of some embodiments of the invention, the method of identifying the pancreatic progenitor cells comprising determining in a population of cells which comprises pancreatic progenitor cells at least one marker that is: (i) negatively associated with the pancreatic differentiation, the marker being selected from the group consisting of: LGR5, CCKBR, CXCR4, FLRT3, TRPA1, SLC40A1, LRIG3, COLEC12, EPSTI1, GPR128, CDH2, SLC5A9, FSHR, SLC30A10, IL13RA1, SLC7A7, PCDH10, SLC1A1, GPR141, LIFR, TMEM27, GPC4, LYPD6B, FOLH1, TRPC4, PCDH7, KEL, KCNJ3, OR2T4, VIPR2, FLRT2, CD34, SLC39A8, CLDN11, CXCR7, ITGA5, ITGAV, CALCR, CLDN18, SLC7A5, TMBIM4, SLCO2A1, CDH10, AMHR2, ASAM, CLDN1, DSCAM, TMEM88, PLXNA2, CD177, TMEM144, GPR37, GJA5, SEMA6D, NIPAL2, GPR151, MCC, TMEM136, KCNG1, LHFPL2, MOSPD1, SLC37A1, LRIT3, EPHA4, GPR177, IL1R1, CST1, CER1, ANKRD1, TRY6, HAS2, DKK1, PRSS2, HP, APOA2, RHOBTB3, BMP2, ACE2, STC1, PDZK1, HHEX, VIL1, PRDM1, EOMES, DNAJC15, TNIK, IGFBP5, RLBP1L2, ADAMTS9, EPSTI1, C5, ARHGAP24, TRY6, ANGPT2, TTR, MYL7, FST, KITLG, GATA3, ST8SIA4, CCDC141, TSPYL5, EGFLAM, TTN, LEFTY2, FOXA2, FAM184A, STMN2, DIO3, FN1, PRSS1, NPPB, RGS5, MANEA, OTX2, CFLAR, FZD5, LOC151009, IFLTD1, PPFIBP2, SYTL5, ARHGAP28, NTS, APOB, CST2, FOLH1B, TFF1, GPAM, RNF152, SEMA3E, OXCT1, DUSP4, RSPO3, EHHADH, DGKK, VEGFA, APOA1, RAB17, LYPD6B, TNNC1, ZNF280A, FRZB, APOA1, SOX17, SAMD3, BMP5, GATA4, MATN3, FAM122C, SPOCK3, PLOD2, NPL, ATP6V0D2, SERHL, SERPINE2, TGFB2, ELMO1, C8orf49, ENC1, COL5A2, FMO5, COL4A1, MGST2, GLUD2, CLIP4, UNC93A, GLIPR2, GSTA2, H2AFY2, ST6GALNAC2, DUSP6, NTN4, LHX1, PUS7L, TNC, BARB, HNF1B, SHISA2, NEXN, MSL3L2, IRAK4, S100A16, CDK6, FAM159B, ELL2, B3GALNT1, ARL4D, ETS2, MUM1L1, VTN, SEMA3A, ZNF611, VWA5A, ZNF518B, NUDT4P1, RASGEF1B, TPK1, ANXA3, MYL4, BMI1, ODC1, ARSE, PTPN13, ZNF321, NODAL, ANKMY2, LPGAT1, PROS1, KLF8, PAX6, GCNT1, S100Z, ZNF702P, CCDC129, LRRFIP1, TBC1D9, YPEL2, GCNT4, F10, ZNF585B, ELMOD2, GLT8D3, ETS1, SALL1, IAH1, RFC1, ZNF214, DPPA3, FGA, B3GNT5, COL4A6, DENND2C, FZD4, MYOCD, DDIT4L, PLCXD3, ELL2, SPOCK1, IGFBP6, SERHL2, SERPINI1 FAM26E, RNF128, RBM24, GALNT4, CTSL2, ALPK2, ANKRD20B, PLCE1, TAGLN, STAT4, SOAT1, MIXL1, HNF4A, IQCA1, KLHL14, MAML3, EGF, LEFTY1, ANKRD20B, RGS8, TAL2, ADI1, SPA17, KHDRBS2, and MCC, wherein a downregulation above a predetermined threshold of an expression level of the marker negatively associated with the pancreatic differentiation as compared to the expression level of the marker in a reference cell, wherein the reference cell is a definite endodermal cell which expresses SOX17, is indicative of a positive identification of a pancreatic progenitor cell and alternatively or additionally; (ii) positively associated with the pancreatic differentiation, the marker being selected from the group consisting of: TACSTD2 (TROP-2), GPR50, BST2, NTRK2, ITGA4, KDR, PTPRN, LGI1, VIPR2, SLC2A1, MUC15, MUC12, LPHN3, MUC16, VTCN1, MMP16, FZD3, ITGB6, GFRA3, ROBO1, NLGN1, MUC12, CNTFR, LPHN1, SULF1, ADAM23, SCUBE3, PLAU, CDON, SLIT2, C7orf68, PLXDC2, CD74, MUC15, GPR56, VTCN1, ITGB6, AREG, BOC, KLRK1, PRTG, PTPRZ1, GABRP, SILV, KIAA1772, PLP1, OVOS, HAPLN1, EPHA7, ENO2, PCDHB5, OVOS, SYT1, DCT, GPAM, SLITRK6, DCC, FREM2, SDK2, CGA, ATP1B2, SEMA3D, PCDHB15, CDH1, WNT8B, LPAR4, NPIPL3, FAM171B, PTN, ABCC2, ADAMTS3, RASA4, CPT1C, SLC6A6, PCDHB3, LRRC37B, RNFT2, KCNG3, TRPC1, ALPPL2, OR4F21, CCL2, KIF5A, OLFM2, CACNG7, MPHOSPH9, SLC13A4, MOXD1, C6orf186, SLC4A8, STX16, AMY2A, SPARCL1, MGP, A2M, DCN, ATP8B1, MMRN1, EMP1, PLA2G2A, PDE3A, TLR3, CYP1B1, PTGIS, RFTN2, PLEKHA2, SMOC1, STOM, JAM2, CHL1, SCG5, IGFBP7, NPR3, IFI6, CR1L, OR2A4, OR2A7, KCNG3, CACNG7, GRID2, CDH1, LPAR3, SEMA6A, PTPRZ1, ATP1A3, CAMKV, SCNN1G, SYT6, SLC18A2, PCDHB5, ABCG2, HLA-DRA, CR1L, HTR2C, EDNRB, PCDH11X, SLC17A7, SCNN1A, CD9, CXCL16, FXYD5, GABRQ, GFRA3, CACNA2D2, CLDN4, PDPN, MMP24, SDK2, GPR176, GPR64, GPR160, PCDH11Y, NKAIN4, ATP1B2, SCN8A, THBS4, CR2, HLA-DQA1, HLA-DRA, HTR7, SLC2A1, HLA-DRA, KCNS3, SLC7A3, HLA-DPB2, CACNA1B, GPR143, ALPPL2, DPPA5, H19, CRYZ, CXCL12, TYW3, ZYG11A, CRABP1, IDO1, POU5F1, HEY2, HIST1H1A, TFAP2C, DPPA2, ZFP42, LECT1, NECAB1, CKMT1A, SAMHD1, FGF2, PLA2G2A, PRDM14, POU5F1, GLI3, GSTT2, OLFML3, DAZL, GALNT3, SOX2, POU5F1B, ACTN3, CPT1A, DCLK1, EDIL3, NANOG, THUMPD3, VASH2, ATCAY, USP44, HIST1H4F, NANOG, PIM2, DNMT3B, ZNF483, FEZF1, SCARNA9L, SCGB3A2, SILV, ENPP1, MYC, NLRP7, TDRD1, HHLA1, MT1G, RASL11B, PYGM, APOBEC3B, COL14A1, PTRF, HOXA1, CYP2S1, NRK, RASGRP2, FBXO2, KIF5A, PLEKHA2, HERC5, TRIML2, ARRDC4, HSPA2, ZNF248, NPTX2, MT1X, SPP1, PHC1, LRAT, NLRP4, GFPT2, ZNF680, MYO1E, B2M, DDX43, FABP3, GRHL2, ACOXL, CDCA7L, LDB2, KIAA1772, SFRP2, TRIM71, PLAU, AIM1, MT1E, STAT3, ZSCAN10, SCG3, VAT1L, HPDL, ZFP57, BNC2, CYP4F22, NOTCH1, LIX1, QPRT, ZNF398, RGS10, MT1G, GAA, PAX3, RHBDL3, POLR3G, FAM46B, MT1F, GLB1L3, RBM46, PRODH, ZNF300, STOM, PLA2G16, GAL, TOX, B3GALT1, MDGA2, MAD2L2, ATP6V0A4, LAMA2, HORMAD1, TRIM22, RNF157, ADD2, UPRT, PSMB8, ZNF562, FAM9C, UGT8, KIF5A, SNRPN, UNC13A, DOC2A, ZNF560, VENTX, RARRES2, CCDC109B, MT2A, ALDOC, STARD9, and AKAP1, wherein an upregulation above a predetermined threshold of an expression level of the marker positively associated with the pancreatic differentiation as compared to the expression level of the marker in a reference cell, wherein the reference cell is a definite endodermal cell which expresses SOX17 is indicative of a positive identification of a pancreatic progenitor cell.

According to some embodiments of the invention, the method further comprising determining in a population of cells which comprises pancreatic progenitor cells at least one marker that is: (i) negatively associated with the pancreatic differentiation, the marker being selected from the group consisting of: TMPRSS11E, LGR5, SLC39A8, TM4SF18, CUZD1, GPC4, SLC22A3, CXCR4, NRN1, TMBIM4, THBS2, SLC7A5, TMEM47, NTS, CER1, CST1, NODAL, PRRX1, KGFLP1, NFIB, GCNT4, MIXL1, CAV1, LUM, RASGRF2, OXCT1, GLIPR1, VSNL1, FST, POSTN, GNA14, CBR1, TNIK, RGS5, KGFLP1, ETS1, MPPED2, ACTA2, SEMA3A, DACT1, ANXA1, COL12A1, KITLG, MMP2, DLEU2, ACE2, ACTG2, PUS7L, RNU5B-1, COL3A1, LEFTY2, NPPB, S1AE, AFP, ZFP42, HAS2, TRY6, NR5A2, EBF1, RPPH1, NFIX, IAH1, RUNX1T1, COL5A2, VCAN, RNU5E, ADAMTS5, CDK6, LRRC17, DBC1, MYL4, ANKRD1, EOMES, LCP1, TAGLN, TNFAIP6, FAM159B, and PGM5P2, wherein a downregulation above a predetermined threshold of an expression level of the marker negatively associated with the pancreatic differentiation as compared to the expression level of the marker in a reference cell, wherein the reference cell is an undifferentiated embryonic stem cell (hESC), is indicative of a positive identification of a pancreatic progenitor cell, and alternatively or additionally; (ii) positively associated with the pancreatic differentiation, the marker being selected from the group consisting of: TACSTD2 (TROP-2), BST2 LRP2, LAMA1, GPC3, LGI1, FN1, LPHN3, FGB, KIT, DPP4, TRO, SPA17, ROBO2, DLK1, PRTG, DKK1, APOA2, AFP, APOB, FLRT3, KIAA1772, FBN2, FRZB, KLK6, FREM2, RSPO3, APOA1, APOA1, SEMA3D, KEL, BMP5, HEPH, STMN2, CACNB3, CHST9, TMC7, FAM171B, ATP5G1, FOLR1, ABCC2, EFNA5, CPT1C, MAGED1, RASA4, KDELR3, ST6GALNAC2, EDNRA, LRRC37B, ABCC4, CLCN5, MBOAT2, SLC44A5, NPC2, ELFN1, MMD, SLC5A3, IMPAD1, OSTC, DSC2, SLC31A1, SLC5A12, LRRN3, NPIPL3, GDPD1, NPIPL3, NPIPL3, LINGO2, LYPD6B, PRTG, LRP2, CACNG7, DLK1, CACNA2D2, CR1L, SCNN1G, HTR2C, LPAR3, THBS3, KCNG3, SDK2, HLA-DRA, SLC18A2, CXCL16, TMEM63C, SLC17A7, GFRA3, DPPA5, ALPPL2, CRYZ, H19, ZYG11A, TYW3, ARRDC4, TXNIP, FOS, NCRNA00173, GSTT2, C3, DDX43, B2M, PYGM, CYP4F22, ZNF578, ENO2, ZNF248, NLRP4, SNORD59B, SNORD113-4, CASZ1, MIR21, IFI16, ZNF560, TDRD1, ZNF680, HSPA1B, HSPA1A, EGR1, RASGRP2, ASMTL, CPT1C, ATCAY, ACSF2, PAMR1, SILV, ACTN3, HORMAD1, ACSM3, RNF157, SAMHD1, and RCN1, wherein an upregulation above a predetermined threshold of an expression level of the marker positively associated with the pancreatic differentiation as compared to the expression level of the marker in a reference cell, wherein the reference cell is an undifferentiated embryonic stem cell (ESC), is indicative of a positive identification of a pancreatic progenitor cell.

According to an aspect of some embodiments of the invention, the method of identifying pancreatic progenitor cells comprising determining in a population of cells which comprises pancreatic progenitor cells at least one marker that is: (i) negatively associated with pancreatic differentiation, the marker being selected from the group consisting of: LGR5, CCKBR, CXCR4, FLRT3, TRPA1, SLC40A1, LRIG3, COLEC12, EPSTI1, GPR128, CDH2, SLC5A9, FSHR, SLC30A10, IL13RA1, SLC7A7, PCDH10, SLC1A1, GPR141, LIFR, TMEM27, GPC4, LYPD6B, FOLH1, TRPC4, PCDH7, KEL, KCNJ3, OR2T4, VIPR2, FLRT2, CD34, SLC39A8, CLDN11, CXCR7, ITGA5, ITGAV, CALCR, CLDN18, SLC7A5, TMBIM4, SLCO2A1, CDH10, AMHR2, ASAM, CLDN1, DSCAM, TMEM88, PLXNA2, CD177, TMEM144, GPR37, GJA5, SEMA6D, NIPAL2, GPR151, MCC, TMEM136, KCNG1, LHFPL2, MOSPD1, SLC37A1, LRIT3, EPHA4, GPR177, and IL1R1, wherein downregulation above a predetermined threshold of an expression level of the marker negatively associated with the pancreatic differentiation as compared to the expression level of the marker in a reference cell, wherein the reference cell is a definite endodermal cell which expresses SOX17, is indicative of a positive identification of a pancreatic progenitor cell, and alternatively or additionally; (ii) positively associated with pancreatic differentiation, the marker being selected from the group consisting of: TACSTD2 (TROP-2), GPR50, BST2, NTRK2, ITGA4, KDR, PTPRN, LGI1, VIPR2, SLC2A1, MUC15, MUC12, LPHN3, MUC16, VTCN1, MMP16, FZD3, ITGB6, GFRA3, ROBO1, NLGN1, MUC12, CNTFR, LPHN1, SULF1, ADAM23, SCUBE3, PLAU, CDON, SLIT2, C7orf68, PLXDC2, CD74, MUC15, GPR56, VTCN1, ITGB6, AREG, BOC, KLRK1, PRTG, PTPRZ1, GABRP, SILV, KIAA1772, PLP1, OVOS, HAPLN1, EPHA7, ENO2, PCDHB5, OVOS, SYT1, DCT, GPAM, SLITRK6, DCC, FREM2, SDK2, CGA, ATP1B2, SEMA3D, PCDHB15, CDH1, WNT8B, LPAR4, NPIPL3, FAM171B, PTN, ABCC2, ADAMTS3, RASA4, CPT1C, SLC6A6, PCDHB3, LRRC37B, RNFT2, KCNG3, TRPC1, ALPPL2, OR4F21, CCL2, KIF5A, OLFM2, CACNG7, MPHOSPH9, SLC13A4, MOXD1, C6orf186, SLC4A8, STX16, AMY2A, SPARCL1, MGP, A2M, DCN, ATP8B1, MMRN1, EMP1, PLA2G2A, PDE3A, TLR3, CYP1B1, PTGIS, RFTN2, PLEKHA2, SMOC1, STOM, JAM2, CHL1, SCG5, IGFBP7, NPR3, IFI6, CR1L, OR2A4, OR2A7, KCNG3, CACNG7, GRID2, CDH1, LPAR3, SEMA6A, PTPRZ1, ATP1A3, CAMKV, SCNN1G, SYT6, SLC18A2, PCDHB5, ABCG2, HLA-DRA, CR1L, HTR2C, EDNRB, PCDH11X, SLC17A7, SCNN1A, CD9, CXCL16, FXYD5, GABRQ, GFRA3, CACNA2D2, CLDN4, PTPRN, PLP1, PDPN, MMP24, SDK2, GPR176, GPR64, GPR160, PCDH11Y, NKAIN4, ATP1B2, SCN8A, THBS4, CR2, HLA-DQA1, HLA-DRA, HTR7, SLC2A1, HLA-DRA, KCNS3, SLC7A3, HLA-DPB2, CACNA1B, and GPR143 wherein upregulation above a predetermined threshold of an expression level of the marker positively associated with the pancreatic differentiation as compared to the expression level of the marker in a reference cell, wherein the reference cell is a definite endodermal cell which expresses SOX17, is indicative of a positive identification of a pancreatic progenitor cells, and alternatively or additionally; (iii) negatively associated with pancreatic differentiation, the marker being selected from the group consisting of: TMPRSS11E, LGR5, SLC39A8, TM4SF18, CUZD1, GPC4, SLC22A3, CXCR4, NRN1, TMBIM4, THBS2, SLC7A5, and TMEM47, wherein downregulation above a predetermined threshold of an expression level of the marker negatively associated with pancreatic differentiation as compared to the expression level of the marker in a reference cell, wherein the reference cell is an undifferentiated embryonic stem cell (ESC), is indicative of a positive identification of a pancreatic progenitor cell, and alternatively or additionally; (iv) positively associated with pancreatic differentiation, the marker being selected from the group consisting of: TACSTD2 (TROP-2), LRP2, LAMA1, GPC3, LGI1, FN1, LPHN3, FGB, KIT, DPP4, TRO, SPA17, ROBO2, DLK1, BST2, PRTG, LRP2, CACNG7, DLK1, CACNA2D2, CR1L, SCNN1G, HTR2C, LPAR3, THBS3, KCNG3, SDK2, HLA-DRA, SLC18A2, CXCL16, TMEM63C, SLC17A7, GFRA3, PRTG, DKK1, APOA2, AFP, APOB, FLRT3, KIAA1772, FBN2, FRZB, KLK6, FREM2, RSPO3, APOA1, APOA1, SEMA3D, KEL, BMP5, HEPH, STMN2, CACNB3, CHST9, TMC7, FAM171B, ATP5G1, FOLR1, ABCC2, EFNA5, CPT1C, MAGED1, RASA4, KDELR3, ST6GALNAC2, EDNRA, LRRC37B, ABCC4, CLCN5, MBOAT2, SLC44A5, NPC2, ELFN1, MMD, SLC5A3, IMPAD1, OSTC, DSC2, SLC31A1, SLC5A12, LRRN3, NPIPL3, GDPD1, NPIPL3, NPIPL3, LINGO2, and LYPD6B, wherein upregulation above a predetermined threshold of an expression level of the marker positively associated with pancreatic differentiation as compared to the expression level of the marker in a reference cell, wherein the reference cell is an undifferentiated ESC, is indicative of a positive identification of a pancreatic progenitor cell; thereby identifying the pancreatic progenitor cells.

As shown in FIG. 4D and described in Example 2 of the Examples section which follows, pancreatic progenitor cells identified according to the method of some embodiments of the invention express typical pancreatic progenitor cell markers, such as PDX1, hlxb9, Hnf6, ngn3, and Pax4.

According to some embodiments of the invention, the population of cells which comprises pancreatic progenitor cells express a transcription factor selected from the group consisting of PDX1, ngn3, pax4, hlxb9, nkx6.1, Hnf6, and sox9.

According to some embodiments of the invention, the definite endodermal cells are identified by a method comprising determining in a population of cells which comprises definite endodermal cells at least one marker that is: (i) negatively associated with definite endodermal cells, the marker being selected from the group consisting of: KDR, PCDHB5, FAT4, FLT1, NRN1, THBS2, PTPRZ1, SLC6A15, GPR176, SEMA6A, THBS1, CDH11, GRID2, SLC7A11, CDH1, LRFN5, EDNRB, THY1, NETO1, KCND2, TMPRSS11E, CD44, PDPN, SLC7A1, KAL1, KCNG3, GPM6B, FXYD5, PCDH18, ICAM3, MCTP1, TACR3, TMEM155, ZFP42, THUMPD3, ANXA1, SPP1, PRDM14, GNA14, EDIL3, CXCL12, PSMD5, PRRX1, NANOG, TRIM22, NANOG, RASGRF2, POU5F1B, POLR3G, HHLA1, POU5F1, VSNL1, SCG3, B3GALT1, LECT1, NTS, MBNL1, CKMT1A, NECAB1, FGF2, SFRP2, DCLK1, DACT1, CRABP1, TFAP2C, SCGB3A2, LRAT, CUZD1, GLB1L3, METTL7A, VAT1L, COL12A1, OLFML3, SOX2, USP44, HIST1H4F, KGFLP1, CPT1A, DBC1, CHAC1, CAV1, MT1G, NFIX, FERMT1, GLIPR1, TOX, SNRPN (SNORD116-6), HEY2, T1MP4, IDO1, MT2A, NR5A2, UPRT, MYC, CCDC109B, GAL, ZNF483, RND3, BNC2, COL3A1, LUM, LDB2, MT1E, SNRPN (SNORD116-23), SNRPN (SNORD116-27), GALNT3, PIPOX, PDK1, PREX2, CYP2S1, NRK, VAV3, TNFAIP6, ENPP1, ADD2, SNRPN (SNORD116-24), SNRPN (SNORD109A), STC2, SNORA22, MPPED2, ZNF562, GAP43, FOXB1, TSHZ3, HPGD, ZDHHC22, ACOXL, GLI3, CDCA7L, ZSCAN10, GFPT2, PLP2, HIST1H1A, CAMKV, HERC5, MT1X, TERF1, RAB31, SNRPN (SNORD116-13), ETV1, MT1G, ACTA1, SNRPN (SNORD116-20), NFIB, ZEB2, CBR1, ATXN7L1, SNRPN (SNORD116-1), MT1F, SNRPN (SNORD116-29), AP1M2, ACTG2, CYP2B6, SERPINE1, GRHL2, SLIT2, PIM2, SMARCA2, and RPPH1, wherein downregulation above a predetermined threshold of an expression level of the marker negatively associated with definite endodermal cells as compared to the expression level of the marker in a reference cell, wherein the reference cell is an undifferentiated embryonic stem cell (hESC), is indicative of a positive identification of a definite endodermal cell, and alternatively or additionally; (ii) positively associated with definite endodermal cells, the marker being selected from the group consisting of: FLRT3, FSHR, LIFR, ROR2, KEL, TRPA1, CD177, CCKBR, APOA1, APOA1, FZD5, FN1, BMP2, ADAMTS9, DPP4, FGA, COLEC12, GPR128, IGFBP5, FZD4, ITGA5, STC1, TNFSF4, CD177, IHH, LRP2, LAMA1, GPC3, LGI1, FN1, LPHN3, FGB, KIT, DPP4, TRO, SPA17, ROBO2, DLK1, CST1, DKK1, HAS2, APOA2, CDH2, LGR5, AFP, APOB, DGKK, HP, SYTL5, SLC7A7, TTR, FRZB, RSPO3, GCNT1, DIO3, TMEM27, APOC1, EPHA4, C4orf18, GLIPR2, PCDH10, F10, BMP5, FMO5, FOLR1, STMN2, SLC5A9, PORCN, EGFLAM, RAB17, CST2, PNPLA3, MOSPD1, ELMO1, CHST9, SLC30A10, TMC7, C8orf49, CDH12, ST8SIA4, SLCO2A1, MANEA, LRIG3, HCN1, ADAMTS12, FOLH1, TMEM144, VTN, CAMK2N1, ABCC4, PCDH7, OR2T4, UNC93A, COL4A6, PAMR1, SLC1A1, PROS1, APOM, APOM, APOM, LYPD6B, TMEM88, ITLN2, BMPER, GPR141, VEGFA, PRTG, DKK1, APOA2, AFP, APOB, FLRT3, KIAA1772, FBN2, FRZB, KLK6, FREM2, RSPO3, APOA1, APOA1, SEMA3D, KEL, BMP5, HEPH, STMN2, CACNB3, CHST9, TMC7, FAM171B, ATP5G1, FOLR1, ABCC2, EFNA5, CPT1C, MAGED1, RASA4, KDELR3, ST6GALNAC2, EDNRA, LRRC37B, ABCC4, CLCN5, MBOAT2, SLC44A5, NPC2, ELFN1, MMD, SLC5A3, IMPAD1, OSTC, DSC2, SLC31A1, SLC5A12, LRRN3, NPIPL3, GDPD1, NPIPL3, NPIPL3, LINGO2, TRPA1, SLC40A1, SLC30A10, CCKBR, VIPR2, COLEC12, FLRT3, LGR5, GPR141, BST2, SLC5A9, GPR128, KEL, LRIG3, LYPD6B, FSHR, LIFR, FOLH1, CXCR4, ITGA5, AMOT, LY6E, SEMA6D, GJA5, PRTG, CD34, TMEM144, ROR2, GPR177, OR2T4, SLC7A7, KCNJ3, CLDN18, GPR151, SLC44A5, CDH10, TMEM27, SLC1A1, TMEM56, CD177, PLXNA2, SLC26A2, DSCAM, TMEM133, IL13RA1, ATP2B1, CD302, MEGF9, EDNRA, CDH2, GPR161, TYRO3, FLRT2, LRIT3, PCDH7, NRCAM, SMAGP, AMHR2, ELTD1, GRPR, EPHA4, CD99, GATA3, SEMA3E, HHEX, ZNF280A, FAM184A, WDR72, PDZK1, RLBP1L2, SHISA2, VIL1, STMN2, APOA2, SERHL, PPFIBP2, DKK1, MUM1L1, IGFBP5, ST6GALNAC2, TSPYL5, STC1, SYTL5, EPSTI1, ANKRD1, ARHGAP24, KRT18P49, PRSS2, RHOBTB3, FRZB, RARB, ADAMTS9, ARL4D, PRDM1, HP, FZD5, TRY6, ATP6V0D2, ANGPT2, DENND2C, BMP5, FOXA2, HAS2, BMP2, S100A16, FOLH1B, FAM122C, FZD4, C5, S100A14, VEGFA, CLIP4, GPAM, HNF1B, APOA1, CFLAR, RBM24, RNF152, TTR, TTN, EGFLAM, APOB, DIO3, IFLTD1, ABCC4, CCDC141, ENC1, NEK2, ELMO1, SPOCK3, SERPINI1, ACSL1, GATM, EHHADH, NUDT4, CST1, GLUD2, NPL, ZNF702P, TRY6, SPOCK1, AGL, TFF1, DGKK, SALL1, MANEA, KIT, KRT19, TNNC1, SEPP1, ST8SIA4, YPEL2, ANKMY2, DNAJC15, RNF128, PTPN13, F10, SAMD3, GCNT1, IPP, PROS1, SV2B, PLOD2, MAGEH1, CHST9, ZNF518B, TMEM106C, SERHL2, NTN4, SOX17, FRRS1, OTX2, RNASEL, ELMOD2, MYCT1, PAX6, MGST2, BBS5, MTSS1, VTN, WBP5, DUB4, CCDC92, BTG2, LPGAT1, FN1, TBX3, PLCE1, KRT19P2, IFI16, PORCN, PRSS1, MYL7, DUSP4, PROS1, ANKRD20B, CTSL2, FMO5, USP27X, LAMA1, ADAM28, ZNF611, ANKRD20B, ZNF137, S100Z, GPSM2, TGFB2, and ARHGAP28, wherein upregulation above a predetermined threshold of an expression level of the marker positively associated with definite endodermal cells as compared to the expression level of the marker in a reference cell, wherein the reference cell is an undifferentiated embryonic stem cell (hESC), is indicative of a positive identification of a definite endodermal cell, thereby identifying the definite endodermal cells.

According to some embodiments of the invention the at least one marker positively associated with pancreatic differentiation is selected from the group consisting of: TACSTD2 (TROP-2), GPR50, BST2, LGI1, VIPR2, SLC2A1, MUC15, MUC12, LPHN3, MUC16, VTCN1, MMP16, FZD3, ITGB6, GFRA3, ROBO1, NLGN1, MUC12, CNTFR, LPHN1, SULF1, ADAM23, SCUBE3, PLAU, CDON, SLIT2, C7orf68, PLXDC2, CD74, MUC15, GPR56, VTCN1, ITGB6, NTRK2, AREG, BOC, ITGA4, KLRK1, PRTG, PTPRZ1, GABRP, SILV, KIAA1772, PLP1, OVOS, HAPLN1, EPHA7, ENO2, PCDHB5, OVOS, SYT1, DCT, GPAM, SLITRK6, DCC, FREM2, SDK2, CGA, ATP1B2, SEMA3D, PCDHB15, CDH1, WNT8B, LPAR4, NPIPL3, FAM171B, PTN, ABCC2, ADAMTS3, RASA4, CPT1C, SLC6A6, PCDHB3, LRRC37B, RNFT2, KCNG3, TRPC1, ALPPL2, OR4F21, CCL2, KIF5A, OLFM2, CACNG7, MPHOSPH9, SLC13A4, MOXD1, C6orf186, SLC4A8, STX16, AMY2A, SPARCL1, MGP, A2M, DCN, ATP8B1, MMRN1, EMP1, PLA2G2A, PDE3A, TLR3, CYP1B1, PTGIS, RFTN2, PLEKHA2, SMOC1, STOM, JAM2, CHL1, SCG5, IGFBP7, NPR3, IFI6, CR1L, OR2A4, and OR2A7.

According to some embodiments of the invention the at least one marker positively associated with pancreatic differentiation is selected from the group consisting of: TACSTD2 (TROP-2), BST2, GPR50, ROBO1, NTRK2, ITGA4, LGI1, VIPR2, SLC2A1, MUC15, MUC12, LPHN3, MUC16, VTCN1, MMP16, FZD3, ITGB6, GFRA3, NLGN1, MUC12, CNTFR, LPHN1, SULF1, ADAM23, SCUBE3, PLAU, CDON, SLIT2, C7orf68, PLXDC2, CD74, MUC15, GPR56, VTCN1, ITGB6, AREG, BOC, and KLRK1.

According to some embodiments of the invention the at least one marker positively associated with pancreatic differentiation is TROP-2.

According to some embodiments of the invention the at least one marker positively associated with pancreatic differentiation is GPR50.

According to some embodiments of the invention the at least one marker positively associated with pancreatic differentiation comprises at least two markers, said at least two markers are TROP-2 and GPR50.

According to some embodiments of the invention the at least one marker positively associated with pancreatic differentiation comprises at least three markers, said at least three markers comprise TROP-2, GPR50 and at least one marker selected from the group consisting of TACSTD2 (TROP-2), GPR50, BST2, NTRK2, ITGA4, KDR, PTPRN, LGI1, VIPR2, SLC2A1, MUC15, MUC12, LPHN3, MUC16, VTCN1, MMP16, FZD3, ITGB6, GFRA3, ROBO1, NLGN1, MUC12, CNTFR, LPHN1, SULF1, ADAM23, SCUBE3, PLAU, CDON, SLIT2, C7orf68, PLXDC2, CD74, MUC15, GPR56, VTCN1, ITGB6, AREG, BOC, KLRK1, PRTG, PTPRZ1, GABRP, SILV, KIAA1772, PLP1, OVOS, HAPLN1, EPHA7, ENO2, PCDHB5, OVOS, SYT1, DCT, GPAM, SLITRK6, DCC, FREM2, SDK2, CGA, ATP1B2, SEMA3D, PCDHB15, CDH1, WNT8B, LPAR4, NPIPL3, FAM171B, PTN, ABCC2, ADAMTS3, RASA4, CPT1C, SLC6A6, PCDHB3, LRRC37B, RNFT2, KCNG3, TRPC1, ALPPL2, OR4F21, CCL2, KIF5A, OLFM2, CACNG7, MPHOSPH9, SLC13A4, MOXD1, C6orf186, SLC4A8, STX16, AMY2A, SPARCL1, MGP, A2M, DCN, ATP8B1, MMRN1, EMP1, PLA2G2A, PDE3A, TLR3, CYP1B1, PTGIS, RFTN2, PLEKHA2, SMOC1, STOM, JAM2, CHL1, SCG5, IGFBP7, NPR3, IFI6, CR1L, OR2A4, OR2A7, KCNG3, CACNG7, GRID2, CDH1, LPAR3, SEMA6A, PTPRZ1, ATP1A3, CAMKV, SCNN1G, SYT6, SLC18A2, PCDHB5, ABCG2, HLA-DRA, CR1L, HTR2C, EDNRB, PCDH11X, SLC17A7, SCNN1A, CD9, CXCL16, FXYD5, GABRQ, GFRA3, CACNA2D2, CLDN4, PLP1, PDPN, MMP24, SDK2, GPR176, GPR64, GPR160, PCDH11Y, NKAIN4, ATP1B2, SCN8A, THBS4, CR2, HLA-DQA1, HLA-DRA, HTR7, SLC2A1, HLA-DRA, KCNS3, SLC7A3, HLA-DPB2, CACNA1B, and GPR143.

According to some embodiments of the invention, the at least one marker positively associated with definite endodermal cells is selected from the group consisting of: COLEC12, ROR2, FLRT3, LGR5, LIFR, KEL, FSHR, TRPA1, FOLR1, LRP2, FOLH1, CD177, CCKBR, ITGA5, FZD5, FN1, BMP2, ADAMTS9, DPP4, FGA, GPR128, IGFBP5, FZD4, STC1, TNFSF4, CD177, IHH, APOA1, APOA1, LAMA1, GPC3, LGI1, FN1, LPHN3, FGB, KIT, DPP4, TRO, SPA17, ROBO2, DLK1, CST1, DKK1, HAS2, APOA2, CDH2, AFP, APOB, DGKK, HP, SYTL5, SLC7A7, TTR, FRZB, RSPO3, GCNT1, DIO3, TMEM27, APOC1, EPHA4, C4orf18, GLIPR2, PCDH10, F10, BMP5, FMO5, STMN2, SLC5A9, PORCN, EGFLAM, RAB17, CST2, PNPLA3, MOSPD1, ELMO1, CHST9, SLC30A10, TMC7, C8orf49, CDH12, ST8SIA4, SLCO2A1, MANEA, LRIG3, HCN1, ADAMTS12, TMEM144, VTN, CAMK2N1, ABCC4, PCDH7, OR2T4, UNC93A, COL4A6, PAMR1, SLC1A1, PROS1, APOM, APOM, APOM, LYPD6B, TMEM88, ITLN2, BMPER, GPR141, VEGFA, PRTG, DKK1, APOA2, AFP, APOB, FLRT3, KIAA1772, FBN2, FRZB, KLK6, FREM2, RSPO3, APOA1, APOA1, SEMA3D, KEL, BMP5, HEPH, STMN2, CACNB3, CHST9, TMC7, FAM171B, ATP5G1, FOLR1, ABCC2, EFNA5, CPT1C, MAGED1, RASA4, KDELR3, ST6GALNAC2, EDNRA, LRRC37B, ABCC4, CLCN5, MBOAT2, SLC44A5, NPC2, ELFN1, MMD, SLC5A3, IMPAD1, OSTC, DSC2, SLC31A1, SLC5A12, LRRN3, NPIPL3, GDPD1, NPIPL3, NPIPL3, LINGO2, and LYPD6B.

According to some embodiments of the invention, the at least one marker positively associated with definite endodermal cells is selected from the group consisting of: FSHR, LIFR, COLEC12, ROR2, ITGA5, CD177, CCKBR, APOA1, APOA1, FZD5, FN1, BMP2, ADAMTS9, DPP4, FGA, GPR128, IGFBP5, FZD4, STC1, TNFSF4, CD177, IHH, LRP2, LAMA1, GPC3, LGI1, FN1, LPHN3, FGB, KIT, DPP4, TRO, SPA17, ROBO2, and DLK1.

The phrase “embryonic stem cell” refers to an embryonic cell capable of differentiating into cells of all three embryonic germ layers (i.e., endoderm, ectoderm and mesoderm), or remaining in an undifferentiated state.

According to some embodiments of the invention, the ESC which is used as reference cell is a human ESC obtained from the embryonic tissue formed after gestation (e.g., blastocyst) before implantation (i.e., a pre-implantation blastocyst).

The embryonic stem cells of the invention can be obtained using well-known cell-culture methods. For example, human embryonic stem cells can be isolated from human blastocysts. Human blastocysts are typically obtained from human in vivo preimplantation embryos or from in vitro fertilized (IVF) embryos. Alternatively, a single cell human embryo can be expanded to the blastocyst stage. For the isolation of human ES cells the zona pellucida is removed from the blastocyst and the inner cell mass (ICM) is isolated by immunosurgery, in which the trophectoderm cells are lysed and removed from the intact ICM by gentle pipetting. The ICM is then plated in a tissue culture flask containing the appropriate medium which enables its outgrowth. Following 9 to 15 days, the ICM derived outgrowth is dissociated into clumps either by a mechanical dissociation or by an enzymatic degradation and the cells are then re-plated on a fresh tissue culture medium. Colonies demonstrating undifferentiated morphology are individually selected by micropipette, mechanically dissociated into clumps, and re-plated. Resulting ES cells are then routinely split every 4-7 days. For further details on methods of preparation human ES cells see Thomson et al., [U.S. Pat. No. 5,843,780; Science 282: 1145, 1998; Curr. Top. Dev. Biol. 38: 133, 1998; Proc. Natl. Acad. Sci. USA 92: 7844, 1995]; Bongso et al., [Hum Reprod 4: 706, 1989]; and Gardner et al., [Fertil. Steril. 69: 84, 1998].

It will be appreciated that commercially available stem cells can also be used with this aspect of the present invention. Human ES cells can be purchased from the NIH human embryonic stem cells registry (www.escr.nih.gov). Non-limiting examples of commercially available embryonic stem cell lines are BG01, BG02, BG03, BG04, CY12, CY30, CY92, CY10, TE03, TE04 and TE06.

According to the method of some embodiments of the invention, the hESCs are undifferentiated hESCs.

According to some embodiments of the invention, the undifferentiated hESCs are characterized by an Oct4+ expression pattern.

According to an aspect of some embodiments of the invention, there is provided a method of isolating pancreatic progenitor cells, comprising: (a) identifying the pancreatic progenitor cells according to the method of some embodiments of the invention, and (b) isolating the pancreatic progenitor cells identified according to step (a) to thereby obtain isolated pancreatic progenitor cells, thereby isolating the pancreatic progenitor cells.

As used herein the term “isolating” refers to at least partially separating from the natural environment e.g., the population of cells.

Isolating the pancreatic progenitor cells from the cell population can be performed by any immunological based method which results in the physical isolation of pancreatic progenitor cells having a specific cell surface marker using an antibody or an antibody fragment which specifically recognizes the marker. Examples include, but are not limited to isolation by fluorescence-activated cell sorting (FACS) using the specific antibodies, magnetic beads coated by the specific antibodies [Magnetic-activated cell sorting (MACS)], columns coated by the specific antibodies or immunopanning.

According to some embodiments of the invention, step (b) of the method of some embodiments of the invention is effected by an immunological isolation assay selected from the group consisting of fluorescent activated cell sorter (FACS), Magnetic-activated cell sorting (MACS) or immunopanning.

For example, for isolation using fluorescence-activated cell sorting, the cells are labeled with a fluorescent antibody (e.g., PE-conjugated anti TROP-2 antibody, or PE-conjugated anti GPR50 antibody) and then inserted into a cell sorter (e.g., FACS Aria sorter).

For isolation using magnetic beads, the cells are labeled with a magnetic bead conjugated antibody anti TROP-2 antibody (Miltenyi Biotec) or anti GPR antibody; alternatively, the cells can be labeled with a non-conjugated antibody and followed by incubation with a match isotype bead conjugated secondary antibody (anti mouse IgG1 bead conjugated). Isolation can be performed using magnetic cell separation column such as MAX (Miltenyi Biotec).

According to an aspect of some embodiments of the invention, there is provided an isolated population of pancreatic progenitor cells obtained according to the method of some embodiments of the invention.

According to some embodiments of the invention, the isolated pancreatic progenitor cell population is characterized by TROP-2+ expression pattern.

According to some embodiments of the invention, the isolated pancreatic progenitor cell population is characterized by TROP-2+/GPR50+ expression pattern.

According to some embodiments of the invention, the isolated pancreatic progenitor cell population is characterized by an ngn3+/pax4+/hlxb9+/nkx6.1+/Hnf6+/sox9+/PDX1+ expression signature.

According to an aspect of some embodiments of the invention, there is provided an isolated population of pancreatic progenitor cells, comprising at least about 50%, at least about 60%, e.g., at least about 75% (e.g., 75%), e.g., at least about 80% (e.g., 80%), e.g., at least about 85% (e.g., 85%), e.g., at least about 90% (e.g., 90%), e.g., at least about 95% (e.g., 95%), e.g., at least about 96%, e.g., at least about 97%, e.g., at least about 98%, e.g., at least about 99%, e.g., 100% of cells having a TROP-2+ and/or TROP-2+/GPR50+ expression pattern.

According to some embodiments of the invention, the isolated population of pancreatic progenitor cells is characterized by a TROP-2+/GPR50+/ngn3+/pax4+/hlxb9+/nkx6.1+/Hnf6+/sox9+/PDX1+ expression signature.

According to an aspect of some embodiments of the invention, there is provided an isolated population of pancreatic progenitor cells, comprising at least about 50%, at least about 60%, e.g., at least about 75% (e.g., 75%), e.g., at least about 80% (e.g., 80%), e.g., at least about 85% (e.g., 85%), e.g., at least about 90% (e.g., 90%), e.g., at least about 95% (e.g., 95%), e.g., at least about 96%, e.g., at least about 97%, e.g., at least about 98%, e.g., at least about 99%, e.g., 100% of cells PDX1+.

According to some embodiments of the invention, the isolated population of pancreatic progenitor cells is characterized by a TROP-2+/ngn3+/pax4+/hlxb9+/nkx6.1+/Hnf6+/sox9+/PDX1+ expression signature.

According to some embodiments of the invention, the pancreatic progenitor cells are genetically unmodified (i.e., were not subjected to genetic manipulation, using e.g., recombinant DNA techniques).

According to some embodiments of the invention, the isolated population of pancreatic progenitor cells can be further expanded to produce an expanded population of cells.

Following are non-limiting protocols for culturing and expanding the pancreatic progenitor cells isolated according to some embodiments of the invention, e.g., the TROP-2+/GPR50+ cells:

Protocol 1: The pancreatic progenitor cells (e.g., TROP-2+/GPR50+ cells) are plated on adherent tissue culture plates and cultured in the presence of the DMEM medium (Invitrogen) supplemented with 1% B27 (Invitrogen) [For further details see Kroon 2008 (Nat Biotechnol. 2008; 26:443-52. Epub 2008 Feb. 20) and D'Amour 2006 (Nat Biotechnol. 2006, 24:1392-401. Epub 2006 Oct. 19), each of which is fully incorporated herein by reference]. The medium can be further supplemented with retinoic acid (RA, all trans retinoic acid at a concentration of 2 μM), CYC (KAAD cyclopamine 0.25 μM), and Nog (noggin 50 ng/ml). Additionally or alternatively, the medium can be supplemented with 2 μM RA, 0.25 μM CYC, and 50 ng/m FGF10. Additionally or alternatively, the medium can be supplemented with 50 ng/ml Exendin 4, with or without 1 μM DAPT. Additionally or alternatively, the medium can be CMRL (Invitrogen) supplemented with 1% BSA, 50 ng/ml IGF-1 and 50 ng/ml HGF with or without 50 ng/ml Exendin 4. Additionally or alternatively, the cells can be cultured in DMEM/F12 (Invitrogen), supplemented with N2 (Invitrogen), BSA (2 mg/ml) and bFGF (10 ng/ml) for 4 days, followed by 8 days of culturing in the presence of nicotinamide (10 mM), or alternatively culturing in DMEM/F12 (Invitrogen) supplemented with nicotinamide (10 mM) for 8 days.

Protocol 2: The pancreatic progenitor cells (e.g., TROP-2+/GPR50+ cells) are plated on adherent tissue culture plates and cultured in the presence of the DMEM medium supplemented with 1% B27, with or without Indolactam V (e.g., at a concentration of 330 mM). Additionally or alternatively, the pancreatic progenitor cells are plated on adherent tissue culture plates and cultured in the presence of the DMEM medium supplemented with 1% B27, 2 μM RA, 50 ng/ml FGF10, 0.25 μM KAAD-cyclopamine and cultured for up to 3 days, followed by culturing the cells in the DMEM medium, in the presence of 1% B27, 50 ng/ml FGF10 with or without ILV (e.g., at a concentration of 300 nM) for up to 4 days [for further details see Borowiak 2009 (Cell Stem Cell. 2009; 4: 348-58) and Chen 2009 (Nat Chem Biol. 2009; 5:258-65. Epub 2009 Mar. 15), each of which is fully incorporated herein by reference].

According to an aspect of some embodiments of the invention, there is provided a method of qualifying a pancreatic progenitor cell population, comprising: determining in a sample of the cell population a percentage of the pancreatic progenitor cells which are identified according to the method of some embodiments of the invention out of the total cells in the sample, thereby qualifying the pancreatic progenitor cell population.

According to some embodiments of the invention, pancreatic progenitor cell population has been isolated according to the method of some embodiments of the invention.

According to some embodiments of the invention, presence of at least a predetermined percentage of the pancreatic progenitor cells in the cell sample indicates the suitability of the pancreatic progenitor cells for transplantation in a subject.

According to some embodiments of the invention, the predetermined percentage of the pancreatic progenitor cells comprises at least about 80%, e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pancreatic progenitor cells.

According to an aspect of some embodiments of the invention, there is provided a method of isolating insulin producing cells, comprising culturing the pancreatic progenitor cells isolated by the method of some embodiments of the invention, or the pancreatic progenitor cell population qualified according to the method of some embodiments of the invention under conditions suitable for maturation of the pancreatic progenitor cells into beta cells, thereby isolating insulin producing cells.

Methods and conditions suitable for maturation of the pancreatic progenitor cells into beta cells are known in the art and described in Kroon 2008 (Nat Biotechnol. 2008; 26:443-52), D'Amour 2006 (Nat Biotechnol. 2006, 24:1392-401), Borowiak 2009 (Cell Stem Cell. 2009; 4: 348-58) and Chen 2009 (Nat Chem Biol. 2009; 5:258-65. Epub 2009 Mar. 15), each of which is fully incorporated herein by reference in its entirety.

