Differentiation of Pluripotent Stem Cells

FIELD OF THE INVENTION

The present invention is directed to methods to differentiate pluripotent stem cells. In particular, the present invention is directed to methods and compositions to differentiate pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage. The present invention also provides methods to generate and purify agents capable of differentiating pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage.

BACKGROUND

Advances in cell-replacement therapy for Type I diabetes mellitus and a shortage of transplantable islets of Langerhans have focused interest on developing sources of insulin-producing cells, or β cells, appropriate for engraftment. One approach is the generation of functional β cells from pluripotent stem cells, such as, for example, embryonic stem cells.

In vertebrate embryonic development, a pluripotent cell gives rise to a group of cells comprising three germ layers (ectoderm, mesoderm, and endoderm) in a process known as gastrulation. Tissues such as, for example, thyroid, thymus, pancreas, gut, and liver, will develop from the endoderm, via an intermediate stage. The intermediate stage in this process is the formation of definitive endoderm. Definitive endoderm cells express a number of markers, such as, for example, HNF-3beta, GATA4, MIXL1, CXCR4 and SOX17.

Formation of the pancreas arises from the differentiation of definitive endoderm into pancreatic endoderm. Cells of the pancreatic endoderm express the pancreatic-duodenal homeobox gene, PDX1. In the absence of PDX1, the pancreas fails to develop beyond the formation of ventral and dorsal buds. Thus, PDX1 expression marks a critical step in pancreatic organogenesis. The mature pancreas contains, among other cell types, exocrine tissue and endocrine tissue. Exocrine and endocrine tissues arise from the differentiation of pancreatic endoderm.

Cells bearing the features of islet cells have reportedly been derived from embryonic cells of the mouse. For example, Lumelsky et al. (Science 292:1389, 2001) report differentiation of mouse embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Soria et al. (Diabetes 49:157, 2000) report that insulin-secreting cells derived from mouse embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice.

In one example, Hori et al. (PNAS 99: 16105, 2002) discloses that treatment of mouse embryonic stem cells with inhibitors of phosphoinositide 3-kinase (LY294002) produced cells that resembled β cells.

In another example, Blyszczuk et al. (PNAS 100:998, 2003) reports the generation of insulin-producing cells from mouse embryonic stem cells constitutively expressing Pax4.

Micallef et al. reports that retinoic acid can regulate the commitment of embryonic stem cells to form PDX1 positive pancreatic endoderm. Retinoic acid is most effective at inducing PDX1 expression when added to cultures at day 4 of embryonic stem cell differentiation during a period corresponding to the end of gastrulation in the embryo (Diabetes 54:301, 2005).

Miyazaki et al. reports a mouse embryonic stem cell line over-expressing Pdx1. Their results show that exogenous Pdx1 expression clearly enhanced the expression of insulin, somatostatin, glucokinase, neurogenin3, p48, Pax6, and HNF6 genes in the resulting differentiated cells (Diabetes 53: 1030, 2004).

Skoudy et al. reports that activin A (a member of the TGF-β superfamily) upregulates the expression of exocrine pancreatic genes (p48 and amylase) and endocrine genes (Pdx1, insulin, and glucagon) in mouse embryonic stem cells.

The maximal effect was observed using 1 nM activin A. They also observed that the expression level of insulin and Pdx1 mRNA was not affected by retinoic acid; however, 3 nM FGF7 treatment resulted in an increased level of the transcript for Pdx1 (Biochem. J. 379: 749, 2004).

Shiraki et al. studied the effects of growth factors that specifically enhance differentiation of embryonic stem cells into PDX1 positive cells. They observed that TGFβ2 reproducibly yielded a higher proportion of PDX1 positive cells (Genes Cells. 2005 June; 10(6): 503-16).

Gordon et al. demonstrated the induction of brachyury [positive]/HNF-3beta [positive] endoderm cells from mouse embryonic stem cells in the absence of serum and in the presence of activin along with an inhibitor of Wnt signaling (US 2006/0003446A 1).