According to an aspect of some embodiments of the invention, there is provided a method of transplanting pancreatic progenitor cells in a subject, (a) qualifying the pancreatic progenitor cells according to the method of some embodiments of the invention, wherein presence of at least a predetermined percentage of the pancreatic progenitor cells in the cell sample indicates the suitability of the pancreatic progenitor cells for transplantation in a subject, to thereby obtain a pancreatic progenitor cell population being suitable for transplantation in a subject, (b) transplanting in the subject the pancreatic progenitor cell population or cells derived therefrom being suitable for transplantation in a subject, thereby transplanting the pancreatic progenitor cells in the subject.

As used herein the phrase “cells derived therefrom” refers to cells which are differentiated from the pancreatic progenitor cells.

According to some embodiments of the invention, the cells which are differentiated from the pancreatic progenitor cells are capable of producing insulin.

According to some embodiments of the invention, the pancreatic progenitor cell population or cells derived therefrom can be encapsulated prior to transplantation in the subject.

The primary goal in encapsulation as a cell therapy is to protect allogeneic and xenogeneic cell transplants from destruction by the host immune system, thereby eliminating or reducing the need for immuno-suppressive drug therapy. Techniques for macro and microencapsulation of cells are known to those of skill in the art (see, for example, Chang, P. et al. 1999; Matthew, H. W. et al. 1991; Yanagi, K. et al. 1989; Cai Z. H. et al. 1988; Chang, T. M. 1992).

Encapsulation techniques are generally classified as microencapsulation, involving small spherical vehicles and macroencapsulation, involving larger flat-sheet and hollow-fiber membranes (Uludag, H. et al. Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000; 42: 29-64).

Methods of preparing microcapsules are known in the arts and include for example those disclosed by Lu M Z, et al., Cell encapsulation with alginate and alpha-phenoxycinnamylidene-acetylated poly(allylamine) Biotechnol Bioeng. 2000, 70: 479-83, Chang T M and Prakash S. Procedures for microencapsulation of enzymes, cells and genetically engineered microorganisms. Mol Biotechnol. 2001, 17: 249-60, and Lu M Z, et al., A novel cell encapsulation method using photosensitive poly(allylamine alpha-cyanocinnamylideneacetate). J. Microencapsul. 2000, 17: 245-51.

For example, microcapsules are prepared by complexing modified collagen with a ter-polymer shell of 2-hydroxyethyl methylacrylate (HEMA), methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in a capsule thickness of 2-5 μm. Such microcapsules can be further encapsulated with additional 2-5 μm ter-polymer shells in order to impart a negatively charged smooth surface and to minimize plasma protein absorption (Chia, S. M. et al. Multi-layered microcapsules for cell encapsulation Biomaterials. 2002 23: 849-56).

Other microcapsules are based on alginate, a marine polysaccharide (Sambanis, A. Encapsulated islets in diabetes treatment. Diabetes Thechnol. Ther. 2003, 5: 665-8) or its derivatives. For example, microcapsules can be prepared by the polyelectrolyte complexation between the polyanions sodium alginate and sodium cellulose sulphate with the polycation poly(methylene-co-guanidine) hydrochloride in the presence of calcium chloride.

It will be appreciated that cell encapsulation is improved when smaller capsules are used. Thus, the quality control, mechanical stability, diffusion properties, and in vitro activities of encapsulated cells improved when the capsule size was reduced from 1 mm to 400 μm (Canaple L. et al., Improving cell encapsulation through size control. J Biomater Sci Polym Ed. 2002; 13: 783-96). Moreover, nanoporous biocapsules with well-controlled pore size as small as 7 nm, tailored surface chemistries and precise microarchitectures were found to successfully immunoisolate microenvironments for cells (Williams D. Small is beautiful: microparticle and nanoparticle technology in medical devices. Med Device Technol. 1999, 10: 6-9; Desai, T. A. Microfabrication technology for pancreatic cell encapsulation. Expert Opin Biol Ther. 2002, 2: 633-46).

Additional methods of cell encapsulation are well known in the art, such as those described in European Patent Publication No. 301,777 or U.S. Pat. Nos. 4,353,888; 4,744,933; 4,749,620; 4,814,274; 5,084,350; 5,089,272; 5,578,442; 5,639,275; and 5,676,943, each of which is incorporated herein by reference. Other methods are described U.S. Pat. No. 6,281,341; Desai 2002 Exp. Opin. Biol. Hortelano et al. 1996 Blood 87:5095-5103; Pelegrin et al. 1998 Gene Ther. 5:828-834; Lohr et al. 2001 Lancet 357:1591-1592; Cirone et al. Hum. Gene Ther. 13: 1157-1166, each of which is hereby incorporated by reference in it's entirety.

The ordinary skilled artisan will select a biocompatible, as well as a mechanically and chemically stable membrane of a suitable permeability cut-off value that provides immune protection to the implant, functional performance, biosafety and long term survival of the graft.

Encapsulated cells generated according to the present teachings can be used in a myriad of research and clinical applications.

As used herein the term “subject” includes mammals, preferably human beings at any age which may benefit from transplantation of pancreatic cells.

According to some embodiments of the invention, the subject suffers from insulin insufficiency.

According to some embodiments of the invention, the subject has diabetes.

According to some embodiments of the invention, the subject has insulin-dependent diabetes such as type I or type II diabetes.

According to some embodiments of the invention, the subject has loss or reduced of insulin production. For example, the subject has an injured pancreas (e.g., due to a trauma), suffers from pancreatitis and/or suffers from a pancreatic tumor.

According to some embodiments of the invention, the subject has pancreatic cancer and being treated with an agent which kills or reduces the number of beta cells, such as Zanosar (streptozotocin).

According to an aspect of some embodiments of the invention, there is provided a method of identifying definite endodermal cells, comprising determining in a population of cells which comprises definite endodermal cells at least one marker that is negatively associated with definite endodermal cells, the marker being selected from the group consisting of: KDR, PCDHB5, FAT4, FLT1, NRN1, THBS2, PTPRZ1, SLC6A15, GPR176, SEMA6A, THBS1, CDH11, GRID2, SLC7A11, CDH1, LRFN5, EDNRB, THY1, NETO1, KCND2, TMPRSS11E, CD44, PDPN, SLC7A1, KAL1, KCNG3, GPM6B, FXYD5, PCDH18, ICAM3, MCTP1, TACR3, and TMEM155, wherein downregulation above a predetermined threshold of an expression level of the marker negatively associated with the definite endodermal cells as compared to the expression level of the marker in a reference cell, wherein the reference cell is an undifferentiated human ESC, is indicative of a positive identification of a definite endodermal cell.

According to some embodiments of the invention, the method further comprising determining in the population of cells which comprises definite endodermal cells at least one marker that is positively associated with definite endodermal cells, the marker being selected from the group consisting of: FSHR, COLEC12, ROR2, LIFR, LIFR, FLRT3, KEL, LGR5, FOLR1, CD177, CCKBR, APOA1, APOA1, FZD5, FN1, BMP2, ADAMTS9, DPP4, FGA, GPR128, IGFBP5, FZD4, ITGA5, STC1, TNFSF4, CD177, IHH, LRP2, LAMA1, GPC3, LGI1, FN1, LPHN3, FGB, KIT, DPP4, TRO, SPA17, ROBO2, DLK1, CST1, DKK1, HAS2, APOA2, CDH2, AFP, APOB, DGKK, HP, SYTL5, SLC7A7, TTR, FRZB, RSPO3, GCNT1, TRPA1, DIO3, TMEM27, APOC1, EPHA4, C4orf18, GLIPR2, PCDH10, F10, BMP5, FMO5, STMN2, SLC5A9, PORCN, EGFLAM, RAB17, CST2, PNPLA3, MOSPD1, ELMO1, CHST9, SLC30A10, TMC7, C8orf49, CDH12, ST8SIA4, SLCO2A1, MANEA, LRIG3, HCN1, ADAMTS12, FOLH1, TMEM144, VTN, CAMK2N1, ABCC4, PCDH7, OR2T4, UNC93A, COL4A6, PAMR1, SLC1A1, PROS1, APOM, APOM, APOM, LYPD6B, TMEM88, ITLN2, BMPER, GPR141, VEGFA, PRTG, DKK1, APOA2, AFP, APOB, FLRT3, KIAA1772, FBN2, FRZB, KLK6, FREM2, RSPO3, APOA1, APOA1, SEMA3D, KEL, BMP5, HEPH, STMN2, CACNB3, CHST9, TMC7, FAM171B, ATP5G1, FOLR1, ABCC2, EFNA5, CPT1C, MAGED1, RASA4, KDELR3, ST6GALNAC2, EDNRA, LRRC37B, ABCC4, CLCN5, MBOAT2, SLC44A5, NPC2, ELFN1, MMD, SLC5A3, IMPAD1, OSTC, DSC2, SLC31A1, SLC5A12, LRRN3, NPIPL3, GDPD1, NPIPL3, NPIPL3, LINGO2, LYPD6B, TRPA1, SLC40A1, SLC30A10, CCKBR, VIPR2, COLEC12, FLRT3, LGR5, GPR141, BST2, SLC5A9, GPR128, KEL, LRIG3, LYPD6B, FSHR, LIFR, FOLH1, CXCR4, ITGA5, AMOT, LY6E, SEMA6D, GJA5, PRTG, CD34, TMEM144, ROR2, GPR177, OR2T4, SLC7A7, KCNJ3, CLDN18, GPR151, SLC44A5, CDH10, TMEM27, SLC1A1, TMEM56, CD177, PLXNA2, SLC26A2, DSCAM, TMEM133, IL13RA1, ATP2B1, CD302, MEGF9, EDNRA, CDH2, GPR161, TYRO3, FLRT2, LRIT3, PCDH7, NRCAM, SMAGP, AMHR2, ELTD1, GRPR, EPHA4, CD99, GATA3, SEMA3E, HHEX, ZNF280A, FAM184A, WDR72, PDZK1, RLBP1L2, SHISA2, VIL1, STMN2, APOA2, SERHL, PPFIBP2, DKK1, MUM1L1, IGFBP5, ST6GALNAC2, TSPYL5, STC1, SYTL5, EPSTI1, ANKRD1, ARHGAP24, KRT18P49, PRSS2, RHOBTB3, FRZB, RARB, ADAMTS9, ARL4D, PRDM1, HP, FZD5, TRY6, ATP6V0D2, ANGPT2, DENND2C, BMP5, FOXA2, HAS2, BMP2, S100A16, FOLH1B, FAM122C, FZD4, C5, S100A14, VEGFA, CLIP4, GPAM, HNF1B, APOA1, CFLAR, RBM24, RNF152, TTR, TTN, EGFLAM, APOB, DIO3, IFLTD1, ABCC4, CCDC141, ENC1, NEK2, ELMO1, SPOCK3, SERPINI1, ACSL1, GATM, EHHADH, NUDT4, CST1, GLUD2, NPL, ZNF702P, TRY6, SPOCK1, AGL, TFF1, DGKK, SALL1, MANEA, KIT, KRT19, TNNC1, SEPP1, ST8SIA4, YPEL2, ANKMY2, DNAJC15, RNF128, PTPN13, F10, SAMD3, GCNT1, IPP, PROS1, SV2B, PLOD2, MAGEH1, CHST9, ZNF518B, TMEM106C, SERHL2, NTN4, SOX17, FRRS1, OTX2, RNASEL, ELMOD2, MYCT1, PAX6, MGST2, BBS5, MTSS1, VTN, WBP5, DUB4, CCDC92, BTG2, LPGAT1, FN1, TBX3, PLCE1, KRT19P2, IFI16, PORCN, PRSS1, MYL7, DUSP4, PROS1, ANKRD20B, CTSL2, FMO5, USP27X, LAMA1, ADAM28, ZNF611, ANKRD20B, ZNF137, S100Z, GPSM2, TGFB2, and ARHGAP28, wherein upregulation above a predetermined threshold of an expression level of the marker positively associated with definite endodermal cells as compared to the expression level of the marker in a reference cell, wherein the reference cell is an undifferentiated hESC, is indicative of a positive identification of a definite endodermal cell.

According to an aspect of some embodiments of the invention, there is provided a method of identifying definite endodermal cells, comprising determining in a population of cells which comprises definite endodermal cells at least one marker that is positively associated with definite endodermal cells, the marker being selected from the group consisting of: FSHR, COLEC12, ROR2 ITGA5, LRP2, CD177, CCKBR, TRPA1, KEL, FOLR1, FOLH1, APOA1, APOA1, FZD5, FN1, BMP2, ADAMTS9, DPP4, FGA, GPR128, IGFBP5, LIFR, FZD4, PRTG, STC1, TNFSF4, CD177, IHH, LAMA1, GPC3, LGI1, FN1, LPHN3, FGB, KIT, DPP4, TRO, SPA17, ROBO2, DLK1, CST1, DKK1, HAS2, APOA2, CDH2, LGR5, AFP, FLRT3, APOB, DGKK, HP, SYTL5, SLC7A7, TTR, FRZB, RSPO3, GCNT1, DIO3, TMEM27, APOC1, EPHA4, C4orf18, GLIPR2, PCDH10, F10, BMP5, FMO5, STMN2, SLC5A9, PORCN, EGFLAM, RAB17, CST2, PNPLA3, MOSPD1, ELMO1, CHST9, SLC30A10, TMC7, C8orf49, CDH12, ST8SIA4, SLCO2A1, MANEA, LRIG3, HCN1, ADAMTS12, TMEM144, VTN, CAMK2N1, ABCC4, PCDH7, OR2T4, UNC93A, COL4A6, PAMR1, SLC1A1, PROS1, APOM, APOM, APOM, LYPD6B, TMEM88, ITLN2, BMPER, GPR141, VEGFA, DKK1, APOA2, AFP, APOB, FLRT3, KIAA1772, FBN2, FRZB, KLK6, FREM2, RSPO3, APOA1, APOA1, SEMA3D, KEL, BMP5, HEPH, STMN2, CACNB3, CHST9, TMC7, FAM171B, ATP5G1, FOLR1, ABCC2, EFNA5, CPT1C, MAGED1, RASA4, KDELR3, ST6GALNAC2, EDNRA, LRRC37B, ABCC4, CLCN5, MBOAT2, SLC44A5, NPC2, ELFN1, MMD, SLC5A3, IMPAD1, OSTC, DSC2, SLC31A1, SLC5A12, LRRN3, NPIPL3, GDPD1, NPIPL3, NPIPL3, LINGO2, LYPD6B, TRPA1, SLC40A1, SLC30A10, CCKBR, VIPR2, COLEC12, FLRT3, LGR5, GPR141, BST2, SLC5A9, GPR128, KEL, LRIG3, LYPD6B, FSHR, LIFR, FOLH1, CXCR4, ITGA5, AMOT, LY6E, SEMA6D, GJA5, PRTG, CD34, TMEM144, ROR2, GPR177, OR2T4, SLC7A7, KCNJ3, CLDN18, GPR151, SLC44A5, CDH10, TMEM27, SLC1A1, TMEM56, CD177, PLXNA2, SLC26A2, DSCAM, TMEM133, IL13RA1, ATP2B1, CD302, MEGF9, EDNRA, CDH2, GPR161, TYRO3, FLRT2, LRIT3, PCDH7, NRCAM, SMAGP, AMHR2, ELTD1, GRPR, EPHA4, and CD99, wherein upregulation above a predetermined threshold of an expression level of the marker positively associated with the definite endodermal cells as compared to the expression level of the marker in a reference cell, wherein the reference cell is an undifferentiated hESCs, is indicative of a positive identification of a definite endodermal cell.

According to some embodiments of the invention, the method further comprising determining in the population of cells which comprises definite endodermal cells at least one marker that is positively associated with definite endodermal cells, the marker being selected from the group consisting of: GATA3, SEMA3E, HHEX, ZNF280A, FAM184A, WDR72, PDZK1, RLBP1L2, SHISA2, VIL1, STMN2, APOA2, SERHL, PPFIBP2, DKK1, MUM1L1, IGFBP5, ST6GALNAC2, TSPYL5, STC1, SYTL5, EPSTI1, ANKRD1, ARHGAP24, KRT18P49, PRSS2, RHOBTB3, FRZB, RARB, ADAMTS9, ARL4D, PRDM1, HP, FZD5, TRY6, ATP6V0D2, ANGPT2, DENND2C, BMP5, FOXA2, HAS2, BMP2, S100A16, FOLH1B, FAM122C, FZD4, C5, S100A14, VEGFA, CLIP4, GPAM, HNF1B, APOA1, CFLAR, RBM24, RNF152, TTR, TTN, EGFLAM, APOB, DIO3, IFLTD1, ABCC4, CCDC141, ENC1, NEK2, ELMO1, SPOCK3, SERPINI1, ACSL1, GATM, EHHADH, NUDT4, CST1, GLUD2, NPL, ZNF702P, TRY6, SPOCK1, AGL, TFF1, DGKK, SALL1, MANEA, KIT, KRT19, TNNC1, SEPP1, ST8SIA4, YPEL2, ANKMY2, DNAJC15, RNF128, PTPN13, F10, SAMD3, GCNT1, IPP, PROS1, SV2B, PLOD2, MAGEH1, CHST9, ZNF518B, TMEM106C, SERHL2, NTN4, SOX17, FRRS1, OTX2, RNASEL, ELMOD2, MYCT1, PAX6, MGST2, BBS5, MTSS1, VTN, WBP5, DUB4, CCDC92, BTG2, LPGAT1, FN1, TBX3, PLCE1, KRT19P2, IFI16, PORCN, PRSS1, MYL7, DUSP4, PROS1, ANKRD20B, CTSL2, FMO5, USP27X, LAMA1, ADAM28, ZNF611, ANKRD20B, ZNF137, S100Z, GPSM2, TGFB2, and ARHGAP28, wherein upregulation above a predetermined threshold of an expression level of the marker positively associated with the definite endodermal cells as compared to the expression level of the marker in a reference cell, wherein the reference cell is an undifferentiated hESC, is indicative of a positive identification of a definite endodermal cell.

According to some embodiments of the invention, the at least one marker positively associated with definite endodermal cells is selected from the group consisting of: FSHR, COLEC12, ROR2 ITGA5, LRP2, CD177, CCKBR, TRPA1, KEL, FOLR1, FOLH1, APOA1, APOA1, FZD5, FN1, BMP2, ADAMTS9, DPP4, FGA, GPR128, IGFBP5, LIFR, FZD4, PRTG, STC1, TNFSF4, CD177, IHH, LAMA1, GPC3, LGI1, FN1, LPHN3, FGB, KIT, DPP4, TRO, SPA17, ROBO2, DLK1, CST1, DKK1, HAS2, APOA2, CDH2, LGR5, AFP, FLRT3, APOB, DGKK, HP, SYTL5, SLC7A7, TTR, FRZB, RSPO3, GCNT1, DIO3, TMEM27, APOC1, EPHA4, C4orf18, GLIPR2, PCDH10, F10, BMP5, FMO5, STMN2, SLC5A9, PORCN, EGFLAM, RAB17, CST2, PNPLA3, MOSPD1, ELMO1, CHST9, SLC30A10, TMC7, C8orf49, CDH12, ST8SIA4, SLCO2A1, MANEA, LRIG3, HCN1, ADAMTS12, TMEM144, VTN, CAMK2N1, ABCC4, PCDH7, OR2T4, UNC93A, COL4A6, PAMR1, SLC1A1, PROS1, APOM, APOM, APOM, LYPD6B, TMEM88, ITLN2, BMPER, GPR141, VEGFA, DKK1, APOA2, AFP, APOB, FLRT3, KIAA1772, FBN2, FRZB, KLK6, FREM2, RSPO3, APOA1, APOA1, SEMA3D, KEL, BMP5, HEPH, STMN2, CACNB3, CHST9, TMC7, FAM171B, ATP5G1, FOLR1, ABCC2, EFNA5, CPT1C, MAGED1, RASA4, KDELR3, ST6GALNAC2, EDNRA, LRRC37B, ABCC4, CLCN5, MBOAT2, SLC44A5, NPC2, ELFN1, MMD, SLC5A3, IMPAD1, OSTC, DSC2, SLC31A1, SLC5A12, LRRN3, NPIPL3, GDPD1, NPIPL3, NPIPL3, LINGO2, and LYPD6B.

According to some embodiments of the invention, the at least one marker positively associated with definite endodermal cells is selected from the group consisting of: FSHR, COLEC12, ROR2 ITGA5, LRP2, CD177, CCKBR, TRPA1, KEL, FOLR1, FOLH1, APOA1, APOA1, FZD5, FN1, BMP2, ADAMTS9, DPP4, FGA, GPR128, IGFBP5, LIFR, FZD4, PRTG, STC1, TNFSF4, CD177, IHH, LAMA1, GPC3, LGI1, FN1, LPHN3, FGB, KIT, DPP4, TRO, SPA17, ROBO2, and DLK1.

According to some embodiments of the invention, the at least one marker positively associated with definite endodermal cells is selected from the group consisting of: CD177, CCKBR, APOA1, APOA1, FSHR, FZD5, FN1, BMP2, ADAMTS9, DPP4, FGA, COLEC12, ROR2, GPR128, IGFBP5, LIFR, FZD4, ITGA5, STC1, TNFSF4, CD177 and IHH.

According to an aspect of some embodiments of the invention, there is provided a method of identifying definite endodermal cells. The method is effected by determining in a population of cells which comprises definite endodermal cells at least one marker that is positively associated with definite endodermal cells, the marker being selected from the group consisting of: TRPA1, SLC40A1, SLC30A10, CCKBR, VIPR2, COLEC12, FLRT3, LGR5, GPR141, BST2, SLC5A9, GPR128, KEL, LRIG3, LYPD6B, FSHR, LIFR, FOLH1, CXCR4, ITGA5, AMOT, LY6E, SEMA6D, GJA5, PRTG, CD34, TMEM144, ROR2, GPR177, OR2T4, SLC7A7, KCNJ3, CLDN18, GPR151, SLC44A5, CDH10, TMEM27, SLC1A1, TMEM56, CD177, PLXNA2, SLC26A2, DSCAM, TMEM133, IL13RA1, ATP2B1, CD302, MEGF9, EDNRA, CDH2, GPR161, TYRO3, FLRT2, LRIT3, PCDH7, NRCAM, SMAGP, AMHR2, ELTD1, GRPR, EPHA4, and CD99, wherein upregulation above a predetermined threshold of an expression level of the marker positively associated with the definite endodermal cells as compared to the expression level of the marker in a reference cell, wherein the reference cell is an undifferentiated hESC, is indicative of a positive identification of a definite endodermal cell.

According to some embodiments of the invention, the method further comprising determining in the population of cells which comprises definite endodermal cells at least one marker that is positively associated with definite endodermal cells, the marker being selected from the group consisting of: GATA3, SEMA3E, HHEX, ZNF280A, FAM184A, WDR72, PDZK1, RLBP1L2, SHISA2, VIL1, STMN2, APOA2, SERHL, PPFIBP2, DKK1, MUM1L1, IGFBP5, ST6GALNAC2, TSPYL5, STC1, SYTL5, EPSTI1, ANKRD1, ARHGAP24, KRT18P49, PRSS2, RHOBTB3, FRZB, RARB, ADAMTS9, ARL4D, PRDM1, HP, FZD5, TRY6, ATP6V0D2, ANGPT2, DENND2C, BMP5, FOXA2, HAS2, BMP2, S100A16, FOLH1B, FAM122C, FZD4, C5, S100A14, VEGFA, CLIP4, GPAM, HNF1B, APOA1, CFLAR, RBM24, RNF152, TTR, TTN, EGFLAM, APOB, DIO3, IFLTD1, ABCC4, CCDC141, ENC1, NEK2, ELMO1, SPOCK3, SERPINI1, ACSL1, GATM, EHHADH, NUDT4, CST1, GLUD2, NPL, ZNF702P, TRY6, SPOCK1, AGL, TFF1, DGKK, SALL1, MANEA, KIT, KRT19, TNNC1, SEPP1, ST8SIA4, YPEL2, ANKMY2, DNAJC15, RNF128, PTPN13, F10, SAMD3, GCNT1, IPP, PROS1, SV2B, PLOD2, MAGEH1, CHST9, ZNF518B, TMEM106C, SERHL2, NTN4, SOX17, FRRS1, OTX2, RNASEL, ELMOD2, MYCT1, PAX6, MGST2, BBS5, MTSS1, VTN, WBP5, DUB4, CCDC92, BTG2, LPGAT1, FN1, TBX3, PLCE1, KRT19P2, IFI16, PORCN, PRSS1, MYL7, DUSP4, PROS1, ANKRD20B, CTSL2, FMO5, USP27X, LAMA1, ADAM28, ZNF611, ANKRD20B, ZNF137, S100Z, GPSM2, TGFB2, and ARHGAP28, wherein upregulation above a predetermined threshold of an expression level of the marker positively associated with the definite endodermal cells as compared to the expression level of the marker in a reference cell, wherein the reference cell is an undifferentiated hESC, is indicative of a positive identification of a definite endodermal cell.

According to some embodiments of the invention, the method further comprising determining in the population of cells which comprises definite endodermal cells at least one marker that is negatively associated with definite endodermal cells, the marker being selected from the group consisting of: KDR, PCDHB5, FAT4, FLT1, NRN1, THBS2, PTPRZ1, SLC6A15, GPR176, SEMA6A, THBS1, CDH11, GRID2, SLC7A11, CDH1, LRFN5, EDNRB, THY1, NETO1, KCND2, TMPRSS11E, CD44, PDPN, SLC7A1, KAL1, KCNG3, GPM6B, FXYD5, PCDH18, ICAM3, MCTP1, TACR3, TMEM155, ZFP42, THUMPD3, ANXA1, SPP1, PRDM14, GNA14, EDIL3, CXCL12, PSMD5, PRRX1, NANOG, TRIM22, NANOG, RASGRF2, POU5F1B, POLR3G, HHLA1, POU5F1, VSNL1, SCG3, B3GALT1, LECT1, NTS, MBNL1, CKMT1A, NECAB1, FGF2, SFRP2, DCLK1, DACT1, CRABP1, TFAP2C, SCGB3A2, LRAT, CUZD1, GLB1L3, METTL7A, VAT1L, COL12A1, OLFML3, SOX2, USP44, HIST1H4F, KGFLP1, CPT1A, DBC1, CHAC1, CAV1, MT1G, NFIX, FERMT1, GLIPR1, TOX, SNRPN (SNORD116-6), HEY2, T1MP4, IDO1, MT2A, NR5A2, UPRT, MYC, CCDC109B, GAL, ZNF483, RND3, BNC2, COL3A1, LUM, LDB2, MT1E, SNRPN (SNORD116-23), SNRPN (SNORD116-27), GALNT3, PIPOX, PDK1, PREX2, CYP2S1, NRK, VAV3, TNFAIP6, ENPP1, ADD2, SNRPN (SNORD116-24), SNRPN (SNORD109A), STC2, SNORA22, MPPED2, ZNF562, GAP43, FOXB1, TSHZ3, HPGD, ZDHHC22, ACOXL, GLI3, CDCA7L, ZSCAN10, GFPT2, PLP2, HIST1H1A, CAMKV, HERC5, MT1X, TERF1, RAB31, SNRPN (SNORD116-13), ETV1, MT1G, ACTA1, SNRPN (SNORD116-20), NFIB, ZEB2, CBR1, ATXN7L1, SNRPN (SNORD116-1), MT1F, SNRPN (SNORD116-29), AP1M2, ACTG2, CYP2B6, SERPINE1, GRHL2, SLIT2, PIM2, SMARCA2, and RPPH1, wherein downregulation above a predetermined threshold of an expression level of the marker negatively associated with definite endodermal cells as compared to the expression level of the marker in a reference cell, wherein the reference cell is an undifferentiated hESC, is indicative of a positive identification of a definite endodermal cell.

According to an aspect of some embodiments of the invention, there is provided a method of isolating definite endodermal cells. The method is effected by (a) identifying the definite endodermal cells according to the method of some embodiments of the invention, (b) isolating the definite endodermal cells identified according to step (a) to thereby obtain an isolated population of the definite endodermal cells, thereby isolating the definite endodermal cells.

According to an aspect of some embodiments of the invention, there is provided an isolated population of definite endodermal cells obtained according to the method of some embodiments of the invention.

According to some embodiments of the invention, the isolated cell population of definite endodermal cells is characterized by a SOX17+/SOX7+ expression pattern.

According to an aspect of some embodiments of the invention, there is provided an isolated population of definite endodermal cells, comprising at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% (e.g., 50%), at least about 55% (e.g., 55%), at least about 60% (e.g., 60%), at least about 65% (e.g., 65%), at least about 70% (e.g., 70%), at least about 75% (e.g., 75%), at least about 80% (e.g., 80%), at least about 85% (e.g., 85%), at least about 90% (e.g., 90%), at least about 95% (e.g., 95%), at least about 96% (e.g., 96%), at least about 97% (e.g., 97%), at least about 98% (e.g., 98%), at least about 99% (e.g., 99%), e.g., 100% cells having a SOX17+/SOX7+ expression pattern.

According to an aspect of some embodiments of the invention, there is provided a method qualifying a definite endodermal cell population. The method is effected by determining in a sample of the cell population a percentage of the definite endodermal cells which are identified according to the method of some embodiments of the invention out of the total cells in the sample, thereby qualifying the definite endodermal cell population.

According to some embodiments of the invention, the pancreatic progenitor cells or the definite endodermal cells are obtained by differentiating stem cells into pancreatic progenitor cells.

According to some embodiments of the invention, the stem cells are undifferentiated pluripotent stem cells.

As used herein the phrase “pluripotent stem cells” refers to cells which are capable of differentiating into cells of all three embryonic germ layers (i.e., endoderm, ectoderm and mesoderm). The phrase “pluripotent stem cells” may read on embryonic stem cells (ESCs) and/or induced pluripotent stem cells (iPS cells).

The ESCs which are used to generate the pancreatic progenitor cells or the definite endodermal cells can be obtained from the embryonic tissue formed after gestation (e.g., blastocyst) before implantation (i.e., a pre-implantation blastocyst); extended blastocyst cells (EBCs) which are obtained from a post-implantation/pre-gastrulation stage blastocyst (see WO2006/040763]; and/or embryonic germ (EG) cells which are obtained from the genital tissue of a fetus any time during gestation, preferably before 10 weeks of gestation.

Extended blastocyst cells (EBCs) can be obtained from a blastocyst of at least nine days post fertilization at a stage prior to gastrulation. Prior to culturing the blastocyst, the zona pellucida is digested [for example by Tyrode's acidic solution (Sigma Aldrich, St Louis, Mo., USA)] so as to expose the inner cell mass. The blastocysts are then cultured as whole embryos for at least nine and no more than fourteen days post fertilization (i.e., prior to the gastrulation event) in vitro using standard embryonic stem cell culturing methods.

Embryonic germ (EG) cells are prepared from the primordial germ cells obtained from fetuses of about 8-11 weeks of gestation (in the case of a human fetus) using laboratory techniques known to anyone skilled in the arts. The genital ridges are dissociated and cut into small chunks which are thereafter disaggregated into cells by mechanical dissociation. The EG cells are then grown in tissue culture flasks with the appropriate medium. The cells are cultured with daily replacement of medium until a cell morphology consistent with EG cells is observed, typically after 7-30 days or 1-4 passages. For additional details on methods of preparation human EG cells see Shamblott et al., [Proc. Natl. Acad. Sci. USA 95: 13726, 1998] and U.S. Pat. No. 6,090,622.

The phrase “induced pluripotent stem (iPS) cell” (or embryonic-like stem cell) as used herein refers to a proliferative and pluripotent stem cell which is obtained by de-differentiation of a somatic cell (e.g., an adult somatic cell).

According to some embodiments of the invention, the iPS cell is characterized by a proliferative capacity which is similar to that of ESCs and thus can be maintained and expanded in culture for an almost unlimited time.

IPS cells can be endowed with pluripotency by genetic manipulation which re-program the cell to acquire embryonic stem cells characteristics. For example, the iPS cells of the invention can be generated from somatic cells by induction of expression of Oct-4, SOX2, Kfl4 and c-Myc in a somatic cell essentially as described in Takahashi and Yamanaka, 2006, Takahashi et al, 2007, Meissner et al, 2007, and Okita K., et al, 2007, Nature 448: 313-318). Additionally or alternatively, the iPS cells of the invention can be generated from somatic cells by induction of expression of Oct4, SOX2, Nanog and Lin28 essentially as described in Yu et al, 2007, and Nakagawa et al, 2008. It should be noted that the genetic manipulation (re-programming) of the somatic cells can be performed using any known method such as using plasmids or viral vectors, or by derivation without any integration to the genome [Yu J, et al., Science. 2009, 324: 797-801].

The iPS cells of the invention can be obtained by inducing de-differentiation of embryonic fibroblasts [Takahashi and Yamanaka, 2006; Meissner et al, 2007], fibroblasts formed from hESCs [Park et al, 2008], Fetal fibroblasts [Yu et al, 2007; Park et al, 2008], foreskin fibroblast [Yu et al, 2007; Park et al, 2008], adult dermal and skin tissues [Hanna et al, 2007; Lowry et al, 2008], b-lymphocytes [Hanna et al 2007] adult liver and stomach cells [Aoi et al, 2008] and beta-cells derived iPSCs (Bar-Nur O., et al., 2011m Cell Stem Cell 9: 1-7).

IPS cell lines are also available via cell banks such as the WiCell bank. Non-limiting examples of commercially available iPS cell lines include the iPS foreskin clone 1 [WiCell Catalogue No. iPS(foreskin)-1-DL-1], the iPSIMR90 clone 1 [WiCell Catalogue No. iPS(IMR90)-1-DL-1], and the iPSIMR90 clone 4 [WiCell Catalogue No. iPS(IMR90)-4-DL-1].

According to some embodiments of the invention, the induced pluripotent stem cells are human induced pluripotent stem cells.

According to some embodiments of the invention, the stem cells are adult stem cells.

The phrase “adult stem cells” (also called “tissue stem cells” or a stem cell from a somatic tissue) refers to any stem cell derived from a somatic tissue [of either a postnatal or prenatal animal (especially the human)]. The adult stem cell is generally thought to be a multipotent stem cell, capable of differentiation into multiple cell types. Adult stem cells can be derived from any adult, neonatal or fetal tissue such as adipose tissue, skin, kidney, liver, prostate, pancreas, intestine, bone marrow and placenta. Non-limiting examples include mesenchymal stem cells (MSCs), cord blood stem cells, fetal stem cells and the like.

Hematopoietic stem cells, which may also referred to as adult tissue stem cells, include stem cells obtained from blood or bone marrow tissue of an individual at any age or from cord blood of a newborn individual. Preferred stem cells according to this aspect of some embodiments of the invention are embryonic stem cells, preferably of a human or primate (e.g., monkey) origin.

Placental and cord blood stem cells may also be referred to as “young stem cells”.

According to some embodiments of the invention, the cells are human cells.

According to some embodiments of the invention, differentiating the undifferentiated pluripotent stem cells into the pancreatic progenitor cells is performed by: (a) differentiating the pluripotent stem cells into definite endodermal cells to thereby obtain a population of cells which comprises definite endodermal cells, and (b) differentiating the population of cells which comprises the definite endodermal cells into the pancreatic progenitor cells, thereby inducing the differentiation of the pluripotent stem cells into the pancreatic progenitor cells.

According to some embodiments of the invention, differentiating the undifferentiated pluripotent stem cells into the definite endodermal cells is performed by culturing the pluripotent stem cells in the presence of activin A, Wnt3A, a small molecule Induce Definitive Endoderm 1 (IDE1) and/or a small molecule Induce Definitive Endoderm 2 (IDE2) as described in the Examples section which follows and in Jiang J., et al., 2007 (Generation of insulin-producing islet-like clusters from human embryonic stem cells. Stem Cells 25:1940-1953) and/or in D'Amour et al., 2006 (Nat Biotechnol 24:1392-1401), each of which is fully incorporated herein by reference in its entirety.

Differentiation of the definite endodermal cells into the pancreatic progenitor cells can be performed by culturing the definite endodermal cells in the presence of bFGF, KGF, FGF10, noggin, cyclopamine, KAAD cyclopamine, B27, Indolactam V, nicotinamide and/or epidermal growth factor.

According to some embodiments of the invention, differentiating the definite endodermal cells into the pancreatic progenitor cells is performed by culturing the definite endodermal cells in the presence of bFGF, noggin and epidermal growth factor (additional description is provided in Jiang J, Stem Cells. 2007; 25(8):1940-53, which is fully incorporated herein by reference in its entirety.

According to some embodiments of the invention, differentiating the undifferentiated pluripotent stem cells into the pancreatic progenitor cells is performed by differentiation of the pluripotent stem cells into embryoid bodies.

As used herein the phrase “embryoid bodies” (EBs) refers to three dimensional multicellular aggregates of differentiated and undifferentiated cells derivatives of three embryonic germ layers.

Embryoid bodies are formed upon the removal of embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) from feeder layers or feeder cells-free culture systems. ESCs and/or iPSCs removal can be effected using type IV Collagenase treatment for a limited time. Following dissociation from the culturing surface, the cells are transferred to tissue culture plates containing a culture medium supplemented with serum and amino acids.

During the culturing period, EBs are further monitored for their differentiation state. Cell differentiation can be determined upon examination of cell or tissue-specific markers which are known to be indicative of differentiation. For example, EB-derived-differentiated cells may express the neurofilament 68 KD which is a characteristic marker of the ectoderm cell lineage.

The differentiation level of the EB cells can be monitored by following the loss of expression of Oct-4, and the increased expression level of other markers such as α-fetoprotein, NF-68 kDa, α-cardiac and albumin. Methods useful for monitoring the expression level of specific genes are well known in the art and include RT-PCR, semi-quantitative RT-PCR, Northern blot, RNA in situ hybridization, Western blot analysis and immunohistochemistry.

Embryoid bodies can be generated from various primates and mammals such as human, monkeys and rodents (e.g., mouse, rat).

According to some embodiments of the invention, the embryoid bodies are obtained from human embryoid bodies.

According to some embodiments of the invention, the embryoid bodies are obtained by spontaneous differentiation of pluripotent stem cells.

According to some embodiments of the invention, for differentiation into pancreatic progenitor cells, the embryoid bodies are differentiated until about day 7-21 of human EBs differentiation.

According to some embodiments of the invention, for differentiation into definite endodermal cells, the embryoid bodies are differentiated until about day 3-7 of human EBs differentiation.

According to an aspect of some embodiments of the invention there is provided a nucleic acid construct comprising a first polynucleotide encoding a reporter protein and a second polynucleotide which comprises a human endogenous SOX17 regulatory sequence, wherein the first polynucleotide being under transcriptional regulation of the SOX17 regulatory sequence, wherein the SOX17 regulatory sequence comprises an upstream sequence and a downstream sequence.

According to some embodiments of the invention, expression of the reporter protein is under the transcriptional regulation of the SOX17 regulatory sequences.

As used herein the phrase “reporter protein” refers to any polypeptide which can be detected in a cell. According to some embodiments of the invention, the reporter polypeptide can be directly detected in the cell (no need for a detectable moiety with an affinity to the reporter) by exerting a detectable signal which can be viewed in living cells (e.g., using a fluorescent microscope). Non-limiting examples of a nucleic acid sequence encoding a reporter polypeptide according to this aspect of the present invention include the red fluorescent protein (RFP), the green fluorescent protein (GFP) (e.g., SEQ ID NO:2) or mCherry (e.g., SEQ ID NO:33).

Alternatively, the reporter polypeptide can be indirectly detected such as when the reporter polypeptide is an epitope tag. Indirect detection can be effected by introducing a detectable moiety (labeled antibody) having an affinity to the reporter or when the reporter is an enzyme by introducing a labeled substrate. For example, the reporter polypeptide can be an antigen which is recognized by and binds to a specific antibody. Preferably, when such a reporter polypeptide is utilized the antibody or the polypeptide capable of binding the reporter protein is labeled (e.g., by covalently attaching to a label such as a fluorescent dye).

Non-limiting examples of suitable SOX17 upstream regulatory sequences which can be used in the nucleic acid construct of some embodiments of the invention include the sequences comprising SEQ ID NO: 7, 36, and 38.

Non-limiting examples of suitable SOX17 downstream regulatory sequences which can be used in the nucleic acid construct of some embodiments of the invention include the sequences comprising SEQ ID NO: 8, 37 and 39.

According to an aspect of some embodiments of the invention there is provided a nucleic acid construct comprising a first polynucleotide encoding a reporter protein and a second polynucleotide which comprises a human endogenous SOX17 regulatory sequence, wherein the first polynucleotide being under transcriptional regulation of the SOX17 regulatory sequence, wherein the SOX17 regulatory sequence comprises an upstream sequence and a downstream sequence, wherein the upstream sequence comprises the nucleotide sequence set forth in SEQ ID NO:38; and wherein the downstream sequence comprises the nucleotide sequence set forth in SEQ ID NO:39.

According to an aspect of some embodiments of the present invention there is provided a nucleic acid construct comprising a first polynucleotide encoding a reporter protein and a second polynucleotide which comprises a human endogenous PDX1 regulatory sequence, wherein the first polynucleotide being under transcriptional regulation of the PDX1 regulatory sequence, wherein the PDX1 regulatory sequence comprises an upstream sequence and a downstream sequence.

Non-limiting examples of suitable PDX1 upstream regulatory sequences which can be used in the nucleic acid construct of some embodiments of the invention include the sequences comprising SEQ ID NO: 16, 19, 23, and 31.

Non-limiting examples of suitable PDX1 downstream regulatory sequences which can be used in the nucleic acid construct of some embodiments of the invention include the sequences comprising SEQ ID NO: 17, 20, 24, and 32.

According to an aspect of some embodiments of the present invention there is provided a nucleic acid construct comprising a first polynucleotide encoding a reporter protein and a second polynucleotide which comprises a human endogenous PDX1 regulatory sequence, wherein the first polynucleotide being under transcriptional regulation of the PDX1 regulatory sequence, wherein the PDX1 regulatory sequence comprises an upstream sequence and a downstream sequence, wherein the upstream sequence comprises the nucleotide sequence set forth in SEQ ID NO:16 (corresponds to nucleotides 1 to 8640 of AL353195.14); and wherein the downstream sequence comprises the nucleotide sequence set forth in SEQ ID NO:17 (corresponds to nucleotides 13321 to 32526 of AL353195.14 (SEQ ID NO:34)). According to some embodiments of the invention, the nucleic acid construct is a bacterial artificial chromosome (BAC).

Non-limiting examples of suitable BACs which comprise the SOX17 genomic sequence include the RP11-53M11 BAC (SEQ ID NO:35; GenBank Accession No. AC091076.7), and the RP11-47H10 BAC (SEQ ID NO:40).

A non-limiting example of a suitable BACs which comprise the PDX1 genomic sequence include the RP11-322P28 (SEQ ID NO:34; GenBank Accession No. AL353195.14).

According to an aspect of some embodiments of the invention there is provided a cell comprising the nucleic acid construct of some embodiments of the invention.

According to some embodiments of the invention, the cell is a stem cell.

According to some embodiments of the invention, the stem cell is an embryonic stem cell or an induced pluripotent stem cell.

According to some embodiments of the invention, the cell is a human cell.

According to an aspect of some embodiments of the invention there is provided a method of screening for markers which differentiate a definite endodermal cell from an undifferentiated pluripotent stem cell, comprising comparing the expression level of markers between the undifferentiated pluripotent stem cell and the cell of some embodiments of the invention (e.g., the cell which comprises the reporter protein under the transcriptional regulation of the SOX17 upstream and downstream regulatory sequence), wherein upregulation or downregulation in the expression level above a predetermined threshold indicates that the markers differentiate the definite endodermal cell from the undifferentiated pluripotent stem cell, thereby screening for markers which differentiate the definite endodermal cell from the undifferentiated pluripotent stem cell.

According to an aspect of some embodiments of the invention there is provided a method of screening for compounds capable of inducing differentiation of undifferentiated pluripotent stem cells to definite endodermal cells, comprising: (a) contacting undifferentiated pluripotent stem cells which comprise the nucleic acid construct of some embodiments of the invention (e.g., the construct which comprises the reporter protein under the transcriptional regulation of the SOX17 upstream and downstream regulatory sequence) with at least one compound of a plurality of candidate compounds, and; (b) monitoring an expression level of the reporter protein in the cells following the contacting, wherein an increase above a predetermined level in the expression level of the reporter protein following the contacting as compared to the expression level prior to the contacting is indicative that the at least one compound is capable of inducing differentiation of the undifferentiated pluripotent stem cells to the definite endodermal cells, thereby screening for the compounds capable of inducing differentiation of undifferentiated pluripotent stem cells to definite endodermal cells.

According to some embodiments of the invention, the method further comprising synthesizing the compound capable of inducing differentiation of the undifferentiated pluripotent stem cells to the definite endodermal cells.

According to an aspect of some embodiments of the invention there is provided a method of screening for markers which differentiate a pancreatic progenitor cell from a definite endodermal cell, comprising comparing the expression level of markers between the cell which comprises the reporter protein under the transcriptional regulation of the SOX17 upstream and downstream regulatory sequence of some embodiments of the invention and the cell which comprises the reporter protein under the transcriptional regulation of the PDX1 upstream and downstream regulatory sequence of some embodiments of the invention, wherein upregulation or downregulation in the expression level above a predetermined threshold indicates that the markers differentiate the pancreatic progenitor cell from the definite endodermal cell, thereby screening for markers which differentiate the pancreatic progenitor cell from the definite endodermal cell.

According to an aspect of some embodiments of the invention there is provided a method of screening for compounds capable of inducing differentiation of definite endodermal cells or undifferentiated pluripotent stem cells to pancreatic progenitor cells, comprising: (a) contacting definite endodermal cells or undifferentiated pluripotent stem cells which comprise the nucleic acid construct of some embodiments of the invention (which comprises the reporter protein under the transcriptional regulation of the PDX1 upstream and downstream regulatory sequences) with at least one compound of a plurality of candidate compounds, and; (b) monitoring an expression level of the reporter protein in the cells following the contacting, wherein an increase above a predetermined level in the expression level of the reporter protein following the contacting as compared to the expression level prior to the contacting is indicative that the at least one compound is capable of inducing differentiation of the definite endodermal cells or undifferentiated pluripotent stem cells to the pancreatic progenitor cells, thereby screening for the compounds capable of inducing differentiation of definite endodermal cells or undifferentiated pluripotent stem cells to the pancreatic progenitor cells.

According to some embodiments of the invention the method further comprising synthesizing the compound capable of inducing differentiation of the definite endodermal cells or undifferentiated pluripotent stem cells to the pancreatic progenitor cells.