Gordon et al. (PNAS, Vol 103, page 16806, 2006) states: “Wnt and TGF beta/nodal/activin signaling simultaneously were required for the generation of the anterior primitive streak.”

However, the mouse model of embryonic stem cell development may not exactly mimic the developmental program in higher mammals, such as, for example, humans.

Thomson et al. isolated embryonic stem cells from human blastocysts (Science 282:114, 1998). Concurrently, Gearhart and coworkers derived human embryonic germ (hEG) cell lines from fetal gonadal tissue (Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). Unlike mouse embryonic stem cells, which can be prevented from differentiating simply by culturing with Leukemia Inhibitory Factor (LIF), human embryonic stem cells must be maintained under very special conditions (U.S. Pat. No. 6,200,806; WO 99/20741; WO 01/51616).

D'Amour et al. describes the production of enriched cultures of human embryonic stem cell-derived definitive endoderm in the presence of a high concentration of activin and low serum (D'Amour K A et al. 2005). Transplanting these cells under the kidney capsule of mice resulted in differentiation into more mature cells with characteristics of some endodermal organs. Human embryonic stem cell-derived definitive endoderm cells can be further differentiated into PDX1 positive cells after addition of FGF-10 (US 2005/0266554A 1).

D'Amour et al. (Nature Biotechnology—24, 1392-1401 (2006)) states: “We have developed a differentiation process that converts human embryonic stem (hES) cells to endocrine cells capable of synthesizing the pancreatic hormones insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursor en route to cells that express endocrine hormones.”

In another example, Fisk et al. reports a system for producing pancreatic islet cells from human embryonic stem cells (US2006/0040387A1). In this case, the differentiation pathway was divided into three stages. Human embryonic stem cells were first differentiated to endoderm using a combination of n-butyrate and activin A. The cells were then cultured with TGFβ antagonists such as Noggin in combination with EGF or betacellulin to generate PDX1 positive cells. The terminal differentiation was induced by nicotinamide.

In one example, Benvenistry et al. states: “We conclude that over-expression of PDX1 enhanced expression of pancreatic enriched genes, induction of insulin expression may require additional signals that are only present in vivo” (Benvenistry et al., Stem Cells 2006; 24:1923-1930).

Activin A is a TGF-β family member that exhibits a wide range of biological activities including regulation of cellular proliferation and differentiation, and promotion of neuronal survival. Activin A is a homo-dimer, consisting of two activin βA subunits, encoded by the inhibin A gene. Other activins are known consisting of homo- or hetero-dimers of βA βC, βD, and βE subunits. For example, activin B consists of a homo-dimer of two βB subunits. The peptides comprising the βA subunit and the βB subunit are 63% identical and the positions of eight cysteines are conserved in both peptide sequences.

Activin A exerts its effect on cells by binding to a receptor. The receptor consists of a heteromeric receptor complex consisting of two types of receptor, type 1 (ActR-I) and type II (ActR-II), each containing an intracellular serine/threonine kinase domain. These receptors are structurally similar with small cysteine-rich extracellular regions and intracellular regions consisting of kinase domains. ActR-I, but not ActR-II, has a region rich in glycine and serine residues (GS domain) in the juxtamembrane domain. Activin A binds first with ActR-II, which is present in the cell membrane as an oligomeric form with a constitutively active kinase. ActR-1, which also exists as an oligomeric form, cannot bind activin A in the absence of ActR-II. ActR-I is recruited into a complex with ActR-II after activin A binding. ActR-II then phosphorylates ActR-I in the GS domain and activates its corresponding kinase.

Isolation and purification of activin A is often complex and can often result in poor yields. For example, Pangas, S. A. and Woodruff, T. K states: “Inhibin and activin are protein hormones with diverse physiological roles including the regulation of pituitary FSH secretion. Like other members of the transforming growth factor-β gene family, they undergo processing from larger precursor molecules as well as assembly into functional dimers. Isolation of inhibin and activin from natural sources can only produce limited quantities of bioactive protein.” (J. Endocrinol. 172 (2002) 199-210).