The agents of some embodiments of the invention which are described hereinabove for screening for markers which differentiate a definite endodermal cell from an undifferentiated pluripotent stem cell, for screening for compounds capable of inducing differentiation of undifferentiated pluripotent stem cells to definite endodermal cells, for screening for markers which differentiate a pancreatic progenitor cell from a definite endodermal cell, and/or for screening for compounds capable of inducing differentiation of definite endodermal cells or undifferentiated pluripotent stem cells to pancreatic progenitor cells may be included in a diagnostic kit/article of manufacture preferably along with appropriate instructions for use and labels indicating FDA approval for the above described use.

Such a kit can include, for example, at least one container including at least one of the above described agents (e.g., the nucleic acid constructs, the cells comprising same) and an imaging reagent packed in another container (e.g., enzymes, antibodies, buffers, chromogenic substrates, fluorogenic material). The kit may also include appropriate buffers and preservatives for improving the shelf-life of the kit.

According to an aspect of some embodiments of the invention there is provided a kit for screening for markers which differentiate a definite endodermal cell from a pluripotent stem cell, comprising the cell of some embodiments of the invention.

According to some embodiments of the invention the kit further comprising a pluripotent stem cell.

According to an aspect of some embodiments of the invention there is provided a kit for screening for markers which differentiate a pancreatic progenitor cell from a definite endodermal cell, comprising the cell of some embodiments of the invention and the cell of some embodiments of the invention.

According to some embodiments of the invention the kit further comprising at least one agent suitable for detecting an expression level of a marker of interest.

According to some embodiments of the invention the expression level is detected by an RNA detection method.

According to some embodiments of the invention the expression level is detected by a protein detection method.

According to some embodiments of the invention the kit further comprising a genetic micro array chip.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., eds. (1985); “Transcription and Translation” Hames, B. D., and Higgins S. J., Eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide To Methods And Applications”, Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategies for Protein Purification and Characterization—A Laboratory Course Manual” CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

General Materials and Experimental Methods

hESC Culture and Differentiation—

H9 (4), I3, I6 (13) and CSES2 (14, 15) cells were routinely cultured on mouse embryonic fibroblasts (MEFs) that have been mitotically inactivated by mitomycin C. The cells were grown in ES cell medium consisting of: 80% knockout Dulbeco's Modified Eagle's Medium (Invitrogen, Paisley, UK), 20% knockout serum replacement (Invitrogen), 1 mM glutamine (Biological Industries, Israel) and 1% non-essential amino acids (Biological Industries, Israel), 0.1 mM 2-mercaptoethanol (Sigma Chemical Co.) and 4 ng/ml basic fibroblast growth factor (bFGF) (PeproTech, Rehovot, Israel). Karyotyping of hESCs and transfected clones was performed as described (16). Embryonic bodies (EBs) were formed by harvesting the cells with a trypsin solution which contains calcium and magnesium (Biological Industries, Israel). When cell clusters began to detach from the MEF feeders, the cells were collected, centrifuged and transferred to plastic non coated UV-irradiated bacteriological Petri dishes in EB medium in order to allow their aggregation. The EB medium used is essentially the same as the ES cell medium except bFGF was not included. The EB medium consisted of: 80% knockout Dulbecco's Modified Eagle's Medium (Invitrogen, Paisley, UK), 20% knockout serum replacement (Invitrogen), 1 mM glutamine (Biological Industries, Israel) and 1% non-essential amino acids (Biological Industries, Israel).

Transfections

BAC Transgenesis of a SOX17-GFP Reporter BAC—

The SOX17 BAC reporter construct was built using recombineering (recombination-mediated genetic engineering) (17) in which the coding sequence of the SOX17 gene was replaced by the coding sequence of the GFP gene together with a floxed neomycin resistance gene. BACs were obtained from the Australian Genome Research Facility and specifically RP11-53M11 and RP11-47H10 were modified. The modified BACs were electroporated into hESCs essentially according to the protocol of Zwaka and Thomson (18). Briefly, hESCs cells were harvested from the plate with a trypsin solution containing calcium and magnesium and were centrifuged and the pellet was resuspended in 500 microliters of ES cell medium. Fifty micrograms of BAC DNA were mixed with PBS to a final volume of 300 microliters and the DNA solution was mixed with the cell suspension and was transferred to a 4 mm cuvette (Bio-Rad, Richmond, Calif.). The cells were electroporated using a Bio-Rad Gene Pulser at 320V and 200 mF. After electroporation the cuvette was allowed to stand for ten minutes and then the contents were transferred to a plate of MEFs in ES cell medium. For selection of resistant colonies, the hESCs were cultured on MEFs harboring the neomycin resistance gene (derived from DR4 mice; Jackson Laboratories USA). To obtain optimal survival of the cells after electroporation, Rho-associated kinase (ROCK) Inhibitor (Alexis Biochemicals, San Diego, Calif., USA) was added to the medium at a concentration of 10 mM. Two days later, selection with G418 (40 microgram/ml effective concentration) was initiated and selection medium was changed every two or three days. At about day 14, G418 resistant clones were isolated and expanded.

The various clones were allowed to form embryoid bodies (EBs) in the presence of activin A (66 ng/ml) (Peprotech) and two days later, the EBs were dissociated into single cells with TrypLE select (Invitrogen) and were analysed by FACS.

BAC Transgenesis of PDX1-GFP Reporter BAC—

A PDX1 BAC reporter construct was built in which the coding sequence of the PDX1 gene was replaced by the coding sequence of the GFP gene together with a floxed Neo gene in a BAC spanning the genomic region of the PDX1 gene (RP11-328P22, GenBank Accession No. AL353195.14 (SEQ ID NO:34)). The BAC included about 8.6 kb of upstream promoter sequence of the PDX1 gene (SEQ ID NO:16) and about 19.2 Kb of the downstream sequence of the PDX1 gene (SEQ ID NO:17).

Similarly to the SOX17-BAC, various hESC clones harbouring the PDX1-GFP reporter BAC were isolated and were allowed to differentiate by forming EBs. At day 14, the EBs were dissociated into single cells and were analysed by FACS.

Cell Culture and Genetic Labeling—

Human Embryonic Stem Cells (HESC) clones carrying either SOX17 or PDX1 BAC GFP reporter protein were generated for the screening. Cells were grown in a tissue culture facility and received standard treatments and medium replacement. Differentiations to definitive endoderm (SOX17⁺) and to pancreatic cells (PDX1⁺) were achieved by applying the appropriate protocols as described below. Positive monitoring of SOX17-GFP⁺ cells and PDX1-GFP⁺ cells enables the isolation of semi-homogenous population for the expression analysis.

Differentiation of Pluripotent Stem Cells into Definite Endodermal Cells—

To differentiate hESCs toward definitive endoderm, the present inventors used the protocol described by D'Amour (D'Amour 2006, Nat Biotechnol 24:1392-1401) which includes culturing the cells in the presence of Activin A or its substitute IDE2 (Borowiak M, 2009 (Cell Stem Cell 4, 348-358)). In brief, 80-90% confluent hESCs were cultured for 1 day in RPMI (Invitrogen) medium supplemented with 1 mM glutamine and either 25 ng/ml Wnt3a (R&D) and 100 ng/ml Activin A (Peprotec), or 5 μM IDE2 (Enzo Life Sciences Inc, Lausen, Switzerland). The medium was then changed to RPMI with 0.2% Foetal bovine serum (FBS) (Invitrogen), 1 mM glutamine and 100 ng/ml Activin A or 5 μM IDE2 for culturing of 2 additional days.

Differentiation of Definite Endodermal Cells into Pancreatic Progenitor Cells—

Definite endodermal cells can be further differentiated into pancreatic progenitor cells by following the differentiation protocol(s) described by D'Amour 2006 (Nat Biotechnol 24:1392-1401), Kroon 2008 (Nat Biotechnol. 2008; 26:443-52. Epub 2008 Feb. 20) and/or Borowiak 2009 (Cell Stem Cell 4, 348-358, Apr. 3, 2009).

Microarray—

RNA was extracted from FACS sorted cell populations using Qiagen micro RNA isolation kit. The integrity of the RNA was confirmed using an RNA Pico Bioanalyzer and microarray analysis was performed on the Human Gene ST1.0 chip (Affymetrix, Santa Clara, Calif.). Three replicas from each differentiation stage (SOX17⁺ and PDX1⁺ cells) were loaded on microarray chips. Analysis was performed on the Human Gene ST1.0 chip (Affymetrix, Santa Clara, Calif.). Evaluation of changes in gene expression was performed by comparing array results to a previous Human Embryonic Stem Cells (HESC) microarray data (3 replicas) and to Pancreas microarray results (3 replicas).

Data Analysis—

In accordance with the aim of the current study, an emphasis was put on genes with known surface characteristics. The Gene Ontology (GO) Cellular Component Term information from the Affymetrix database was analyzed, enabling an efficient prioritization of the relevant genes and sequences. The present inventors used the Partek® Genomics Suite™ (Partek Incorporated, St. Louis, Mo., U.S.A) for the bioinformatic analysis. Following the first analysis, a refinement of each selected gene was done by crossing the results with the comprehensive GeneCards database (http://www(dot)genecards(dot)org/). Following the cross check, genes were divided into two groups: cell surface genes; and putative surface and membranal genes.

Statistical Analysis—

The Fold Change (FC) threshold was set on FC=2.0. Expression comparisons were calculated by using Student's test. Significance was taken as P<0.05.

Selection of PDX1 Expressing Cells—

14 day-old EBs or older EBs were used to select for TROP-2 and GPR50 expressing cells.

FACS Sorting—

FACS sorting was performed on single cells that had been dissociated using TrypLE select (Invitrogen) using a FACS ARIA (Becton Dickinson, Bedford, Mass.). GFP positive (GFP+) and GFP negative (GFP−) cells were isolated. FACS sorting was also performed using allophycocyanin (APC) conjugated mouse monoclonal anti human TROP-2 (1:100) and mouse monoclonal anti human GPR50 (1:200) (both from R&D Systems Inc., Minneapolis, Minn.) followed by secondary anti-mouse immunoglobulin G (IgG) fluorescence isothiocyanate (FITC) conjugated antibody 1:100 (Chemicon/Millipore, Billerica, Mass.).

RT-PCR—

Total RNA was isolated from differentiated hESCs using RNeasy micro kit (Qiagen, Hilden, GmbH) according to the manufacturer's recommended protocol. cDNA was synthesized using Superscript reverse transcriptase (Invitrogen). Quantitative Real Time (QRT) PCR (RT-qPCR) analysis was performed in triplicate and normalized to the internal endogene GAPDH gene expression. The reaction was performed in an ABI Prism 7000 (Applied Biosystems, Warrington, UK) with the TaqMan Universal PCR Master Mix (Applied Biosystems) using Taqman probes (Applied Biosystems) and analyzed using the relative Quantification (RQ) study in the Sequence Detection Software (V. 1.2; Applied Biosystems).

Immunofluorescence—

The cells were seeded on 13 mm glass cover slides in E-well culture plates. Forty-eight hours after seeding, cells were fixed for 20 minutes in 4% paraformaldehyde in PBS; permeabilized using 0.5% TritonX-100 in PBS/1% fetal bovine serum, and incubated overnight with the primary antibodies: goat anti-SOX17 1:400 (R&D), goat anti-OCT4 1:200 (Santa Cruz), rabbit anti-green florescent protein (GFP) polyclonal antibody 1:100 (Chemicon/Millipore, Billerica, Mass.), or goat anti-PDX1 1:10,000 (Abcam plc, Cambridge, UK). After rinsing, secondary anti-mouse or anti-goat IgG Cy3 and anti-rabbit IgG Cy2 (Jackson Laboratories, West Grove, Pa.) were added to the samples, which were then incubated for an additional hour. Finally, the cells were rinsed once more and mounted with mounting media (VECTASHIELD, Vector Lab, CA). The slides were analyzed using a confocal microscope (Bio-Rad MRC 1024, Richmond Calif., USA).

Example 1

The differentiation of hESCs into pancreatic beta cells is a stepwise process by which the initially pluripotent cell gradually becomes more committed towards the final cell fate of a functional insulin-producing cell. Initially, the pluripotent stem cells differentiate via mesendoderm into definitive endoderm. The definitive endoderm then commits towards a pancreatic cell fate, and these cells in turn differentiate towards an endocrine pancreatic cell fate, after which they commit to beta cells. FIG. 5A depicts a schematic illustration of differentiation of stem cells into pancreatic cells.

In order to fully characterize the differentiation of hESCs into insulin-producing cells it is important to identify and isolate these stage specific progenitor cells and characterize their properties on the molecular level. By obtaining a transcriptional profile of these cells and by identifying transcription factors that they express, a clearer picture of the differentiation process can be obtained. The present inventors took the approach of genetically labeling hESCs with stage specific fluorescent reporter constructs, thus generating hESCs reporter clones. These provide a simple, accurate and sensitive readout of the appearance of the progenitors thus enabling optimization of conditions for inducing their differentiation. The fluorescent reporter lines also provide valuable tools for isolating progenitor cells by FACS and further studying their gene profile.

The present inventors chose two key transcription factors expressed during differentiation towards beta cells: SOX17 and PDX1. SOX17 is expressed early at the definitive endoderm stage while PDX1 is expressed at a critical point in the pathway at which the cells decide to differentiate into pancreas or non-pancreas endodermal derivatives. Using genetically modified hESCs expressing EGFP under the control of SOX17 or PDX1 promoters, the present inventors were able to isolate the stage specific cells by FACS and characterize their transcriptional profile by qPCR and microarray analysis.

Experimental Results

BAC Transgenesis of a SOX17-GFP Reporter BAC

The simplest method to generate reporter cells is to introduce a reporter plasmid expressing EGFP under a minimal promoter. The problem with this approach is that the random integration of a plasmid is subject to position effects, and also that the minimal promoter might not mimic the exact in vivo expression pattern of the gene. A different approach that alleviates some of these problems is to modify a BAC that spans the genomic region of the gene by inserting IRES-EGFP into the 3′UTR of the gene or by replacing the coding sequence of the gene with the coding sequence of EGFP. In such a way, EGFP is expressed under the control of a more extensive genomic sequence that presumably contains all or most of the critical transcriptional regulatory elements of the gene. Furthermore, the large size of the BAC may, to some extent, buffer the reporter construct from position effects.

A SOX17-BAC reporter construct was built in which the coding sequence of the SOX17 gene was replaced by the coding sequence of the GFP gene together with a floxed neo gene.

Preparation of RP11-53M11 BAC SOX17-Reporter:

EGFP and floxed neo was knocked into the SOX17 gene locus in a human genomic BAC RP11-53M11 by replacing the coding sequence of SOX17 (ATG to TGA) with the coding sequence of GFP and also the floxed neo. FIG. 1A schematically illustrates the structure of the recombinant construct. The BAC includes approximately 28.3 kb of SOX17 upstream sequence (SEQ ID NO:36) and approximately 132 kb of the SOX17 downstream sequence (SEQ ID NO:37). The sequence of the recombinant BAC (SEQ ID NO:1) includes the GFP coding sequence (SEQ ID NO:2), along with the SV40 poly A sequence (SEQ ID NO:27), a loxP site (SEQ ID NO:4), the neomycin resistance gene sequence (SEQ ID NO:3) with the SV40 early promoter/enhancer (SEQ ID NO:28), and the TK polyadenylation site (SEQ ID NO:29), a second loxP site (SEQ ID NO:4), encompassed by the human endogenous SOX17 upstream and downstream regulatory sequences.

Preparation of SOX17-GFP Knock in (KI) Plasmid Construct—

The present inventors have then subcloned out a region of the recombinant RP11-53M11 BAC SOX17-reporter BAC into a plasmid in order to generate a gene targeting vector (SEQ ID NO:6). The genomic sequence encompassing the SOX17-GFP sequence (from nucleotide 15487 of SEQ ID NO:1 to nucleotide 32492 of SEQ ID NO:1) was subcloned into the Bluescript plasmid (Stratagene) (FIG. 1B, the sequence marked as 5′-arm, EGFP, Floxed Neo and 3′-arm). The 5′-arm includes about 9 kb of SOX17 upstream regulatory sequence (SEQ ID NO:7) and the 3′-arm includes about 5 kb of SOX17 downstream regulatory sequence (SEQ ID NO:8).

Two 3′-external probes were prepared (SEQ ID NOs:9 and 10) in order to detect correct targeting of the vector into the genomic DNA of the host cell. For example, Southern blot screening of targeting at the 3′ end, the 3′-external probe (2) (SEQ ID NO:10) is used on XbaI digested genomic DNA (gDNA). The expected wild type band is 16.7 kb and the expected targeted band is 10.3 kb. The vector can be linearized with NruI. The targeted clone should be ampicillin and kanamycin resistant.

Preparation of BAC SOX17-Reporter RP11-47H10:

EGFP and floxed neo was knocked into the SOX17 gene locus in a human genomic BAC RP11-47H10 by replacing the coding sequence of SOX17 (ATG to TGA) with the coding sequence of GFP and the floxed neo. FIG. 1C schematically illustrates the structure of the recombinant construct. There is approximately 74 kb upstream of the SOX17 upstream regulatory sequence (SEQ ID NO:38) and approximately 73 kb of the downstream regulatory sequence (SEQ ID NO:39). The sequence of the recombinant BAC (SEQ ID NO:5) includes the GFP coding sequence (SEQ ID NO:2), along with the SV40 poly A sequence (SEQ ID NO:27), a loxP site (SEQ ID NO:4), the neomycin resistance gene sequence (SEQ ID NO:3) with the SV40 early promoter/enhancer (SEQ ID NO:28), and the TK polyadenylation site (SEQ ID NO:29), a second loxP site (SEQ ID NO:4), encompassed by the human endogenous SOX17 upstream (SEQ ID NO:38) and downstream (SEQ ID NO:39) regulatory sequences.

Preparation of SOX17-GFP Knock-in hESC Clones and Embryoid Bodies Differentiated Therefrom—

The RP11-47H10 GFP reporter BAC was electroporated into hESCs and G418 resistant clones were isolated. The various clones were allowed to form EBs in the presence of activin A (66 ng/ml) and at day 2, the EBs were dissociated into single cells and were analysed by FACS (FIG. 2A) and by fluorescent microscopy (FIGS. 2B-2E). It was previously shown by several groups that high concentrations of activin A induced hESCs to differentiate into definitive endoderm. SOX17 is one of the endodermal genes that are induced using such a protocol, which initiates the path toward insulin producing cell differentiation (19, 20). As shown, there was a clear population of GFP expressing cells in the different clones which varied from about 5% to about 40% (FIG. 2A). Next, an 80% confluent monolayer of the SOX17-GFP reporter line was treated with activin A (66 ng/ml) for three days after which immunohistochemistry was performed. The staining was carried out with anti-SOX17 (red) and anti-GFP (green) and demonstrated a clear correlation between SOX17 and GFP expression (FIG. 2E) thus confirming that the GFP⁺ cells indeed represent the SOX17⁺ population. The present inventors further stained the cells for both OCT4 (green), which marks undifferentiated cells, and SOX17 (red), marking the definitive endoderm population (FIG. 2G). Cells expressing OCT4 indicating the pluripotent state, were not positive for SOX17 and conversely, cells expressing SOX17 had turned off their OCT4 expression.

Gene Expression Analysis of SOX17+ Cells—

The SOX17-GFP reporter cells were grown as a monolayer in the presence of activin A for two days and then they were FACS-sorted into GFP⁺ and GFP⁻ populations. RNA was extracted from both populations and cDNA was synthesized. qPCR was performed comparing the relative expression of various genes between the GFP⁺ and the GFP⁻ cell populations (FIG. 2H). The present inventors were able to demonstrate that the GFP⁺ cells were enriched for SOX17 expression as compared to the GFP⁻ cell population, thus confirming that the GFP positive cells indeed represent a SOX17 positive cell population. Moreover, in the GFP⁺ cell population, an upregulation of genes which are related to definitive endoderm or mesendoderm such as chemokine receptor 4 (CXCR4), Cerebrus (CER), Goosecoid (GSC), CD34, and hepatocyte nuclear factor 3 beta (FOXA2) was observed, while expression of genes, such as NANOG which is primarily a marker of undifferentiated cells, was clearly decreased in the GFP⁺ population. These results further validate that the GFP+ sorted population indeed represent definitive endoderm or mesendoderm.

Transcriptional Profiling of SOX17⁺ Cells—

Microarray analysis was performed on the SOX17⁺ fraction using an Affymetrix Human Gene ST1.0 chip to obtain a transcriptional profile of this precursor population. Tables 1-2 hereinbelow summarize the findings of the microarray analysis, with information regarding membrane-associated genes (membranal and cell surface markers) which are upregulated (Table 1) and downregulated (Table 2) in the SOX17+ subpopulation of cells as compared to the genetically un-modified hESCs. Tables 3-4 hereinbelow summarize the findings of the microarray analysis, with information regarding non-membrane-associated genes (e.g., non-membranal, intracellular, secreted, transcription factors and the like) which are upregulated (Table 3) and downregulated (Table 4) in the SOX17+ subpopulation of cells as compared to the genetically un-modified hESCs. Examples of transcription factors and growth factors that were enriched in the SOX17⁺ expressing cells include FOXA2, bone morphogenetic protein 2 (BMP2), frizzled homolog 5 (FZD5), HNF1 homeobox B (HNF1b), GATA binding protein 3 (GATA3), paired box 3 (PAX3), vascular endothelial growth factor A (VEGFA) and Kruppel-like factor 8 (KLF8). Examples of secreted proteins that were enriched in the SOX17+ expressing cells include insulin-like growth factor binding protein 5 (IGFBP5), Apolipoprotein A-I (ApoA1) and apolipoprotein B (APOB). Examples of transmembrane proteins enriched in this population include CXCR4 (as was previously reported), CLAUDIN 18, cholecystokinin B receptor (CCKBR), leukemia inhibitory factor receptor alpha (LIFR), G protein-coupled receptor 141 (GPR141), G protein-coupled receptor 128 (GPR128), follicle stimulating hormone receptor (FSHR), CD34, and bone marrow stromal cell antigen 2 (BST2).

TABLE 1
Membranal genes which are upregulated in SOX17+ cells as compared to genetically un-modified hESCs
				SEQ ID
				NO: (Of
		Affy. target nucl.		Rep. Public		Log 2 fold change
Gene Symbol	Probe Set ID	SEQ ID NO:	Rep. Public ID	ID)	Polyn. SEQ ID NOs: rep. by target	[SOX17-HESC]
TRPA1	8151341	44	NM_007332	107	4060, 8269, 5433,	+4.18
SLC40A1	8057677	45	NM_014585	108	3154, 4368, 6969, 5406, 7045,	+3.69
					8144, 8627, 7674, 6595, 6989,
SLC30A10	7924342	46	NM_018713	109	3493, 3718, 6268, 6130, 8273,	+3.65
					8216,
CCKBR	7938090	47	NM_176875	110	2809, 5307, 5676,	+3.50
VIPR2	8165658	48	X59268	111	2553, 3426, 3422, 6269,	+3.45
COLEC12	8021946	49	NM_130386	112	2891, 4601, 6579,	+3.12
FLRT3	8065071	50	NM_198391	113	2812, 5172, 5173, 5918, 7056,	+3.11
					6380,
LGR5	7957140	51	NM_003667	114	2732, 3193, 6795, 5509,	+3.09
GPR141	8132347	52	NM_181791	115	3313, 3318, 6342, 5701, 6692,	+3.04
BST2	8035304	53	NM_004335	116	3090, 2805, 5403, 7124,	+3.02
SLC5A9	7901316	54	NM_001011547	117	4682, 4626, 4627, 6649, 6403,	+2.94
					7403, 7829, 6781, 6361,
GPR128	8081298	55	NM_032787	118	3995, 4847, 5470, 7733, 8308,	+2.93
					8445,
KEL	8143534	56	NM_000420	119	2886, 2987, 6934, 8187, 8288,	+2.91
					6818, 6144, 8559,
LRIG3	7964602	57	NM_153377	120	3457, 3537, 4639, 5656, 6683,	+2.89
					7048,
LYPD6B	8045664	58	NM_177964	121	3039, 3538, 8714, 5464, 7286,	+2.83
					6455, 7123, 7806, 7494, 8341,
					7067, 6613,
FSHR	8052072	59	NM_000145	122	4265, 5021, 5022, 6252, 5713,	+2.73
					7612, 7221, 6397,
LIFR	8111677	60	NM_001127671	123	2551, 4403, 4404, 5491, 7187,	+2.72
					7919, 8314, 7758,
FOLH1	7948058	61	NM_153696	124	3060, 3615, 2779, 2843, 3871,	+2.70
					2598, 4703, 4705, 4140, 4138,
					2773, 3026, 3280, 5177, 5176,
					2766, 5178, 3821, 6173, 5921,
					6307, 5425, 6469, 6968,
CXCR4	8055465	62	NM_001008540	125	2782, 3775, 3776, 5384, 6834,	+2.69
					7645,
ITGA5	7963786	63	NM_002205	126	3341, 3761, 5661, 6880,	+2.58
AMOT	8174576	64	NM_001113490	127	2826, 4332, 3837, 7904, 5933,	+2.55
					6751, 5641, 6093, 6295,
LY6E	8148572	65	NM_001127213	128	2668, 4370, 4371, 8116, 5582,	+2.47
					7083, 8656, 8711, 8353, 7871,
					7878,
SEMA6D	7983527	66	NM_024966	129	3078, 3079, 3080, 3081, 3082,	+2.46
					4192, 4279, 3184, 3186, 3187,
					3185, 3188, 3183, 6040, 7595,
					5709, 6809, 6877, 7470, 6289,
					5994,
GJA5	7919340	67	NM_005266	130	3364, 4104, 4105, 7691, 6103,	+2.46
					6550, 5474,
PRTG	7989073	68	NM_173814	131	3710, 4778, 6379,	+2.42
CD34	7923978	69	NM_001773	132	2563, 3891, 3854, 5956, 6086,	+2.40
					5785, 6059, 8087,
TMEM144	8098041	70	NM_018342	133	2802, 3163, 3170, 3261, 3319,	+2.40
					3506, 3267, 4584, 8697, 8424,
					5686, 8259, 8744, 8370, 7977,
					8192, 7923, 7849, 8039, 7954,
					8413, 7830,
ROR2	8162283	71	NM_004560	134	3804, 4102, 2608, 3151, 3287,	+2.35
					5241, 6393, 7306, 6756, 8135,
GPR177	7916862	72	NM_024911	135	3473, 5169, 5171, 5170, 6985,	+2.33
					7944, 8040, 5423, 6217, 5957,
					7720, 6063, 8128, 6006,
OR2T4	7911273	73	NM_001004696	136	3717, 6107,	+2.33
SLC7A7	7977786	74	NM_001126105	137	2789, 5292, 5291, 7158, 7255,	+2.32
					5870, 8255, 6471, 7642,
KCNJ3	8045795	75	NM_002239	138	2667, 3201, 5594, 8178,	+2.31
CLDN18	8082928	76	NM_016369	139	4183, 3788, 3787, 8686, 5317,	+2.29
					6211,
TMEM106C	7955063	77	NM_024056	140	3447, 4690, 4693, 4691, 4692,	+2.22
					5419, 6591, 7332,
GPR151	8114896	78	NM_194251	141	3379, 3780, 5572,	+2.21
SLC44A5	7917052	79	NM_152697	142	3153, 4453, 4454, 7183, 6087,	+2.18
					6160,
CDH10	8111255	80	NM_006727	143	2788, 5142, 8497, 5507, 8253,	+2.18
					8371,
TMEM27	8171472	81	NM_020665	144	2960, 4942, 6430,	+2.17
SLC1A1	8154135	82	NM_004170	145	3070, 5025, 5401, 7218, 8331,	+2.13
TMEM56	7903162	83	NM_152487	146	4133, 4607, 6302, 7020, 6966,	+2.11
					6405,
CD177	8037298	84	NM_020406	147	3744, 4005, 6408, 7480, 7666,	+2.09
PLXNA2	7923991	85	NM_025179	148	4111, 4029, 6303, 8403, 6499,	+2.08
SLC26A2	8109194	86	NM_000112	149	3484, 3972, 5711, 7134,	+2.05
DSCAM	8070421	87	NM_001389	150	2796, 3591, 6842,	+2.04
TMEM133	7943369	88	NM_032021	151	2860, 3175, 5703,	+2.02
IL13RA1	8169580	89	NM_001560	152	2683, 3219, 6256, 6220, 8730,	+2.00
					6320,
ATP2B1	7965359	90	NM_001001323	153	2655, 3664, 3663, 6331, 5399,	+1.92
					6047, 6021, 6425,
CD302	8056102	91	NM_014880	154	3314, 3316, 3317, 3215, 2603,	+1.92
					4536, 3431, 3435, 4256, 3004,
					4660, 5416, 6685, 8428, 8482,
					7955,
MEGF9	8163775	92	NM_001080497	155	4704, 5185, 6235, 6965,	+1.91
EDNRA	8097692	93	NM_001957	156	2600, 4920, 4921, 4922, 8628,	+1.89
					5591, 5960, 8235, 5962, 8220,
					6628, 7962,
CDH2	8022674	94	NM_001792	157	3134, 4668, 5546,	+1.88
GPR161	7922108	95	NM_153832	158	3044, 3194, 7409, 6232, 7729,	+1.86
					6084, 8332, 5530, 5999,
TYRO3	7982938	96	NM_006293	159	2646, 5109, 5565,	+1.85
FLRT2	7976073	97	NM_013231	160	4059, 3816, 5667,	+1.82
LRIT3	8096839	98	NM_198506	161	3439, 4440, 5693, 7042, 6600,	+1.82
PCDH7	8094520	99	NM_032457	162	2723, 5014, 5015, 2937, 5262,	+1.80
					5768, 5971,
NRCAM	8142270	100	NM_001037132	163	3565, 3859, 3993, 3989, 3309,	+1.79
					3102, 3441, 4484, 5180, 5181,
					5182, 5183, 5184, 8267, 7609,
					7127, 6412, 6732, 7550, 6919,
					8165, 7672, 6704, 6312, 7407,
					7189, 7606, 6991, 7159, 6420,
					6482, 6987,
SMAGP	7963280	101	NM_001033873	164	3242, 3886, 3885, 6421, 7267,	+1.73
AMHR2	7955797	102	NM_020547	165	4056, 4904, 4903, 4905, 7400,	+1.73
					5395,
ELTD1	7917182	103	NM_022159	166	3027, 4175, 6126, 7630,	+1.73
GRPR	8166202	104	NM_005314	167	2579, 3797, 6967,	+1.73
EPHA4	8059279	105	NM_004438	168	3926, 3577, 6427, 7497, 5543,	+1.72
CD99	8165794	106	NM_001122898	169	3639, 2591, 2693, 3374, 2887,	+1.70
					3376, 3010, 3030, 4292, 4358,
					3378, 8134, 6730, 8768, 6667,
					6606, 7512, 7524,
Table 1. Provided are the description of the membranal genes along with the sequence identifiers of the Affymetrix probes, the target sequence identified by the Affymetrix probe (Affy. target nucl. SEQ ID NO:), and the mRNA sequences represented by the target sequences. Also shown are the log 2 fold change in expression levels of the indicated genes between the SOX17 GFP cells and the genetically unmodified HESCs. The (+) or (−) sign means that the genes are upregulated or downregulated, respectively, in the SOX17-GFP cells as compared to the genetically unmodified HESCs.

TABLE 2
Membranal genes which are downregulated in SOX17+ cells as compared to genetically un-modified ESCs
		Affy.				Log 2 fold
		target		SEQ ID NO:		change
Gene		nucl. SEQ		(of Rep.		[SOX17-
Symbol	Probe Set ID	ID NO:	Rep. Public ID	Public ID)	Polyn. SEQ ID NOs: rep. by target	HESC]
KDR	8100393	170	NM_002253	204	2708, 4588, 7921, 5498, 8228,	−4.30
PCDHB5	8108697	171	NM_015669	205	2872, 2931, 5379,	−3.42
FAT4	8097288	172	NM_024582	206	3507, 4331, 5697, 7491,	−2.99
FLT1	7970763	173	NM_001159920	207	3763, 4816, 4818, 4819, 4817, 5510,	−2.88
NRN1	8123739	174	NM_016588	208	3230, 3454, 7755, 5387,	−2.87
THBS2	8130867	175	NM_003247	209	4184, 3540, 8063, 7661, 5992, 6904,	−2.85
PTPRZ1	8135774	176	NM_002851	210	2599, 3943, 7457, 8030, 7815, 8011, 8237, 7646,	−2.79
SLC6A15	7965206	177	NM_001146335	211	3618, 4807, 4688, 4689, 5536, 5746, 5761, 7293,	−2.77
GPR176	7987439	178	NM_007223	212	3580, 2783, 5852,	−2.72
SEMA6A	8113666	179	NM_020796	213	2844, 4722, 3122, 3148, 4310, 4145, 8137, 8152,	−2.68
					7900, 5499, 8724, 8667, 8467, 8111, 5437, 8321,
					7896, 5978, 8764,
THBS1	7982597	180	NM_003246	214	4759, 3539, 7996, 5434, 8028, 7563, 6593, 8344,	−2.63
CDH11	8001800	181	NM_001797	215	2639, 2970, 5559, 6928,	−2.51
GRID2	8096440	182	NM_001510	216	2719, 4235, 8429, 5557, 7933, 8154,	−2.51
SLC7A11	8102800	183	NM_014331	217	2916, 3899, 8531, 5555,	−2.46
CDH1	7996837	184	NM_004360	218	4149, 4182, 2614, 4278, 4556, 2794, 2795, 4415,	−2.46
					4294, 4315, 4347, 5506, 7453, 5404, 7133,
LRFN5	7974109	185	NM_152447	219	4563, 4583, 5622,	−2.45
EDNRB	7972157	186	NM_003991	220	2606, 5247, 5246, 5248, 5597, 7819, 7369,	−2.44
THY1	7952268	187	NM_006288	221	3096, 4729, 5694,	−2.36
NETO1	8023828	188	NM_138999	222	3269, 3045, 5252, 5646, 6813, 5692,	−2.28
KCND2	8135705	189	NM_012281	223	3907, 3225, 5604, 8488,	−2.28
TMPRSS11E	8095380	190	NM_014058	224	3472, 4222, 5579, 8653,	−2.21
TMPRSS11E	8095364	191	NM_014058	225	3472, 4222, 5579, 8653,	−2.19
CD44	7939341	192	NM_000610	226	3067, 3068, 2739, 3276, 4538, 2566, 2586, 2564,	−2.17
					2565, 2660, 4282, 4552, 4163, 4631, 2890, 3277,
					3579, 3658, 3659, 3660, 3661, 3662, 6650, 7581,
					7244, 6542, 7096, 5461, 6031, 7241, 6225, 7493,
					6898, 5515, 6679, 5545, 5516,
PDPN	7898057	193	NM_001006624	227	3236, 2746, 2747, 3084, 3519, 2965, 3133, 4305,	−2.08
					4306, 3747, 3748, 3745, 3746, 8458, 8786, 8213,
					5840, 7528, 8392, 8050, 7659, 7822, 8446,
SLC7A1	7970810	194	NM_003045	228	3517, 4636, 6881,	−2.08
KAL1	8171248	195	NM_000216	229	2607, 4088, 5560, 8200,	−2.07
KCNG3	8051785	196	NM_133329	230	3647, 5227, 5228, 5783, 7561,	−2.05
GPM6B	8171359	197	NM_005278	231	4714, 3686, 3685, 3687, 3684, 6150, 8294, 6043,	−2.04
					5590, 7105,
FXYD5	8027778	198	NM_144779	232	3228, 3470, 2828, 3419, 3286, 4528, 3008, 3388,	−1.96
					4895, 4896, 4898, 4897, 6381, 7395, 6565, 5906,
					7311, 8512,
PCDH18	8102792	199	NM_019035	233	4262, 4225, 5888, 7284,	−1.93
ICAM3	8033987	200	NM_002162	234	3247, 4340, 5318,	−1.92
MCTP1	8113130	201	NM_024717	235	3688, 3689, 4710, 3372, 4621, 4622, 8584, 8536,	−1.89
					8020, 8738, 8599, 7737, 8789, 5624, 7945, 7981,
					7997, 8146, 6686, 7715, 8769, 8630, 7018,
TACR3	8102127	202	NM_001059	236	2592, 5166, 5672,	−1.82
TMEM155	8102636	203	NM_152399	237	3259, 4659, 4024, 8590, 7444, 8029, 8685, 7009,	−1.79
					7201, 8783, 5815, 7282,
Table 2. Provided are the description of the membranal genes along with the sequence identifiers of the Affymetrix probes, the target sequence identified by the Affymetrix probe, and the mRNA sequences represented by the target sequences. Also shown are the log 2 fold change in expression levels of the indicated genes between the SOX17 GFP cells and the genetically unmodified HESCs. The (+) or (−) sign means that the genes are upregulated or downregulated, respectively, in the SOX17-GFP cells as compared to the genetically unmodified HESCs.