In another example, Arai, K. Y. et al states: “Activins are multifunctional growth factors belonging to the transforming growth factor-β superfamily. Isolation of activins from natural sources requires many steps and only produces limited quantities. Even though recombinant preparations have been used in recent studies, purification of recombinant activins still requires multiple steps.” (Protein Expression and Purification 49 (2006) 78-82).

There have been considerable efforts to develop a more potent or cheaper alternative to activin A. For example, U.S. Pat. No. 5,215,893 discloses methods for making proteins in recombinant cell culture which contain the β or β chains of inhibin. In particular, it relates to methods for obtaining and using DNA which encodes inhibin, and for making inhibin variants that depart from the amino acid sequence of natural animal or human inhibins and the naturally-occurring alleles thereof.

In another example, U.S. Pat. No. 5,716,810 discloses methods for making proteins in recombinant cell culture which contain the α or β chains of inhibin. In particular, it relates to methods for obtaining and using DNA which encodes inhibin, and for making inhibin variants that depart from the amino acid sequence of natural animal or human inhibins and the naturally-occurring alleles thereof.

In another example, U.S. Pat. No. 5,525,488 discloses methods for making proteins in recombinant cell culture which contain the α or β chains of inhibin. In particular, it relates to methods for obtaining and using DNA which encodes inhibin, and for making inhibin variants that depart from the amino acid sequence of natural animal or human inhibins and the naturally-occurring alleles thereof.

In another example, U.S. Pat. No. 5,665,568 discloses methods for making proteins in recombinant cell culture which contain the α or β chains of inhibin. In particular, it relates to methods for obtaining and using DNA which encodes inhibin, and for making inhibin variants that depart from the amino acid sequence of natural animal or human inhibins and the naturally-occurring alleles thereof.

In another example, U.S. Pat. No. 4,737,578 discloses proteins with inhibin activity having a weight of about 32,000 daltons. The molecule is composed of two chains having molecular weights of about 18,000 and about 14,000 daltons, respectively, which are bound together by disulfide bonding. The 18K chain is obtained from the human inhibin gene and has the formula: H-Ser-Thr-Pro-Leu-Met-Ser-Trp-Pro-Trp-Ser-Pro-Ser-Ala-Leu-Arg-Leu-Leu-Gln-Arg-Pro-Pro-Glu-Glu-Pro-Ala-Ala-His-Ala-Asn-Cys-His-Arg-Val-Ala-Leu-Asn-Ile-Ser-Phe-Gln-Glu-Leu-Gly-Trp-Glu-Arg-Trp-Ile-Val-Tyr-Pro-Pro-Ser-Phe-R.sub.6 5-Phe-His-Tyr-Cys-His-Gly-Gly-Cys-Gly-Leu-His-Ile-Pro-Pro-Asn-Leu-Ser-Leu-Pro-Val-Pro-Gly-Ala-Pro-Pro-Thr-Pro-Ala-Gln-Pro-Tyr-Ser-Leu-Leu-Pro-Gly-Ala-Gln-Pro-Cys-Cys-Ala-Ala-Leu-Pro-Gly-Thr-Met-Arg-Pro-Leu-His-Val-Arg-Thr-Thr-Ser-Asp-Gly-Gly-Tyr-Ser-Phe-Lys-Tyr-Glu-Thr-Val-Pro-Asn-Leu-Leu-Thr-Gln-His-Cys-Ala-Cys-Ile-OH, wherein R.sub.65 is Ile or Arg. The 18K chain is connected by disulfide bonding to the 14K chain.

Therefore, there still remains a significant need for cheaper, more potent alternatives for activin A to facilitate the differentiation of pluripotent stem cells.

SUMMARY

The present invention provides compounds capable of differentiating pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage. In one embodiment, the compounds capable of differentiating pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage are peptides comprising the amino acid sequence of activin A containing at least one point mutation.