TABLE 3
Non-membranal genes which are up-regulated in SOX17+ cells as compared to genetically un-modified ESCs
		Affy. target		SEQ ID NO:		Log 2 fold
Gene	Probe	nucl. SEQ	Rep.	(Of Rep.	Polyn. SEQ ID NOs:	change
Symbol	Set ID	ID NO:	Public ID	Public ID)	rep. by target	[SOX17-HESC]
GATA3	7926105	238	NM_002051	530	2915, 3695, 3696, 6780, 6049, 7381,	+4.48
SEMA3E	8140650	239	NM_012431	531	4218, 5112, 5113, 5625, 7193,	+4.11
HHEX	7929282	240	NM_002729	532	2961, 4124, 7867, 5797, 8612,	+3.94
ZNF280A	8074853	241	NM_080740	533	3304, 3570, 5866,	+3.88
FAM184A	8129231	242	NM_024581	534	3503, 4208, 4209, 8021, 7766, 5900, 8495,	+3.82
					6262,
WDR72	7988990	243	NM_182758	535	3418, 4240, 6598, 6055,	+3.80
PDZK1	7904843	244	NM_002614	536	3336, 5243, 4204, 7461, 7260, 6532, 6226,	+3.75
					7090, 6900, 7707, 7472, 6465,
RLBP1L2	8121803	245	NM_001010852	537	3116, 3869, 5483, 6248,	+3.66
SHISA2	7970676	246	NM_001007538	538	4110, 3758, 5588,	+3.57
VIL1	8048319	247	NM_007127	539	4477, 4578, 5439,	+3.51
STMN2	8147030	248	NM_007029	540	4711, 5225, 5342,	+3.51
APOA2	7921834	249	NM_001643	541	2901, 2758, 6253, 8161, 7460, 7113, 8544,	+3.44
					6696, 7530, 7963, 6627, 6449,
SERHL	8073585	250	NM_014509	542	4046, 3113, 3203, 4870, 6789, 7257, 7696,	+3.42
					6190, 7885, 6148, 8055, 5575,
PPFIBP2	7938231	251	NM_003621	543	3011, 4355, 5626,	+3.39
DKK1	7927631	252	NM_012242	544	3471, 3796, 7543, 8463, 8242, 5959,	+3.38
MUM1L1	8169145	253	NM_152423	545	3142, 4991, 4993, 6316, 5894, 5576,	+3.33
IGFBP5	8058857	254	NM_000599	546	2583, 4357, 8723, 5360,	+3.28
ST6GALNAC2	8018761	255	NM_006456	547	2638, 4437, 5345,	+3.27
TSPYL5	8151931	256	NM_033512	548	3241, 3675, 5757,	+3.26
STC1	8149825	257	NM_003155	549	2725, 3793, 5771,	+3.25
SYTL5	8166747	258	NM_001163335	550	3411, 4887, 4885, 4886, 5912, 6888, 5629,	+3.19
EPSTI1	7971296	259	NM_033255	551	3367, 3694, 3693, 5738, 5732, 7664, 7742,	+3.18
ANKRD1	7934979	260	NM_014391	552	2995, 3511, 5932,	+3.18
ARHGAP24	8096160	261	NM_001025616	553	3856, 4013, 4014, 4012, 8287, 7220, 7970,	+3.08
					6456, 8329, 8596, 5517, 8693, 8490,
KRT18P49	8169233	262	ENST00000427083	554	7599,	+3.07
PRSS2	8136790	263	—			+3.05
RHOBTB3	8106986	264	NM_014899	555	3394, 4041, 7121, 8530, 7782, 8683, 8248,	+3.05
					7828, 8218, 8489,
FRZB	8057506	265	NM_001463	556	2679, 5106, 5695,	+3.05
RARB	8078286	266	NM_016152	557	2681, 3505, 4596, 4598, 8684, 7033, 8333,	+3.03
					6707, 5820, 6360, 7180,
ADAMTS9	8088560	267	NM_182920	558	4100, 3333, 8622, 8439, 8457, 6636, 7795,	+3.01
					8233, 8784,
ARL4D	8007493	268	NM_001661	559	3037, 3901, 5663,	+3.00
PRDM1	8121257	269	NM_182907	560	3253, 5303, 4361, 6228, 6168, 8184, 8624,	+2.96
					7700,
HP	7997188	270	NM_005143	561	3893, 4016, 4018, 2580, 2622, 2977, 2992,	+2.96
					3453, 3617, 4365, 4366, 6921, 6310, 6000,
					6001,
FZD5	8058498	271	NM_003468	562	2934, 4015, 5838,	+2.95
TRY6	8136807	272	—			+2.94
ATP6V0D2	8147145	273	NM_152565	563	3548, 3146, 7959, 8150, 5593,	+2.93
ANGPT2	8149071	274	NM_001147	564	2696, 4344, 4345, 4346, 5744, 7577,	+2.92
DENND2C	7918768	275	NM_198459	565	3586, 4103, 6796, 6943, 8236, 8046, 6164,	+2.88
					7594,
BMP5	8127193	276	NM_021073	566	2611, 3202, 5997, 7150,	+2.87
FOXA2	8065344	277	NM_021784	567	2770, 4577, 4466, 5823, 7393, 7364, 6571,	+2.83
HAS2	8152617	278	NM_005328	568	2676, 4348, 5843, 6409,	+2.80
BMP2	8060850	279	NM_001200	569	3900, 6567,	+2.79
S100A16	7920291	280	NM_080388	570	3782, 2999, 5985, 6066, 6285, 6298, 8082,	+2.79
FOLH1B	7942998	281	NM_153696	571	3060, 2843, 3280, 5177, 2766, 5178, 6173,	+2.78
					5921, 6307, 5425, 6469,
FAM122C	8169995	282	NM_001170779	572	4513, 4955, 4956, 4957, 4958, 4959, 4960,	+2.77
					4954, 6451, 7031, 8034, 6865, 6135, 8059,
					8191, 6912, 8587, 6236,
FZD4	7950885	283	NM_012193	573	2784, 5289,	+2.77
C5	8163839	284	NM_001735	574	3997, 3497, 8045, 5344, 6954, 6376,	+2.77
S100A14	7920297	285	NM_020672	575	2827, 2832, 8484, 5892, 5948, 6180, 7538,	+2.76
					6036,
VEGFA	8119898	286	NM_001025366	576	3808, 4966, 4967, 4968, 4969, 4970, 4971,	+2.74
					4972, 4973, 4974, 4975, 4976, 4977, 4978,
					4979, 4980, 4981, 8712, 5968, 6175, 7640,
					5961, 5690, 7243, 8549, 6759, 6136, 7716,
					8136, 8355, 8159, 8018, 7079, 8270, 8120,
					7866, 8507, 8066, 7946,
CLIP4	8041179	287	NM_024692	577	4055, 4666, 6910, 6677, 7982, 7631, 5684,	+2.72
					7192, 6576, 6387, 7420, 8380,
GPAM	8165663	288	AK172782	578	3609,	+2.70
HNF1B	8014591	289	NM_000458	579	2989, 4226, 4915, 5347, 6709, 6537,	+2.69
APOA1	7951865	290	NM_000039	580	3902, 2762, 6306, 5375, 6654, 6990,	+2.68
CFLAR	8047381	291	NM_003879	581	2698, 2700, 2701, 2702, 2703, 2697, 2727,	+2.67
					2728, 2729, 2714, 2715, 2716, 2717, 2718,
					3809, 3282, 4540, 4723, 2706, 2695, 2707,
					4275, 4518, 2881, 4319, 5254, 5255, 7718,
					7181, 7213, 6901, 7210, 7050, 6868, 7797,
					7093, 6246, 6301, 6416, 6026, 8088, 7173,
					6972, 6669, 7157, 7540, 7179, 6019, 5721,
RBM24	8117045	292	NM_153020	582	3092, 4733, 4735, 4734, 6592, 6373, 5606,	+2.66
					8008, 8572, 8110,
RNF152	8023598	293	NM_173557	583	3123, 3946, 5605,	+2.66
TTR	8020795	294	NM_000371	584	2722, 4730, 6739, 5362,	+2.66
TTN	8057056	295	NM_133378	585	3308, 2656, 3424, 4106, 4107, 3344, 3048,	+2.63
					5008, 5009, 5011, 5007, 5010, 7368, 5887,
					5945, 7339, 6830, 8194, 6950, 6315, 6321,
					6481, 7366, 6697, 6793, 7546, 6131, 5681,
EGFLAM	8105013	296	NM_152403	586	3530, 5277, 5276, 5275, 6119, 5859, 8466,	+2.62
					5742, 6783, 8384, 6717, 7825, 8629, 8422,
					7942, 6265,
APOB	8050619	297	NM_000384	587	2558, 3976, 5351,	+2.62
DIO3	7976858	298	NM_001362	588	2990, 4161, 8265, 6088,	+2.61
IFLTD1	7961875	299	NM_152590	589	4498, 4781, 4782, 4784, 4785, 4783, 5790,	+2.60
					7143, 6544, 6853,
ABCC4	7972297	300	NM_005845	590	3298, 4237, 4238, 6825, 6506, 6826, 6736,	+2.58
					7298,
CCDC141	8057377	301	NM_173648	591	3425, 5152, 5914, 5666, 7929,	+2.58
ENC1	8112615	302	NM_003633	592	2731, 4418, 7756, 5853, 8481,	+2.57
NEK2	7924096	303	NM_002497	593	3232, 5258, 5965, 7142, 6196,	+2.57
ELMO1	8139057	304	NM_014800	594	2968, 3692, 4045, 2861, 3438, 4258, 4520,	+2.56
					4479, 2801, 2880, 3542, 5280, 5279, 8359,
					7086, 5936, 6939, 6791, 6584, 7972, 7197,
					7051, 6279, 5770, 6974, 6770, 7488, 7401,
					7549, 6438, 7349,
SPOCK3	8103544	305	NM_016950	595	3468, 4487, 4491, 4493, 4496, 4500, 4505,	+2.54
					4506, 4522, 2959, 3354, 2879, 3351, 3951,
					3952, 7390, 8151, 7745, 8674, 7862, 8555,
					8291, 8542, 7775, 8670, 8212, 7911, 5944,
					8680, 8411, 8508, 6200, 8249, 7909, 8528,
					8673, 7951, 8415, 8750, 7722,
SERPINI1	8083779	306	NM_005025	596	4280, 4350, 4351, 7428, 8534, 7747, 5615,	+2.54
					7839, 7903,
ACSL1	8103951	307	NM_001995	597	2601, 4521, 2572, 4269, 4707, 4709, 3264,	+2.52
					3180, 3549, 8277, 8532, 8386, 8421, 8369,
					8009, 8062, 6545, 8440, 5566, 8524, 7826,
					7554,
GATM	7988414	308	NM_001482	598	3346, 4123, 6982,	+2.52
EHHADH	8092523	309	NM_001166415	599	3206, 4927, 4926, 5368, 8762,	+2.52
NUDT4	7957536	310	NM_199040	600	3968, 3967,	+2.50
CST1	8065412	311	NM_001898	601	3369, 3033, 5734, 6511,	+2.50
GLUD2	8169711	312	NM_012084	602	2899, 3400, 5864,	+2.47
NPL	7908003	313	NM_030769	603	3446, 5244, 8301, 8566, 7329, 6079, 6297,	+2.47
					6185, 6208, 6540, 5453, 5873, 5974, 6575,
ZNF702P	8039025	314	NR_003578	604	3075, 4190, 5550,	+2.45
TRY6	8136801	315	NM_002770	605	3326, 3327, 4043, 4044, 3896, 4082, 2633,	+2.45
					3056, 4031, 3057, 4349, 5199, 5294, 5200,
					4875, 7372, 5743, 6222, 8379,
SPOCK1	8114287	316	NM_004598	606	2804, 3903, 8553, 8505, 6817, 7957, 5469,	+2.41
					8014, 7754,
AGL	7903239	317	NM_000028	607	2687, 4063, 4068, 4064, 4066, 4065, 4067,	+2.40
					6243, 5987, 6127, 5817, 5995, 5972, 8735,
					6008,
TFF1	8070579	318	NM_003225	608	2552, 3668, 5847,	+2.39
DGKK	8172670	319	NM_001013742	609	3778, 5274, 6444,	+2.37
SALL1	8001387	320	NM_001127892	610	4497, 4411, 4078, 6646, 5432, 7163,	+2.36
MANEA	8121144	321	NM_024641	611	4179, 5290, 6156, 5952, 8006,	+2.35
KIT	8095110	322	NM_000222	612	3646, 4153, 4152, 5648, 8162,	+2.35
KRT19	8015349	323	NM_002276	613	3524, 4126, 7422, 8625, 5885, 6794, 6946,	+2.35
TNNC1	8087925	324	NM_003280	614	3054, 4613, 5365, 7708,	+2.35
SEPP1	8111915	325	NM_001093726	615	3112, 3121, 2969, 3211, 3248, 3487, 4155,	+2.33
					4154, 3820, 8621, 7799, 8519, 8258, 7809,
					8140, 7833, 8546, 8284, 8435, 8309, 7973,
					7743, 7917, 8097,
ST8SIA4	8113358	326	NM_005668	616	3302, 4599, 3244, 7055, 7738, 5386,	+2.32
YPEL2	8008819	327	NM_001005404	617	3716, 3768, 5719,	+2.32
ANKMY2	8138370	328	NM_020319	618	3159, 5132, 7035, 7445, 7172, 5729,	+2.31
DNAJC15	7968872	329	NM_013238	619	3842, 3846, 7013, 8073,	+2.31
RNF128	8169174	330	NM_024539	620	3527, 3478, 3479, 5441, 5712, 6719,	+2.31
PTPN13	8096176	331	NM_006264	621	2621, 4579, 4580, 4581, 4582, 6886, 7770,	+2.31
					7790, 7383, 8720, 7840, 6014, 5863, 7589,
F10	7970241	332	NM_000504	622	2567, 2573, 3210, 3246, 4308, 4309, 3935,	+2.30
					7894, 6670, 7728, 6624, 7953, 7352, 6493,
SAMD3	8129482	333	NM_001017373	623	3098, 4120, 4122, 7473, 7107, 6192, 5825,	+2.30
					7041, 6688,
GCNT1	8155930	334	NM_001490	624	2578, 4156, 4157, 4158, 4159, 4160, 8460,	+2.29
					6349, 8001, 7226, 6641,
IPP	7915775	335	NM_016486	625	2772, 4084, 4775, 4774, 7978, 7647, 6202,	+2.28
					6163,
PROS1	8089015	336	NM_000313	626	2597, 4770, 6690, 8232,	+2.27
SV2B	7986195	337	NM_001167580	627	3448, 4936, 4935, 5668, 6488,	+2.26
PLOD2	8091283	338	NM_182943	628	3144, 3823, 3824, 7950, 7025, 6032, 8398,	+2.24
					5670, 7845,
MAGEH1	8167887	339	NM_014061	629	2950, 3007, 6141,	+2.24
CHST9	8022666	340	NM_031422	630	3460, 4937, 5865,	+2.23
ZNF518B	8099364	341	NM_053042	631	3218, 3781, 5718, 8226, 8286, 7880, 7968,	+2.23
TMEM106C	7955063	342	NM_024056	632	3447, 4690, 4693, 4691, 4692, 5419, 6591,	+2.22
					7332,
SERHL2	8073596	343	NM_014509	633	3645, 4046, 4023, 4076, 4075, 4492, 4499,	+2.20
					3203, 7885, 6148, 6161, 6501, 7434, 5747,
					5575,
NTN4	7965573	344	NM_021229	634	2846, 3957, 6056, 5886,	+2.20
SOX17	8146462	345	NM_022454	635	3009, 4141, 5778,	+2.20
FRRS1	7917954	346	NM_001013660	636	3386, 4150, 5573, 8650, 7934, 6212, 6764,	+2.20
OTX2	7979357	347	NM_021728	637	3073, 3223, 3224, 6604, 6242,	+2.19
RNASEL	7922707	348	NM_021133	638	3790, 5239, 6286, 6523,	+2.19
ELMOD2	8097529	349	NM_153702	639	3416, 5196, 8425, 7800, 5802, 8102, 8387,	+2.18
					8257, 8391,
MYCT1	8122860	350	NM_025107	640	3129, 4215, 6145,	+2.17
PAX6	7947338	351	NM_000280	641	3805, 4400, 4401, 4402, 6733, 7304, 6876,	+2.16
					7135, 7188, 7299, 6953, 7333, 7281, 8690,
					7478, 7234, 8432, 7017, 6745, 7558, 5383,
MGST2	8097513	352	NM_002413	642	3025, 5261, 8016, 7820, 8094, 5528, 8127,	+2.15
BBS5	8046147	353	NM_152384	643	3608, 3739, 8230, 6450, 7499, 7824, 5869,	+2.14
					6872,
MTSS1	8152764	354	NM_014751	644	2790, 4171, 7149, 7038, 8535, 8296, 7813,	+2.13
					8241, 8289, 8581, 5899,
VTN	8013606	355	NM_000638	645	2897, 3932, 5348,	+2.13
WBP5	8169022	356	NM_001006613	646	3375, 3752, 3755, 3753, 3751, 6158, 6146,	+2.12
DUB4	8094134	357	AY533200	647	3572, 3865, 4780, 7780, 7776, 8514, 8351,	+2.12
CCDC92	7967486	358	NM_025140	648	3339, 2893, 5390,	+2.12
BTG2	7908917	359	NM_006763	649	2680, 3257, 8005, 5749,	+2.12
LPGAT1	7924107	360	NM_014873	650	3152, 4614, 7821, 6002, 6174,	+2.11
FN1	8058765	361	NM_054034	651	3732, 3409, 3983, 4677, 4679, 3284, 3310,	+2.11
					3403, 3404, 3405, 3406, 4727, 3110, 4551,
					4571, 4572, 4576, 3822, 3494, 2852, 3620,
					3621, 3622, 3623, 3624, 3625, 5996, 7280,
					7649, 6915, 8364, 8529, 6768, 7398, 5846,
					6424, 8736, 8430, 7036, 5993, 8153, 8487,
					6324, 6771, 6223, 6205, 5539, 6620, 6121,
					6206, 8147, 5687,
TBX3	7966690	362	NM_016569	652	3024, 3627, 3626, 6215, 5414, 6072,	+2.11
PLCE1	7929388	363	NM_016341	653	4217, 4071, 4918, 6138, 5440, 6045, 6266,	+2.11
KRT19P2	7957608	364	NM_002276	654	4928, 4126, 5201, 5885, 6917,	+2.10
IFI16	7906400	365	NM_005531	655	3138, 2735, 2792, 2582, 3114, 4519, 2986,	+2.10
					4028, 5903, 7297, 7065, 5990, 6176, 8454,
					7984, 8195, 7186, 7588, 5913, 5739,
PORCN	8167287	366	NM_203473	656	2854, 2855, 2856, 2857, 4728, 2616, 4468,	+2.09
					2979, 2983, 3366, 3574, 3575, 3573, 3576,
					5975, 8479, 5883, 6961, 5689, 8317, 8547,
					6074, 7887, 5982, 7832, 6053, 6535,
PRSS1	8136795	367	NM_002769	657	4044, 4083, 5224, 7372, 8647, 5627, 7412,	+2.09
					7969,
MYL7	8139307	368	NM_021223	658	3043, 3698, 7148, 5334, 7843, 7318, 7526,	+2.08
DUSP4	8150076	369	NM_001394	659	2649, 5265, 5266, 5393,	+2.06
PROS1	8089011	370	NM_000313	660	2597, 4770, 6690,	+2.06
ANKRD20B	8020349	371	ENST00000400842	661	0	+2.05
CTSL2	8162652	372	NM_001333	662	3462, 5253, 8071, 5450,	+2.05
FMO5	7919314	373	NM_001461	663	3169, 4740, 4742, 4741, 5452, 5926, 6568,	+2.05
					7792, 6924,
USP27X	8167601	374	NM_001145073	664	4754,	+2.04
LAMA1	8022176	375	NM_005559	665	4685, 5278, 8564, 7980, 8761, 6083,	+2.04
					7657,
ADAM28	8145293	376	NM_014265	666	4384, 3969, 3970, 7848, 8743, 8310, 7519,	+2.03
					7029, 7897, 7794, 7884, 5486, 8614,
ZNF611	8038981	377	NM_030972	667	3136, 4861, 4862, 4863, 4864, 5722,	+2.03
ANKRD20B	7970381	378	AK057820	668	2984, 4169, 6891, 6432, 6336, 7704, 6356,	+2.02
					7272, 7347,
ZNF137	8030954	379	U09414	669	2628, 4364,	+2.02
S100Z	8106411	380	NM_130772	670	3017, 4191, 8752, 5748,	+2.01
GPSM2	7903565	381	NM_013296	671	3143, 4876, 3650, 2672, 4515, 3370, 4289,	+2.01
					7669, 5503, 6737, 6476, 6581, 7296, 6065,
					7166, 5954, 5917, 5991,
TGFB2	7909789	382	NM_003238	672	2610, 5188, 5189, 7936, 5882, 8554, 5876,	+2.00
ARHGAP28	8019964	383	NM_001010000	673	2803, 5198, 5415, 6442, 5664, 6551, 7534,	+2.00
					7585,
Table 3. Provided are the description of the non-membranal genes along with the sequence identifiers of the Affymetrix probes, the target sequence identified by the Affymetrix probe, and the mRNA sequences represented by the target sequences. Also shown are the log 2 fold change in expression levels of the indicated genes between the SOX17 GFP cells and the genetically unmodified HESCs. The (+) or (−) sign means that the genes are upregulated or downregulated, respectively, in the SOX17-GFP cells as compared to the genetically unmodified HESCs.

TABLE 4
Non-membranal genes which are down-regulated in SOX17+ cells as compared to genetically un-modified ESCs
		Affy. target		SEQ ID NO:		Log 2 fold
Gene	Probe	nucl. SEQ	Rep.	(Of Rep.	Polyn. SEQ ID NOs:	change
Symbol	Set ID	ID NO:	Public ID	Public ID)	rep. by target	[SOX17-HESC]
ZFP42	8098690	384	NM_174900	674	3498, 3937, 5793, 8300,	−5.87
THUMPD3	8077513	385	NM_015453	675	2882, 2944, 4333, 4334, 6458, 6573, 7246, 8510,	−4.50
					6238, 6855, 6555, 8394, 7095, 6527,
ANXA1	8155849	386	NM_000700	676	3181, 2757, 6327, 5394, 7974, 6460, 7578, 7227,	−4.42
					8129, 8580,
SPP1	8096301	387	NM_001040058	677	4706, 3942, 3501, 3944, 8278, 7992, 5370, 6716,	−4.21
					8452, 8651, 6109, 6075, 7831, 7801,
PRDM14	8151240	388	NM_024504	678	2858, 3552, 5534,	−4.18
GNA14	8161892	389	NM_004297	679	2753, 4750, 6030, 7575,	−4.16
EDIL3	8112980	390	NM_005711	680	2720, 3295, 8700, 8201, 7504, 5630,	−4.05
CXCL12	7933194	391	NM_199168	681	2665, 5012, 5013, 5117, 5003, 6588, 8491, 7914,	−3.84
					6051, 6656, 6859,
PSMD5	8163795	392	NM_005047	682	4517, 4254, 2800, 2963, 3212, 8729, 8252, 5958,	−3.78
					6078, 8378, 5320, 5967,
PRRX1	7907222	393	NM_022716	683	4004, 3771, 3772, 5361, 6117, 8431, 7764, 8243,	−3.77
					8582,
NANOG	7953675	394	NM_024865	684	3571, 3299, 4206, 5371,	−3.72
TRIM22	7938035	395	NM_006074	685	2821, 2822, 2823, 4283, 4533, 4544, 4481, 3016,	−3.56
					3162, 4213, 4312, 4320, 4326, 5229, 7088, 6634,
					6762, 8083, 6459, 8567, 6533, 6911, 6474, 7269,
					7353,
NANOG	7987365	396	NM_024865	686	3571, 3299, 4206, 5371,	−3.53
RASGRF2	8106660	397	NM_006909	687	4058, 5302, 8643, 5518, 8502, 8537, 8352,	−3.52
POU5F1B	8148315	398	NM_001159542	688	5084, 4815, 6578, 6639,	−3.50
POLR3G	8106820	399	NM_006467	689	4507, 4049, 6583, 8418,	−3.44
HHLA1	8152938	400	NM_001145095	690	2750, 4755, 7527, 8041,	−3.36
POU5F1	8124889	401	NM_203289	691	2865, 2866, 2867, 2868, 3988, 3986, 4587, 5026,	−3.36
					5027, 5028, 5029, 5030, 5031, 5032, 5033, 5034,
					5035, 5036, 5037, 5038, 5039, 5040, 5041, 5042,
					5043, 5044, 5045, 5046, 5047, 5048, 5049, 5050,
					5051, 5052, 5053, 5054, 5055, 5056, 5057, 5058,
					5059, 5060, 5061, 5062, 5063, 5064, 5065, 5066,
					5067, 5068, 5069, 5070, 5071, 5072, 5073, 5074,
					5075, 5076, 5077, 5078, 5079, 5080, 5081, 5082,
					5083, 5084, 5085, 5086, 5087, 5088, 5089, 5090,
					5091, 5092, 5093, 5094, 5095, 5096, 5097, 5098,
					5099, 5100, 5101, 5102, 5103, 5104, 4227, 4228,
					4229, 4230, 4231, 3958, 3959, 4369, 5016, 5017,
					4052, 5175, 5179, 8740, 7043, 6463, 8204, 6434,
					6854, 6431, 8068, 6882, 7219, 7760, 8266, 8247,
					6495, 7073, 7834, 7275, 7155, 8483, 6801, 7986,
					6354, 8509, 6963, 7415, 7877, 6325, 7358, 5405,
					7117, 6852, 8575, 6970, 7641, 7552,
VSNL1	8040430	402	NM_003385	692	3384, 3838, 6777, 6629, 8659, 5837, 6389, 6436,	−3.33
SCG3	7983718	403	NM_001165257	693	3014, 4909, 4908, 5333,	−3.33
B3GALT1	8046078	404	NM_020981	694	2763, 5191, 6587, 5756,	−3.28
LECT1	7971838	405	NM_007015	695	3028, 3784, 3785, 7153, 6673,	−3.25
NTS	7957458	406	NM_006183	696	2948, 5242, 5429,	−3.19
MBNL1	8083429	407	NM_021038	697	3270, 3597, 3598, 3599, 3600, 3601, 3602, 3603,	−3.18
					6944, 7767, 5777, 8388, 7338, 5574, 7958, 5685,
					5890, 6259, 8038, 5830,
CKMT1A	7983239	408	NM_020990	698	4017, 4725, 3845, 3844, 5618, 8459, 7658, 6700,	−3.18
					6805, 7001, 6390, 7746, 7418, 6964, 8012, 7084,
					7264, 7777, 8717, 7315, 6930, 7503, 6473, 7092,
					7417, 7902, 7582,
NECAB1	8147244	409	NM_022351	699	3290, 4737, 6800, 7785, 7823, 8669, 7989, 8696,	−3.14
FGF2	8097256	410	NM_198041	700	2576, 5304, 4197, 5305, 5478, 5980, 5822,	−3.12
SFRP2	8103254	411	NM_003013	701	2933, 3724, 5471,	−3.07
DCLK1	7970954	412	NM_004734	702	4221, 5192, 5193, 5194, 5195, 5448, 7317, 6024,	−3.07
					6492, 7076,
DACT1	7974689	413	NM_016651	703	2815, 4080, 4079,	−3.06
CRABP1	7985159	414	NM_004378	704	3015, 4439, 5645,	−3.02
TFAP2C	8063536	415	NM_003222	705	3168, 3531, 5324,	−3.01
SCGB3A2	8108995	416	NM_054023	706	3022, 4995, 8754, 8215, 5614,	−2.89
LRAT	8097920	417	NM_004744	707	3566, 3595, 8748, 8069, 7763, 8070, 5620,	−2.88
CUZD1	7936798	418	NM_022034	708	3275, 5260, 6276, 6229, 6120, 6165, 6557, 6134,	−2.86
GLB1L3	7945296	419	NM_001080407	709	3430, 4288, 6702, 8356, 8044, 7094, 7185, 6706,	−2.86
METTL7A	7955441	420	NM_014033	710	3523, 3936, 5745, 6365,	−2.85
VAT1L	7997336	421	NM_020927	711	3166, 3189, 5813,	−2.81
COL12A1	8127563	422	NM_080645	712	4761, 3954, 3953, 5758, 6009, 7798, 8346, 8170,	−2.80
					5484, 6823,
OLFML3	7904158	423	NM_020190	713	3467, 3697, 6147, 5677, 8035, 6986,	−2.78
SOX2	8084165	424	NM_003106	714	3338, 5264, 5632,	−2.76
USP44	7965565	425	NM_032147	715	3062, 3978, 3979, 6775, 5436,	−2.72
HIST1H4F	8117422	426	NM_003540	716	3825, 3059, 7626,	−2.70
KGFLP1	8067839	427	NM_002009	717	3477, 2587, 4695, 4203, 5525, 7263,	−2.69
CPT1A	7949971	428	NM_001876	718	2870, 4398, 4399, 7075, 5473,	−2.69
DBC1	8163716	429	NM_014618	719	2726, 4469, 4474, 3012, 3546, 3649, 4164, 6292,	−2.64
					6191, 5480,
CHAC1	7982868	430	NM_024111	720	3003, 4671, 4672, 7389, 6822, 8727,	−2.63
CAV1	8135594	431	NM_001753	721	4248, 4999, 5000, 5001, 5002, 6085, 6753, 8406,	−2.63
MT1G	8001531	432	NM_005950	722	3006, 2840, 6392, 7114, 7170, 6815,	−2.63
NFIX	8026139	433	NM_002501	723	4021, 4022, 3743, 3307, 2629, 2644, 2645, 4501,	−2.62
					4543, 4907, 3764, 5981, 6837, 5556, 6879, 5925,
FERMT1	8064904	434	NM_017671	724	3567, 4061, 7556, 7678, 8349, 5970, 5336,	−2.59
GLIPR1	7957260	435	NM_007043	725	2953, 3451, 4074, 4007, 6328, 5493, 5382,	−2.59
TOX	8150962	436	NM_014729	726	2740, 3709, 6856, 6017,	−2.58
SNRPN //	7981960	437	NR_003321	727	4118,	−2.57
SNORD116-6
HEY2	8121850	438	NM_012259	728	4276, 3975, 5989, 6023,	−2.57
TIMP4	8085360	439	NM_003256	729	2945, 5251, 5824,	−2.56
IDO1	8146092	440	NM_002164	730	2584, 5259, 5421, 8523, 8757, 8169, 8149, 8114,	−2.53
					8319, 7915, 8778,
MT2A	7995783	441	NM_005953	731	2919, 4617, 5366,	−2.52
NR5A2	7908597	442	NM_003822	732	2737, 3582, 3583, 6058, 5356, 8618, 7477, 6293,	−2.52
					5367,
UPRT	8168438	443	NM_145052	733	3674, 4272, 2966, 4297, 4940, 4941, 7089, 8335,	−2.50
					6323, 6105, 6157,
MYC	8148317	444	NM_002467	734	2869, 4851, 6776, 7274,	−2.48
CCDC109B	8096808	445	NM_032992	735	3120, 3873, 3874, 4445, 8500, 7859, 7100, 6201,	−2.48
GAL	7942064	446	NM_015973	736	3055, 3933, 5477,	−2.47
ZNF483	8157193	447	NM_133464	737	4219, 4428, 4429, 7118, 6162, 5698,	−2.45
RND3	8055688	448	NM_005168	738	3040, 3678, 3679, 3283, 3713, 4557, 4566, 2954,	−2.45
					4291, 4318, 4304, 3770, 7689, 7080, 7920, 6797,
					7108, 7603, 8181, 5569, 8123,
BNC2	8160260	449	NM_017637	739	3551, 4555, 3100, 4545, 4032, 4033, 4034, 4035,	−2.45
					4036, 4037, 2799, 3963, 6528, 6375, 7529, 7058,
					6914, 7266, 7235, 7564, 8608, 7382, 8765,
COL3A1	8046922	450	NM_000090	740	3042, 4000, 7681, 5702, 7724, 5577,	−2.42
LUM	7965403	451	NM_002345	741	2920, 3798, 5519,	−2.42
LDB2	8099524	452	NM_001290	742	2733, 4590, 4591, 8679, 8166, 7837, 8661, 8298,	−2.41
					5669, 8605, 7719, 7814, 8032, 8473, 7455, 8734,
MT1E	7995797	453	NM_175617	743	3381, 3905, 5861, 5789,	−2.40
SNRPN //	7981994	454	NR_003337	744	4131,	−2.37
SNORD116-23
SNRPN //	7982002	455	NR_003341	745	4127,	−2.37
SNORD116-27
GALNT3	8056408	456	NM_004482	746	3996, 3990, 3410, 3332, 4301, 4196, 6671, 7016,	−2.36
					6517, 7277, 7565, 6510, 6479, 6804, 6507,
PIPOX	8006005	457	NM_016518	747	3342, 3789, 7690, 7748, 5638, 6426, 6464,	−2.35
PDK1	8046408	458	NM_002610	748	3198, 3485, 7350, 7468, 6847, 5563, 6903,	−2.34
PREX2	8146794	459	NM_024870	749	3640, 3643, 3644, 6885, 8788, 6153, 5651,	−2.33
CYP2S1	8028991	460	NM_030622	750	3461, 3715, 5647, 5708,	−2.33
NRK	8169115	461	NM_198465	751	3311, 4081, 5354,	−2.33
VAV3	7918157	462	NM_006113	752	2741, 4095, 4094, 8118, 7879, 6112, 7502, 5897,	−2.32
					6080, 6942,
TNFAIP6	8045688	463	NM_007115	753	3052, 5234, 5352, 6810,	−2.29
ENPP1	8122099	464	NM_006208	754	3492, 4353, 5924, 6245, 8263, 8565, 7205,	−2.27
ADD2	8052882	465	NM_001617	755	3312, 2549, 4252, 4541, 3221, 3555, 5136, 5137,	−2.26
					5138, 5139, 5140, 7591, 8079, 5513, 5527, 8334,
					8155, 6282, 7652, 6883, 7881, 7694, 6520, 6309,
					7614, 7098,
SNRPN //	7981996	466	NR_003338	756	4129,	−2.25
SNORD116-24
SNRPN //	7981947	467	NR_001289	757	3262, 3274,	−2.25
SNORD109A
STC2	8115851	468	NM_003714	758	3356, 3794, 8437, 5481,	−2.22
SNORA22	8133106	469	NR_002961	759	3945,	−2.22
MPPED2	7947274	470	NM_001584	760	2671, 4776, 4777, 6358, 6060,	−2.21
ZNF562	8033801	471	NM_001130031	761	4039, 4450, 4451, 4452, 6833, 5584, 6691,	−2.20
GAP43	8081810	472	NM_002045	762	2575, 4459, 4460, 5773,	−2.20
FOXB1	7984011	473	NM_012182	763	4249, 3974, 6779,	−2.19
TSHZ3	8035896	474	NM_020856	764	4257, 4125, 5381,	−2.18
HPGD	8103769	475	NM_000860	765	2662, 2663, 2690, 4708, 4570, 4471, 4475, 3508,	−2.17
					3534, 4794, 4795, 4796, 7891, 7937, 8399, 8639,
					8591, 5581, 7846, 5659, 8577, 7975, 6419, 7812,
ZDHHC22	7980344	476	NM_174976	766	4047, 4139, 5735,	−2.17
ACOXL	8044353	477	NM_001142807	767	4470, 4673, 6005, 7357, 6866, 6663, 6681,	−2.17
GLI3	8139212	478	NM_000168	768	3991, 3982, 2577, 4553, 3392, 4592, 6480, 7171,	−2.16
					6577, 6439, 8751, 6708,
CDCA7L	8138489	479	NM_018719	769	3585, 4801, 4802, 4803, 7510, 6116, 7011, 6541,	−2.14
					6183, 8558,
ZSCAN10	7998921	480	NM_032805	770	3149, 2932, 5400,	−2.13
GFPT2	8116418	481	NM_005110	771	2751, 4135, 8471, 5407, 8222, 8775, 7999,	−2.13
PLP2	8167449	482	NM_002668	772	2555, 4965, 7651, 6875, 7509,	−2.13
HIST1H1A	8124380	483	NM_005325	773	3881, 4053, 5377,	−2.12
CAMKV	8087530	484	NM_024046	774	3407, 4516, 4542, 4547, 4568, 3355, 2883, 3337,	−2.12
					3368, 3000, 3165, 3910, 8007, 8299, 6547, 8504,
					8573, 8756, 8739, 7861, 8075, 7731, 6769, 7182,
					8603, 8099, 7254, 5602,
HERC5	8096361	485	NM_016323	775	3489, 4008, 8649, 5458, 8138, 8244,	−2.12
MT1X	7995838	486	NM_005952	776	3371, 4455, 6391,	−2.12
TERF1	8102789	487	NM_003218	777	2691, 4416, 4417, 6010, 5544, 5494,	−2.12
RAB31	8020110	488	NM_006868	778	2829, 4352, 5595, 6339,	−2.12
SNRPN //	7981974	489	NR_003328	779	4128,	−2.10
SNORD116-13
ETV1	8138289	490	NM_004956	780	3858, 3887, 3888, 4724, 2640, 2658, 4696, 4698,	−2.10
					4701, 4715, 3245, 4328, 4314, 4877, 4878, 4879,
					4880, 4881, 4882, 4883, 6616, 7632, 7308, 7476,
					7190, 7710, 7373, 7697, 7749, 8199, 6672, 6566,
					5364, 6410, 7966, 7584, 8722, 7292, 7655, 6209,
					7888, 8053, 8570, 6394,
MT1G	8162531	491	ENST00000396160	781	7570,	−2.10
ACTA1	7924910	492	NM_001100	782	3288, 3669, 3677, 4719, 2955, 3612, 5805, 6231,	−2.10
					5889, 6187, 6159,
SNRPN //	7981988	493	AF241255	783	2839, 4130,	−2.10
SNORD116-20
NFIB	8160138	494	NM_005596	784	3285, 2688, 2685, 4244, 4562, 2874, 3417, 5146,	−2.09
					5145, 5147, 7613, 7638, 6784, 7684, 7327, 6514,
					7007, 8444, 7463,
ZEB2	8055624	495	NM_014795	785	3737, 3738, 4072, 4073, 4702, 2907, 3889, 3164,	−2.08
					3616, 3504, 4311, 4982, 4983, 4984, 7620, 7739,
					7312, 6428, 7804, 5578, 7411, 7695, 7432, 6411,
					6906, 7751, 8052, 7712, 6862, 6562, 7983, 7245,
					7643, 6372, 7130, 7002, 6978, 7523,
CBR1	8068401	496	NM_001757	786	3890, 3754, 7622, 5848,	−2.08
ATXN7L1	8142087	497	NM_138495	787	4574, 4661, 4286, 7346, 7677, 7827,	−2.07
SNRPN //	7981949	498	NR_003316	788	4117,	−2.06
SNORD116-1
MT1F	7995825	499	NM_005949	789	3051, 4447, 5801, 6580,	−2.06
SNRPN //	7982006	500	NR_003360	790	4121,	−2.06
SNORD116-29
AP1M2	8034084	501	NM_005498	791	2896, 4739, 5398,	−2.05
ACTG2	8042788	502	NM_001615	792	2956, 3832, 6377, 7341, 6703, 6599, 7930, 5891,	−2.05
CYP2B6	8028963	503	NM_000767	793	2569, 3046, 5598, 5779,	−2.05
SERPINE1	8135069	504	NM_000602	794	2947, 4910, 4911, 5338,	−2.03
GRHL2	8147697	505	NM_024915	795	4098, 4097, 4270, 4529, 3637, 3606, 3638, 4299,	−2.03
					4354, 5422, 7907, 7656, 8209, 8281, 7516,
SLIT2	8094301	506	NM_004787	796	2748, 2765, 3985, 4678, 3436, 2744, 2767, 8366,	−2.03
					7786, 6676, 7740, 5522, 8033, 7772, 7956, 8641,
					8725,
PIM2	8172471	507	NM_006875	797	2994, 4637, 7671, 8024, 6884,	−2.02
SMARCA2	8154059	508	NM_139045	798	2631, 2619, 3109, 4525, 4559, 4564, 4569, 3205,	−2.02
					4322, 3654, 3655, 7236, 8645, 6437, 7334, 7214,
					5617, 7026, 6958, 5795, 5880, 7544, 7653, 6932,
					6870, 6536, 7608, 5988, 7032,
RPPH1	7977507	509	NR_002312	799	3839,	−2.01
Table 4. Provided are the description of the non-membranal genes along with the sequence identifiers of the Affymetrix probes, the target sequence identified by the Affymetrix probe, and the mRNA sequences represented by the target sequences. Also shown are the log 2 fold change in expression levels of the indicated genes between the SOX17 GFP cells and the genetically unmodified HESCs. The (+) or (−) sign means that the genes are upregulated or downregulated, respectively, in the SOX17-GFP cells as compared to the genetically unmodified HESCs.

Example 2

Generation of a PDX1-GFP BAC Construct and Isolation of PDX1-Expressing ESCS

Experimental Results

BAC Transgenesis of PDX1-GFP Reporter BAC—

An important gene in the commitment of definitive endoderm cells towards pancreas is PDX1. Thus, a fluorescent reporter construct was built to study precursor cells at this later stage of the differentiation process. The present inventors generated PDX1-reporter constructs in which the coding sequence of the PDX1 gene was replaced by the coding sequence of the EGFP gene together with a floxed Neo gene, as follows.

Preparation of PDX1 ATG-GFP-Knockin Construct:

EGFP and floxed neo was knocked into the PDX1 gene locus in BAC BAC RP11-322P28 by replacing the coding sequence of PDX1 (ATG to TGA) with the coding sequence of GFP and the floxed neomycin resistance gene. FIGS. 3A-B schematically illustrate the structure of the recombinant BAC RP11-322P28 PDX1-GFP construct (SEQ ID NO:15), which includes the PDX1 upstream regulatory sequence (SEQ ID NO:16, about 8.7 kb), the GFP coding sequence (SEQ ID NO:2) followed by the SV40 polyA sequence (SEQ ID NO:27), a loxP site (SEQ ID NO:4), the neomycin resistance gene sequence (SEQ ID NO:3) with the SV40 early promoter/enhancer (SEQ ID NO:28), and the TK polyadenylation site (SEQ ID NO:29), a second loxP site (SEQ ID NO:4), the PDX1 downstream regulatory sequence (SEQ ID NO:17, about 19.5 kb).

Preparation of PDX1 ATG-GFP-Knockin Plasmid Construct:

Part of the BAC PDX1-GFP construct (SEQ ID NO:15) was subcloned into the Bluescript plasmid (Stratagene) to generate the recombinant PDX1-GFP plasmid construct (SEQ ID NO:18) as follows. FIG. 3C schematically illustrates the structure of the recombinant plasmid PDX1-GFP construct (SEQ ID NO:18), which includes the 5′-arm (SEQ ID NO:19), the GFP coding sequence (SEQ ID NO:2) followed by the SV40 polyA sequence (SEQ ID NO:27), a loxP site (SEQ ID NO:4), the neomycin resistance gene sequence (SEQ ID NO:3) with the SV40 early promoter/enhancer (SEQ ID NO:28), and the TK polyadenylation site (SEQ ID NO:29), a second loxP site (SEQ ID NO:4), and a 3′-arm (SEQ ID NO:20).

A 3′-external probe was prepared (SEQ ID NO:21) in order to detect correct targeting of the vector into the genomic DNA of the host cell.

Preparation of PSC1x: PDX1 IRES-GFP-Knockin Plasmid Construct—

IRES-eGFP and floxed neo was knocked into the 3′ UTR of the PDX1 gene. FIG. 3D schematically illustrates the structure of the recombinant plasmid construct (SEQ ID NO:22), which includes the 5′-arm (SEQ ID NO:23) which includes the PDX1 exons, the PDX1 5′-UTR (5′-untranslated region), the 3′-UTR, an IRES sequence (SEQ ID NO:25), the GFP coding sequence (SEQ ID NO:2) followed by the SV40 polyA sequence (SEQ ID NO:27), a loxP site (SEQ ID NO:4), the neomycin resistance gene sequence (SEQ ID NO:3) with the SV40 early promoter/enhancer (SEQ ID NO:28), and the TK polyadenylation site (SEQ ID NO:29), a second loxP site (SEQ ID NO:4), and a 3′-arm (SEQ ID NO:24).

A 3′-external probe was prepared (SEQ ID NO:26) in order to detect correct targeting of the vector into the genomic DNA of the host cell.

Preparation of PDX1 ATG-mCherry-Knockin BAC—

mCherry and a floxed neo was knocked into the PDX1 gene locus in BAC RP11-322P28. FIG. 3D schematically illustrates the structure of the recombinant BAC construct (SEQ ID NO:30), which includes the 5′-arm (SEQ ID NO: 31) which includes PDX1 upstream regulatory sequence, the mCherry coding sequence (SEQ ID NO:33) followed by the SV40 polyA sequence (SEQ ID NO:27), a loxP site (SEQ ID NO:4), the neomycin resistance gene sequence (SEQ ID NO:3) with the SV40 early promoter/enhancer (SEQ ID NO:28), and the TK polyadenylation site (SEQ ID NO:29), a second loxP site (SEQ ID NO:4), and a 3′-arm (SEQ ID NO:32).

Transfection of the Recombinant Construct of Some Embodiments of the Invention into Human ESCs—

Various hESC clones harbouring the PDX1 GFP reporter BAC (SEQ ID NO:15) were isolated and were allowed to differentiate by forming EBs. At day 14, the EBs were dissociated into single cells and were analysed by FACS. The number of GFP+ cells in the different clones varied from about 0.5% to about 3% (FIG. 4C). 14 day-old EBs from a reporter clone were seen to contain clusters of GFP⁺ cells. The EBs were dissociated into single cells and were put onto cover slips using the cytospin, fixed and immunostained for PDX1. The cells clearly co-expressed PDX1 and GFP demonstrating that the GFP⁺ population indeed represent PDX1 positive cells (FIG. 4D).

Next, the PDX1⁺ cells were sorted by FACS and RNA was extracted from the GFP⁺ and GFP⁻ populations. RT-qPCR was performed to compare expression of various markers in the two different cell populations. As can be seen in FIG. 4E, PDX1 and other pancreatic differentiation markers were enriched in the GFP⁺ population. Some of the enriched genes include NGN3, paired box 4 (PAX4), hepatocyte nuclear factor 6 (HNF6) and homeobox gene HB9 (HLXB9)). These results further validate the identity of the PDX+ population as pancreatic progenitor cells.

TABLE 5
Membranal genes which are upregulated in PDX1 cells as compared to genetically un-modified ESCs
	Affy. target		SEQ ID NO:			Log 2 fold
Probe	nucl. SEQ	Rep.	(Of Rep.	Polyn. SEQ ID NOs:	Gene	change
Set ID	ID NO:	Public ID	Public ID)	rep. by target	Symbol	[PDX1-HESC]
8035304	510	NM_004335	800	3090, 2805, 5403, 7124,	BST2	3.99
7989073	511	NM_173814	801	3710, 4778, 6379,	PRTG	3.28
8056611	512	NM_004525	802	2684, 4119, 5456, 8262,	LRP2	2.83
8031076	513	NM_031896	803	3827, 5226, 7212, 5339,	CACNG7	2.71
7976783	514	NM_003836	804	4273, 4994, 6774, 5796, 6319,	DLK1	2.66
8087691	515	NM_001174051	805	2721, 2711, 2712, 4220, 4298, 5019, 3735,	CACNA2D2	2.56
				3736, 6362, 6812, 5540, 6239, 8681, 7132,
				7985, 7734, 6957, 7200,
7909390	516	NM_175710	806	3897, 4185, 8470, 6548, 5851,	CR1L	2.56
7994058	517	NM_001039	807	3481, 4177, 5784,	SCNN1G	2.54
8169447	518	NM_000868	808	2609, 4287, 5920, 5564, 5986,	HTR2C	2.44
7917276	519	NM_012152	809	2780, 4363, 7551, 8539, 6011,	LPAR3	2.27
7920664	520	NM_007112	810	2652, 3541, 6070, 7928, 8701, 6699, 6720,	THBS3	2.18
				8067, 8010, 7294, 6846,
8051785	521	NM_133329	811	3647, 5227, 5228, 5783, 7561,	KCNG3	2.15
8018114	522	NM_001144952	812	2814, 4748, 7774, 5765, 6851, 6326,	SDK2	2.13
8178193	523	NM_019111	813	3652, 4537, 2590, 2589, 4532, 4478, 4721,	HLA-DRA	2.10
				4482, 3074, 3666, 5174, 6840, 8271, 7495,
				7301, 6340, 7125, 7667, 6973, 6698, 6694,
				7895, 6462, 7070, 8427, 7321, 6682, 7906,
				7421, 7648, 7487, 6525,
7930837	524	NM_003054	814	3141, 5107, 7960, 5655,	SLC18A2	2.09
8011713	525	NM_022059	815	2851, 4210, 4211, 5728,	CXCL16	1.96
7916584	526	NM_002353	816	2675, 4337, 6094,	TACSTD2	1.91
7975932	527	NM_020431	817	4392, 4432, 5791,	TMEM63C	1.85
8038367	528	NM_020309	818	3483, 4743, 5332,	SLC17A7	1.83
8114415	529	NM_001496	819	3167, 3584, 7363, 5508,	GFRA3	1.83
Table 5: Provided are the description of the membranal genes along with the sequence identifiers of the Affymetrix probes, the target sequence identified by the Affymetrix probe, and the mRNA sequences represented by the target sequences. Also shown are the log 2 fold change in expression levels of the indicated genes between the PDX1-GFP cells and the genetically unmodified HESCs. The (+) or (−) sign means that the genes are upregulated or downregulated, respectively, in the PDX1-GFP cells as compared to the genetically unmodified HESCs.

TABLE 6
Membranal genes which are downregulated in PDX1 cells as compared to genetically un-modified ESCs
	Affy. target		SEQ ID NO:			Log 2 fold
Probe	nucl. SEQ	Rep.	(Of Rep.	Polyn. SEQ ID NOs:	Gene	change
Set ID	ID NO:	Public ID	Public ID)	rep. by target	Symbol	[PDX1-HESC]
8095364	820	NM_014058	1411	3472, 4222, 5579, 8653,	TMPRSS11E	−3.58
8095380	821	NM_014058	1412	3472, 4222, 5579, 8653,	TMPRSS11E	−3.60
7957140	822	NM_003667	1413	2732, 3193, 6795, 5509,	LGR5	−3.03
8101992	823	NM_001135146	1414	2875, 4623, 4624, 3783, 4625, 7838, 6311, 8464,	SLC39A8	−2.82
				6341, 7047,
8091402	824	NM_001184723	1415	2962, 5118, 5119, 5621, 7779, 6927, 6623, 8441,	TM4SF18	−2.73
7936798	825	NM_022034	1416	3275, 5260, 6276, 6229, 6120, 6165, 6557, 6134,	CUZD1	−2.27
8175217	826	NM_001448	1417	2738, 3072, 6154,	GPC4	−2.20
8123246	827	NM_021977	1418	3195, 5535, 7120,	SLC22A3	−2.20
8055465	828	NM_001008540	1419	2782, 3775, 3776, 5384, 6834, 7645,	CXCR4	−2.19
8123739	829	NM_016588	1420	3230, 3454, 7755, 5387,	NRN1	−2.16
7964745	830	NM_016056	1421	3443, 2942, 4069, 7063, 6210, 5607, 7161,	TMBIM4	−2.11
8130867	831	NM_003247	1422	4184, 3540, 8063, 7661, 5992, 6904,	THBS2	−2.05
8003298	832	NM_003486	1423	2755, 3870, 5397,	SLC7A5	−1.92
8172022	833	NM_031442	1424	3197, 4343, 5466,	TMEM47	−1.90
Table 6: Provided are the description of the membranal genes along with the sequence identifiers of the Affymetrix probes, the target sequence identified by the Affymetrix probe, and the mRNA sequences represented by the target sequences. Also shown are the log 2 fold change in expression levels of the indicated genes between the PDX1-GFP cells and the genetically unmodified HESCs. The (+) or (−) sign means that the genes are upregulated or downregulated, respectively, in the PDX1-GFP cells as compared to the genetically unmodified HESCs.

TABLE 7
Non-membranal genes which are upregulated in PDX1 cells as compared to genetically un-modified ESCs
	Affy. target		SEQ ID NO:			Log 2 fold
Probe	nucl. SEQ	Rep.	(Of Rep.	Polyn. SEQ ID NOs:	Gene	change
Set ID	ID NO:	Public ID	Public ID)	rep. by target	Symbol	[PDX1-HESC]
8127522	834	NM_001025290	1425	4388, 5232, 5916,	DPPA5	5.21
8049128	835	NM_031313	1426	2557, 4233, 5696,	ALPPL2	5.18
7917037	836	NM_001889	1427	3420, 2605, 3199, 3619, 4456, 4457, 4458,	CRYZ	4.64
				4612, 6114, 6052, 6863, 6288, 6530, 6018,
				8189,
7945680	837	NR_030533	1428	3526, 4930, 3779, 6619, 7610, 6652, 6726,	H19	4.26
				6561, 7131, 6743, 7010, 6680, 6895, 7340,
				7012, 6491,
7901497	838	NM_001004339	1429	3328, 4808, 6317, 5872,	ZYG11A	3.74
7902345	839	NM_001162916	1430	3648, 4873, 4874, 4872, 8405, 6496, 6385,	TYW3	3.33
				8689, 7110, 8780, 6273,
7986350	840	NM_183376	1431	3385, 4436, 5479,	ARRDC4	3.28
7904726	841	NM_006472	1432	2648, 4356, 6092, 8025, 8646, 8023,	TXNIP	3.08
7975779	842	NM_005252	1433	3348, 4888, 5787,	FOS	3.07
7959016	843	NR_027345	1434	4799, 4800, 7778, 8557,	NCRNA00173	2.72
8071809	844	NM_000854	1435	2877, 3667, 4143, 4144, 5682, 5316,	GSTT2	2.63
8074962	845	NM_000854	1436	2877, 3667, 4143, 4144, 5682, 5316,	GSTT2	2.63
8033257	846	NM_000064	1437	4194, 4038, 7636, 8397, 8664, 8376, 5358,	C3	2.60
				6890, 6959, 7466, 8745,
8120679	847	NM_018665	1438	2820, 2957, 4749, 6125, 7145, 8551, 6076,	DDX43	2.58
7983360	848	NM_004048	1439	3076, 3490, 6485, 6270,	B2M	2.53
7949124	849	NM_001164716	1440	2730, 4906, 4290, 8065, 5323,	PYGM	2.47
8026424	850	NM_173483	1441	3826, 4241, 5547,	CYP4F22	2.46
8030944	851	NM_001099694	1442	4546, 4201,	ZNF578	2.44
7953532	852	NM_001975	1443	3363, 2973, 5374,	ENO2	2.44
7933092	853	NM_021045	1444	4114, 3320, 8456, 7285, 8408, 7542, 7283,	ZNF248	2.43
				7447, 6137,
8031576	854	NM_134444	1445	3031, 4444, 5611, 5951,	NLRP4	2.39
7964246	855	NR_003046	1446	3964,	SNORD59B	2.34
7976812	856	NR_003232	1447	4042,	SNORD113-4	2.34
7912347	857	NM_001079843	1448	3894, 4091, 8716, 7616,	CASZ1	2.28
8008885	858	NR_029493	1449	3773, 4929,	MIR21	2.27
7906400	859	NM_005531	1450	3138, 2735, 2792, 2582, 3114, 4519, 2986,	IFI16	2.24
				4028, 5903, 7297, 7065, 5990, 6176, 8454,
				7984, 8195, 7186, 7588, 5913, 5739,
8033767	860	NM_152476	1451	2982, 4116, 5683,	ZNF560	2.22
7930631	861	NM_198795	1452	3518, 3513, 6133, 6452, 5445, 6004, 5919,	TDRD1	2.22
8139820	862	NM_178558	1453	3061, 4448, 4449, 8320, 6601, 8790, 5658,	ZNF680	2.20
8118314	863	NM_005346	1454	2997, 3529, 4339, 4461, 7351, 7433, 6971,	HSPA1B	2.15
				6486, 6386, 6633, 6951, 7233, 6772, 7683,
				6519, 6662,
8118310	864	NM_005345	1455	4504, 3360, 4461, 6563, 7351, 6971, 6630,	HSPA1A	2.14
				7449, 7498, 6386, 6951, 6668, 6772, 7683,
				7557, 6519,
8108370	865	NM_001964	1456	2571, 3296, 5376, 7583,	EGR1	2.09
7949104	866	NM_001098670	1457	2786, 2787, 2742, 3987, 2749, 3104, 3106,	RASGRP2	2.07
				3428, 4186, 4187, 3200, 6996, 7253, 7251,
				6422, 6110, 6605, 7167, 6721, 6400, 7783,
				7408, 7101, 6992, 8419, 6612, 7675, 7336,
				6494, 7993, 7692, 6747, 7413,
8177011	867	NM_004192	1458	3533, 5004, 5005, 5006, 7168, 8058, 7484,	ASMTL	2.06
				7559, 6621, 7587,
8030448	868	NM_001136052	1459	3049, 5236, 5237, 7371, 6022, 7446, 5780,	CPT1C	2.05
				5691,
8024712	869	NM_033064	1460	3258, 3101, 4560, 3035, 4405, 5929, 7505,	ATCAY	2.05
				5849, 7325,
8008321	870	NM_025149	1461	4485, 4216, 8568, 8657, 7865, 8078, 8132,	ACSF2	2.05
				8703, 8080, 8098, 8518, 8485, 5631, 8002,
				8231, 8654, 8238, 8203, 8085,
7947512	871	NM_015430	1462	3786, 3701, 3702, 6790, 5537, 6920,	PAMR1	2.04
7963970	872	NM_006928	1463	3105, 5245, 6941, 6287,	SILV	2.03
7941662	873	NM_001104	1464	3432, 2761, 8043, 7856, 8550,	ACTN3	2.03
7919787	874	NM_032132	1465	3665, 3069, 3254, 3722, 5240, 8304, 6029,	HORMAD1	2.03
				6113, 5998, 7030, 5874, 5867, 8731, 6466,
				5973,
7993756	875	NM_005622	1466	4281, 2813, 3634, 3635, 4670, 6949, 5609,	ACSM3	2.02
				7003, 6291,
8018652	876	NM_052916	1467	4746, 5562, 5570, 5845,	RNF157	2.02
8066117	877	NM_015474	1468	3449, 5023, 3830, 5349, 5541, 7106, 6100,	SAMHD1	2.01
7968928	878	NM_002901	1469	3429, 3373, 4745, 4663, 7615, 8433, 8540,	RCN1	2.01
				8791, 5816, 7507, 7679, 7940, 5326,
Table 7: Provided are the description of the non-membranal genes along with the sequence identifiers of the Affymetrix probes, the target sequence identified by the Affymetrix probe, and the mRNA sequences represented by the target sequences. Also shown are the log 2 fold change in expression levels of the indicated genes between the PDX1-GFP cells and the genetically unmodified HESCs. The (+) or (−) sign means that the genes are upregulated or downregulated, respectively, in the PDX1-GFP cells as compared to the genetically unmodified HESCs.

TABLE 8
Non-membranal genes which are down regulated in PDX1 cells as compared to genetically un-modified ESCs
	Affymetrix					Log 2
	target nucl.		SEQ ID	Polyn.		fold change
Probe	sequence	Rep.	NO: (Of Rep.	SEQ ID NOs:	Gene	[PDX1-
Set ID	SEQ ID NO:	Public ID	Public ID)	rep. by target	Symbol	HESC]
7957458	879	NM_006183	1470	2948, 5242, 5429,	NTS	−6.28
8160163	880	NM_005454	1471	3880, 3883, 6763,	CER1	−5.61
8065412	881	NM_001898	1472	3369, 3033, 5734, 6511,	CST1	−5.17
7934156	882	NM_018055	1473	3088, 4747, 7082, 5642,	NODAL	−4.21
7907222	883	NM_022716	1474	4004, 3771, 3772, 5361, 6117, 8431, 7764,	PRRX1	−3.87
				8243, 8582,
8067839	884	NM_002009	1475	3477, 2587, 4695, 4203, 5525, 7263,	KGFLP1	−3.72
8160138	885	NM_005596	1476	3285, 2688, 2685, 4244, 4562, 2874, 3417,	NFIB	−3.49
				5146, 5145, 5147, 7613, 7638, 6784, 7684,
				7327, 6514, 7007, 8444, 7463,
8112668	886	NM_016591	1477	2806, 4600, 5633,	GCNT4	−3.42
7910134	887	NM_031944	1478	2853, 2923, 6314,	MIXL1	−3.41
8135594	888	NM_001753	1479	4248, 4999, 5000, 5001, 5002, 6085, 6753,	CAV1	−3.37
				8406,
7965403	889	NM_002345	1480	2920, 3798, 5519,	LUM	−3.36
8106660	890	NM_006909	1481	4058, 5302, 8643, 5518, 8502, 8537, 8352,	RASGRF2	−3.34
8111892	891	NM_000436	1482	2674, 4026, 8292, 5313, 8538, 8361, 8084,	OXCT1	−3.25
				7753,
7957260	892	NM_007043	1483	2953, 3451, 4074, 4007, 6328, 5493, 5382,	GLIPR1	−3.25
8040430	893	NM_003385	1484	3384, 3838, 6777, 6629, 8659, 5837, 6389,	VSNL1	−3.24
				6436,
8105302	894	NM_013409	1485	3343, 5282, 5281, 7924, 7913, 6382, 5408,	FST	−3.15
7971077	895	NM_006475	1486	2617, 4640, 4641, 4642, 4649, 7522, 8474,	POSTN	−2.97
				8449, 7103, 8183, 7375, 6642,
8161892	896	NM_004297	1487	2753, 4750, 6030, 7575,	GNA14	−2.96
8068401	897	NM_001757	1488	3890, 3754, 7622, 5848,	CBR1	−2.95
8092095	898	NM_015028	1489	4193, 4852, 4853, 4854, 4855, 4856, 4857,	TNIK	−2.95
				4858, 4859, 4860, 8054, 5939, 6549, 7883,
				5603, 6786, 7448, 7215, 8447, 8382, 7882,
				6219,
7921916	899	NM_001195303	1490	3306, 5190, 5110, 7452, 5828, 6057,	RGS5	−2.93
8155487	900	NM_002009	1491	3476, 2587, 2975, 4695, 4203, 5525, 7263,	KGFLP1	−2.89
				7112, 6748,
7952601	901	NM_001162422	1492	3402, 4871, 4686, 4687, 7425, 5612, 5931,	ETS1	−2.88
7947274	902	NM_001584	1493	2671, 4776, 4777, 6358, 6060,	MPPED2	−2.79
7934906	903	NM_001141945	1494	3670, 3812, 3774, 2556, 3442, 4717, 4473,	ACTA2	−2.79
				2836, 3380, 4664, 4665, 5340, 8239, 7483,
				8264, 7450, 7803, 6754,
8140668	904	NM_006080	1495	4019, 3973, 5554, 7046, 6980,	SEMA3A	−2.72
7974689	905	NM_016651	1496	2815, 4080, 4079,	DACT1	−2.67
8155849	906	NM_000700	1497	3181, 2757, 6327, 5394, 7974, 6460, 7578,	ANXA1	−2.66
				7227, 8129, 8580,
8127563	907	NM_080645	1498	4761, 3954, 3953, 5758, 6009, 7798, 8346,	COL12A1	−2.59
				8170, 5484, 6823,
7965322	908	NM_000899	1499	4054, 5149, 5148, 6216, 5328, 6318, 7574,	KITLG	−2.58
7995681	909	NM_004530	1500	3362, 4409, 4410, 5329, 6518,	MMP2	−2.57
7971653	910	NR_002612	1501	2849, 3911, 7223, 6445, 7650, 7147, 7126,	DLEU2	−2.57
				7028, 6609,
8171449	911	NM_021804	1502	3847, 4462, 6787, 7949, 7759, 7328, 5427,	ACE2	−2.56
8042788	912	NM_001615	1503	2956, 3832, 6377, 7341, 6703, 6599, 7930,	ACTG2	−2.56
				5891,
7962427	913	NM_031292	1504	3592, 4173, 4172, 4174, 6664, 6050, 6684,	PUS7L	−2.52
7984259	914	NR_002757	1505	4615,	RNU5B-1	−2.40
8046922	915	NM_000090	1506	3042, 4000, 7681, 5702, 7724, 5577,	COL3A1	−2.37
7924682	916	NM_003240	1507	3558, 4989, 4988, 8493, 7506, 6104,	LEFTY2	−2.35
7912520	917	NM_002521	1508	3029, 3909, 7941, 6388,	NPPB	−2.35
7944867	918	NM_170601	1509	3593, 5256, 5538,	SIAE	−2.30
8095646	919	NM_001134	1510	3041, 2756, 7886, 7273, 8047, 8272,	AFP	−2.29
8098690	920	NM_174900	1511	3498, 3937, 5793, 8300,	ZFP42	−2.29
8152617	921	NM_005328	1512	2676, 4348, 5843, 6409,	HAS2	−2.28
8136807	922	—			TRY6	−2.28
7908597	923	NM_003822	1513	2737, 3582, 3583, 6058, 5356, 8618, 7477,	NR5A2	−2.27
				6293, 5367,
8115543	924	NM_024007	1514	3190, 4342, 8173, 8061, 8377, 5613, 8417,	EBF1	−2.27
				6926, 7796, 8496, 7892,
7977507	925	NR_002312	1515	3839,	RPPH1	−2.26
8026139	926	NM_002501	1516	4021, 4022, 3743, 3307, 2629, 2644, 2645,	NFIX	−2.21
				4501, 4543, 4907, 3764, 5981, 6837, 5556,
				6879, 5925,
8040163	927	NM_001039613	1517	3912, 3107, 4261, 4549, 3207, 3482, 3588,	IAH1	−2.20
				3934, 6534, 8330, 8409, 8205, 6128, 8770,
				8556, 7901, 8340, 8747, 8755, 7721, 8381,
				8091, 7817, 5835,
8151768	928	NM_004349	1518	2705, 3651, 2626, 2602, 2625, 2624, 2654,	RUNX1T1	−2.20
				4142, 3323, 2686, 4712, 2904, 3581, 5209,
				5210, 5211, 5212, 5213, 5214, 5215, 5216,
				5217, 5218, 5219, 5220, 5221, 5222, 5223,
				8013, 6461, 5898, 7238, 8282, 8648, 8373,
				8771, 8423, 8210, 8363, 8306, 8545, 8741,
				8410, 8779, 8219, 7874, 8339, 8400, 8131,
				8197, 8357, 7910, 8124, 8636, 7994, 7908,
				7863, 8687, 8792, 8763, 7842, 8594, 8665,
				8145, 8274, 8404, 8092, 8089, 8443, 7757,
				8719, 5496, 6644,
8057620	929	NM_000393	1519	3762, 3939, 7320, 7380, 7573,	COL5A2	−2.18
8106743	930	NM_004385	1520	2643, 4889, 4890, 4891, 4892, 6221, 7925,	VCAN	−2.17
				8699, 7773, 8196, 5495, 8365, 6091,
7897801	931	M77839	1521	2554, 4136,	RNU5E	−2.14
8069689	932	NM_007038	1522	2778, 4585, 5725,	ADAMTS5	−2.14
8140955	933	NM_001145306	1523	3279, 4773, 4772, 5552, 8113, 8064, 7668,	CDK6	−2.11
				6346,
8135218	934	NM_005824	1524	2666, 4848, 4849, 5940, 5451, 7410, 8602,	LRRC17	−2.11
8163716	935	NM_014618	1525	2726, 4469, 4474, 3012, 3546, 3649, 4164,	DBC1	−2.11
				6292, 6191, 5480,
8007921	936	NM_001002841	1526	3053, 3703, 2998, 6007, 6931,	MYL4	−2.10
7934979	937	NM_014391	1527	2995, 3511, 5932,	ANKRD1	−2.09
8085946	938	NM_005442	1528	3171, 3140, 5868, 7137, 7635,	EOMES	−2.07
7971461	939	NM_002298	1529	2593, 2594, 3108, 3126, 3127, 4472, 2928,	LCP1	−2.05
				2972, 4589, 6343, 5723, 6824, 7508, 6347,
				6695, 8668, 6816,
7944082	940	NM_003186	1530	2895, 3656, 3657, 3038, 7198, 5531, 6894,	TAGLN	−2.04
				5715,
8045688	941	NM_007115	1531	3052, 5234, 5352, 6810,	TNFAIP6	−2.04
8105607	942	NM_001164442	1532	4893, 7805, 7948, 7918, 8707, 6538,	FAM159B	−2.03
8161520	943	NM_021965	1533	3077, 4232, 3852, 6909, 6516,
Table 8: Provided are the description of the non-membranal genes along with the sequence identifiers of the Affymetrix probes, the target sequence identified by the Affymetrix probe, and the mRNA sequences represented by the target sequences. Also shown are the log 2 fold change in expression levels of the indicated genes between the PDX1-GFP cells and the genetically unmodified HESCs. The (+) or (−) sign means that the genes are upregulated or downregulated, respectively, in the PDX1-GFP cells as compared to the genetically unmodified HESCs.

Example 3

Genes which are Upregulated or Downregulated in Pancreatic Progenitor (PDX1+) Versus Definite Endoderm (SOX17+) Cells

TABLE 9
Membranal genes which are upregulated in PDX1 cells as compared to SOX17 cells
			SEQ ID
	Affy. target		NO: (Of			Log 2
Probe	nucl. SEQ	Rep.	Rep. Pub-	Polyn. SEQ ID NOs:	Gene	fold change
Set ID	ID NO:	Public ID	lic ID)	rep. by target	Symbol	[PDX-SOX]
8100393	944	NM_002253	1534	2708, 4588, 7921, 5498, 8228,	KDR	4.62
8051785	945	NM_133329	1535	3647, 5227, 5228, 5783, 7561,	KCNG3	4.19
8031076	946	NM_031896	1536	3827, 5226, 7212, 5339,	CACNG7	3.80
8096440	947	NM_001510	1537	2719, 4235, 8429, 5557, 7933, 8154,	GRID2	3.63
7996837	948	NM_004360	1538	4149, 4182, 2614, 4278, 4556, 2794, 2795,	CDH1	3.45
				4415, 4294, 4315, 4347, 5506, 7453, 5404,
				7133,
7917276	949	NM_012152	1539	2780, 4363, 7551, 8539, 6011,	LPAR3	3.33
8113666	950	NM_020796	1540	2844, 4722, 3122, 3148, 4310, 4145, 8137,	SEMA6A	3.29
				8152, 7900, 5499, 8724, 8667, 8467, 8111,
				5437, 8321, 7896, 5978, 8764,
8135774	951	NM_002851	1541	2599, 3943, 7457, 8030, 7815, 8011, 8237,	PTPRZ1	3.15
				7646,
8037079	952	NM_152296	1542	3345, 3672, 8109, 6417, 5616, 7816,	ATP1A3	3.09
8087530	953	NM_024046	1543	3407, 4516, 4542, 4547, 4568, 3355, 2883,	CAMKV	3.04
				3337, 3368, 3000, 3165, 3910, 8007, 8299,
				6547, 8504, 8573, 8756, 8739, 7861, 8075,
				7731, 6769, 7182, 8603, 8099, 7254, 5602,
7994058	954	NM_001039	1544	3481, 4177, 5784,	SCNN1G	3.00
7918716	955	NM_205848	1545	2981, 3239, 4010, 6234, 6167, 7976, 6283,	SYT6	2.92
				6937, 6744, 6657,
7930837	956	NM_003054	1546	3141, 5107, 7960, 5655,	SLC18A2	2.87
8108697	957	NM_015669	1547	2872, 2931, 5379,	PCDHB5	2.84
8101675	958	NM_004827	1548	3811, 3819, 7943, 5391,	ABCG2	2.61
8178193	959	NM_019111	1549	3652, 4537, 2590, 2589, 4532, 4478, 4721,	HLA-DRA	2.60
				4482, 3074, 3666, 5174, 6840, 8271, 7495,
				7301, 6340, 7125, 7667, 6973, 6698, 6694,
				7895, 6462, 7070, 8427, 7321, 6682, 7906,
				7421, 7648, 7487, 6525,
7916584	960	NM_002353	1550	2675, 4337, 6094,	TACSTD2	2.57
7909390	961	NM_175710	1551	3897, 4185, 8470, 6548, 5851,	CR1L	2.54
8169447	962	NM_000868	1552	2609, 4287, 5920, 5564, 5986,	HTR2C	2.48
7972157	963	NM_003991	1553	2606, 5247, 5246, 5248, 5597, 7819, 7369,	EDNRB	2.47
8168657	964	NM_001168362	1554	3001, 2888, 4950, 4951, 2807, 4945, 4946,	PCDH11X	2.45
				4947, 4948, 4949, 2939, 2940, 2938, 5762,
				6189, 5311, 6640, 6106, 6184, 6241, 5877,
				7081, 5601, 6247, 8389,
8038367	965	NM_020309	1555	3483, 4743, 5332,	SLC17A7	2.44
7960529	966	NM_001159575	1556	3514, 4813, 4814, 4812, 5353, 6068, 6294,	SCNN1A	2.35
				6143, 6414,
7953291	967	NM_001769	1557	3817, 5250, 7598, 7601, 7771, 7211, 6952,	CD9	2.35
				5319,
8011713	968	NM_022059	1558	2851, 4210, 4211, 5728,	CXCL16	2.29
8027778	969	NM_144779	1559	3228, 3470, 2828, 3419, 3286, 4528, 3008,	FXYD5	2.25
				3388, 4895, 4896, 4898, 4897, 6381, 7395,
				6565, 5906, 7311, 8512,
8170538	970	NM_018558	1560	2811, 4939, 6037, 5850,	GABRQ	2.25
8114415	971	NM_001496	1561	3167, 3584, 7363, 5508,	GFRA3	2.23
8087691	972	NM_001174051	1562	2721, 2711, 2712, 4220, 4298, 5019, 3735,	CACNA2D2	2.23
				3736, 6362, 6812, 5540, 6239, 8681, 7132,
				7985, 7734, 6957, 7200,
8133360	973	NM_001305	1563	3437, 3401, 7342, 6124, 7250,	CLDN4	2.21
8059186	974	NM_002846	1564	2647, 5233, 5755, 7545, 6502, 6839, 8004,	PTPRN	2.20
				8090, 8318, 8312, 7916,
8169061	975	NM_001128834	1565	3671, 3836, 3741, 3742, 2596, 2595, 2588,	PLP1	2.20
				4267, 4502, 4503, 2878, 4324, 4329, 4438,
				3554, 3553, 8469, 6590, 7115, 7231, 7458,
				6933, 7196, 7209, 8108, 8781, 8623, 8141,
				8486, 7855, 5832, 8385, 5923, 8694, 6335,
				7709, 6348, 8343, 7178, 7629,
7898057	976	NM_001006624	1566	3236, 2746, 2747, 3084, 3519, 2965, 3133,	PDPN	2.13
				4305, 4306, 3747, 3748, 3745, 3746, 8458,
				8786, 8213, 5840, 7528, 8392, 8050, 7659,
				7822, 8446,
8062123	977	NM_006690	1567	2775, 3872, 5355,	MMP24	2.13
8018114	978	NM_001144952	1568	2814, 4748, 7774, 5765, 6851, 6326,	SDK2	2.12
7987439	979	NM_007223	1569	3580, 2783, 5852,	GPR176	2.10
8171624	980	NM_005756	1570	3176, 3177, 3178, 3179, 3216, 3862, 3994,	GPR64	2.09
				3992, 4253, 3172, 3173, 3174, 3093, 5123,
				5124, 5125, 5126, 5127, 5128, 5129, 5130,
				5131, 6034, 6617, 6614, 8617, 6938, 6296,
				6048, 6115, 6013, 6956, 6659, 6099,
8083839	981	NM_014373	1571	2885, 4317, 4609, 8290, 8552, 8316, 8057,	GPR160	2.07
				7844, 7988, 6244, 7922,
8176400	982	NM_001168362	1572	3001, 2889, 4950, 2807, 4945, 4947, 2939,	PCDH11Y	2.07
				2940, 2938, 5762, 6189, 5311, 6640, 6184,
				6241, 5877, 7081,
8067602	983	NM_152864	1573	3226, 4323, 4809, 6602, 6260, 6027, 8666,	NKAIN4	2.06
				7541, 6149, 6332, 8610, 6274,
8004545	984	NM_001678	1574	2604, 3681, 5449,	ATP1B2	2.05
7955502	985	NM_001177984	1575	2769, 5108, 4009, 6054, 6237, 6251,	SCN8A	2.03
8106573	986	NM_003248	1576	3271, 5018, 8753, 6398, 8163, 6313,	THBS4	2.01
7909350	987	NM_001006658	1577	4488, 4916, 4917, 6275, 5909, 8223, 8585,	CR2	2.01
				6171, 6814,
8118556	988	NM_002122	1578	3810, 3721, 7313, 8208, 7034, 6552, 7074,	HLA-DQA1	1.98
				5946, 8475,
8118548	989	NM_019111	1579	3652, 4537, 2590, 2589, 4532, 4478, 4721,	HLA-DRA	1.97
				4482, 3074, 3666, 5174, 6840, 8271, 7495,
				7301, 6340, 7125, 7667, 6973, 6698, 6694,
				7895, 6462, 7070, 8427, 7321, 6682, 7906,
				7421, 7648, 7487, 6525,
7934970	990	NM_000872	1580	3256, 4604, 4605, 4606, 5950, 6214, 5736,	HTR7	1.92
				5523,
7915472	991	NM_006516	1581	4268, 4338, 5901, 6869, 6399, 8613, 6344,	SLC2A1	1.92
				6364, 5884, 7600,
8179481	992	NM_019111	1582	3652, 4537, 2590, 2589, 4532, 4478, 4721,	HLA-DRA	1.92
				4482, 3074, 3666, 5174, 6840, 8271, 7495,
				7301, 6340, 7125, 7667, 6973, 6698, 6694,
				7895, 6462, 7070, 8427, 7321, 6682, 7906,
				7421, 7648, 7487, 6525,
8040458	993	NM_002252	1583	3347, 3213, 6867, 6984, 8229, 5665, 7423,	KCNS3	1.91
8173414	994	NM_032803	1584	3087, 5135, 5134, 5854, 7474,	SLC7A3	1.86
8118607	995	NR_001435	1585	4480, 2993, 3281, 6755, 8176, 8031, 7000,	HLA-DPB2	1.86
				5720, 7237, 8785, 7391, 8295, 6363, 7378,
				8526, 7569, 6929, 7023,
8159734	996	NM_000718	1586	2559, 2560, 5298, 6089, 5532, 5955, 6280,	CACNA1B	1.85
				6151, 5911, 5558, 6198,
8171284	997	NM_000273	1587	3596, 4944, 6448, 6849, 7049, 7224,
Table 9: Provided are the description of the membranal genes along with the sequence identifiers of the Affymetrix probes, the target sequence identified by the Affymetrix probe, and the mRNA sequences represented by the target sequences. Also shown are the log 2 fold change in expression levels of the indicated genes between the PDX1-GFP cells and the SOX17-GFP cells (PDX1-SOX17). The (+) or (−) sign means that the genes are upregulated or downregulated, respectively, in the PDX1-GFP cells as compared to the SOX17-GFP cells.

TABLE 10
Membranal genes which are downregulated in PDX1 cells as compared to SOX17 cells
			SEQ ID			Log 2
	Affy. target		NO: (Of			fold change
Probe Set	nucl. SEQ	Rep.	Rep. Pub-	Polyn. SEQ ID NOs:	Gene	[PDX-
ID	ID NO:	Public ID	lic ID)	rep. by target	Symbol	SOX17]
7957140	998	NM_003667	1588	2732, 3193, 6795, 5509,	LGR5	−6.13
7938090	999	NM_176875	1589	2809, 5307, 5676,	CCKBR	−4.88
8055465	1000	NM_001008540	1590	2782, 3775, 3776, 5384, 6834, 7645,	CXCR4	−4.88
8065071	1001	NM_198391	1591	2812, 5172, 5173, 5918, 7056, 6380,	FLRT3	−4.44
8151341	1002	NM_007332	1592	4060, 8269, 5433,	TRPA1	−4.19
8057677	1003	NM_014585	1593	3154, 4368, 6969, 5406, 7045, 8144, 8627, 7674, 6595,	SLC40A1	−4.12
				6989,
7964602	1004	NM_153377	1594	3457, 3537, 4639, 5656, 6683, 7048,	LRIG3	−3.98
8021946	1005	NM_130386	1595	2891, 4601, 6579,	COLEC12	−3.86
7971296	1006	NM_033255	1596	3367, 3694, 3693, 5738, 5732, 7664, 7742,	EPSTI1	−3.83
8081298	1007	NM_032787	1597	3995, 4847, 5470, 7733, 8308, 8445,	GPR128	−3.63
8022674	1008	NM_001792	1598	3134, 4668, 5546,	CDH2	−3.38
7901316	1009	NM_001011547	1599	4682, 4626, 4627, 6649, 6403, 7403, 7829, 6781, 6361,	SLC5A9	−3.36
8052072	1010	NM_000145	1600	4265, 5021, 5022, 6252, 5713, 7612, 7221, 6397,	FSHR	−3.33
7924342	1011	NM_018713	1601	3493, 3718, 6268, 6130, 8273, 8216,	SLC30A10	−3.27
8169580	1012	NM_001560	1602	2683, 3219, 6256, 6220, 8730, 6320,	IL13RA1	−3.20
7977786	1013	NM_001126105	1603	2789, 5292, 5291, 7158, 7255, 5870, 8255, 6471, 7642,	SLC7A7	−3.14
8097449	1014	NM_032961	1604	2914, 2936, 2935, 7099, 5512, 8691,	PCDH10	−3.11
8154135	1015	NM_004170	1605	3070, 5025, 5401, 7218, 8331,	SLC1A1	−3.07
8132347	1016	NM_181791	1606	3313, 3318, 6342, 5701, 6692,	GPR141	−3.05
8111677	1017	NM_001127671	1607	2551, 4403, 4404, 5491, 7187, 7919, 8314, 7758,	LIFR	−2.98
8171472	1018	NM_020665	1608	2960, 4942, 6430,	TMEM27	−2.95
8175217	1019	NM_001448	1609	2738, 3072, 6154,	GPC4	−2.93
8045664	1020	NM_177964	1610	3039, 3538, 8714, 5464, 7286, 6455, 7123, 7806, 7494,	LYPD6B	−2.91
				8341, 7067, 6613,
7948058	1021	NM_153696	1611	3060, 3615, 2779, 2843, 3871, 2598, 4703, 4705, 4140,	FOLH1	−2.90
				4138, 2773, 3026, 3280, 5177, 5176, 2766, 5178, 3821,
				6173, 5921, 6307, 5425, 6469, 6968,
7971104	1022	NM_016179	1612	3415, 4643, 4644, 4645, 4646, 4647, 4648, 7469, 7085,	TRPC4	−2.88
				8143, 6069, 7054, 6227, 6264, 7998, 7627, 6564, 7066,
8094520	1023	NM_032457	1613	2723, 5014, 5015, 2937, 5262, 5768, 5971,	PCDH7	−2.88
8143534	1024	NM_000420	1614	2886, 2987, 6934, 8187, 8288, 6818, 6144, 8559,	KEL	−2.78
8045795	1025	NM_002239	1615	2667, 3201, 5594, 8178,	KCNJ3	−2.72
7911273	1026	NM_001004696	1616	3717, 6107,	OR2T4	−2.71
8165658	1027	X59268	1617	2553, 3426, 3422, 6269,	VIPR2	−2.68
7976073	1028	NM_013231	1618	4059, 3816, 5667,	FLRT2	−2.66
7923978	1029	NM_001773	1619	2563, 3891, 3854, 5956, 6086, 5785, 6059, 8087,	CD34	−2.64
8101992	1030	NM_001135146	1620	2875, 4623, 4624, 3783, 4625, 7838, 6311, 8464, 6341,	SLC39A8	−2.63
				7047,
8083887	1031	NM_001185056	1621	2958, 5141, 4593, 7870, 5315, 6845, 7064, 7947, 8104,	CLDN11	−2.57
8049471	1032	NM_020311	1622	3396, 4030, 5457,	CXCR7	−2.52
7963786	1033	NM_002205	1623	3341, 3761, 5661, 6880,	ITGA5	−2.48
8046861	1034	NM_002210	1624	2615, 4762, 4763, 4764, 8678, 6893, 8003, 7665, 5446,	ITGAV	−2.44
				6625,
8140995	1035	NM_001742	1625	2561, 4912, 4913, 4914, 6611, 6073, 7384, 7536, 6539,	CALCR	−2.43
8082928	1036	NM_016369	1626	4183, 3788, 3787, 8686, 5317, 6211,	CLDN18	−2.41
8003298	1037	NM_003486	1627	2755, 3870, 5397,	SLC7A5	−2.32
7964745	1038	NM_016056	1628	3443, 2942, 4069, 7063, 6210, 5607, 7161,	TMBIM4	−2.32
8090823	1039	NM_005630	1629	2724, 4396, 7489, 6355, 8401, 8414, 8074, 5640,	SLCO2A1	−2.29
8111255	1040	NM_006727	1630	2788, 5142, 8497, 5507, 8253, 8371,	CDH10	−2.29
7955797	1041	NM_020547	1631	4056, 4904, 4903, 4905, 7400, 5395,	AMHR2	−2.28
7952341	1042	NM_024769	1632	3321, 3556, 7605, 6402,	ASAM	−2.26
8092726	1043	NM_021101	1633	2752, 5133, 5652, 7810,	CLDN1	−2.26
8070421	1044	NM_001389	1634	2796, 3591, 6842,	DSCAM	−2.15
8004691	1045	NM_203411	1635	3456, 3564, 5821,	TMEM88	−2.15
7923991	1046	NM_025179	1636	4111, 4029, 6303, 8403, 6499,	PLXNA2	−2.14
8037298	1047	NM_020406	1637	3744, 4005, 6408, 7480, 7666,	CD177	−2.14
8098041	1048	NM_018342	1638	2802, 3163, 3170, 3261, 3319, 3506, 3267, 4584, 8697,	TMEM144	−2.13
				8424, 5686, 8259, 8744, 8370, 7977, 8192, 7923, 7849,
				8039, 7954, 8413, 7830,
8142687	1049	NM_005302	1639	3208, 3301, 5833,	GPR37	−2.09
7919340	1050	NM_005266	1640	3364, 4104, 4105, 7691, 6103, 6550, 5474,	GJA5	−2.09
7983527	1051	NM_024966	1641	3078, 3079, 3080, 3081, 3082, 4192, 4279, 3184, 3186,	SEMA6D	−2.08
				3187, 3185, 3188, 3183, 6040, 7595, 5709, 6809, 6877,
				7470, 6289, 5994,
8151952	1052	NM_024759	1642	4112, 2892, 8672, 8517, 8125, 6122, 8051, 8637,	NIPAL2	−2.08
8114896	1053	NM_194251	1643	3379, 3780, 5572,	GPR151	−2.07
8113551	1054	NM_002387	1644	3412, 4147, 4148, 8175, 8246, 5792, 7893,	MCC	−2.00
7944554	1055	NM_174926	1645	4534, 5202, 5203, 5204, 5205, 5206, 5207, 5208, 6407,	TMEM136	−1.98
				5814,
8067029	1056	NM_002237	1646	2710, 3445, 4245, 2913, 3399, 4162, 8728, 6738, 7204,	KCNG1	−1.94
				6263, 7217, 6415,
8112803	1057	NM_005779	1647	3315, 4380, 4381, 3977, 8589, 8081, 8592, 8563, 6988,	LHFPL2	−1.89
				8101,
8175288	1058	NM_019556	1648	2903, 2824, 6997, 8283, 6003, 6290, 7788, 7752, 5881,	MOSPD1	−1.87
8068810	1059	NM_018964	1649	2845, 3683, 6723, 7548, 6976, 5984, 7385, 8095,	SLC37A1	−1.86
8096839	1060	NM_198506	1650	3439, 4440, 5693, 7042, 6600,	LRIT3	−1.86
8059279	1061	NM_004438	1651	3926, 3577, 6427, 7497, 5543,	EPHA4	−1.84
7916862	1062	NM_024911	1652	3473, 5169, 5171, 5170, 6985, 7944, 8040, 5423, 6217,	GPR177	−1.83
				5957, 7720, 6063, 8128, 6006,
8043995	1063	NM_000877	1653	2585, 2550, 2838, 3587, 3589, 3590, 3704, 3705, 4327,	IL1R1	−1.80
				3237, 7104, 5389, 7004, 6741, 7479, 7102, 7053, 7439,
				6674, 6715, 7705, 7500, 6803, 6643,
Table 10: Provided are the description of the membranal genes along with the sequence identifiers of the Affymetrix probes, the target sequence identified by the Affymetrix probe, and the mRNA sequences represented by the target sequences. Also shown are the log 2 fold change in expression levels of the indicated genes between the PDX1-GFP cells and the SOX17-GFP cells (PDX1-SOX17). The (+) or (−) sign means that the genes are upregulated or downregulated, respectively, in the PDX1-GFP cells as compared to the SOX17-GFP cells.

TABLE 11
Non-membranal genes which are upregulated in PDX1 cells as compared to SOX17 cells
			SEQ ID			Log 2
	Affy. target		NO: (Of			fold change
Probe	nucl. SEQ	Rep.	Rep. Pub-	Polyn. SEQ ID NOs:	Gene	[PDX-
Set ID	ID NO:	Public ID	lic ID)	rep. by target	Symbol	SOX17]
8049128	1064	NM_031313	1654	2557, 4233, 5696,	ALPPL2	5.61
8127522	1065	NM_001025290	1655	4388, 5232, 5916,	DPPA5	5.51
7945680	1066	NR_030533	1656	3526, 4930, 3779, 6619, 7610, 6652, 6726, 6561, 7131,	H19	5.26
				6743, 7010, 6680, 6895, 7340, 7012, 6491,
7917037	1067	NM_001889	1657	3420, 2605, 3199, 3619, 4456, 4457, 4458, 4612, 6114,	CRYZ	4.85
				6052, 6863, 6288, 6530, 6018, 8189,
7933194	1068	NM_199168	1658	2665, 5012, 5013, 5117, 5003, 6588, 8491, 7914, 6051,	CXCL12	4.67
				6656, 6859,
7902345	1069	NM_001162916	1659	3648, 4873, 4874, 4872, 8405, 6496, 6385, 8689, 7110,	TYW3	4.48
				8780, 6273,
7901497	1070	NM_001004339	1660	3328, 4808, 6317, 5872,	ZYG11A	4.16
7985159	1071	NM_004378	1661	3015, 4439, 5645,	CRABP1	3.92
8146092	1072	NM_002164	1662	2584, 5259, 5421, 8523, 8757, 8169, 8149, 8114, 8319,	IDO1	3.92
				7915, 8778,
8178470	1073	NM_203289	1663	2865, 2866, 2867, 2868, 3988, 3986, 4587, 5026, 5027,	POU5F1	3.76
				5028, 5029, 5030, 5031, 5032, 5033, 5034, 5035, 5036,
				5037, 5038, 5039, 5040, 5041, 5042, 5043, 5044, 5045,
				5046, 5047, 5048, 5049, 5050, 5051, 5052, 5053, 5054,
				5055, 5056, 5057, 5058, 5059, 5060, 5061, 5062, 5063,
				5064, 5065, 5066, 5067, 5068, 5069, 5070, 5071, 5072,
				5073, 5074, 5075, 5076, 5077, 5078, 5079, 5080, 5081,
				5082, 5083, 5084, 5085, 5086, 5087, 5088, 5089, 5090,
				5091, 5092, 5093, 5094, 5095, 5096, 5097, 5098, 5099,
				5100, 5101, 5102, 5103, 5104, 4227, 4228, 4229, 4230,
				4231, 3958, 3959, 4369, 5016, 5017, 4052, 5175, 5179,
				8740, 7043, 6463, 8204, 6434, 6854, 6431, 8068, 6882,
				7219, 7760, 8266, 8247, 6495, 7073, 7834, 7275, 7155,
				8483, 6801, 7986, 6354, 8509, 6963, 7415, 7877, 6325,
				7358, 5405, 7117, 6852, 8575, 6970, 7641, 7552,
8121850	1074	NM_012259	1664	4276, 3975, 5989, 6023,	HEY2	3.74
8124380	1075	NM_005325	1665	3881, 4053, 5377,	HIST1H1A	3.70
8063536	1076	NM_003222	1666	3168, 3531, 5324,	TFAP2C	3.69
8089438	1077	NM_138815	1667	3289, 4865, 8060,	DPPA2	3.63
8098690	1078	NM_174900	1668	3498, 3937, 5793, 8300,	ZFP42	3.59
7971838	1079	NM_007015	1669	3028, 3784, 3785, 7153, 6673,	LECT1	3.54
8147244	1080	NM_022351	1670	3290, 4737, 6800, 7785, 7823, 8669, 7989, 8696,	NECAB1	3.48
7983239	1081	NM_020990	1671	4017, 4725, 3845, 3844, 5618, 8459, 7658, 6700, 6805,	CKMT1A	3.47
				7001, 6390, 7746, 7418, 6964, 8012, 7084, 7264, 7777,
				8717, 7315, 6930, 7503, 6473, 7092, 7417, 7902, 7582,
8066117	1082	NM_015474	1672	3449, 5023, 3830, 5349, 5541, 7106, 6100,	SAMHD1	3.46
8097256	1083	NM_198041	1673	2576, 5304, 4197, 5305, 5478, 5980, 5822,	FGF2	3.44
7913216	1084	NM_000300	1674	2906, 4866, 4867, 4868, 4869, 6945, 6892, 8598, 8048,	PLA2G2A	3.42
				8615, 6396, 7663,
8151240	1085	NM_024504	1675	2858, 3552, 5534,	PRDM14	3.39
8124889	1086	NM_203289	1676	2865, 2866, 2867, 2868, 3988, 3986, 4587, 5026, 5027,	POU5F1	3.39
				5028, 5029, 5030, 5031, 5032, 5033, 5034, 5035, 5036,
				5037, 5038, 5039, 5040, 5041, 5042, 5043, 5044, 5045,
				5046, 5047, 5048, 5049, 5050, 5051, 5052, 5053, 5054,
				5055, 5056, 5057, 5058, 5059, 5060, 5061, 5062, 5063,
				5064, 5065, 5066, 5067, 5068, 5069, 5070, 5071, 5072,
				5073, 5074, 5075, 5076, 5077, 5078, 5079, 5080, 5081,
				5082, 5083, 5084, 5085, 5086, 5087, 5088, 5089, 5090,
				5091, 5092, 5093, 5094, 5095, 5096, 5097, 5098, 5099,
				5100, 5101, 5102, 5103, 5104, 4227, 4228, 4229, 4230,
				4231, 3958, 3959, 4369, 5016, 5017, 4052, 5175, 5179,
				8740, 7043, 6463, 8204, 6434, 6854, 6431, 8068, 6882,
				7219, 7760, 8266, 8247, 6495, 7073, 7834, 7275, 7155,
				8483, 6801, 7986, 6354, 8509, 6963, 7415, 7877, 6325,
				7358, 5405, 7117, 6852, 8575, 6970, 7641, 7552,
8139212	1087	NM_000168	1677	3991, 3982, 2577, 4553, 3392, 4592, 6480, 7171, 6577,	GLI3	3.38
				6439, 8751, 6708,
8071809	1088	NM_000854	1678	2877, 3667, 4143, 4144, 5682, 5316,	GSTT2	3.36
7904158	1089	NM_020190	1679	3467, 3697, 6147, 5677, 8035, 6986,	OLFML3	3.31
8085676	1090	NM_001351	1680	3383, 5153, 5154, 6778,	DAZL	3.30
8056408	1091	NM_004482	1681	3996, 3990, 3410, 3332, 4301, 4196, 6671, 7016, 6517,	GALNT3	3.29
				7277, 7565, 6510, 6479, 6804, 6507,
8084165	1092	NM_003106	1682	3338, 5264, 5632,	SOX2	3.28
8148315	1093	NM_001159542	1683	5084, 4815, 6578, 6639,	POU5F1B	3.27
7941662	1094	NM_001104	1684	3432, 2761, 8043, 7856, 8550,	ACTN3	3.27
7949971	1095	NM_001876	1685	2870, 4398, 4399, 7075, 5473,	CPT1A	3.26
7970954	1096	NM_004734	1686	4221, 5192, 5193, 5194, 5195, 5448, 7317, 6024, 6492,	DCLK1	3.26
				7076,
8112980	1097	NM_005711	1687	2720, 3295, 8700, 8201, 7504, 5630,	EDIL3	3.23
7953675	1098	NM_024865	1688	3571, 3299, 4206, 5371,	NANOG	3.23
8077513	1099	NM_015453	1689	2882, 2944, 4333, 4334, 6458, 6573, 7246, 8510, 6238,	THUMPD3	3.22
				6855, 6555, 8394, 7095, 6527,
7909642	1100	NM_001136475	1690	3791, 4510, 4251, 3161, 3278, 3303, 4655, 4653, 4654,	VASH2	3.19
				6082, 5905, 6123, 6062, 6304, 5489, 8315, 6152, 8644,
				8692,
8024712	1101	NM_033064	1691	3258, 3101, 4560, 3035, 4405, 5929, 7505, 5849, 7325,	ATCAY	3.18
7965565	1102	NM_032147	1692	3062, 3978, 3979, 6775, 5436,	USP44	3.18
8117422	1103	NM_003540	1693	3825, 3059, 7626,	HIST1H4F	3.12
7987365	1104	NM_024865	1694	3571, 3299, 4206, 5371,	NANOG	3.09
8172471	1105	NM_006875	1695	2994, 4637, 7671, 8024, 6884,	PIM2	3.08
8061746	1106	NM_006892	1696	3002, 3249, 3250, 3251, 5288, 6284, 6608, 5831, 5310,	DNMT3B	3.05
				6240, 7520, 7394, 6299,
8157193	1107	NM_133464	1697	4219, 4428, 4429, 7118, 6162, 5698,	ZNF483	3.04
8142580	1108	NM_001024613	1698	4758, 4836, 4837, 7698, 6675, 7454, 5751,	FEZF1	3.04
8171758	1109	NR_023358	1699	4408,	SCARNA9L	3.04
8108995	1110	NM_054023	1700	3022, 4995, 8754, 8215, 5614,	SCGB3A2	3.00
7963970	1111	NM_006928	1701	3105, 5245, 6941, 6287,	SILV	2.98
8122099	1112	NM_006208	1702	3492, 4353, 5924, 6245, 8263, 8565, 7205,	ENPP1	2.94
8148317	1113	NM_002467	1703	2869, 4851, 6776, 7274,	MYC	2.90
8039280	1114	NM_139176	1704	4509, 4372, 4373, 4374, 7316, 5964, 5724, 7405, 7354,	NLRP7	2.88
				7602,
7930631	1115	NM_198795	1705	3518, 3513, 6133, 6452, 5445, 6004, 5919,	TDRD1	2.86
8152938	1116	NM_001145095	1706	2750, 4755, 7527, 8041,	HHLA1	2.86
8001531	1117	NM_005950	1707	3006, 2840, 6392, 7114, 7170, 6815,	MT1G	2.85
8095043	1118	NM_023940	1708	3792, 3377, 8732, 5428, 8096,	RASL11B	2.79
7949124	1119	NM_001164716	1709	2730, 4906, 4290, 8065, 5323,	PYGM	2.78
8073062	1120	NM_004900	1710	2835, 3155, 5168, 5750, 5753,	APOBEC3B	2.78
8148070	1121	NM_021110	1711	4389, 2612, 2613, 2692, 2964, 3160, 4321, 3750, 8105,	COL14A1	2.71
				5673, 5636, 7386, 8362, 7397, 5443,
8015635	1122	NM_012232	1712	3561, 4938, 6271, 5910,	PTRF	2.69
8138708	1123	NM_005522	1713	3389, 3921, 3920, 5895, 5930,	HOXA1	2.67
8028991	1124	NM_030622	1714	3461, 3715, 5647, 5708,	CYP2S1	2.66
8169115	1125	NM_198465	1715	3311, 4081, 5354,	NRK	2.65
7949104	1126	NM_001098670	1716	2786, 2787, 2742, 3987, 2749, 3104, 3106, 3428, 4186,	RASGRP2	2.64
				4187, 3200, 6996, 7253, 7251, 6422, 6110, 6605, 7167,
				6721, 6400, 7783, 7408, 7101, 6992, 8419, 6612, 7675,
				7336, 6494, 7993, 7692, 6747, 7413,
7912473	1127	NM_183412	1717	3680, 3848, 3023, 4316, 3818, 5249, 8328, 6632, 6172,	FBXO2	2.62
				7087, 6489, 6785, 5411,
7956488	1128	NM_004984	1718	4336, 3578, 5671, 7555,	KIF5A	2.62
8145977	1129	NM_021623	1719	4259, 4207, 8302, 7202, 6843, 7732, 8390,	PLEKHA2	2.61
8096361	1130	NM_016323	1720	3489, 4008, 8649, 5458, 8138, 8244,	HERC5	2.61
8104107	1131	NM_173553	1721	3502, 3928, 3929, 4676, 4718, 3234, 7761, 8133, 7793,	TRIML2	2.60
				5580, 8122,
7986350	1132	NM_183376	1722	3385, 4436, 5479,	ARRDC4	2.60
7975076	1133	NM_021979	1723	3158, 4151, 7440, 5369,	HSPA2	2.59
7933092	1134	NM_021045	1724	4114, 3320, 8456, 7285, 8408, 7542, 7283, 7447, 6137,	ZNF248	2.59
8134463	1135	NM_002523	1725	3340, 4771, 5502, 6922,	NPTX2	2.57
7995838	1136	NM_005952	1726	3371, 4455, 6391,	MT1X	2.55
8096301	1137	NM_001040058	1727	4706, 3942, 3501, 3944, 8278, 7992, 5370, 6716, 8452,	SPP1	2.55
				8651, 6109, 6075, 7831, 7801,
7963869	1138	NM_002355	1728	3673, 5287, 3831, 7078, 5809, 6441, 5420, 7521, 5321,	PHC1	2.53
8097920	1139	NM_004744	1729	3566, 3595, 8748, 8069, 7763, 8070, 5620,	LRAT	2.51
8031576	1140	NM_134444	1730	3031, 4444, 5611, 5951,	NLRP4	2.49
8116418	1141	NM_005110	1731	2751, 4135, 8471, 5407, 8222, 8775, 7999,	GFPT2	2.47
8139820	1142	NM_178558	1732	3061, 4448, 4449, 8320, 6601, 8790, 5658,	ZNF680	2.47
7989277	1143	NM_004998	1733	3855, 3757, 5660,	MYO1E	2.45
7983360	1144	NM_004048	1734	3076, 3490, 6485, 6270,	B2M	2.44
8120679	1145	NM_018665	1735	2820, 2957, 4749, 6125, 7145, 8551, 6076,	DDX43	2.44
7914342	1146	NM_004102	1736	2918, 3829, 8327, 8609, 8516, 5937,	FABP3	2.44
8147697	1147	NM_024915	1737	4098, 4097, 4270, 4529, 3637, 3606, 3638, 4299, 4354,	GRHL2	2.43
				5422, 7907, 7656, 8209, 8281, 7516,
8044353	1148	NM_001142807	1738	4470, 4673, 6005, 7357, 6866, 6663, 6681,	ACOXL	2.41
8138489	1149	NM_018719	1739	3585, 4801, 4802, 4803, 7510, 6116, 7011, 6541, 6183,	CDCA7L	2.40
				8558,
8099524	1150	NM_001290	1740	2733, 4590, 4591, 8679, 8166, 7837, 8661, 8298, 5669,	LDB2	2.39
				8605, 7719, 7814, 8032, 8473, 7455, 8734,
8020384	1151	NM_001142966	1741	4467, 2876, 4295, 4675, 7359, 7061, 5529, 7232,	KIAA1772	2.39
8103254	1152	NM_003013	1742	2933, 3724, 5471,	SFRP2	2.38
8078435	1153	NM_001039111	1743	3892, 3922, 6993,	TRIM71	2.38
7928429	1154	NM_002658	1744	3397, 4752, 4753, 6308, 6729, 8322, 7727, 8611, 6028,	PLAU	2.37
				6300, 8374,
8121277	1155	NM_001624	1745	4575, 4051, 6064, 6887, 8412, 5596,	AIM1	2.36
7995797	1156	NM_175617	1746	3381, 3905, 5861, 5789,	MT1E	2.36
8015607	1157	NM_139276	1747	4260, 3613, 3614, 3636, 7860, 7019, 6610, 8049, 5511,	STAT3	2.34
				7592, 8190, 6553,
7998921	1158	NM_032805	1748	3149, 2932, 5400,	ZSCAN10	2.34
7983718	1159	NM_001165257	1749	3014, 4909, 4908, 5333,	SCG3	2.33
7997336	1160	NM_020927	1750	3166, 3189, 5813,	VAT1L	2.32
7901087	1161	NM_032756	1751	2922, 3690, 5860,	HPDL	2.31
8124684	1162	NM_001109809	1752	4390, 4684, 4850, 7287, 7191, 7216, 6705, 6979, 6515,	ZFP57	2.31
				7376, 8185, 6500, 6658,
8160260	1163	NM_017637	1753	3551, 4555, 3100, 4545, 4032, 4033, 4034, 4035, 4036,	BNC2	2.30
				4037, 2799, 3963, 6528, 6375, 7529, 7058, 6914, 7266,
				7235, 7564, 8608, 7382, 8765,
8026424	1164	NM_173483	1754	3826, 4241, 5547,	CYP4F22	2.30
8165217	1165	NM_017617	1755	2850, 4176, 5567,	NOTCH1	2.30
8113278	1166	NM_153234	1756	3387, 4341, 5548,	LIX1	2.30
7994609	1167	NM_014298	1757	3509, 3569, 6447, 6560, 5343,	QPRT	2.29
8137091	1168	NM_170686	1758	3019, 4464, 4465, 8588, 6484, 7701, 5871, 8626, 7762,	ZNF398	2.29
				7857,
7936673	1169	NM_002925	1759	3525, 3730, 3731, 8042, 7377, 6524, 6267, 6015,	RGS10	2.27
8162531	1170	ENST00000396160	1760	7570,	MT1G	2.26
8010354	1171	NM_000152	1761	3217, 4085, 4086, 4087, 5704, 6841,	GAA	2.25
8059301	1172	NM_181460	1762	4255, 5159, 5161, 5162, 5163, 5164, 5165, 5158, 5160,	PAX3	2.23
				7119, 5969, 5904, 7673, 5842, 7706, 6334, 5413,
8006367	1173	NM_138328	1763	3863, 3884, 5472, 7404,	RHBDL3	2.23
8106820	1174	NM_006467	1764	4507, 4049, 6583, 8418,	POLR3G	2.23
7914015	1175	NM_052943	1765	3427, 4638, 5752,	FAM46B	2.23
7995825	1176	NM_005949	1766	3051, 4447, 5801, 6580,	MT1F	2.22
7945296	1177	NM_001080407	1767	3430, 4288, 6702, 8356, 8044, 7094, 7185, 6706,	GLB1L3	2.21
8097928	1178	NM_144979	1768	3391, 4586, 5462,	RBM46	2.21
8074335	1179	NM_001195226	1769	2764, 3807, 3998, 4025, 2793, 2709, 3097, 3594, 5186,	PRODH	2.21
				5187, 5737, 7271, 5953, 7991, 7069, 6983, 7547, 8254,
				6370, 7730, 6607, 5836,
8115196	1180	NM_052860	1770	2952, 4996, 4997, 4998, 5526, 6512, 6897,	ZNF300	2.21
8163896	1181	NM_004099	1771	2946, 3496, 3495, 6038, 5639,	STOM	2.20
7948987	1182	NM_007069	1772	3359, 4422, 4423, 5706, 6689, 7225,	PLA2G16	2.20
7942064	1183	NM_015973	1773	3055, 3933, 5477,	GAL	2.18
8150962	1184	NM_014729	1774	2740, 3709, 6856, 6017,	TOX	2.17
8046078	1185	NM_020981	1775	2763, 5191, 6587, 5756,	B3GALT1	2.17
7978801	1186	NM_182830	1776	3421, 4330, 5167, 6351, 6182, 7688, 8193, 6799, 7207,	MDGA2	2.17
7912481	1187	NM_006341	1777	2774, 2791, 2768, 2798, 3111, 2967, 4393, 4394, 6406,	MAD2L2	2.16
				7367, 7044, 5373, 7566, 7290, 7330, 6832, 7175,
8143221	1188	NM_130840	1778	4271, 3924, 3925, 3923, 8501, 8715, 6101, 5610, 7586,	ATP6V0A4	2.16
8121949	1189	NM_000426	1779	3433, 4089, 4090, 6848, 5928, 7289, 6035,	LAMA2	2.16
7919787	1190	NM_032132	1780	3665, 3069, 3254, 3722, 5240, 8304, 6029, 6113, 5998,	HORMAD1	2.15
				7030, 5874, 5867, 8731, 6466, 5973,
7938035	1191	AL360187	1781	2821, 2822, 2823, 4283, 4533, 4544, 4481, 3016, 3162,	TRIM22	2.15
				4213, 4312, 4320, 4326, 5229, 7088, 6634, 6762, 8083,
				6459, 8567, 6533, 6911, 6474, 7269, 7353,
8018652	1192	NM_052916	1782	4746, 5562, 5570, 5845,	RNF157	2.15
8052882	1193	NM_001617	1783	3312, 2549, 4252, 4541, 3221, 3555, 5136, 5137, 5138,	ADD2	2.15
				5139, 5140, 7591, 8079, 5513, 5527, 8334, 8155, 6282,
				7652, 6883, 7881, 7694, 6520, 6309, 7614, 7098,
8168438	1194	NM_145052	1784	3674, 4272, 2966, 4297, 4940, 4941, 7089, 8335, 6323,	UPRT	2.15
				6105, 6157,
8125500	1195	NM_004159	1785	3676, 2641, 2642, 4720, 3358, 3875, 3876, 6366, 7256,	PSMB8	2.15
				7662, 7787, 7258, 6923, 7465, 7711, 6603, 7071, 8698,
				6850, 6638, 7406, 7295, 8663, 7850, 7345, 6440, 6333,
				7938, 7517, 6750, 8634, 6788, 7146, 8416, 7360, 7022,
				6858, 8225, 6666, 7687, 7230, 6844, 6589, 8360, 7319,
				8354, 7670,
8033801	1196	NM_001130031	1786	4039, 4450, 4451, 4452, 6833, 5584, 6691,	ZNF562	2.14
8171338	1197	NM_174901	1787	3150, 5120, 5679, 6572, 7485, 7604,	FAM9C	2.13
8097017	1198	NM_003360	1788	2689, 4419, 4420, 5810, 8336, 6908,	UGT8	2.13
7956522	1199	AF063608	1789	2859, 7555,	KIF5A	2.11
7981945	1200	BC043194	1790	3229,	SNRPN	2.09
8035318	1201	NM_001080421	1791	4963, 8704, 7597, 8506, 5412,	UNC13A	2.09
8000757	1202	NM_003586	1792	3325, 3610, 3233, 5788, 5896,	DOC2A	2.09
8033767	1203	NM_152476	1793	2982, 4116, 5683,	ZNF560	2.09
7931556	1204	NM_014468	1794	2743, 3330, 5776,	VENTX	2.07
8143772	1205	NM_002889	1795	3103, 4674, 8675, 7835, 7515, 5341,	RARRES2	2.07
8096808	1206	NM_032992	1796	3120, 3873, 3874, 4445, 8500, 7859, 7100, 6201,	CCDC109B	2.06
7995783	1207	NM_005953	1797	2919, 4617, 5366,	MT2A	2.05
8013660	1208	NM_005165	1798	3335, 3853, 6651, 5330, 6714, 7379,	ALDOC	2.03
7983143	1209	NM_020759	1799	4943, 5714,	STARD9	2.03
8008664	1210	NM_003488	1800	3357, 3981, 5585, 8407, 7660, 5628,	AKAP1	2.03
Table 11: Provided are the description of the non-membranal genes along with the sequence identifiers of the Affymetrix probes, the target sequence identified by the Affymetrix probe, and the mRNA sequences represented by the target sequences. Also shown are the log 2 fold change in expression levels of the indicated genes between the PDX1-GFP cells and the SOX17-GFP cells (PDX1-SOX17). The (+) or (−) sign means that the genes are upregulated or downregulated, respectively, in the PDX1-GFP cells as compared to the SOX17-GFP cells.

TABLE 12
Non-membranal genes which are downregulated in PDX1 cells as compared to SOX17 cells
	Affy.		SEQ ID			Log 2
	target		NO: (Of			fold change
Probe	nucl. SEQ	Rep.	Rep. Pub-	Polyn. SEQ ID NOs:	Gene	[PDX-
Set ID	ID NO:	Public ID	lic ID)	rep. by target	Symbol	SOX17]
8065412	1211	NM_001898	1801	3369, 3033, 5734, 6511,	CST1	−7.67
8160163	1212	NM_005454	1802	3880, 3883, 6763,	CER1	−7.42
7934979	1213	NM_014391	1803	2995, 3511, 5932,	ANKRD1	−5.27
8136807	1214	—			TRY6	−5.22
8152617	1215	NM_005328	1804	2676, 4348, 5843, 6409,	HAS2	−5.08
7927631	1216	NM_012242	1805	3471, 3796, 7543, 8463, 8242, 5959,	DKK1	−5.03
8136790	1217	—			PRSS2	−4.58
7997188	1218	NM_005143	1806	3893, 4016, 4018, 2580, 2622, 2977, 2992, 3453,	HP	−4.53
				3617, 4365, 4366, 6921, 6310, 6000, 6001,
7921834	1219	NM_001643	1807	2901, 2758, 6253, 8161, 7460, 7113, 8544, 6696,	APOA2	−4.50
				7530, 7963, 6627, 6449,
8106986	1220	NM_014899	1808	3394, 4041, 7121, 8530, 7782, 8683, 8248, 7828, 8218, 8489,	RHOBTB3	−4.44
8060850	1221	NM_001200	1809	3900, 6567,	BMP2	−4.43
8171449	1222	NM_021804	1810	3847, 4462, 6787, 7949, 7759, 7328, 5427,	ACE2	−4.39
8149825	1223	NM_003155	1811	2725, 3793, 5771,	STC1	−4.32
7904843	1224	NM_002614	1812	3336, 5243, 4204, 7461, 7260, 6532, 6226, 7090,	PDZK1	−4.29
				6900, 7707, 7472, 6465,
7929282	1225	NM_002729	1813	2961, 4124, 7867, 5797, 8612,	HHEX	−4.18
8048319	1226	NM_007127	1814	4477, 4578, 5439,	VIL1	−4.16
8121257	1227	NM_182907	1815	3253, 5303, 4361, 6228, 6168, 8184, 8624, 7700,	PRDM1	−4.07
8085946	1228	NM_005442	1816	3171, 3140, 5868, 7137, 7635,	EOMES	−4.01
7968872	1229	NM_013238	1817	3842, 3846, 7013, 8073,	DNAJC15	−3.99
8092095	1230	NM_015028	1818	4193, 4852, 4853, 4854, 4855, 4856, 4857, 4858,	TNIK	−3.97
				4859, 4860, 8054, 5939, 6549, 7883, 5603, 6786,
				7448, 7215, 8447, 8382, 7882, 6219,
8058857	1231	NM_000599	1819	2583, 4357, 8723, 5360,	IGFBP5	−3.95
8121803	1232	NM_001010852	1820	3116, 3869, 5483, 6248,	RLBP1L2	−3.92
8088560	1233	NM_182920	1821	4100, 3333, 8622, 8439, 8457, 6636, 7795, 8233,	ADAMTS9	−3.90
				8784,
7971296	1234	NM_033255	1822	3367, 3694, 3693, 5738, 5732, 7664, 7742,	EPSTI1	−3.83
8163839	1235	NM_001735	1823	3997, 3497, 8045, 5344, 6954, 6376,	C5	−3.80
8096160	1236	NM_001025616	1824	3856, 4013, 4014, 4012, 8287, 7220, 7970, 6456,	ARHGAP24	−3.76
				8329, 8596, 5517, 8693, 8490,
8136801	1237	NM_002770	1825	3326, 3327, 4043, 4044, 3896, 4082, 2633, 3056,	TRY6	−3.73
				4031, 3057, 4349, 5199, 5294, 5200, 4875, 7372,
				5743, 6222, 8379,
8149071	1238	NM_001147	1826	2696, 4344, 4345, 4346, 5744, 7577,	ANGPT2	−3.73
8020795	1239	NM_000371	1827	2722, 4730, 6739, 5362,	TTR	−3.71
8139307	1240	NM_021223	1828	3043, 3698, 7148, 5334, 7843, 7318, 7526,	MYL7	−3.71
8105302	1241	NM_013409	1829	3343, 5282, 5281, 7924, 7913, 6382, 5408,	FST	−3.66
7965322	1242	NM_000899	1830	4054, 5149, 5148, 6216, 5328, 6318, 7574,	KITLG	−3.65
7926105	1243	NM_002051	1831	2915, 3695, 3696, 6780, 6049, 7381,	GATA3	−3.65
8113358	1244	NM_005668	1832	3302, 4599, 3244, 7055, 7738, 5386,	ST8SIA4	−3.60
8057377	1245	NM_173648	1833	3425, 5152, 5914, 5666, 7929,	CCDC141	−3.57
8151931	1246	NM_033512	1834	3241, 3675, 5757,	TSPYL5	−3.56
8105013	1247	NM_152403	1835	3530, 5277, 5276, 5275, 6119, 5859, 8466, 5742,	EGFLAM	−3.55
				6783, 8384, 6717, 7825, 8629, 8422, 7942, 6265,
8057056	1248	NM_133378	1836	3308, 2656, 3424, 4106, 4107, 3344, 3048, 5008,	TTN	−3.52
				5009, 5011, 5007, 5010, 7368, 5887, 5945, 7339,
				6830, 8194, 6950, 6315, 6321, 6481, 7366, 6697,
				6793, 7546, 6131, 5681,
7924682	1249	NM_003240	1837	3558, 4989, 4988, 8493, 7506, 6104,	LEFTY2	−3.52
8065344	1250	NM_021784	1838	2770, 4577, 4466, 5823, 7393, 7364, 6571,	FOXA2	−3.38
8129231	1251	NM_024581	1839	3503, 4208, 4209, 8021, 7766, 5900, 8495, 6262,	FAM184A	−3.38
8147030	1252	NM_007029	1840	4711, 5225, 5342,	STMN2	−3.35
7976858	1253	NM_001362	1841	2990, 4161, 8265, 6088,	DIO3	−3.33
8058765	1254	NM_054034	1842	3732, 3409, 3983, 4677, 4679, 3284, 3310, 3403,	FN1	−3.33
				3404, 3405, 3406, 4727, 3110, 4551, 4571, 4572,
				4576, 3822, 3494, 2852, 3620, 3621, 3622, 3623,
				3624, 3625, 5996, 7280, 7649, 6915, 8364, 8529,
				6768, 7398, 5846, 6424, 8736, 8430, 7036, 5993,
				8153, 8487, 6324, 6771, 6223, 6205, 5539, 6620,
				6121, 6206, 8147, 5687,
8136795	1255	NM_002769	1843	4044, 4083, 5224, 7372, 8647, 5627, 7412, 7969,	PRSS1	−3.32
7912520	1256	NM_002521	1844	3029, 3909, 7941, 6388,	NPPB	−3.32
7921916	1257	NM_001195303	1845	3306, 5190, 5110, 7452, 5828, 6057,	RGS5	−3.30
8121144	1258	NM_024641	1846	4179, 5290, 6156, 5952, 8006,	MANEA	−3.29
7979357	1259	NM_021728	1847	3073, 3223, 3224, 6604, 6242,	OTX2	−3.28
8047381	1260	NM_003879	1848	2698, 2700, 2701, 2702, 2703, 2697, 2727, 2728,	CFLAR	−3.28
				2729, 2714, 2715, 2716, 2717, 2718, 3809, 3282,
				4540, 4723, 2706, 2695, 2707, 4275, 4518, 2881,
				4319, 5254, 5255, 7718, 7181, 7213, 6901, 7210,
				7050, 6868, 7797, 7093, 6246, 6301, 6416, 6026,
				8088, 7173, 6972, 6669, 7157, 7540, 7179, 6019,
				5721,
8058498	1261	NM_003468	1849	2934, 4015, 5838,	FZD5	−3.27
8054517	1262	AK095678	1850	3119, 2978, 6378, 5623, 6529,	LOC151009	−3.24
7961875	1263	NM_152590	1851	4498, 4781, 4782, 4784, 4785, 4783, 5790, 7143,	IFLTD1	−3.23
				6544, 6853,
7938231	1264	NM_003621	1852	3011, 4355, 5626,	PPFIBP2	−3.18
8166747	1265	NM_001163335	1853	3411, 4887, 4885, 4886, 5912, 6888, 5629,	SYTL5	−3.13
8019964	1266	NM_001010000	1854	2803, 5198, 5415, 6442, 5664, 6551, 7534, 7585,	ARHGAP28	−3.12
7957458	1267	NM_006183	1855	2948, 5242, 5429,	NTS	−3.09
8050619	1268	NM_000384	1856	2558, 3976, 5351,	APOB	−3.08
8065416	1269	NM_001322	1857	3515, 3034, 5782,	CST2	−3.05
7942998	1270	NM_153696	1858	3060, 2843, 3280, 5177, 2766, 5178, 6173, 5921,	FOLH1B	−3.04
				6307, 5425, 6469,
8070579	1271	NM_003225	1859	2552, 3668, 5847,	TFF1	−3.04
8165663	1272	AK172782	1860	3609,	GPAM	−3.04
8023598	1273	NM_173557	1861	3123, 3946, 5605,	RNF152	−3.04
8140650	1274	NM_012431	1862	4218, 5112, 5113, 5625, 7193,	SEMA3E	−3.04
8111892	1275	NM_000436	1863	2674, 4026, 8292, 5313, 8538, 8361, 8084, 7753,	OXCT1	−3.03
8150076	1276	NM_001394	1864	2649, 5265, 5266, 5393,	DUSP4	−3.02
8121916	1277	NM_032784	1865	3018, 3878, 6102, 6281, 8688,	RSPO3	−3.01
8092523	1278	NM_001966	1866	3206, 4927, 4926, 5368, 8762,	EHHADH	−3.00
8172670	1279	NM_001013742	1867	3778, 5274, 6444,	DGKK	−2.99
8119898	1280	NM_001025366	1868	3808, 4966, 4967, 4968, 4969, 4970, 4971, 4972,	VEGFA	−2.98
				4973, 4974, 4975, 4976, 4977, 4978, 4979, 4980,
				4981, 8712, 5968, 6175, 7640, 5961, 5690, 7243,
				8549, 6759, 6136, 7716, 8136, 8355, 8159, 8018,
				7079, 8270, 8120, 7866, 8507, 8066, 7946,
8077185	1281	NM_000039	1869	3902, 2762, 6306, 5375, 6654, 6990,	APOA1	−2.94
8059955	1282	NM_022449	1870	3268, 4986, 4985, 7579, 8393, 6896, 6505, 7240,	RAB17	−2.92
				5533,
8045664	1283	NM_177964	1871	3039, 3538, 8714, 5464, 7286, 6455, 7123, 7806,	LYPD6B	−2.91
				7494, 8341, 7067, 6613,
8087925	1284	NM_003280	1872	3054, 4613, 5365, 7708,	TNNC1	−2.90
8074853	1285	NM_080740	1873	3304, 3570, 5866,	ZNF280A	−2.90
8057506	1286	NM_001463	1874	2679, 5106, 5695,	FRZB	−2.89
7951865	1287	NM_000039	1875	3902, 2762, 6306, 5375, 6654, 6990,	APOA1	−2.89
8146462	1288	NM_022454	1876	3009, 4141, 5778,	SOX17	−2.86
8129482	1289	NM_001017373	1877	3098, 4120, 4122, 7473, 7107, 6192, 5825, 7041,	SAMD3	−2.85
				6688,
8127193	1290	NM_021073	1878	2611, 3202, 5997, 7150,	BMP5	−2.85
8144643	1291	NM_002052	1879	2632, 4360, 5839, 5435,	GATA4	−2.81
8050537	1292	NM_002381	1880	4246, 4011, 7511, 6871, 8601,	MATN3	−2.80
8169995	1293	NM_001170779	1881	4513, 4955, 4956, 4957, 4958, 4959, 4960, 4954,	FAM122C	−2.80
				6451, 7031, 8034, 6865, 6135, 8059, 8191, 6912,
				8587, 6236,
8103544	1294	NM_016950	1882	3468, 4487, 4491, 4493, 4496, 4500, 4505, 4506,	SPOCK3	−2.78
				4522, 2959, 3354, 2879, 3351, 3951, 3952, 7390,
				8151, 7745, 8674, 7862, 8555, 8291, 8542, 7775,
				8670, 8212, 7911, 5944, 8680, 8411, 8508, 6200,
				8249, 7909, 8528, 8673, 7951, 8415, 8750, 7722,
8091283	1295	NM_182943	1883	3144, 3823, 3824, 7950, 7025, 6032, 8398, 5670,	PLOD2	−2.76
				7845,
7908003	1296	NM_030769	1884	3446, 5244, 8301, 8566, 7329, 6079, 6297, 6185,	NPL	−2.74
				6208, 6540, 5453, 5873, 5974, 6575,
8147145	1297	NM_152565	1885	3548, 3146, 7959, 8150, 5593,	ATP6V0D2	−2.73
8073585	1298	NM_014509	1886	4046, 3113, 3203, 4870, 6789, 7257, 7696, 6190	SERHL	−2.73
				7885, 6148, 8055, 5575,
8059376	1299	NM_006216	1887	3235, 4656, 4657, 4658, 6475, 5410, 8093, 7735,	SERPINE2	−2.70
				6860,
7909789	1300	NM_003238	1888	2610, 5188, 5189, 7936, 5882, 8554, 5876,	TGFB2	−2.68
8139057	1301	NM_014800	1889	2968, 3692, 4045, 2861, 3438, 4258, 4520, 4479,	ELMO1	−2.68
				2801, 2880, 3542, 5280, 5279, 8359, 7086, 5936,
				6939, 6791, 6584, 7972, 7197, 7051, 6279, 5770,
				6974, 6770, 7488, 7401, 7549, 6438, 7349,
8144656	1302	BC132945	1890	4115, 2976,	C8orf49	−2.66
8112615	1303	NM_003633	1891	2731, 4418, 7756, 5853, 8481,	ENC1	−2.65
8057620	1304	NM_000393	1892	3762, 3939, 7320, 7380, 7573,	COL5A2	−2.64
7919314	1305	NM_001461	1893	3169, 4740, 4742, 4741, 5452, 5926, 6568, 7792	FMO5	−2.63
				6924,
7972750	1306	NM_001845	1894	4195, 4166, 6498, 6735,	COL4A1	−2.61
8097513	1307	NM_002413	1895	3025, 5261, 8016, 7820, 8094, 5528, 8127,	MGST2	−2.60
8169711	1308	NM_012084	1896	2899, 3400, 5864,	GLUD2	−2.59
8041179	1309	NM_024692	1897	4055, 4666, 6910, 6677, 7982, 7631, 5684, 7192,	CLIP4	−2.58
				6576, 6387, 7420, 8380,
8123388	1310	NM_018974	1898	3851, 4738, 4736, 5385, 8261, 6041, 8180, 6272,	UNC93A	−2.57
8155192	1311	NM_022343	1899	3083, 3132, 7426, 7611, 7741, 8325, 6925,	GLIPR2	−2.54
8127065	1312	NM_000846	1900	4266, 4667, 8541,	GSTA2	−2.53
7928107	1313	NM_018649	1901	2818, 3965, 5976, 7242,	H2AFY2	−2.53
8018761	1314	NM_006456	1902	2638, 4437, 5345,	ST6GALNAC2	−2.52
7965335	1315	NM_001946	1903	2898, 3562, 3563, 5485, 5764,	DUSP6	−2.52
7965573	1316	NM_021229	1904	2846, 3957, 6056, 5886,	NTN4	−2.50
8006681	1317	NM_005568	1905	2635, 5231, 5430,	LHX1	−2.50
7962427	1318	NM_031292	1906	3592, 4173, 4172, 4174, 6664, 6050, 6684,	PUS7L	−2.50
8163637	1319	NM_002160	1907	2581, 5293, 6635, 7873, 8072, 6170, 6096, 6061,	TNC	−2.47
				5879, 7429, 6261, 6994,
8078286	1320	NM_016152	1908	2681, 3505, 4596, 4598, 8684, 7033, 8333, 6707	RARB	−2.47
				5820, 6360, 7180,
8014591	1321	NM_000458	1909	2989, 4226, 4915, 5347, 6709, 6537,	HNF1B	−2.46
7970676	1322	NM_001007538	1910	4110, 3758, 5588,	SHISA2	−2.45
7902495	1323	NM_144573	1911	3950, 4178, 4987, 5775, 6960, 6998, 8177, 6811,	NEXN	−2.45
				5834, 5619, 7685, 5942,
8059852	1324	AK301707	1912	4489, 4652, 6889,	MSL3L2	−2.43
7954969	1325	NM_001114182	1913	3491, 4765, 4766, 4767, 4768, 4769, 7247, 7490,	IRAK4	−2.42
				6878, 6277,
7920291	1326	NM_080388	1914	3782, 2999, 5985, 6066, 6285, 6298, 8082,	S100A16	−2.40
8140955	1327	NM_001145306	1915	3279, 4773, 4772, 5552, 8113, 8064, 7668, 6346,	CDK6	−2.40
8105607	1328	NM_001164442	1916	4893, 7805, 7948, 7918, 8707, 6538,	FAM159B	−2.40
7921344	1329	NM_012081	1917	4573, 4214, 5359, 7416,	ELL2	−2.39
8091780	1330	NM_003781	1918	3474, 2819, 3653, 4514, 2847, 2848, 3036, 3135,	B3GALNT1	−2.39
				3255, 3915, 3916, 3917, 3918, 3919, 8586, 8499,
				8324, 5811, 6661, 6367, 8420, 8708, 8676, 8027,
				7430, 8305, 8658, 7607, 8455, 6477, 8126, 8442,
				7637,
8007493	1331	NM_001661	1919	3037, 3901, 5663,	ARL4D	−2.38
8068593	1332	NM_005239	1920	3390, 3760, 7144, 5963, 7355, 6033,	ETS2	−2.36
8169145	1333	NM_152423	1921	3142, 4991, 4993, 6316, 5894, 5576,	MUM1L1	−2.36
8013606	1334	NM_000638	1922	2897, 3932, 5348,	VTN	−2.35
8140668	1335	NM_006080	1923	4019, 3973, 5554, 7046, 6980,	SEMA3A	−2.35
8038981	1336	NM_030972	1924	3136, 4861, 4862, 4863, 4864, 5722,	ZNF611	−2.34
7944803	1337	NM_014622	1925	3486, 3642, 3641, 4463, 5983, 7513, 6905, 7593,	VWA5A	−2.33
				6204, 7228,
8099364	1338	NM_053042	1926	3218, 3781, 5718, 8226, 8286, 7880, 7968,	ZNF518B	−2.32
7919193	1339	NM_199040	1927	2810, 4694, 3968, 3967, 6935, 5781,	NUDT4P1	−2.32
8101304	1340	NM_152545	1928	4526, 3145, 8395, 5654, 5568, 7781, 8214, 6622,	RASGEF1B	−2.31
8143643	1341	NM_022445	1929	3220, 4001, 4002, 8245, 6258, 8436, 7518, 7791,	TPK1	−2.30
				6711,
8095986	1342	NM_005139	1930	2871, 3966, 8438, 8323, 7932, 5460, 8713, 8202,	ANXA3	−2.29
				7789,
8007921	1343	NM_001002841	1931	3053, 3703, 2998, 6007, 6931,	MYL4	−2.29
7926609	1344	NM_005180	1932	3522, 5257, 6836, 7177, 8311, 6490, 6981, 7037,	BMI1	−2.29
				8773, 6757, 6570,
8050240	1345	NM_002539	1933	3273, 2760, 5350, 7567, 7441, 6418,	ODC1	−2.28
8171161	1346	NM_000047	1934	4099, 4234, 6631, 6722,	ARSE	−2.28
8096176	1347	NM_006264	1935	2621, 4579, 4580, 4581, 4582, 6886, 7770, 7790,	PTPN13	−2.27
				7383, 8720, 7840, 6014, 5863, 7589,
8039013	1348	NM_203307	1936	3243, 5855,	ZNF321	−2.26
7934156	1349	NM_018055	1937	3088, 4747, 7082, 5642,	NODAL	−2.26
8138370	1350	NM_020319	1938	3159, 5132, 7035, 7445, 7172, 5729,	ANKMY2	−2.24
7924107	1351	NM_014873	1939	3152, 4614, 7821, 6002, 6174,	LPGAT1	−2.24
8089011	1352	NM_000313	1940	2597, 4770, 6690,	PROS1	−2.23
8167912	1353	NM_007250	1941	3520, 4810, 4811, 7702, 8280, 6724, 6350, 6020,	KLF8	−2.23
7947338	1354	NM_000280	1942	3805, 4400, 4401, 4402, 6733, 7304, 6876, 7135,	PAX6	−2.23
				7188, 7299, 6953, 7333, 7281, 8690, 7478, 7234,
				8432, 7017, 6745, 7558, 5383,
8155930	1355	NM_001490	1943	2578, 4156, 4157, 4158, 4159, 4160, 8460, 6349,	GCNT1	−2.23
				8001, 7226, 6641,
8106411	1356	NM_130772	1944	3017, 4191, 8752, 5748,	S100Z	−2.23
8039025	1357	NR_003578	1945	3075, 4190, 5550,	ZNF702P	−2.21
8132167	1358	NM_194300	1946	4108, 4535, 3117, 4495, 4212, 7015, 6902, 6655,	CCDC129	−2.21
				7399, 7356, 7062, 8117, 5731, 6766,
8049542	1359	NM_001137550	1947	4662, 7654,	LRRFIP1	−2.20
8102912	1360	NM_015130	1948	4180, 4132, 6423,	TBC1D9	−2.20
8008819	1361	NM_001005404	1949	3716, 3768, 5719,	YPEL2	−2.19
8112668	1362	NM_016591	1950	2806, 4600, 5633,	GCNT4	−2.18
7970241	1363	NM_000504	1951	2567, 2573, 3210, 3246, 4308, 4309, 3935, 7894,	F10	−2.18
				6670, 7728, 6624, 7953, 7352, 6493,
8036389	1364	NM_152279	1952	3196, 4990, 5862, 6596,	ZNF585B	−2.17
8097529	1365	NM_153702	1953	3416, 5196, 8425, 7800, 5802, 8102, 8387, 8257,	ELMOD2	−2.16
				8391,
7962349	1366	NM_173601	1954	3139, 4199, 4198, 7252, 5482,	GLT8D3	−2.16
7952601	1367	NM_001162422	1955	3402, 4871, 4686, 4687, 7425, 5612, 5931,	ETS1	−2.16
8001387	1368	NM_001127892	1956	4497, 4411, 4078, 6646, 5432, 7163,	SALL1	−2.16
8040163	1369	NM_001039613	1957	3912, 3107, 4261, 4549, 3207, 3482, 3588, 3934,	IAH1	−2.16
				6534, 8330, 8409, 8205, 6128, 8770, 8556, 7901,
				8340, 8747, 8755, 7721, 8381, 8091, 7817, 5835,
8165672	1370	L23320	1958	2618,	RFC1	−2.15
7946288	1371	NM_013249	1959	2781, 4224, 5492,	ZNF214	−2.15
7953665	1372	NM_199286	1960	3455, 3720, 6044,	DPPA3	−2.15
8103311	1373	NM_000508	1961	2574, 3860, 3861, 6647, 5699, 6582,	FGA	−2.15
8084206	1374	NM_032047	1962	2926, 3099, 3434, 3020, 2912, 3182, 3956, 6802,	B3GNT5	−2.13
				6453, 8375, 8383, 8256, 7811, 7501, 5794, 8221,
				7151,
8174389	1375	NM_001847	1963	2623, 4165, 4167, 6197, 5733, 6413, 8100, 7288,	COL4A6	−2.13
				7442, 7535,
7918768	1376	NM_198459	1964	3586, 4103, 6796, 6943, 8236, 8046, 6164, 7594,	DENND2C	−2.13
7950885	1377	NM_012193	1965	2784, 5289,	FZD4	−2.12
8005048	1378	NM_001146313	1966	3740, 4806, 4804, 4805, 7259, 6792, 7438, 6345	MYOCD	−2.12
				6178, 6713,
8101952	1379	NM_145244	1967	3382, 5197, 8631, 5514, 8733,	DDIT4L	−2.12
8111887	1380	NM_001005473	1968	4020, 5772, 7331,	PLCXD3	−2.11
8113220	1381	NM_012081	1969	3398, 4214, 5359, 7416, 8279, 8525,	ELL2	−2.10
8114287	1382	NM_004598	1970	2804, 3903, 8553, 8505, 6817, 7957, 5469, 8014,	SPOCK1	−2.10
				7754
7955694	1383	NM_002178	1971	3353, 3682, 5662,	IGFBP6	−2.09
8073596	1384	NM_014509	1972	3645, 4046, 4023, 4076, 4075, 4492, 4499, 3203	SERHL2	−2.09
				7885, 6148, 6161, 6501, 7434, 5747, 5575,
8083779	1385	NM_005025	1973	4280, 4350, 4351, 7428, 8534, 7747, 5615, 7839,	SERPINI1	−2.09
				7903,
8121601	1386	NM_153711	1974	3931, 6155,	FAM26E	−2.08
8169174	1387	NM_024539	1975	3527, 3478, 3479, 5441, 5712, 6719,	RNF128	−2.08
8117045	1388	NM_153020	1976	3092, 4733, 4735, 4734, 6592, 6373, 5606, 8008,	RBM24	−2.08
				8572, 8110,
7965357	1389	NM_003774	1977	4550, 3393, 5235, 6995,	GALNT4	−2.08
8162652	1390	NM_001333	1978	3462, 5253, 8071, 5450,	CTSL2	−2.08
8023528	1391	NM_052947	1979	3413, 4170, 6142,	ALPK2	−2.07
8069508	1392	ENST00000457997	1980	6891, 6940,	ANKRD20B	−2.06
7929388	1393	NM_016341	1981	4217, 4071, 4918, 6138, 5440, 6045, 6266,	PLCE1	−2.06
7944082	1394	NM_003186	1982	2895, 3656, 3657, 3038, 7198, 5531, 6894, 5715,	TAGLN	−2.06
8057771	1395	NM_003151	1983	3066, 5299, 7451, 8037, 7396, 6374, 6129, 7059	STAT4	−2.06
				8595, 6687, 8616,
7907702	1396	NM_003101	1984	3047, 5308, 5878, 7270,	SOAT1	−2.05
7910134	1397	NM_031944	1985	2853, 2923, 6314,	MIXL1	−2.04
8062823	1398	NM_000457	1986	3706, 3707, 3708, 4669, 4387, 4383, 2677, 2664,	HNF4A	−2.04
				3125, 4751, 3292, 3293, 3294, 3867, 3868, 3866,
				6660, 5774, 5653, 6081, 7077, 5571, 7633,
8059878	1399	NM_024726	1987	4003, 4483, 2974, 3095, 3137, 3231, 3536, 8451,	IQCA1	−2.04
				6443, 7562, 7057, 8660, 5402, 7302, 7122, 6806,
				5760, 7624, 7362,
8022817	1400	NM_020805	1988	4494, 3749, 6140, 6012,	KLHL14	−2.04
8102862	1401	NM_018717	1989	4377, 4379, 2704, 4531, 2925, 4284, 7322, 8019,	MAML3	−2.03
				7858, 5635,
8096845	1402	NM_001963	1990	3828, 5114, 5115, 5116, 5500, 7841, 7967, 7927,	EGF	−2.03
7924663	1403	NM_020997	1991	2777, 5273, 5561, 8017,	LEFTY1	−2.03
7970381	1404	AK057820	1992	2984, 4169, 6891, 6432, 6336, 7704, 6356, 7272,	ANKRD20B	−2.03
				7347,
7922727	1405	NM_001102450	1993	3879, 4223, 3904, 8112, 8121, 6042, 8709, 7965,	RGS8	−2.03
				5447, 5943,
8157090	1406	NM_005421	1994	4057, 4779, 5674,	TAL2	−2.02
8050071	1407	NM_018269	1995	3559, 5111, 5637,	ADI1	−2.02
7944869	1408	NM_017425	1996	2833, 2894, 4744, 5335,	SPA17	−2.01
8127370	1409	NM_152688	42	4263, 4412, 8227, 5521,	KHDRBS2	−2.01
8113551	1410	NM_002387	43	3412, 4147, 4148, 8175, 8246, 5792, 7893,	MCC	−2.00
Table 12: Provided are the description of the non-membranal genes along with the sequence identifiers of the Affymetrix probes, the target sequence identified by the Affymetrix probe, and the mRNA sequences represented by the target sequences. Also shown are the log 2 fold change in expression levels of the indicated genes between the PDX1-GFP cells and the SOX17-GFP cells (PDX1-SOX17). The (+) or (−) sign means that the genes are upregulated or downregulated, respectively, in the PDX1-GFP cells as compared to the SOX17-GFP cells.

Identification of Clusters of Genes which are Overexpressed During Differentiation into Pancreatic Cells—

The transcriptional profiles of the SOX17⁺ and PDX1⁺ progenitor cells were compared to profiles of undifferentiated ES cells and of differentiated pancreatic cells. Partek analysis enabled to define different clusters of genes according to their expression patterns (FIGS. 5B-E).

The present inventors have further studied several surface markers that were found to be enriched in the PDX1⁺ cells in the microarray analysis and were validated by qPCR analysis. These are the G protein-coupled receptor 50 (GPR50) and tumor-associated calcium signal transducer 2 (TROP-2), whose expression showed a good correlation with PDX1 expression (FIG. 6A). Cells from 25 day old EBs were dissociated and were stained with antibodies specific to TROP-2 and GPR50 and were FACS sorted. Four different populations were obtained: GPR50⁺TROP-2⁺ (1.9%), GPR50⁺TROP-2⁻ (20%), GPR50⁻TROP-2⁺ (0.6%) and a negative population which did not express either of these markers (GPR50⁻/TROP-2⁻). Quantitative-PCR analysis revealed an increase in mature pancreatic markers, especially in the GPR50⁺TROP-2⁺ population, including an induction in INSULIN, NGN3, PAX4, HLXB9, NK6 homeobox 1(NKX6.1), and SRY box 9 (SOX9).

TROP-2 and GPR50 are two exemplary markers of the large number of cell surface markers that were revealed by the Affymetrix study including markers enriched in the populations of interest as well as markers depleted in the populations of interest. As shown, these markers can be used in positive selection to enrich for the desired cells. They can also be used in negative selection to remove cells other than the cells that are desired.

The cell surface markers that were identified based on comparative analysis between pluripotent undifferentiated hESC, the GFP+ population from the cells bearing the SOX17-GFP construct and the GFP+ population from the cells bearing the PDX1-GFP construct fall into a number of categories, all of which can potentially be used for immuno-purification/immuno-isolation. Following is a short description of these marker groups:

I. Markers that could be Useful to Enrich for Endodermal Progenitor Cells:

Cell surface markers enriched in the SOX17 population relative to undifferentiated hESC;

Cell surface markers depleted in the SOX17 population relative to undifferentiated hESC;

II. Markers that could be Useful to Enrich for Pancreatic Progenitor Cells:

Cell surface markers enriched in the PDX1 population relative to undifferentiated hESC;

Cell surface markers depleted in the PDX1 population relative to undifferentiated hESC;

III. Cell Surface Markers Enriched in the PDX1 Population Relative to the SOX17 Population.

Without being bound by any theory, these markers would be useful in the event that two sorting procedures are incorporated into the process of manufacturing the therapeutic cell population—one sorting procedure at the stage of SOX17 expression and a second sorting procedure at the stage of PDX1 expression.

IV. Cell Surface Markers Depleted in the PDX1 Population Relative to the SOX17 Population.

In addition, all the cell surface markers above and additional non-cell surface markers that exhibit similar expression patterns can be used for quality control of replacement islet beta cells for transplant-based diabetes treatment during their manufacture.

Example 4

Identification of Stage Specific Markers Using the Partek Cluster Analysis

The bioinformatic analysis of cell specific markers was a central part of the entire experiment. Key steps in this part were: A. To assign those genes to a stage specific category; B. To analyze genes related to the relevant cell component. The present inventors have used the Gene Ontology data to include only genes with cell surface and membranal information. Stage specific genes were assigned to one of four categories: (i). SOX17⁺ specific; (ii). PDX1⁺ specific; (iii). SOX17⁺/PDX1⁺ specific; (iv). PDX1+/Pancreas specific. The generation of those specific lists was done by using the PARTEK partition clustering analysis (FIG. 7).

Stage and Compartment Specific Gene Identification—

Following the PARTEK cluster analysis a further prioritization of relevant genes was needed. For this purpose a standard excel analysis was used. A threshold of Fold Change=2.0 was set (at it is an acceptable threshold in microarray studies). In addition, the present inventors have used the Student's test for the gene selection. FIGS. 8-11 present selected genes for each stage. The entire list appears in Tables 13-20 below.

TABLE 13
SOX17 specific cell surface genes
			SEQ			SOX17/
	Affy. target		ID NO:	Polyn. SEQ		HESC
Probe	nucl.SEQ		(of Rep.	ID NOs: rep.	Gene	fold
set ID	ID NO:	Rep. Public ID	Public ID)	by target	Symbol	change
8029280	1997	NM_020406	2273	3465, 2825, 3947, 3744,	CD177	9.45
				4962, 4250, 3064, 3545,
				3050, 4005, 6408, 6835,
				7480,
7938090	1998	NM_176875	2274	2809, 5307, 5676,	CCKBR	5.81
8077185	1999	NM_000039	2275	3902, 2762, 6306, 5375,	APOA1	7.79
				6654, 6990,
7951865	2000	NM_000039	2276	3902, 2762, 6306, 5375,	APOA1	7.44
				6654, 6990,
8052072	2001	NM_000145	2277	4265, 5021, 5022, 6252,	FSHR	5.19
				5713, 7612, 7221, 6397,
8058498	2002	NM_003468	2278	2934, 4015, 5838,	FZD5	2.38
8058765	2003	NM_212482	2279	3732, 3409, 3983, 4677,	FN1	4.30
				4679, 3284, 3310, 3403,
				3404, 3405, 3406, 4727,
				3110, 4551, 4571, 4572,
				4576, 3822, 3494, 2852,
				3620, 3621, 3622, 3623,
				3624, 3625, 5996, 7280,
				7649, 6915, 8364, 8529,
				6768, 7398, 5846, 6424,
				8736, 8430, 7036, 5993,
				8153, 8487, 6324, 6771,
				6223, 6205, 5539, 6620,
				6121, 6206, 8147, 5687,
8060850	2004	NM_001200	2280	3900, 6567,	BMP2	3.36
8088560	2005	NM_182920	2281	4100, 3333, 8622, 8439,	ADAMTS9	7.20
				8457, 6636, 7795, 8233,
				8784,
8056222	2006	NM_001935	2282	3604, 3605, 4611, 2562,	DPP4	4.32
				2570, 3550, 4303, 3611,
				6918, 6975, 6558, 7617,
				8396, 7176, 6249, 7784,
				8632, 8468,
8103311	2007	NM_000508	2283	2574, 3860, 3861, 6647,	FGA	7.36
				5699, 6582,
8021946	2008	NM_130386	2284	2891, 4601, 6579,	COLEC12	5.08
8162283	2009	NM_004560	2285	3804, 4102, 2608, 3151,	ROR2	3.51
				3287, 5241, 6393, 7306,
				6756, 8135,
8081298	2010	NM_032787	2286	3995, 4847, 5470, 7733,	GPR128	4.64
				8308, 8445,
8058857	2011	NM_000599	2287	2583, 4357, 8723, 5360,	IGFBP5	5.34
8111677	2012	NM_002310	2288	2551, 4403, 4404, 5491,	LIFR	4.97
				7187, 7919, 8314, 7758,
7950885	2013	NM_012193	2289	2784, 5289,	FZD4	4.47
7963786	2014	NM_002205	2290	3341, 3761, 5661, 6880,	ITGA5	4.42
8149825	2015	NM_003115	2291	2725, 3793, 5771,	STC1	3.21
7922343	2016	NM_003326	2292	3222, 4594, 5549, 8171,	TNFSF4	2.79
				6071,
8029280	2017	NM_020406	2293	3465, 2825, 3947, 3744,	CD177	2.43
				4962, 4250, 3064, 3545,
				3050, 4005, 6408, 6835,
				7480,
8059067	2018	NM_002181	2294	4757, 4992, 5587,	IHH	2.06
		SOX17/	SOX17/
		PDX1	PANC	fold	SOX17	SOX17	SOX17
	Probe	fold	fold	change	vs.	vs.	vs.
	set ID	change	change	average	HESC	PDX1	PANC
	8029280	11.14	6.93	9.17	3.9E−04	2.4E−04	1.0E−03
	7938090	8.69	9.10	7.87	1.6E−03	4.5E−04	3.9E−04
	8077185	4.01	8.83	6.88	1.2E−02	1.0E−01	1.0E−02
	7951865	3.85	8.49	6.59	1.2E−02	1.0E−01	9.6E−03
	8052072	5.53	6.22	5.65	1.1E−03	8.9E−04	5.8E−04
	8058498	4.11	9.65	5.38	1.7E−01	6.3E−02	2.5E−02
	8058765	2.39	8.73	5.14	2.9E−04	3.0E−01	1.2E−03
	8060850	5.86	5.66	4.96	1.4E−02	1.8E−03	2.1E−03
	8088560	3.76	3.07	4.67	2.0E−05	1.8E−03	2.8E−04
	8056222	2.07	6.97	4.45	9.4E−02	3.7E−01	3.6E−02
	8103311	3.04	2.03	4.15	7.9E−02	4.1E−02	6.3E−01
	8021946	3.50	3.74	4.10	1.1E−02	3.4E−02	2.7E−02
	8162283	2.68	5.90	4.03	4.5E−03	1.1E−02	2.0E−03
	8081298	4.55	2.52	3.90	4.5E−02	4.7E−02	1.9E−01
	8058857	2.55	3.36	3.75	2.3E−02	1.6E−01	7.7E−02
	8111677	4.00	1.50	3.49	4.8E−02	7.3E−02	5.8E−01
	7950885	3.91	1.76	3.38	3.0E−02	4.2E−02	5.0E−01
	7963786	3.10	2.50	3.34	4.3E−04	2.4E−03	7.7E−03
	8149825	2.62	3.23	3.02	8.2E−02	1.4E−01	8.1E−02
	7922343	2.70	2.89	2.79	7.8E−03	9.2E−03	6.5E−03
	8029280	2.40	2.51	2.44	4.5E−03	4.2E−03	3.4E−03
	8059067	2.09	2.13	2.09	9.2E−02	8.7E−02	8.1E−02
	Table 13. Provided are the description of the cell surface genes along with the sequence identifiers of the Affymetrix probes, the target sequence identified by the Affymetrix probe, the polynucleotide represented by the target sequence and the Public ID (and SEQ ID NO:) of the mRNA sequence representative of the target sequence. “Polyn.” = polynucleotide; “Rep.” = representative; Also shown are the fold change and T-test p-values for change in expression levels of the indicated genes between the SOX17-GFP cells and undifferentiated hESCs (SOX17/HESC); the SOX17-GFP cells and PDX1-GFP cells (SOX17/PDX1); and the SOX17-GFP cells and the Pancreas cells (SOX17/PANC).

TABLE 14
PDX1 specific cell surface genes
			SEQ			PDX1/
	Affy. target		ID NO:	Polyn. SEQ		HESC
Probe	nucl. SEQ	Rep.	(of Rep.	ID NOs: rep.	Gene	fold
set ID	ID NO:	Public ID	Public ID)	by target	Symbol	change
7929373	2019	NM_005097	2295	2745, 4359, 8710, 8593,	LGI1	9.41
				6230, 6132,
8165658	2020	U18810	2296	2553, 3426, 3422, 6269,	VIPR2	4.01
7915472	2021	NM_006516	2297	4268, 4338, 5901, 6869,	SLC2A1	1.77
				6399, 8613, 6344, 6364,
				5884, 7600,
7947156	2022	NM_001135091	2298	3463, 4618, 4620, 4619,	MUC15	8.35
				5504, 6821, 7005,
8135033	2023	ENST00000379442	2299	4894, 7027,	MUC12	7.36
8095303	2024	NM_015236	2300	2842, 4699, 4713, 4296,	LPHN3	3.77
				4285, 8560, 7869, 8477,
				8293, 8434, 5644, 8571,
				8574, 8160, 8174, 5553,
				7807, 8297, 8402, 8511,
				8619, 8139, 8480,
8033674	2025	NM_024690	2301	3192, 3906, 7402, 7014,	MUC16	4.82
				7634, 7481,
7918936	2026	NM_024626	2302	2837, 3971, 6218, 6213,	VTCN1	6.12
				7307, 8522, 5804, 6025,
8151684	2027	AL136588	2303	5826,	MMP16	2.44
8145611	2028	NM_017412	2304	2808, 5230, 8548, 5392,	FZD3	2.33
8056184	2029	NM_000888	2305	3091, 2830, 8224, 8635,	ITGB6	5.53
				5643, 7596, 6710,
8114415	2030	NM_001496	2306	3167, 3584, 7363, 5508,	GFRA3	3.92
8170476	2031	NM_004224	2307	2669, 4964, 5346,	GPR50	2.51
8088919	2032	NM_133631	2308	2713, 3930, 3948, 4146,	ROBO1	1.93
				3949, 4391, 4683, 3115,
				4548, 4716, 4797, 4798,
				5105, 6728, 8746, 5966,
				7836, 8600, 7464, 8561,
				6404, 8251, 6433, 7621,
8083968	2033	NM_014932	2309	4961, 2776, 3071, 4307,	NLGN1	2.49
				3297, 6467, 7268, 7261,
				7427, 6874, 7249, 6559,
				6760, 8521, 5935,
8035304	2034	NM_004335	2310	3090, 2805, 5403, 7124,	BST2	4.49
8135033	2035	ENST00000379442	2311	4894, 7027,	MUC12	4.59
8160823	2036	NM_147164	2312	2568, 5285, 5284, 6097,	CNTFR	4.25
				6693, 6678,
8034783	2037	NM_001008701	2313	2841, 4650, 4651, 5915,	LPHN1	1.72
				5875,
8146863	2038	NM_001128205	2314	3240, 4424, 4425, 4426,	SULF1	3.46
				4427, 6483, 8782, 7482,
				6383, 5417,
8047788	2039	NM_003812	2315	4109, 4113, 4530, 2862,	ADAM23	1.50
				2734, 3877, 7431, 7068,
				6740, 7531, 5501,
8118890	2040	NM_152753	2316	3214, 3300, 5524, 6429,	SCUBE3	3.05
7928429	2041	NM_002658	2317	3397, 4752, 4753, 6308,	PLAU	2.31
				6729, 8322, 7727, 8611,
				6028, 6300, 8374,
7952526	2042	NM_016952	2318	3857, 4137, 6357, 5459,	CDON	2.51
8094301	2043	NM_004787	2319	2748, 2765, 3985, 4678,	SLIT2	2.35
				3436, 2744, 2767, 8366,
				7786, 6676, 7740, 5522,
				8033, 7772, 7956, 8641,
				8725,
8135915	2044	NM_013332	2320	2771, 4188, 4189, 8677,	C7orf68	2.08
				7435, 5442, 6618, 7939,
7926545	2045	NM_032812	2321	3459, 3535, 6338, 7091,	PLXDC2	2.13
				7374,
		PDX1/	PDX1/
		SOX17	PANC		PDX1	PDX1	PDX1
	Probe	fold	fold	FC	vs.	vs.	vs.
	set ID	change	change	average	HESC	SOX17	PANC
	7929373	4.10	10.86	8.12	2.7E−02	1.0E−01	2.2E−02
	8165658	4.29	13.53	7.27	2.1E−01	2.9E−01	5.1E−02
	7915472	2.04	16.02	6.61	5.1E−02	2.6E−01	5.2E−04
	7947156	7.47	2.59	6.14	4.0E−02	4.6E−02	4.1E−01
	8135033	4.01	2.56	4.64	3.5E−02	9.5E−02	2.2E−01
	8095303	2.02	8.14	4.64	4.0E−02	2.5E−01	1.6E−02
	8033674	3.99	4.97	4.60	9.6E−02	1.4E−01	9.1E−02
	7918936	5.23	2.07	4.47	2.0E−02	4.0E−02	3.9E−01
	8151684	2.18	8.56	4.40	2.5E−02	1.2E−01	6.4E−04
	8145611	2.96	7.13	4.14	3.9E−03	2.4E−02	1.5E−02
	8056184	4.77	1.82	4.04	1.4E−01	1.8E−01	6.1E−01
	8114415	4.05	3.49	3.82	1.5E−02	1.8E−02	2.2E−02
	8170476	1.70	7.24	3.81	2.2E−03	1.1E−01	9.7E−04
	8088919	3.79	4.74	3.49	3.6E−02	9.4E−03	1.3E−02
	8083968	3.63	4.30	3.48	1.5E−02	7.4E−03	3.8E−03
	8035304	2.36	3.52	3.46	5.8E−02	3.7E−01	1.1E−01
	8135033	2.70	2.87	3.39	4.1E−02	1.5E−01	1.1E−01
	8160823	2.81	2.87	3.31	2.3E−02	1.4E−01	1.8E−01
	8034783	1.83	6.32	3.29	1.0E−03	1.2E−01	3.9E−05
	8146863	4.15	1.86	3.16	1.1E−01	1.2E−01	3.9E−01
	8047788	1.55	6.27	3.11	2.7E−02	1.1E−01	1.4E−03
	8118890	2.37	3.23	2.88	1.0E−01	2.0E−01	9.0E−02
	7928429	3.69	2.63	2.88	3.7E−04	4.8E−03	2.0E−02
	7952526	2.00	4.00	2.84	5.4E−02	1.1E−01	7.5E−02
	8094301	3.57	2.55	2.82	4.4E−01	3.2E−01	4.1E−01
	8135915	2.50	2.60	2.40	2.1E−02	3.1E−02	8.3E−02
	7926545	2.18	2.09	2.13	1.7E−01	2.1E−01	2.2E−01
	Table 14. Provided are the description of the cell surface genes along with the sequence identifiers of the Affymetrix probes, the target sequence identified by the Affymetrix probe, the polynucleotide represented by the target sequence and the Public ID (and SEQ ID NO:) of the mRNA sequence representative of the target sequence. “Polyn.” = polynucleotide; “Rep.” = representative; Also shown are the fold change and T-test p-values for change in expression levels of the indicated genes between the PDX1-GFP cells and undifferentiated hESCs (PDX1/HESC); the PDX1-GFP cells and SOX17-GFP cells (PDX1/SOX17); and the PDX1-GFP cells and the Pancreas cells (PDX1/PANC).

TABLE 15
SOX17 and PDX1 specific cell surface
				Polyn.		PDX1/	SOX17/	SOX17/
	Affy. target		SEQ ID	SEQ ID		PANC	PANC	HESC
Probe	nucl.SEQ	Rep.	NO: (of Rep	NOs: rep.	Gene	fold	fold	fold
set ID	ID NO:	Public ID	Public ID)	by target	Symbol	change	change	change
8056611	2046	NM_004525	2322	2684,	LRP2	11.62	8.04	5.82
				4119,
				5456,
				8262,
8022176	2047	NM_005559	2323	4685,	LAMA1	9.23	16.26	3.68
				5278,
				8564,
				7980,
				8761,
				6083,
				7657,
8175234	2048	NM_004484	2324	3157,	GPC3	6.29	12.26	6.24
				4899,
				4900,
				4901,
				4902,
				7303,
				7576,
				6111,
7929373	2049	NM_005097	2325	2745,	LGI1	10.86	2.65	2.29
				4359,
				8710,
				8593,
				6230,
				6132,
8058765	2050	NM_212482	2326	3732,	FN1	3.65	8.73	4.30
				3409,
				3983,
				4677,
				4679,
				3284,
				3310,
				3403,
				3404,
				3405,
				3406,
				4727,
				3110,
				4551,
				4571,
				4572,
				4576,
				3822,
				3494,
				2852,
				3620,
				3621,
				3622,
				3623,
				3624,
				3625,
				5996,
				7280,
				7649,
				6915,
				8364,
				8529,
				6768,
				7398,
				5846,
				6424,
				8736,
				8430,
				7036,
				5993,
				8153,
				8487,
				6324,
				6771,
				6223,
				6205,
				5539,
				6620,
				6121,
				6206,
				8147,
				5687,
8095303	2051	NM_015236	2327	2842,	LPHN3	8.14	4.02	1.86
				4699,
				4713,
				4296,
				4285,
				8560,
				7869,
				8477,
				8293,
				8434,
				5644,
				8571,
				8574,
				8160,
				8174,
				5553,
				7807,
				8297,
				8402,
				8511,
				8619,
				8139,
				8480,
8097910	2052	NM_005141	2328	3895,	FGB	3.34	4.95	5.54
				5121,
				5122,
				8372,
				7847,
				5858,
				8448,
				6585,
				8337,
8095110	2053	NM_000222	2329	3646,	KIT	4.19	8.30	3.08
				4153,
				4152,
				5648,
				8162,
8056222	2054	NM_001935	2330	3604,	DPP4	3.37	6.97	4.32
				3605,
				4611,
				2562,
				2570,
				3550,
				4303,
				3611,
				6918,
				6975,
				6558,
				7617,
				8396,
				7176,
				6249,
				7784,
				8632,
				8468,
8167835	2055	NM_001039705	2331	2908,	TRO	3.40	4.69	2.20
				2909,
				2910,
				2911,
				3329,
				4523,
				4561,
				2927,
				3265,
				3266,
				3938,
				6828,
				7971,
				8198,
				6749,
				7539,
				6497,
				5600,
				6368,
				6521,
				6543,
				7262,
				6752,
				7979,
				7021,
				6782,
				7644,
				6999,
				7279,
				6597,
				5857,
				7072,
				7725,
				5327,
				8749,
				7136,
7944869	2056	NM_017425	2332	2833,	SPA17	2.63	3.76	3.08
				2894,
				4744,
				5335,
8081001	2057	NM_002942	2333	4181,	ROBO2	3.08	2.63	2.60
				5144,
				5143,
				7229,
				7140,
				7162,
				7040,
				8742,
				7889,
7976783	2058	NM_003836	2334	4273,	DLK1	3.61	2.14	1.89
				4994,
				6774,
				5796,
				6319,
		PDX1/
		HESC		PDX11	PDX1	SOX17	SOX17
	Probe	fold	FC	vs.	vs.	vs.	vs.
	set ID	change	average	HESC	PANC	HESC	PANC
	8056611	8.40	8.47	4.0E−05	1.1E−04	1.2E−01	7.9E−02
	8022176	2.09	7.82	3.1E−02	2.6E−03	2.8E−02	3.6E−03
	8175234	3.20	7.00	7.0E−02	3.7E−02	5.5E−02	3.2E−02
	7929373	9.41	6.30	2.7E−02	2.2E−02	9.0E−03	3.6E−03
	8058765	1.80	4.62	4.7E−01	1.7E−01	2.9E−04	1.2E−03
	8095303	3.77	4.45	4.0E−02	1.6E−02	1.5E−01	3.7E−02
	8097910	3.74	4.39	2.4E−01	3.3E−01	2.3E−01	2.9E−01
	8095110	1.56	4.28	4.9E−03	2.4E−04	5.9E−02	8.4E−03
	8056222	2.09	4.19	2.2E−01	1.6E−01	1.5E−01	1.1E−01
	8167835	1.60	2.97	5.1E−04	6.3E−05	7.6E−02	1.0E−02
	7944869	2.15	2.90	2.3E−01	1.4E−01	3.2E−02	1.6E−02
	8081001	3.05	2.84	2.5E−02	2.4E−02	5.4E−02	5.3E−02
	7976783	3.19	2.71	6.3E−04	2.0E−02	1.2E−03	8.4E−02
	Table 15. Provided are the description of the cell surface genes along with the sequence identifiers of the Affymetrix probes, the target sequence identified by the Affymetrix probe, the polynucleotide represented by the target sequence and the Public ID (and SEQ ID NO:) of the mRNA sequence representative of the target sequence. “Polyn.” = polynucleotide; “ Rep.” = representative; Also shown are the fold change and T-test p-values for change in expression levels of the indicated genes between the PDX1-GFP cells and the Pancreas cells (PDX1/PANC); SOX17-GFP cells and the Pancreas cells (SOX17/PANC); SOX17-GFP cells and undifferentiated hESCs (PDX1/HESC); the PDX1-GFP cells and undifferentiated hESCs (PDX1/HESC).

TABLE 16
PDX1 and Pancreas specific cell surface
						PDX1/	PDX1/
	Affy. target			Polyn. SEQ		HESC	SOX17
Probe	nucl. SEQ	Rep.	SEQ	ID NOs: rep.	Gene	fold	fold
Set ID	ID NO:	Public ID	ID NO:	by target	Symbol	change	change
8115147	2059	NM_001025159	2335	2996, 5297,	CD74	2.79	2.22
				5295, 5296,
				7571, 5325,
				8303, 8172,
				8179, 8156,
				6067, 8367,
				8576, 7868,
7947156	2060	NM_001135091	2336	3463, 4618,	MUC15	8.35	7.47
				4620, 4619,
				5504, 6821,
				7005,
7996081	2061	NM_201524	2337	3334, 4786,	GPR56	4.74	3.21
				4787, 4788,
				4789, 4790,
				4791, 4792,
				4793, 6472,
				7052, 6947,
				6626, 7618,
7916584	2062	NM_002353	2338	2675, 4337,	TACSTD2	2.77	2.65
				6094,	(TROP-
					2)
7918936	2063	NM_024626	2339	2837, 3971,	VTCN1	6.12	5.23
				6218, 6213,
				7307, 8522,
				5804, 6025,
8056184	2064	NM_000888	2340	3091, 2830,	ITGB6	5.53	4.77
				8224, 8635,
				5643, 7596,
				6710,
8156134	2065	NM_006180	2341	3803, 3756,	NTRK2	3.42	3.52
				4629, 4630,
				3840, 3841,
				7335, 5675,
				5488, 5700,
				5977, 6773,
				7194, 6916,
				6615,
8095744	2066	NM_001657	2342	2941, 3130,	AREG	3.27	2.86
				6586, 5463,
				6936,
8081686	2067	NM_033254	2343	2900, 3458,	BOC	4.26	2.87
				4375, 3408,
				2980, 3094,
				3128, 3691,
				4325, 3512,
				7851, 7097,
				8462, 8015,
				8182, 8307,
				8026, 7628,
				7853, 5922,
				7736, 5497,
				8579,
8046695	2068	NM_000885	2344	2653, 3850,	ITGA4	2.24	2.71
				4838, 4839,
				4840, 4841,
				4842, 4843,
				4844, 7580,
				7623, 8157,
				8345, 8766,
				7537, 7987,
				8342, 8492,
7961151	2069	NM_007360	2345	4264, 2991,	KLRK1	2.51	2.29
				3980, 5238,
				5378, 6831,
				5634,
	PANC/	PANC/
	SOX17	HESC		PDX1	PDX1	PANC	PANC
Probe	fold	fold	FC	vs.	vs.	vs.	vs.
Set ID	change	change	average	HESC	SOX17	HESC	SOX17
8115147	12.88	16.18	8.52	2.8E−02	2.8E−02	2.6E−03	4.9E−03
7947156	2.89	3.22	5.48	4.6E−02	4.6E−02	1.9E−01	2.3E−01
7996081	5.30	7.82	5.27	1.5E−01	1.5E−01	8.2E−04	5.7E−03
7916584	6.30	6.58	4.57	1.9E−02	1.9E−02	7.8E−02	9.1E−02
7918936	2.52	2.95	4.21	4.0E−02	4.0E−02	1.4E−01	2.3E−01
8056184	2.62	3.04	3.99	1.8E−01	1.8E−01	1.1E−01	1.7E−01
8156134	4.32	4.20	3.87	1.5E−01	1.5E−01	2.6E−04	4.0E−03
8095744	3.81	4.36	3.57	1.7E−01	1.7E−01	4.9E−02	7.2E−02
8081686	2.32	3.44	3.22	1.0E−01	1.0E−01	4.1E−02	1.8E−01
8046695	3.61	3.00	2.89	2.9E−02	2.9E−02	2.2E−02	2.1E−02
7961151	3.05	3.34	2.80	7.9E−02	7.9E−02	6.9E−02	8.2E−02
Table 16. Provided are the description of the cell surface genes along with the sequence identifiers of the Affymetrix probes, the target sequence identified by the Affymetrix probe, the polynucleotide represented by the target sequence and the Public ID (and SEQ ID NO:) of the mRNA sequence representative of the target sequence. “Polyn.” = polynucleotide; “Rep.” = representative; Also shown are the fold change and T-test p-values for change in expression levels of the indicated genes between the PDX1-GFP cells and undifferentiated hESCs (PDX1/HESC); the PDX1-GFP cells and SOX17-GFP cells (PDX1/SOX17); the Pancreas cells and SOX17 cells (PANC/SOX17); and the Pancreas cells and the hESCs (PANC/HESC);

TABLE 17
SOX17 specific putative cell surface genes
						SOX17/
	Affy. target		SEQ ID	Polyn. SEQ		HESC
Probe	nucl. SEQ	Rep.	NO: (of Rep.	ID NOs: rep.	Gene	fold
set ID	ID NO:	Public ID	Public ID)	by target	Symbol	change
8065412	2070	NM_001898	2346	3369, 3033, 5734,	CST1	8.15
				6511,
7927631	2071	NM_012242	2347	3471, 3796, 7543,	DKK1	12.95
				8463, 8242, 5959,
8152617	2072	NM_005328	2348	2676, 4348, 5843,	HAS2	4.17
				6409,
7921834	2073	NM_001643	2349	2901, 2758, 6253,	APOA2	17.82
				8161, 7460, 7113,
				8544, 6696, 7530,
				7963, 6627, 6449,
8022674	2074	NM_001792	2350	3134, 4668, 5546,	CDH2	2.39
7957140	2075	NM_003667	2351	2732, 3193, 6795,	LGR5	7.91
				5509,
8095646	2076	NM_001134	2352	3041, 2756, 7886,	AFP	8.81
				7273, 8047, 8272,
8065071	2077	NM_198391	2353	2812, 5172, 5173,	FLRT3	9.61
				5918, 7056, 6380,
8050619	2078	NM_000384	2354	2558, 3976, 5351,	APOB	17.42
8172670	2079	NM_001013742	2355	3778, 5274, 6444,	DGKK	9.55
7997188	2080	NM_005143	2356	3893, 4016, 4018,	HP	7.70
				2580, 2622, 2977,
				2992, 3453, 3617,
				4365, 4366, 6921,
				6310, 6000, 6001,
8166747	2081	NM_138780	2357	3411, 4887, 4885,	SYTL5	13.15
				4886, 5912, 6888,
				5629,
7977786	2082	NM_003982	2358	2789, 5292, 5291,	SLC7A7	6.69
				7158, 7255, 5870,
				8255, 6471, 7642,
8020795	2083	NM_000371	2359	2722, 4730, 6739,	TTR	14.97
				5362,
8057506	2084	NM_001463	2360	2679, 5106, 5695,	FRZB	3.96
8121916	2085	NM_032784	2361	3018, 3878, 6102,	RSPO3	9.02
				6281, 8688,
8155930	2086	NM_001490	2362	2578, 4156, 4157,	GCNT1	3.13
				4158, 4159, 4160,
				8460, 6349, 8001,
				7226, 6641,
8151341	2087	NM_007332	2363	4060, 8269, 5433,	TRPA1	6.01
8143534	2088	NM_000420	2364	2886, 2987, 6934,	KEL	5.85
				8187, 8288, 6818,
				6144, 8559,
7976858	2089	NM_001362	2365	2990, 4161, 8265,	DIO3	4.61
				6088,
8171472	2090	NM_020665	2366	2960, 4942, 6430,	TMEM27	4.68
8029536	2091	NM_001645	2367	3322, 3714, 5431,	APOC1	3.42
8059279	2092	NM_004438	2368	3926, 3577, 6427,	EPHA4	3.81
				7497, 5543,
8103415	2093	NM_001128424	2369	3395, 4433, 4434,	C4orf18	7.34
				4435, 7152, 7195,
				5766, 5649,
8155192	2094	NM_022343	2370	3083, 3132, 7426,	GLIPR2	3.24
				7611, 7741, 8325,
				6925,
8097449	2095	NM_032961	2371	2914, 2936, 2935,	PCDH10	2.82
				7099, 5512, 8691,
7970241	2096	NM_000504	2372	2567, 2573, 3210,	F10	6.85
				3246, 4308, 4309,
				3935, 7894, 6670,
				7728, 6624, 7953,
				7352, 6493,
8127193	2097	NM_021073	2373	2611, 3202, 5997,	BMP5	7.19
				7150,
7919314	2098	NM_001461	2374	3169, 4740, 4742,	FMO5	6.01
				4741, 5452, 5926,
				6568, 7792, 6924,
7968872	2099	NM_013238	2375	3842, 3846, 7013,	DNAJC15	3.99
				8073,
7942332	2100	NM_016730	2376	2548, 4931, 4932,	FOLR1	5.10
				4933, 4934, 5730,
				7111, 7703, 7300,
8147030	2101	NM_007029	2377	4711, 5225, 5342,	STMN2	4.91
7901316	2102	NM_001135181	2378	4682, 4626, 4627,	SLC5A9	4.12
				6649, 6403, 7403,
				7829, 6781, 6361,
8167287	2103	NM_022825	2379	2854, 2855, 2856,	PORCN	4.41
				2857, 4728, 2616,
				4468, 2979, 2983,
				3366, 3574, 3575,
				3573, 3576, 5975,
				8479, 5883, 6961,
				5689, 8317, 8547,
				6074, 7887, 5982,
				7832, 6053, 6535,
8105013	2104	NM_152403	2380	3530, 5277, 5276,	EGFLAM	4.32
				5275, 6119, 5859,
				8466, 5742, 6783,
				8384, 6717, 7825,
				8629, 8422, 7942,
				6265,
8059955	2105	NM_022449	2381	3268, 4986, 4985,	RAB17	2.97
				7579, 8393, 6896,
				6505, 7240, 5533,
8065416	2106	NM_001322	2382	3515, 3034, 5782,	CST2	4.29
8073633	2107	NM_025225	2383	3547, 2988, 7337,	PNPLA3	2.32
				5314, 8107, 8426,
8175288	2108	NM_019556	2384	2903, 2824, 6997,	MOSPD1	2.05
				8283, 6003, 6290,
				7788, 7752, 5881,
8139057	2109	NM_014800	2385	2968, 3692, 4045,	ELMO1	5.57
				2861, 3438, 4258,
				4520, 4479, 2801,
				2880, 3542, 5280,
				5279, 8359, 7086,
				5936, 6939, 6791,
				6584, 7972, 7197,
				7051, 6279, 5770,
				6974, 6770, 7488,
				7401, 7549, 6438,
				7349,
8022666	2110	NM_031422	2386	3460, 4937, 5865,	CHST9	3.68
7924342	2111	NM_018713	2387	3493, 3718, 6268,	SLC30A10	4.77
				6130, 8273, 8216,
7993588	2112	NM_024847	2388	3156, 4846, 4845,	TMC7	3.45
				5727, 7486,
8144656	2113	BC132945	2389	4115, 2976,	C8orf49	4.28
8111234	2114	NM_004061	2390	3260, 4595, 7174,	CDH12	4.25
				8759, 8207, 8652,
				8503, 7723, 8276,
8113358	2115	NM_005668	2391	3302, 4599, 3244,	ST8SIA4	3.69
				7055, 7738, 5386,
8090823	2116	NM_005630	2392	2724, 4396, 7489,	SLCO2A1	5.26
				6355, 8401, 8414,
				8074, 5640,
8121144	2117	NM_024641	2393	4179, 5290, 6156,	MANEA	4.38
				5952, 8006,
7964602	2118	NM_153377	2394	3457, 3537, 4639,	LRIG3	3.70
				5656, 6683, 7048,
8111998	2119	NM_021072	2395	3252, 5301, 5807,	HCN1	3.60
8111387	2120	NM_030955	2396	2863, 3711, 8350,	ADAMTS12	3.27
				8347, 7769, 6108,
				8583,
7948058	2121	NM_004476	2397	3060, 3615, 2779,	FOLH1	3.84
				2843, 3871, 2598,
				4703, 4705, 4140,
				4138, 2773, 3026,
				3280, 5177, 5176,
				2766, 5178, 3821,
				6173, 5921, 6307,
				5425, 6469, 6968,
8098041	2122	NM_018342	2398	2802, 3163, 3170,	TMEM144	4.83
				3261, 3319, 3506,
				3267, 4584, 8697,
				8424, 5686, 8259,
				8744, 8370, 7977,
				8192, 7923, 7849,
				8039, 7954, 8413,
				7830,
8013606	2123	NM_000638	2399	2897, 3932, 5348,	VTN	5.44
7913237	2124	NM_018584	2400	3291, 4040, 7514,	CAMK2N1	5.38
				8167,
7972297	2125	NM_005845	2401	3298, 4237, 4238,	ABCC4	3.94
				6825, 6506, 6826,
				6736, 7298,
8094520	2126	NM_032456	2402	2723, 5014, 5015,	PCDH7	2.10
				2937, 5262, 5768,
				5971,
7911273	2127	NM_001004696	2403	3717, 6107,	OR2T4	3.58
8123388	2128	NM_018974	2404	3851, 4738, 4736,	UNC93A	2.39
				5385, 8261, 6041,
				8180, 6272,
7902235	2129	NM_020794	2405	2985, 3272, 3712,	LRRC7	4.47
				4096, 4697, 4027,
				5312, 5716, 5818,
				7008, 6195, 6139,
8145736	2130	NM_013958	2406	2651, 4050, 4820,	NRG1	2.76
				4824, 4825, 4826,
				4827, 4828, 4829,
				4830, 4823, 4821,
				4822, 4831, 4832,
				4833, 4834, 4835,
				5786, 8022, 8533,
				5741, 6039, 6077,
				7952, 8077, 7456,
				5800, 5979, 7905,
				8268, 8671,
8174389	2131	NM_033641	2407	2623, 4165, 4167,	COL4A6	2.45
				6197, 5733, 6413,
				8100, 7288, 7442,
				7535,
7947512	2132	NM_015430	2408	3786, 3701, 3702,	PAMR1	4.04
				6790, 5537, 6920,
8154135	2133	NM_004170	2409	3070, 5025, 5401,	SLC1A1	2.42
				7218, 8331,
8089011	2134	NM_000313	2410	2597, 4770, 6690,	PROS1	3.68
8118209	2135	NM_019101	2411	2785, 3005, 2797,	APOM	3.46
				3131, 7568, 7419,
				7154, 6913, 7443,
				7676, 7525, 6531,
				7361, 7365, 7639,
				7323, 7713, 7039,
				6734, 7139, 6637,
				6503, 6767, 6861,
				6765,
8045664	2136	NM_177964	2412	3039, 3538, 8714,	LYPD6B	3.68
				5464, 7286, 6455,
				7123, 7806, 7494,
				8341, 7067, 6613,
8004691	2137	NM_203411	2413	3456, 3564, 5821,	TMEM88	2.64
7921702	2138	NM_080878	2414	3464, 3488, 6169,	ITLN2	2.45
				7935, 8767,
8132250	2139	NM_133468	2415	3324, 4608, 8606,	BMPER	2.95
				5589, 7995, 6712,
				6435, 7169, 8472,
8132347	2140	NM_181791	2416	3313, 3318, 6342,	GPR141	2.93
				5701, 6692,
8119898	2141	NM_001025366	2417	3808, 4966, 4967,	VEGFA	3.81
				4968, 4969, 4970,
				4971, 4972, 4973,
				4974, 4975, 4976,
				4977, 4978, 4979,
				4980, 4981, 8712,
				5968, 6175, 7640,
				5961, 5690, 7243,
				8549, 6759, 6136,
				7716, 8136, 8355,
				8159, 8018, 7079,
				8270, 8120, 7866,
				8507, 8066, 7946,
		SOX17/	SOX17/
		PDX1	PANC		SOX17	SOX17	SOX17
	Probe	fold	fold	FC	vs.	vs.	vs.
	set ID	change	change	average	HESC	PDX1	PANC
	8065412	10.57	155.94	58.22	0.0006	0.1703	0.0001
	7927631	7.52	73.19	31.22	0.0004	0.0534	0.0002
	8152617	5.70	73.73	27.86	0.0191	0.0616	0.0009
	7921834	4.25	45.73	22.60	0.0032	0.3017	0.0010
	8022674	2.80	61.48	22.22	0.0457	0.1237	0.0002
	7957140	8.46	32.45	16.28	0.0013	0.0713	0.0001
	8095646	2.45	30.64	13.97	0.1268	0.6168	0.0390
	8065071	5.74	24.40	13.25	0.0051	0.0774	0.0017
	8050619	3.68	15.61	12.24	0.0141	0.3073	0.0164
	8172670	8.75	14.66	10.99	0.0227	0.0287	0.0133
	7997188	14.45	4.02	8.72	0.0000	0.0003	0.0848
	8166747	7.21	4.94	8.44	0.0348	0.0865	0.1644
	7977786	6.71	5.99	6.46	0.0520	0.0694	0.0685
	8020795	2.62	1.70	6.43	0.0295	0.5580	0.7566
	8057506	2.12	13.08	6.39	0.0323	0.2374	0.0095
	8121916	2.83	6.85	6.24	0.0361	0.4235	0.0693
	8155930	2.82	12.65	6.20	0.0322	0.0469	0.0024
	8151341	5.25	6.96	6.07	0.0138	0.0181	0.0118
	8143534	3.84	7.89	5.86	0.0101	0.0277	0.0058
	7976858	5.01	7.09	5.57	0.0164	0.0135	0.0069
	8171472	4.58	7.38	5.55	0.0062	0.0088	0.0031
	8029536	3.05	9.63	5.37	0.1049	0.1256	0.0172
	8059279	2.77	9.52	5.36	0.0070	0.0234	0.0083
	8103415	5.08	3.39	5.27	0.0084	0.1540	0.0567
	8155192	6.10	6.17	5.17	9.69E−02	2.99E−02	3.47E−02
	8097449	2.41	9.33	4.85	2.75E−02	2.95E−01	2.51E−03
	7970241	3.59	3.89	4.77	1.83E−02	9.73E−02	5.48E−02
	8127193	2.33	4.68	4.73	1.45E−02	3.43E−01	4.07E−02
	7919314	6.14	1.82	4.66	2.69E−02	2.24E−02	4.69E−01
	7968872	4.94	4.96	4.63	1.82E−02	3.52E−02	1.17E−02
	7942332	2.93	5.60	4.54	1.56E−02	7.53E−02	1.31E−02
	8147030	2.31	6.26	4.49	4.21E−03	2.20E−01	1.10E−02
	7901316	4.66	4.70	4.49	3.29E−02	2.49E−02	2.98E−02
	8167287	3.25	5.77	4.47	5.07E−03	1.25E−02	3.33E−03
	8105013	4.61	4.31	4.41	2.36E−02	2.67E−02	6.88E−02
	8059955	5.62	4.64	4.41	9.80E−02	2.73E−02	5.52E−02
	8065416	3.10	5.27	4.22	1.11E−03	8.10E−02	6.18E−04
	8073633	2.76	7.56	4.21	1.20E−01	8.06E−02	1.41E−02
	8175288	3.50	6.98	4.18	6.67E−02	1.46E−02	2.67E−03
	8139057	4.60	2.36	4.18	1.38E−02	1.85E−02	2.21E−01
	8022666	2.03	6.76	4.16	9.17E−02	2.98E−01	4.17E−02
	7924342	3.28	4.38	4.15	3.27E−02	7.14E−02	4.01E−02
	7993588	2.13	6.80	4.13	8.15E−02	2.29E−01	2.94E−02
	8144656	4.29	3.78	4.12	1.72E−02	1.62E−02	2.23E−02
	8111234	3.48	4.28	4.01	4.39E−02	6.74E−02	4.30E−02
	8113358	5.57	2.70	3.99	4.46E−02	2.36E−02	1.69E−01
	8090823	3.69	2.94	3.96	5.59E−02	1.74E−01	1.69E−01
	8121144	3.33	4.07	3.93	4.95E−03	4.71E−02	4.99E−03
	7964602	4.41	3.36	3.82	5.09E−02	4.70E−02	7.91E−02
	8111998	3.20	4.59	3.80	3.66E−02	5.55E−02	2.73E−02
	8111387	2.68	5.03	3.66	3.37E−02	6.13E−02	3.57E−02
	7948058	3.34	3.70	3.63	5.45E−02	8.44E−02	5.93E−02
	8098041	2.59	3.36	3.59	4.76E−02	1.51E−01	8.13E−02
	8013606	2.52	2.78	3.58	3.57E−02	2.92E−01	1.39E−01
	7913237	2.04	2.93	3.45	2.99E−03	1.68E−01	1.56E−02
	7972297	2.02	4.33	3.43	3.04E−02	1.61E−01	2.42E−02
	8094520	2.44	5.60	3.38	6.57E−03	3.94E−02	3.10E−04
	7911273	3.35	3.17	3.37	1.66E−02	2.69E−02	2.30E−02
	8123388	3.04	4.59	3.34	3.61E−02	2.35E−02	5.64E−03
	7902235	2.42	2.94	3.27	4.41E−03	1.18E−01	1.57E−02
	8145736	2.44	4.60	3.26	3.01E−03	3.23E−02	2.10E−03
	8174389	2.49	4.84	3.26	8.73E−02	8.17E−02	1.62E−02
	7947512	2.34	3.37	3.25	1.98E−02	1.39E−01	4.14E−02
	8154135	3.71	3.59	3.24	2.52E−01	1.18E−01	1.28E−01
	8089011	3.53	2.45	3.22	5.10E−02	5.29E−02	1.40E−01
	8118209	2.22	3.77	3.15	2.08E−01	3.99E−01	1.84E−01
	8045664	2.06	3.60	3.12	3.42E−02	2.45E−01	3.90E−02
	8004691	2.18	4.46	3.09	1.78E−01	3.55E−01	1.06E−01
	7921702	2.26	4.52	3.08	2.29E−02	5.63E−02	3.65E−03
	8132250	2.80	3.43	3.06	1.67E−02	1.55E−02	6.52E−02
	8132347	2.87	2.84	2.88	1.22E−01	1.27E−01	1.25E−01
	8119898	2.14	2.02	2.66	8.92E−03	1.74E−01	8.31E−02
	Table 17. Provided are the description of the putative cell surface genes along with the sequence identifiers of the Affymetrix probes, the target sequence identified by the Affymetrix probe, the polynucleotide represented by the target sequence and the Public ID (and SEQ ID NO:) of the mRNA sequence representative of the target sequence. “Polyn.” = polynucleotide; “Rep.” = representative; Also shown are the fold change and T-test p-values for change in expression levels of the indicated genes between the SOX17-GFP cells and undifferentiated hESCs (SOX17/HESC); the SOX17-GFP cells and PDX1-GFP cells (SOX17/PDX1); and the SOX17-GFP cells and the Pancreas cells (SOX17/PANC).

TABLE 18
PDX1 specific putative cell surface genes
	Affy. target		SEQ ID NO:
Probe	nucl. SEQ	Rep.	(of Rep.	Polyn. SEQ ID NOs:	Gene	PDX1/HESC
set ID	ID NO:	Public ID	Public ID)	rep. by target	Symbol	fold change
7989073	2142	NM_173814	2418	3710, 4778, 6379,	PRTG	17.99
8135774	2143	NM_002851	2419	2599, 3943, 7457,	PTPRZ1	1.82
				8030, 7815, 8011,
				8237, 7646,
8109926	2144	NM_014211	2420	3898, 4597, 7818,	GABRP	10.28
				8036, 8604, 8494,
				8777, 8597, 5487,
7963970	2145	NM_006928	2421	3105, 5245, 6941,	SILV	3.32
				6287,
8020384	2146	AK293321	2422	4467, 2876, 4295,	KIAA1772	12.92
				4675, 7359, 7061,
				5529, 7232,
8169061	2147	NM_000533	2423	3671, 3836, 3741,	PLP1	3.49
				3742, 2596, 2595,
				2588, 4267, 4502,
				4503, 2878, 4324,
				4329, 4438, 3554,
				3553, 8469, 6590,
				7115, 7231, 7458,
				6933, 7196, 7209,
				8108, 8781, 8623,
				8141, 8486, 7855,
				5832, 8385, 5923,
				8694, 6335, 7709,
				6348, 8343, 7178,
				7629,
7953873	2148	BX647938	2424	3414, 3204, 8476,	OVOS	3.13
				8450,
8112971	2149	NM_001884	2425	2661, 3452, 4443,	HAPLN1	4.27
				8313, 8478, 8578,
				8217, 5490, 8326,
				8275,
8128284	2150	NM_004440	2426	2636, 4616, 6203,	EPHA7	2.00
				6224,
7953532	2151	NM_001975	2427	3363, 2973, 5374,	ENO2	2.71
8108697	2152	NM_015669	2428	2872, 2931, 5379,	PCDHB5	1.92
7953873	2153	BX647938	2429	3414, 3204, 8476,	OVOS	2.96
				8450,
7957338	2154	NM_005639	2430	3480, 4632, 4633,	SYT1	2.87
				4634, 7165, 5424,
				6857,
7972259	2155	NM_001922	2431	2627, 4441, 4442,	DCT	6.21
				6478, 8000, 7156,
				7726, 6808, 8056,
8113356	2156	AK172782	2432	3609,	GPAM	5.84
7972239	2157	NM_032229	2433	3864, 3532, 6395,	SLITRK6	5.14
8021245	2158	NM_005215	2434	4508, 4919, 7276,	DCC	5.58
				5767, 6546,
7968678	2159	NM_207361	2435	3444, 4093, 5476,	FREM2	5.40
				7899,
8018114	2160	NM_001144952	2436	2814, 4748, 7774,	SDK2	5.29
				5765, 6851, 6326,
8128001	2161	NM_000735	2437	2949, 5306, 5947,	CGA	5.24
8004545	2162	NM_001678	2438	2604, 3681, 5449,	ATP1B2	5.83
8140686	2163	NM_152754	2439	3466, 3557, 8721,	SEMA3D	5.79
				5583,
8108753	2164	NM_018935	2440	3191, 2929, 5380,	PCDHB15	4.40
7996837	2165	NM_004360	2441	4149, 4182, 2614,	CDH1	2.09
				4278, 4556, 2794,
				2795, 4415, 4294,
				4315, 4347, 5506,
				7453, 5404, 7133,
7929822	2166	NM_003393	2442	3013, 4006, 6207,	WNT8B	4.75
8168517	2167	NM_005296	2443	3834, 4756, 7854,	LPAR4	1.95
				5927,
7994026	2168	NM_130464	2444	3833, 4524, 5020,	NPIPL3	5.42
				4635, 3908, 7496,
				7414, 7388, 6046,
				6948, 7475, 6819,
				8569, 5650, 6371,
				7765, 6554, 6829,
				5907, 7686, 6199,
				8130, 8706, 8211,
				7864,
8046895	2169	NM_177454	2445	4274, 4070, 5467,	FAM171B	3.56
				5763,
8143144	2170	NM_002825	2446	2905, 3560, 6250,	PTN	2.21
7929779	2171	NM_000392	2447	2682, 4397, 7717,	ABCC2	5.16
				5949, 5908,
8100870	2172	NM_014243	2448	4101, 5286, 8348,	ADAMTS3	3.79
				5586,
8141843	2173	NM_006989	2449	5151, 5150, 7699,	RASA4	5.77
				5542, 6353,
8030448	2174	NM_001136052	2450	3049, 5236, 5237,	CPT1C	3.10
				7371, 6022, 7446,
				5780, 5691,
8078014	2175	NM_003043	2451	3914, 4382, 4385,	SLC6A6	1.83
				4527, 2650, 2637,
				3349, 4300, 4293,
				4603, 3999, 4602,
				6095, 6731, 7138,
				7680, 7533, 7619,
				7898, 7128, 6509,
				7006,
8108688	2176	NM_018937	2452	2924, 2930, 5388,	PCDHB3	3.94
8006336	2177	NM_052888	2453	3521, 3984, 4680,	LRRC37B	4.03
				4511, 4567, 4302,
				3733, 5844, 6194,
				7424,
7959025	2178	NM_001109903	2454	3352, 4242, 4243,	RNFT2	2.20
				5418, 5592, 7222,
				7129,
8051785	2179	NM_133329	2455	3647, 5227, 5228,	KCNG3	1.99
				5783, 7561,
8083166	2180	NM_003304	2456	2670, 3955, 8655,	TRPC1	2.51
				8076, 5551, 6798,
				7437,
8049128	2181	NM_031313	2457	2557, 4233, 5696,	ALPPL2	3.77
8148962	2182	NM_001005504	2458	4378, 4092, 3719,	OR4F21	3.52
				3723, 3734, 5799,
				7532, 5680, 6665,
				7961,
8006433	2183	NM_002982	2459	2971, 3759, 5331,	CCL2	4.78
7956522	2184	AF063608	2460	2859, 7555,	KIF5A	1.94
8033818	2185	NM_058164	2461	2754, 4134, 5475,	OLFM2	3.68
8031076	2186	NM_031896	2462	3827, 5226, 7212,	CACNG7	2.65
				5339,
7967386	2187	NM_022782	2463	3124, 3475, 5829,	MPHOSPH9	2.00
				5803, 7370,
8143110	2188	NM_012450	2464	3802, 3305, 7278,	SLC13A4	2.72
				8106, 8640, 7462,
				8737, 6233, 7314,
				7467,
8129573	2189	NM_015529	2465	2831, 4077, 8607,	MOXD1	3.57
				6186,
8128837	2190	NM_001123364	2466	4362,	C6orf186	3.86
7955469	2191	NM_001039960	2467	4335, 3940, 3941,	SLC4A8	1.83
				6177, 6955, 5856,
				6574,
8063636	2192	NM_001001433	2468	4565, 5270, 5269,	STX16	1.57
				5271, 5272, 6016,
				6278, 6873, 8633,
				8620, 8642, 7876,
				6090, 8358, 7184,
				6337, 8702, 6864,
				5934, 6359, 5902,
				6322,
	Probe	PDX1/SOX17	PDX1/PANC	FC	PDX1/	PDX1/	PDX1/
	set ID	fold change	fold change	average	HESC	SOX17	PANC
	7989073	1.67	44.84	21.50	2.18E−03	4.65E−01	1.27E−03
	8135774	7.84	43.05	17.57	2.84E−02	9.92E−04	8.98E−05
	8109926	14.17	13.85	12.77	6.22E−02	4.32E−02	4.56E−02
	7963970	8.34	26.38	12.68	2.55E−03	1.25E−03	8.28E−05
	8020384	3.53	11.66	9.37	1.98E−05	1.24E−02	8.94E−04
	8169061	6.21	13.94	7.88	4.42E−04	1.55E−03	3.02E−04
	7953873	4.15	13.46	6.91	1.90E−02	2.03E−02	2.06E−03
	8112971	5.91	10.17	6.78	1.95E−02	9.53E−03	3.52E−03
	8128284	2.17	15.05	6.41	2.20E−01	1.86E−01	4.99E−03
	7953532	3.64	11.81	6.05	3.42E−02	8.95E−02	2.06E−03
	8108697	11.87	3.96	5.92	3.08E−01	1.83E−02	1.25E−01
	7953873	4.05	10.68	5.90	1.67E−02	1.84E−02	2.26E−03
	7957338	4.38	10.29	5.85	1.17E−02	5.02E−03	5.53E−04
	7972259	6.19	4.67	5.69	1.32E−01	1.33E−01	1.99E−01
	8113356	5.67	5.49	5.67	1.15E−01	1.24E−01	1.27E−01
	7972239	5.03	5.97	5.38	1.07E−01	1.10E−01	8.58E−02
	8021245	4.92	5.31	5.27	1.01E−01	1.39E−01	1.08E−01
	7968678	1.62	8.14	5.06	1.60E−02	4.62E−01	2.29E−02
	8018114	3.87	6.00	5.05	7.13E−03	2.36E−02	6.24E−03
	8128001	4.37	5.35	4.99	2.50E−02	3.39E−02	2.05E−02
	8004545	4.82	4.24	4.96	2.18E−03	8.00E−03	8.78E−03
	8140686	2.31	6.55	4.88	5.75E−02	4.03E−01	7.48E−02
	8108753	5.61	3.46	4.49	1.30E−02	6.15E−03	3.65E−02
	7996837	7.44	3.84	4.45	5.03E−04	5.81E−04	3.27E−01
	7929822	3.80	4.79	4.45	1.08E−01	1.55E−01	1.06E−01
	8168517	3.31	7.09	4.12	1.71E−01	5.48E−02	8.05E−03
	7994026	2.63	3.75	3.93	2.06E−02	2.06E−01	3.92E−02
	8046895	1.84	6.16	3.85	2.89E−02	2.67E−01	7.34E−03
	8143144	3.53	5.72	3.82	1.00E−01	8.57E−02	1.75E−02
	7929779	1.91	4.16	3.74	1.77E−01	6.36E−01	2.27E−01
	8100870	3.04	4.35	3.73	3.65E−02	6.50E−02	3.23E−02
	8141843	1.96	3.11	3.61	1.73E−02	1.93E−01	4.40E−02
	8030448	1.73	5.83	3.55	9.71E−04	5.50E−02	2.61E−04
	8078014	2.25	6.42	3.50	2.76E−02	3.80E−02	2.83E−03
	8108688	3.21	3.27	3.47	3.06E−02	5.11E−02	6.64E−02
	8006336	2.00	4.37	3.46	2.70E−02	2.13E−01	3.59E−02
	7959025	2.09	5.91	3.40	6.84E−03	1.43E−02	1.87E−03
	8051785	4.61	3.32	3.31	2.11E−01	3.59E−02	6.45E−02
	8083166	1.92	5.44	3.29	1.48E−04	2.85E−02	1.01E−03
	8049128	3.01	2.98	3.25	2.85E−01	3.75E−01	3.66E−01
	8148962	2.75	3.48	3.25	1.01E−01	1.78E−01	1.06E−01
	8006433	2.60	2.30	3.22	7.29E−02	2.46E−01	2.62E−01
	7956522	3.39	4.30	3.21	6.96E−02	1.39E−02	6.49E−03
	8033818	2.33	3.58	3.20	6.17E−03	1.08E−01	9.26E−03
	8031076	2.97	3.68	3.10	1.49E−02	1.44E−01	5.39E−03
	7967386	2.05	5.23	3.09	3.11E−02	1.48E−01	9.67E−04
	8143110	1.89	4.63	3.08	2.25E−02	1.00E−01	6.15E−03
	8129573	2.63	2.94	3.05	1.08E−01	1.97E−01	1.57E−01
	8128837	3.24	1.97	3.02	9.06E−02	1.28E−01	3.35E−01
	7955469	2.07	5.16	3.02	1.77E−04	4.25E−03	4.18E−05
	8063636	1.54	1.76	1.62	2.08E−01	3.28E−01	1.30E−01
	Table 18. Provided are the description of the putative cell surface genes along with the sequence identifiers of the Affymetrix probes, the target sequence identified by the Affymetrix probe, the polynucleotide represented by the target sequence and the Public ID (and SEQ ID NO:) of the mRNA sequence representative of the target sequence. “Polyn.” = polynucleotide; “Rep.” = representative; Also shown are the fold change and T-test p-values for change in expression levels of the indicated genes between the PDX1-GFP cells and undifferentiated hESCs (PDX1/HESC); the PDX1-GFP cells and the Pancreas cells (PDX1/PANC); and the PDX1-GFP cells and SOX17-GFP cells (PDX1/SOX17).

TABLE 19
SOX17 and PDX1 specific putative cell surface genes
	Affy. target		SEQ ID NO:
Probe	nucl. SEQ	Rep.	(of Rep.	Polyn. SEQ ID NOs:	Gene	PDX1/PANC	SOX17/PANC
set ID	ID NO:	Public ID	Public ID)	rep. by target	Symbol	fold change	fold change
7989073	2193	NM_173814	2469	3710, 4778,	PRTG	44.84	26.86
				6379,
7927631	2194	NM_012242	2470	3471, 3796,	DKK1	9.73	73.19
				7543, 8463,
				8242, 5959,
7921834	2195	NM_001643	2471	2901, 2758,	APOA2	10.76	45.73
				6253, 8161,
				7460, 7113,
				8544, 6696,
				7530, 7963,
				6627, 6449,
8095646	2196	NM_001134	2472	3041, 2756,	AFP	12.50	30.64
				7886, 7273,
				8047, 8272,
8050619	2197	NM_000384	2473	2558, 3976,	APOB	4.24	15.61
				5351,
8065071	2198	NM_198391	2474	2812, 5172,	FLRT3	4.25	24.40
				5173, 5918,
				7056, 6380,
8020384	2199	AK293321	2475	4467, 2876,	KIAA1772	11.66	3.30
				4295, 4675,
				7359, 7061,
				5529, 7232,
8113800	2200	NM_001999	2476	2620, 3843,	FBN2	9.22	12.60
				5455, 7802,
				7852, 8234,
8057506	2201	NM_001463	2477	2679, 5106,	FRZB	6.16	13.08
				5695,
8038683	2202	NM_002774	2478	2673, 3799,	KLK6	4.93	6.26
				3800, 3801,
				7560, 5608,
				6746, 7343,
				6725, 6526,
7968678	2203	NM_207361	2479	3444, 4093,	FREM2	8.14	5.01
				5476, 7899,
8121916	2204	NM_032784	2480	3018, 3878,	RSPO3	2.42	6.85
				6102, 6281,
				8688,
7951865	2205	NM_000039	2481	3902, 2762,	APOA1	2.20	8.83
				6306, 5375,
				6654, 6990,
7951865	2206	NM_000039	2482	3902, 2762,	APOA1	2.21	8.49
				6306, 5375,
				6654, 6990,
8140686	2207	NM_152754	2483	3466, 3557,	SEMA3D	6.55	2.84
				8721, 5583,
8143534	2208	NM_000420	2484	2886, 2987,	KEL	2.06	7.89
				6934, 8187,
				8288, 6818,
				6144, 8559,
8127193	2209	NM_021073	2485	2611, 3202,	BMP5	2.01	4.68
				5997, 7150,
8167973	2210	NM_138737	2486	3469, 2884,	HEPH	6.78	6.77
				4726, 4486,
				4512, 3021,
				2834, 2951,
				4313, 3058,
				4952, 4953,
				5726, 7553,
				7348, 6504,
				7890, 6899,
				5938, 6468,
				6569,
8147030	2211	NM_007029	2487	4711, 5225,	STMN2	2.71	6.26
				5342,
7955142	2212	NM_000725	2488	3227, 5283,	CACNB3	5.25	4.99
				5827,
8022666	2213	NM_031422	2489	3460, 4937,	CHST9	3.33	6.76
				5865,
7993588	2214	NM_024847	2490	3156, 4846,	TMC7	3.19	6.80
				4845, 5727,
				7486,
8046895	2215	NM_177454	2491	4274, 4070,	FAM171B	6.16	3.35
				5467, 5763,
8008132	2216	NM_005175	2492	3118, 3700,	ATP5G1	6.24	3.00
				3699, 7206,
				8638, 7714,
				8758, 6255,
				7875, 7750,
				8115,
7942332	2217	NM_016730	2493	2548, 4931,	FOLR1	1.91	5.60
				4932, 4933,
				4934, 5730,
				7111, 7703,
				7300,
7929779	2218	NM_000392	2494	2682, 4397,	ABCC2	4.16	2.18
				7717, 5949,
				5908,
8113433	2219	NM_001962	2495	2657, 4446,	EFNA5	5.21	5.44
				8787, 5710,
				8776, 8760,
8030448	2220	NM_001136052	2496	3049, 5236,	CPT1C	5.83	3.37
				5237, 7371,
				6022, 7446,
				5780, 5691,
8167656	2221	NM_001005333	2497	3365, 3727,	MAGED1	3.13	5.78
				3728, 3726,
				8338, 8682,
				7309, 7060,
				6807, 7682,
				8515, 8260,
				6701, 5806,
				7744,
8141843	2222	NM_006989	2498	5151, 5150,	RASA4	3.11	1.59
				7699, 5542,
				6353,
8073015	2223	NM_006855	2499	2873, 2817,	KDELR3	2.26	3.38
				2816, 5309,
				7392, 7239,
8018761	2224	NM_006456	2500	2638, 4437,	ST6GALNAC2	1.54	2.34
				5345,
8097692	2225	NM_001957	2501	2600, 4920,	EDNRA	2.63	2.16
				4921, 4922,
				8628, 5591,
				5960, 8235,
				5962, 8220,
				6628, 7962,
8006336	2226	NM_052888	2502	3521, 3984,	LRRC37B	4.37	2.19
				4680, 4511,
				4567, 4302,
				3733, 5844,
				6194, 7424,
7972297	2227	NM_005845	2503	3298, 4237,	ABCC4	2.14	4.33
				4238, 6825,
				6506, 6826,
				6736, 7298,
8167603	2228	NM_001127899	2504	2659, 4413,	CLCN5	2.79	4.87
				4414, 4062,
				7768, 5769,
				6329, 6401,
				6907, 7160,
8050160	2229	NM_138799	2505	4760, 3543,	MBOAT2	4.88	3.47
				5812, 8164,
				8465, 8453,
				7310, 8461,
				8368, 6454,
				7808,
7917052	2230	NM_152697	2506	3153, 4453,	SLC44A5	2.71	3.64
				4454, 7183,
				6087, 6160,
7980146	2231	NM_006432	2507	3361, 3263,	NPC2	2.91	3.66
				7305, 5357,
8131096	2232	NM_001128636	2508	4884,	ELFN1	3.03	2.52
8016832	2233	NM_012329	2509	3499, 3725,	MMD	4.53	3.01
				5396,
8068353	2234	NM_006933	2510	3065, 4367,	SLC5A3	3.32	3.03
				4168, 7291,
				8103, 8705,
				8188, 8498,
				6457,
8150906	2235	NM_017813	2511	4236, 5520,	IMPAD1	2.34	4.01
8096771	2236	NM_021227	2512	3331, 3544,	OSTC	3.36	3.77
				8513, 8662,
				6166,
8022711	2237	NM_024422	2513	3516, 4406,	DSC2	3.66	4.14
				4407, 5438,
				5505, 6352,
				7116,
8157270	2238	NM_001859	2514	2699, 4628,	SLC31A1	4.11	4.15
				6193, 6305,
7947165	2239	NM_178498	2515	3423, 4239,	SLC5A12	4.13	3.22
				7203,
8135488	2240	NM_001099660	2516	3450, 4205,	LRRN3	2.69	1.97
				4202, 4200,
				6645, 7164,
				5798,
7994026	2241	NM_130464	2517	3833, 4524,	NPIPL3	3.51	2.04
				5020, 4635,
				3908, 7496,
				7414, 7388,
				6046, 6948,
				7475, 6819,
				8569, 5650,
				6371, 7765,
				6554, 6829,
				5907, 7686,
				6199, 8130,
				8706, 8211,
				7864,
8008802	2242	NM_182569	2518	3086, 4924,	GDPD1	3.19	2.86
				4925, 4923,
				5759,
7994026	2243	NM_130464	2519	3833, 4524,	NPIPL3	3.44	2.08
				5020, 4635,
				3908, 7496,
				7414, 7388,
				6046, 6948,
				7475, 6819,
				8569, 5650,
				6371, 7765,
				6554, 6829,
				5907, 7686,
				6199, 8130,
				8706, 8211,
				7864,
7994026	2244	NM_130464	2520	3833, 4524,	NPIPL3	3.48	2.07
				5020, 4635,
				3908, 7496,
				7414, 7388,
				6046, 6948,
				7475, 6819,
				8569, 5650,
				6371, 7765,
				6554, 6829,
				5907, 7686,
				6199, 8130,
				8706, 8211,
				7864,
8160546	2245	NM_152570	2521	4386, 3147,	LINGO2	2.80	2.77
				5841, 7024,
8045664	2246	NM_177964	2522	3039, 3538,	LYPD6B	1.75	3.60
				8714, 5464,
				7286, 6455,
				7123, 7806,
				7494, 8341,
				7067, 6613,
					PDX1	PDX1	SOX17	SOX17
	Probe	SOX17/HESC	PDX1/HESC	FC	vs.	vs.	vs.	vs.
	set ID	fold change	fold change	average	HESC	PANC	HESC	PANC
	7989073	10.78	17.99	25.12	2.18E−03	1.27E−03	9.95E−03	4.28E−03
	7927631	12.95	1.72	24.40	4.85E−01	3.70E−02	4.00E−04	1.77E−04
	7921834	17.82	4.19	19.63	2.79E−01	1.06E−01	3.17E−03	9.95E−04
	8095646	8.81	3.59	13.89	3.53E−01	1.07E−01	1.27E−01	3.90E−02
	8050619	17.42	4.73	10.50	1.55E−01	1.80E−01	1.41E−02	1.64E−02
	8065071	9.61	1.67	9.98	4.67E−01	9.17E−02	5.11E−03	1.73E−03
	8020384	3.66	12.92	7.89	1.98E−05	8.94E−04	1.07E−02	3.58E−02
	8113800	3.03	2.22	6.77	5.94E−02	2.49E−03	1.98E−02	1.36E−03
	8057506	3.96	1.87	6.27	1.38E−01	1.99E−02	3.23E−02	9.55E−03
	8038683	6.87	5.41	5.87	5.13E−02	5.92E−02	1.34E−01	1.49E−01
	7968678	3.33	5.40	5.47	1.60E−02	2.29E−02	4.98E−02	5.29E−02
	8121916	9.02	3.19	5.37	2.83E−01	4.23E−01	3.61E−02	6.93E−02
	7951865	7.79	1.94	5.19	2.33E−01	1.74E−01	1.18E−02	1.00E−02
	7951865	7.44	1.94	5.02	2.29E−01	1.67E−01	1.16E−02	9.63E−03
	8140686	2.50	5.79	4.42	5.75E−02	7.48E−02	2.06E−01	2.31E−01
	8143534	5.85	1.53	4.33	1.95E−02	2.85E−03	1.01E−02	5.80E−03
	8127193	7.19	3.09	4.24	1.49E−01	3.51E−01	1.45E−02	4.07E−02
	8167973	1.56	1.56	4.16	1.17E−01	7.23E−03	4.00E−01	2.71E−02
	8147030	4.91	2.12	4.00	2.20E−01	1.74E−01	4.21E−03	1.10E−02
	7955142	2.69	2.83	3.94	4.21E−02	9.05E−03	7.93E−03	1.19E−03
	8022666	3.68	1.82	3.90	2.88E−02	1.92E−02	9.17E−02	4.17E−02
	7993588	3.45	1.62	3.76	2.02E−02	1.03E−02	8.15E−02	2.94E−02
	8046895	1.94	3.56	3.75	2.89E−02	7.34E−03	1.36E−01	2.25E−02
	8008132	1.73	3.61	3.65	6.59E−02	2.56E−02	5.24E−01	2.42E−01
	7942332	5.10	1.74	3.59	4.99E−02	3.32E−02	1.56E−02	1.31E−02
	7929779	2.70	5.16	3.55	1.77E−01	2.27E−01	2.68E−01	3.68E−01
	8113433	1.78	1.70	3.53	2.74E−01	3.02E−02	3.25E−02	6.71E−03
	8030448	1.79	3.10	3.52	9.71E−04	2.61E−04	2.27E−02	2.18E−03
	8167656	3.33	1.80	3.51	1.86E−03	6.73E−03	2.72E−02	1.27E−02
	8141843	2.95	5.77	3.35	1.73E−02	4.40E−02	2.15E−02	8.07E−02
	8073015	4.63	3.09	3.34	5.00E−02	1.47E−01	2.55E−02	6.57E−02
	8018761	5.70	3.73	3.33	1.28E−01	5.74E−01	1.89E−02	1.49E−01
	8097692	3.77	4.59	3.29	6.31E−02	2.87E−01	1.87E−03	2.30E−01
	8006336	2.02	4.03	3.15	2.70E−02	3.59E−02	8.81E−02	1.18E−01
	7972297	3.94	1.95	3.09	2.00E−03	7.17E−04	3.04E−02	2.42E−02
	8167603	2.90	1.66	3.06	4.81E−04	5.85E−05	4.81E−02	1.41E−02
	8050160	1.60	2.26	3.05	6.44E−02	9.59E−03	1.64E−01	1.42E−02
	7917052	3.22	2.40	2.99	3.64E−03	2.74E−02	9.92E−02	9.89E−02
	7980146	3.01	2.39	2.99	2.93E−02	5.07E−02	1.71E−02	3.13E−02
	8131096	2.91	3.50	2.99	7.80E−03	1.14E−02	8.80E−02	1.23E−01
	8016832	1.61	2.41	2.89	6.75E−03	1.87E−01	1.50E−01	3.20E−01
	8068353	2.47	2.71	2.88	6.65E−02	1.10E−01	4.94E−04	6.50E−02
	8150906	3.26	1.91	2.88	3.17E−01	2.27E−01	1.13E−01	8.71E−02
	8096771	2.27	2.03	2.86	4.86E−02	2.51E−02	1.02E−01	4.36E−02
	8022711	1.85	1.64	2.82	2.46E−01	3.49E−02	1.35E−01	2.09E−02
	8157270	1.52	1.50	2.82	2.50E−01	1.64E−02	3.33E−01	2.82E−02
	7947165	1.68	2.16	2.80	1.62E−02	1.45E−03	4.66E−02	2.41E−03
	8135488	2.75	3.76	2.79	1.53E−03	3.07E−02	1.35E−04	5.92E−02
	7994026	2.06	3.55	2.79	2.47E−02	2.52E−02	1.04E−01	1.07E−01
	8008802	2.40	2.68	2.78	6.34E−05	3.96E−04	2.05E−02	1.40E−02
	7994026	2.10	3.49	2.78	2.59E−02	2.62E−02	9.45E−02	9.69E−02
	7994026	2.07	3.48	2.78	2.55E−02	2.52E−02	9.97E−02	9.87E−02
	8160546	2.73	2.76	2.76	1.09E−02	1.44E−02	3.38E−02	3.79E−02
	8045664	3.68	1.79	2.71	1.63E−01	1.89E−01	3.42E−02	3.90E−02
	Table 19. Provided are the description of the putative cell surface genes along with the sequence identifiers of the Affymetrix probes, the target sequence identified by the Affymetrix probe, the polynucleotide represented by the target sequence and the Public ID (and SEQ ID NO:) of the mRNA sequence representative of the target sequence. “Polyn.” = polynucleotide; “Rep.” = representative; Also shown are the fold change and T-test p-values for change in expression levels of the indicated genes between the PDX1-GFP cells and the Pancreas cells (PDX1/PANC); SOX17-GFP cells and the Pancreas cells (SOX17/PANC); SOX17-GFP cells and undifferentiated hESCs (PDX1/HESC); the PDX1-GFP cells and undifferentiated hESCs (PDX1/HESC).

TABLE 20
PDX1 and Pancreas specific putative cell surface genes
	Affy. target		SEQ ID NO:
Probe	nucl. SEQ	Rep.	(of Rep.	Polyn. SEQ ID NOs:	Gene	PDX1/	PDX1/
set ID	ID NO:	Public ID	Public ID)	rep. by target	Symbol	HESC	SOX17
7903414	2247	NM_000699	2523	3814, 2634,	AMY2A	3.79	2.83
				2921, 3063,
				3766, 3767,
				3777, 3765,
				3769, 6181,
				8186, 6513,
				8562, 7109,
				6188, 7492,
				7387, 8726,
				6179, 8240,
				6758, 6648,
				5740, 6977,
				6257, 5819,
8101659	2248	NM_001128310	2524	3607, 4539,	SPARCL1	3.14	2.98
				4554, 4700,
				4490, 4558,
				4476, 3089,
				2943, 4431,
				4430, 6384,
				8285, 5454,
				8543, 7693,
				7326, 8142,
				7471,
7961514	2249	NM_000900	2525	3815, 5155,	MGP	2.07	2.10
				5156, 5372,
7960947	2250	NM_000014	2526	3209, 3849,	A2M	7.83	4.71
				5717, 8772,
				8168, 6820,
				8158, 6446,
				6742,
7965410	2251	NM_001920	2527	2902, 3628,	DCN	2.02	4.12
				3629, 3630,
				3631, 3632,
				3633, 7344,
				7248, 6470,
				7199, 5678,
				5337, 5322,
				6594,
8023497	2252	NM_005603	2528	2736, 5024,	ATP8B1	2.34	2.80
				5707,
8096415	2253	NM_007351	2529	3528, 3568,	MMRN1	7.09	5.79
				6962, 7912,
				6838, 5465,
7954090	2254	NM_001423	2530	2678, 4610,	EMP1	2.26	2.14
				6653, 7265,
				5426,
7913216	2255	NM_000300	2531	2906, 4866,	PLA2G2A	2.85	5.25
				4867, 4868,
				4869, 6945,
				6892, 8598,
				8048, 8615,
				6396, 7663,
7954293	2256	NM_000921	2532	3510, 5300,	PDE3A	2.94	2.38
				5941,
8098611	2257	NM_003265	2533	3835, 3032,	TLR3	2.25	2.60
				8206, 5688,
8051583	2258	NM_000104	2534	2630, 4421,	CYP1B1	3.41	2.85
				6727, 8527,
				5409,
8066925	2259	NM_000961	2535	3882, 3795,	PTGIS	4.05	5.49
				5363, 8250,
				8695,
8058063	2260	NM_144629	2536	3913, 4395,	RFTN2	4.30	3.80
				5599, 7926,
8145977	2261	NM_021623	2537	4259, 4207,	PLEKHA2	2.02	2.85
				8302, 7202,
				6843, 7732,
				8390,
7975390	2262	NM_001034852	2538	4277, 5268,	SMOC1	3.45	3.29
				5267, 7459,
				5893,
8163896	2263	NM_004099	2539	2946, 3496,	STOM	2.23	2.77
				3495, 6038,
				5639,
8068024	2264	NM_021219	2540	2864, 3085,	JAM2	3.47	3.81
				8520, 7625,
				6556, 8774,
				7964, 7436,
				6761, 5705,
8077270	2265	NM_006614	2541	2694, 3806,	CHL1	2.67	2.03
				3927, 4681,
				3440, 3238,
				6369, 6718,
				6522, 7572,
				7141, 5444,
				7590, 6487,
				6827, 7208,
7982366	2266	NM_001144757	2542	3813, 4731,	SCG5	3.05	3.07
				4732, 8086,
				6330, 8119,
				8148, 5657,
8100541	2267	NM_001553	2543	3500, 2759,	IGFBP7	2.65	2.26
				5754, 7872,
				7990,
8104746	2268	NM_000908	2544	4247, 5263,	NPR3	2.87	2.48
				6508, 8718,
				5468, 7324,
7914127	2269	NM_002038	2545	3350, 3961,	IFI6	2.10	2.58
				3962, 3960,
				6118, 6098,
				6254,
7909390	2270	NM_175710	2546	3897, 4185,	CR1L	2.16	2.05
				8470, 6548,
				5851,
8129558	2271	NM_030908	2547	4048, 4376,	OR2A4	2.17	2.16
				2917, 5157,
				3729, 5808,
				7931,
8143633	2272	NM_001005328	8793	4048, 4376,	OR2A7	2.15	2.14
				2917, 5157,
				3729, 5808,
				7931,
					PDX1	PDX1	PANC	PANC
	Probe	PANC/	PANC/	FC	vs.	vs.	vs.	vs.
	set ID	SOX17	HESC	average	HESC	SOX17	HESC	SOX17
	7903414	63.39	84.98	38.75	6.94E−03	6.94E−03	3.93E−02	4.82E−02
	8101659	59.17	62.31	31.90	7.73E−02	7.73E−02	7.74E−04	8.61E−04
	7961514	51.99	51.42	26.89	2.49E−01	2.49E−01	1.55E−03	1.73E−03
	7960947	35.60	59.17	26.83	2.35E−01	2.35E−01	2.79E−04	4.65E−04
	7965410	42.02	20.66	17.21	1.69E−01	1.69E−01	8.61E−03	4.37E−03
	8023497	10.73	8.96	6.21	4.46E−03	4.46E−03	2.60E−02	2.05E−02
	8096415	4.78	5.86	5.88	1.60E−01	1.60E−01	2.49E−02	3.48E−02
	7954090	9.03	9.56	5.75	2.69E−01	2.69E−01	1.32E−02	1.54E−02
	7913216	8.17	4.44	5.18	1.13E−02	1.13E−02	7.39E−02	4.19E−02
	7954293	5.02	6.18	4.13	2.66E−01	2.66E−01	5.56E−04	2.20E−02
	8098611	6.01	5.20	4.02	2.63E−02	2.63E−02	1.79E−04	6.80E−04
	8051583	4.07	4.87	3.80	3.83E−02	3.83E−02	4.62E−02	7.35E−02
	8066925	3.11	2.30	3.74	9.29E−04	9.29E−04	6.62E−02	2.86E−02
	8058063	2.58	2.92	3.40	1.45E−01	1.45E−01	7.03E−02	1.21E−01
	8145977	5.08	3.60	3.39	4.17E−02	4.17E−02	3.87E−02	4.15E−02
	7975390	3.18	3.34	3.31	1.41E−01	1.41E−01	4.59E−03	3.14E−02
	8163896	4.38	3.53	3.23	3.49E−02	3.49E−02	1.08E−02	2.35E−02
	8068024	2.75	2.50	3.14	1.36E−01	1.36E−01	9.78E−02	8.38E−02
	8077270	3.09	4.08	2.97	2.29E−01	2.29E−01	3.29E−03	2.04E−02
	7982366	2.83	2.81	2.94	1.24E−02	1.24E−02	2.50E−01	2.63E−01
	8100541	2.94	3.45	2.82	4.45E−01	4.45E−01	5.51E−03	1.56E−01
	8104746	2.05	2.37	2.44	2.26E−01	2.26E−01	3.10E−01	4.14E−01
	7914127	2.75	2.25	2.42	1.47E−02	1.47E−02	5.76E−02	3.14E−02
	7909390	2.06	2.17	2.11	2.28E−01	2.28E−01	3.57E−02	5.29E−02
	8129558	2.03	2.04	2.10	2.92E−03	2.92E−03	1.24E−01	1.32E−01
	8143633	2.02	2.03	2.09	6.13E−03	6.13E−03	1.43E−01	1.56E−01
	Table 20. Provided are the description of the putative cell surface genes along with the sequence identifiers of the Affymetrix probes, the target sequence identified by the Affymetrix probe, the polynucleotide represented by the target sequence and the Public ID (and SEQ ID NO:) of the mRNA sequence representative of the target sequence. “Polyn.” = polynucleotide; “Rep.” = representative; Also shown are the fold change and T-test p-values for change in expression levels of the indicated genes between the PDX1-GFP cells and undifferentiated hESCs (PDX1/HESC); the PDX1-GFP cells and SOX17-GFP cells (PDX1/SOX17); the Pancreas cells and SOX17 cells (PANC/SOX17); and the Pancreas cells and the hESCs (PANC/HESC);

Example 5

TROP-2 FACS Sorting of Cells Derived from Embryoid Bodies Results in an Isolated Popupation of PDX1-Positive Cells (at Least 84% PDX1 Positive)

Experimental Results

Separation of TROP-2 Expressing Cells by FACS ARIA and Staining with PDX1—

Embryoid bodies were prepared from H9.2 hESCs and grown for 28 days in EBs medium (DMEM, 20% serum replacement, 1 mM Glutamax and 1% nonessential amino acid—all from GIBCO Invitrogen) with a change of medium every 3 days. The EBs were separated to single cells by treating for 20-30 minutes with TrypLE Select (Invitrogen) in 37° C. The cells were washed once with EBs medium, once more in FACS buffer (0.5% BSA, 10 mM EDTA, 25 mM Hepes in PBS). 10⁷ cells were diluted in 950 μl FACS buffer with 50 μl of anti-human TROP-2-APC (R&D Systems Inc., Minneapolis, Minn.) and incubated for 30 minutes at room temperature in the dark with shaking every 5 minutes. The cells were washed twice with PBS and centrifuged for 3 minutes at 1500 rpm. The cells were filtered with a 40 μm strainer (BD) and resuspended in FACS buffer. The FACS ARIA (BD) sorter was used to separate TROP-2 positive cells from the whole EB population. 1.3% of the cells were positive for allophycocyanin (APC) and were collected into DMEM with 10% FBS. The cells were spun down by Cytospin onto glass slides and fixed with cold acetone.

In order to stain the cells with PDX1 antibody, the cells were fixed again with 4% paraformaldehyde for 15 minutes and washed twice with PBS. The cells were blocked for one hour by adding 5% Normal Goat Serum (NGS), 1% BSA (bovine serum albumin), 0.5% Triton in PBS (phosphate buffered saline). The cells were washed twice with PBS and stained with goat polyclonal PDX1 (ABcam, Cambridge, UK) at a dilution of 1:1000 in PBS with 1% Triton, 0.5% BSA and 1% FBS over night in 4° C. The cells were washed twice with PBS and stained with anti-goat Cy3 secondary antibody 1:100 (Jackson Laboratories, West Grove, Pa.). Cells were rinsed twice with PBS and the nuclei were stained with DAPI 1:1000 (Sigma) and then the cells were mounted in fluorescent mounting media (Dako). The slides were analyzed using a confocal microscope (LSM 700, Zeiss).

Experimental Results

As shown in FIGS. 14A-C (representative confocal microscopy images) 84% of the cells which were isolated using an anti-human TROP-2 antibody were positive for PDX1 expression. These results demonstrate the isolation, for the first time, of a population of cells comprising at least 84% PDX1-positive cells which are not genetically modified.

Example 6

Endoderm Cells Expressing BST2, FLRT3, COLEC12, GPR49/LGR5 OR LIF-R can be Separated by FACs from Cell Populations Derived from Young Embryoid Bodies

Seven days old EBs contain cells of mesoderm, ectoderm and endoderm lineages but not fully differentiated tissues. These cells were tested for the expression of several surface markers which were found to be increased in the SOX17 expressing definitive endoderm cells by Affymetrix expression analysis.

Experimental Results

Sorting with Endoderm Markers Using FACS with Single Markers on Young EBs

H9.2 ESCs were grown for 7 days in EBs medium (DMEM, 20% serum replacement, 1 mM Glutamax and 1% nonessential amino acid—all from GIBCO Invitrogen) with a change of medium every 3 days. The EBs were separated to single cells by treating for 20-30 minutes with TrypLE Select (Invitrogen) in 37° C. The cells were washed once with EBs medium and once more in FACS buffer (0.5% BSA, 10 mM EDTA, 25 mM Hepes in PBS). The following antibodies were tested:

1. Rabbit anti BST-2 (Santa Cruz) 1:2002. Rabbit anti FLRT-3 (Santa Cruz) 1:503. Goat anti CL-P1/COLEC12 (R&D) 1:404. Rabbit anti GPR-49/LGR5 (Novus) 1:1005. Anti human LIF-R-PE (R&D) 1:106. Control non treated cells

The cells were incubated for 30 minutes at room temperature with the antibodies in FACS buffer (0.5% BSA, 10 mM EDTA, 25 mM Hepes in PBS) and washed twice with PBS, followed by secondary antibodies anti rabbit Cy3 (for BST2, FLRT-3 and GPR-49) or anti goat Cy3 (COLEC12) for 15 minutes at room temperature. The cells were washed twice more with PBS and resuspended in FACS buffer. The expression levels of these markers in the EBS was compared to those in undifferentiated hESCs cells by FACS calibrator (BD) using Flow-Jo analysis.

Experimental Results

The results are presented in FIGS. 12A-E and are summarized in Table 21 hereinbelow.

TABLE 21
Expression analysis of antibodies in undifferentiated embryonic stem cells
and in 7-day old embryoid bodies
Antibody	ESCs	7 days EBs
BST-2	2.25%	43.3%
FLRT-3*	1.56%	1.71%
COLEC12	0.26%	70.19%
GPR-49/LGR5	1.34%	38.2%
LIF-R	2.28%	16.13%
*The result with FLRT3 may change after optimization of the antibody which is not usually used in FACS analysis.

The results presented in FIGS. 12A-E and in Table 21 above show that in young embryoid bodies there is increase in cells expressing the endodermal specific markers identified in the SOX17 expressing cells as compared to undifferentiated hESC. These results point to the potential usefulness of these markers for characterization and isolation of populations enriched in definitive endoderm.

Example 7

Negative Selection with Underexpressed Markers can be Used to Enrich for Definitive Endoderm

Experimental Methods

Induction of Differentiation of Undifferentiated hESCs into Definite Endodermal Cells Using the IDE1/IDE2 Protocol

Undifferentiated H9.2 cells were treated with IDE1 or IDE2 as follows: The cells were washed once with PBS followed by incubation for 24 hours in RPMI with 5 μM IDE1/IDE2. The next day the medium was changed to RPMI with 0.2% FBS and 5 μM IDE1/IDE2 was added for 2 days. The cells were separated to single cells by treating the cells for 20-30 minutes with TrypLE Select (Invitrogen) in 37° C. The cells were washed once with EBs medium and once more in FACS buffer (0.5% BSA, 10 mM EDTA, 25 mM Hepes in PBS).

BST2 Expression on Adherent Cells

The cells which were induced by IDE1/IDE2 as described above were stained 1:100-1:200 with anti rabbit BST-2 (Santa Cruz) for 30 minutes in room temperature followed by secondary antibody Cy3 anti rabbit for 15 minutes at room temperature. Finally the cells were resuspended in FACS buffer and were tested by FACS calibrator.

BST2 Positive/KDR Negative and CXCR4 Positive/KDR Negative

The cells which were induced by IDE1/IDE2 as described above were stained 1:100-1:200 with anti rabbit BST-2 (Santa-Cruz) or 1:10 anti-human CXCR4-PerCP and 1:10 anti-human VEGF R2/KDR-FITC (both from R&D) for 30 min in room temperature followed by secondary antibody Cy3 anti rabbit for 15 min at room temperature (for the samples with BST2). Finally the cells were resuspended in FACS buffer and were tested by FACS calibrator.

Experimental Results

Cells which are Induced to Differentiate into Definite Endodermal Cells Express BST2—

The results of the FACS analysis showed that 11.9% of the cells in IDE1/IDE2 treated population express BST-2, and that only a subset of these, 10.7%, are BST2⁺/KDR⁻ (FIGS. 13A-C). These results demonstrate that combined use of the BST-2 marker with the KDR marker, which is underexpressed in definitive endoderm, may enable greater purification of an enriched definite endodermal cell population

Cells which are Induced to Differentiate into Definite Endodermal Cells Exhibit a BST2+/KDR− or CXCR4+/KDR− Expression Pattern—

The results of the FACS analysis showed that 1.05% of the cells treated with IDE1/2 express CXCR4 but not KDR (i.e., they exhibit the CXCR4+/KDR− expression signature) (FIG. 13D). As above, the combined use of positive and negative selection may yield a more highly enriched definitive endoderm population.

Altogether, these results show that the markers identified in the present study, individually and in combinations that include both overexpressed and underexpressed markers, can be used to isolate definite endodermal cells from a non-genetically modified population of cells that are differentiated from pluripotent stem cells.

ANALYSIS AND DISCUSSION

Differentiation of cells towards pancreatic beta—cells is still a challenging task. Based on genetic labeling approach the present inventors have succeeded in isolating from HESCs an enriched population of endoderm cells and pancreatic progenitor cells. These populations were then used in microarray analysis to identify candidate markers for defining (alone or in combination with other markers) specific precursor cell types characteristic of particular stages of the differentiation process. From the present analysis, a PDX1 specific gene GPR50 (FIG. 9, Table 14) and PDX1/Pancreas specific gene TROP2 (FIG. 11, Table 16) were selected for further study as promising genes. Particular emphasis has been placed on cell surface markers that by replacing genetic modification provide a more robust and stable approach for defining and isolating cell precursor populations. The information gained through these studies will permit more efficient generation of populations enriched with functional beta cells derived from stem cells. These protocols will rely on more efficient production of pancreatic progenitor cells and on their enrichment to enable improved treatment of diabetes.

hESCs represent one source of pluripotent cells capable of differentiating into almost any cell type (2). As a result, the therapeutic potential of these and other renewable stem cells for treating various diseases has generated much excitement. The present inventors disclose a method for selecting differentiating cells that will develop into functional insulin-producing beta cells. Such enriched populations may eventually be used in replacement therapy (e.g., cell transplantation) for the treatment of insulin dependent diabetes.

Selection and enrichment processes are needed because presently available protocols for differentiation of hESCs to definitive endoderm and pancreatic progenitors cells (5, 19, 21-24), do not yield pure populations. The importance of isolating the progenitors lies in the facts that, in contrast to mature beta cells, the progenitor cells have a proliferative capacity, and in contrast to hESCs they are non-tumorigenic. Thus purification or enrichment of progenitors followed by their continued culture would yield a more suitable transplant population.

The present inventors generated hESC clones harboring SOX17 and PDX1 BAC reporter constructs and identified a subpopulation of GFP⁺ cells.

SOX17 was originally identified as a stage-specific transcription activator during mouse spermatogenesis (26). Members of this gene family encode transcription factors that regulate the specification of cell types and tissue differentiation. Consistent with the general role of SOX genes in lineage specification, SOX17 is expressed specifically in the endoderm during gastrulation and plays a key role in endoderm formation.

PDX1 is expressed broadly in the pancreas during the first several days of pancreatic development, as the organ grows and branches. PDX1 regulates the insulin gene and from E15.5 (mouse embryonic days) onwards its expression becomes mainly restricted to β-cells. The transitions of PDX1 expression coincide with the overall conversion of progenitors to mature endocrine and exocrine cells (27).

The hESC clones harboring the SOX17 or PDX1⁺ BAC reporter and expressing GFP were isolated by FACS sorting and were analyzed by qPCR and microarray to identify cell surface and other markers expressed at these particular stages of the differentiation process.

The present inventors chose to use BACs, which are composed of relatively large stretches of genomic DNA, in order to render the transgenes less susceptible to mosaic or position effect variation. The larger size of BACs might also provide a more complete set of regulatory sequences (28).

Using this approach, the present inventors have succeeded in isolating enriched populations of endoderm and, for the first time pancreatic progenitor cells derived from human ESCs. Cell sorting based on SOX17 expression in reporter lines revealed markers which were previously correlated with definitive endoderm, thus validating the approach. Similarly, cells sorted base on PDX1 expression, revealed pancreatic progenitor markers. Affymetrix analysis then identified stage specific cell surface markers that enable cell type enrichment without the need for genetically modified hESCs. These markers provide a more robust, stable and clinically relevant approach for defining and isolating cell precursor populations.

To exemplify the above approach, use was made of two surface markers which are overexpressed in the PDX1⁺ cells, GPR50 and TROP-2. The latter was also found to be overexpressed in the pancreas array. Little is known about GPR50. The X-linked orphan receptor GPR50 shares 45% homology with the melatonin receptors, yet its ligand and physiological function remain mostly unknown. It is an orphan GPCR which has no affinity for melatonin, but as a dimer with MT1, it inhibits melatonin signaling (29, 30). Other reports have shown that GPR50 is also an important regulator of energy metabolism (31) The role of TROP-2 is not well understood and the physiological ligand is still unknown. TROP-2, the human trophoblast cell-surface glycoprotein, has been shown to play a role in regulating the growth of variety epithelial cancers including pancreatic tumors (32-35). Cell sorting of unmodified hESCs using these two markers enabled isolation of a cell population with a high relative expression of pancreatic progenitor transcription factors. These results exemplify the potential usefulness of the newly identified markers each alone, in combination with one another or in combination with other markers, in enabling isolation of enriched endoderm and pancreas progenitor populations. Other uses of these markers include quality control of candidate replacement therapy cell populations.

The availability of stage specific reporter constructs for key stages of pancreatic beta cell differentiation enables systematic assessment of the effect of signaling factors, small molecules or other compounds on growth, differentiation and survival of these progenitor cells. These constructs also provide valuable tools for efficient isolation of cell populations enriched for endoderm progenitor or pancreatic progenitor cells and their subsequent characterization by strategies such as microarray expression profiling. The discovery, through such an analysis, of new stage specific cell markers, including cell surface markers, opens the possibility for purification or enrichment of critical cell populations undergoing beta cell differentiation without the need for genetic modification. They also provide information about a “molecular signature” that can be used for “quality control” in assessing potential islet replacement cells developed from non-pancreatic sources.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

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Claims

1. A method of isolating a population of cells enriched in pancreatic progenitor cells, comprising: (a) detecting in a population of cells comprising pancreatic progenitor cells, cells that express at least one marker selected from the group consisting of TROP-2, BST2, GPR50, ROBO1, NTRK2, ITGA4, LGI1, VIPR2, SLC2A1, MUC15, MUC12, LPHN3, MUC16, VTCN1, MMP16, FZD3, ITGB6, GFRA3, NLGN1, CNTFR, LPHN1, SULF1, ADAM23, SCUBE3, PLAU, CDON, SLIT2, C7orf68, PLXDC2, CD74, GPR56, AREG, BOC, and KLRK1; and(b) isolating the cells expressing said at least one marker detected according to step (a) to thereby obtain an isolated population of cells enriched in pancreatic progenitor cells, thereby isolating the population of cells enriched in pancreatic progenitor cells.

2. The method of claim 1, further comprising detecting an additional marker selected from the group consisting of KDR, PTPRN, PRTG, PTPRZ1, GABRP, SILV, KIAA1772, PLP1, OVOS, HAPLN1, EPHA7, ENO2, PCDHB5, SYT1, DCT, GPAM, SLITRK6, DCC, FREM2, SDK2, CGA, ATP1B2, SEMA3D, PCDHB15, CDH1, WNT8B, LPAR4, NPIPL3, FAM171B, PTN, ABCC2, ADAMTS3, RASA4, CPT1C, SLC6A6, PCDHB3, LRRC37B, RNFT2, KCNG3, TRPC1, ALPPL2, OR4F21, CCL2, KIF5A, OLFM2, CACNG7, MPHOSPH9, SLC13A4, MOXD1, C6orf186, SLC4A8, STX16, AMY2A, SPARCL1, MGP, A2M, DCN, ATP8B1, MMRN1, EMP1, PLA2G2A, PDE3A, TLR3, CYP1B1, PTGIS, RFTN2, PLEKHA2, SMOC1, STOM, JAM2, CHL1, SCG5, IGFBP7, NPR3, IFI6, CR1L, OR2A4, OR2A7, GRID2, LPAR3, SEMA6A, ATP1A3, CAMKV, SCNN1G, SYT6, SLC18A2, ABCG2, HLA-DRA, HTR2C, EDNRB, PCDH11X, SLC17A7, SCNN1A, CD9, CXCL16, FXYD5, GABRQ, CACNA2D2, CLDN4, DPN, MMP24, GPR176, GPR64, GPR160, PCDH11Y, NKAIN4, SCN8A, THBS4, CR2, HLA-DQA1, HTR7, KCNS3, SLC7A3, HLA-DPB2, CACNA1B and GPR143.

3. The method of claim 1, wherein said population of cells enriched in pancreatic progenitor cells expresses a transcription factor selected from the group consisting of PDX1, ngn3, pax4, hlxb9, nkx6.1, Hnf6, and sox9.

4. The method of claim 1, wherein step (b) is effected by an immunological isolation technique selected from the group consisting of fluorescent activated cell sorter (FACS), Magnetic-activated cell sorting (MACS) or immunopanning.

5. The method of claim 1, wherein presence of at least a predetermined percentage of the pancreatic progenitor cells in said cell population indicates the suitability of the pancreatic progenitor cells for transplantation in a subject.

6. A method of isolating endocrine progenitors or insulin producing cells, comprising culturing the population of cells enriched in pancreatic progenitor cells isolated by the method of claim 1, under conditions suitable for maturation of the pancreatic progenitor cells into endocrine progenitors or beta cells, thereby isolating insulin producing cells.

7. An isolated population of cells enriched in pancreatic progenitor cells obtained according to the method of claim 1.

8. The isolated population of cells enriched in pancreatic progenitor cells of claim 7, wherein said pancreatic progenitor cells are genetically unmodified.

9. An isolated population of pancreatic progenitor cells, comprising at least about 50% of cells having a TROP-2+ and/or TROP-2+/GPR50+ expression pattern.

10. The isolated population of pancreatic progenitor cells of claim 9, wherein said cells are further characterized by the expression of at least one transcription factor selected from the group consisting of PDX1, ngn3, pax4, hlxb9, nkx6.1, Hnf6, and sox9.

11. The isolated population of pancreatic progenitor cells of claim 9, wherein said cells are obtained according to the method of claim 1.

12. The isolated population of pancreatic progenitor cells of claim 9, wherein said pancreatic progenitor cells are genetically unmodified.

13. A method of isolating a population of cells enriched in definite endodermal cells, comprising: (a) detecting in a population of cells comprising definite endodermal cells, cells that express at least one marker selected from the group consisting of LIFR, LGR5, FLRT3, BST2, COLEC12, FSHR, ROR2, ITGA5, CD177, CCKBR, APOA1, FZD5, FN1, BMP2, ADAMTS9, DPP4, FGA, GPR128, IGFBP5, FZD4, STC1, TNFSF4, IHH, LRP2, LAMA1, GPC3, LPHN3, FGB, KIT, TRO, SPA17, ROBO2, and DLK1; and(b) isolating the cells expressing said at least one marker detected according to step (a) to thereby obtain an isolated population of cells enriched in definite endodermal cells, thereby isolating a population of cells enriched in definite endodermal cells.

14. The method of claim 13, wherein said definite endodermal cells are further characterized by a SOX17+ or SOX17+/SOX7+ expression signature.

15. The method of claim 13, wherein said definite endodermal cells are further characterized by the expression of at least one marker selected from the group consisting of SOX17, SOX7, GSC, CER, FOXA2, CD34 and CXCR4; and a NANOG− expression signature.

16. The method of claim 13, wherein step (b) is by an immunological isolation assay technique selected from the group consisting of fluorescent activated cell sorter (FACS), Magnetic-activated cell sorting (MACS) or immunopanning.

17. An isolated population of cells enriched in definite endodermal cells obtained according to the method of claim 13.

18. The isolated population of cells enriched in definite endodermal cells of claim 17, comprising at least about 50% of cells having a SOX17+ or SOX17+/SOX7+ expression pattern.

19. The isolated population of cells enriched in definite endodermal cells of claim 17, wherein said definite endodermal cells are characterized by the expression of at least one marker selected from the group consisting of SOX17, SOX7, GSC, CER, FOXA2, CD34 and CXCR4; and a NANOG− expression signature.