In one embodiment, the present invention provides a method to differentiate pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage, comprising treating the pluripotent stem cells with a medium containing a peptide comprising the amino acid sequence of activin A containing at least one point mutation, for a period of time sufficient for the pluripotent stem cells to differentiate into cells expressing markers characteristic of the definitive endoderm lineage.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the phylogenetic tree of peptides ACTN 2 to ACTN 48.

FIG. 2 shows the phylogenetic tree of peptides ACTN 49 to ACTN 94.

FIG. 3 shows the nucleic acid sequence of the pro-region of wildtype activin A that was cloned into pcDNA3.1(−).

FIG. 4 shows the nucleic acid sequence of the mature region of ACTN 1, cloned into pcDNA3.1(−).

FIG. 5 shows the nucleic acid sequence of the full-length gene for ACTN 1, containing the pro-region and the mature region, cloned into pcDNA3.1(−).

FIG. 6 shows the ability of ACTN 1 (♦ ACTN 1 WT) and a control activin A (▪ and ▴ OriGene WT) to differentiate human embryonic stem cells into cells expressing markers characteristic of the definitive endoderm lineage. ACTN 1 (♦ ACTN 1 WT) and a wildtype control activin A (▪ OriGene WT), as cloned into their respective mammalian expression vectors, were transfected into HEK293-E cells, and supernatants obtained neat (not conc) or concentrated (conc) were added to human embryonic stem cells at the assay dilutions shown. Differentiation was determined by measuring SOX17 intensity expression relative to untreated control cells.

FIG. 7 shows the expression constructs used to obtain the peptides of the present invention. Panel A shows the nucleic acid sequence of the full-length gene for ACTN 1, containing the pro-region and the mature region, cloned into pUNDER. A pictorial representation of the expression vector is shown in Panel B.

FIG. 8 shows the ability of ACTN 1 and a wildtype activin A control cloned into their respective mammalian expression vectors to differentiate human embryonic stem cells into cells expressing markers characteristic of the definitive endoderm lineage. Panel A shows the effect of supernatants on assay cell number. ACTN 1 cloned into pUNDER and a wildtype activin A control (OriGene) cloned into pCMV6-XL4 were transfected into HEK293-F cells (white bars) and CHO-S cells (black bars), and supernatants collected neat, or concentrated 10-fold were tested at the dilutions shown in the definitive endoderm bioassay. Data shown represent changes relative to untreated cells. Panel B shows the effect of supernatants on SOX17 expression. ACTN 1 cloned into pUNDER and a wildtype activin A control (OriGene) cloned into pCMV6-XL4 were transfected into HEK293-F cells (white bars) and CHO-S cells (black bars), and supernatants collected neat, or concentrated 10-fold were tested at the dilutions shown in the definitive endoderm bioassay. Data shown represent changes relative to untreated cells.

FIG. 9 shows the expression of the peptides of the present invention in supernatants of HEK293-F cells transfected with pUNDER vectors containing the genes encoding the full length peptides indicated (ACTN 2, ACTN 4, ACTN 5, ACTN 6, ACTN 7, and ACTN 8). Supernatants were obtained, and analyzed by Western blot; the membrane was probed with an anti-activin A antibody.

FIG. 10 shows the expression of the peptides of the present invention in supernatants of HEK293-F cells transfected with pUNDER vectors containing the genes encoding the full length peptides indicated (ACTN 9, ACTN 10, ACTN 11, ACTN 12, ACTN 14, ACTN 16, ACTN 17, ACTN 18, ACTN 19, ACTN 20, ACTN 21, ACTN 22 and ACTN 23). Supernatants were obtained, and analyzed by Western blot; the membrane was probed with an anti-activin A antibody.

FIG. 11 shows the expression of peptides of the present invention that were further modified to contain histidine substitutions. HEK293-F cells were transfected with pUNDER vectors containing the genes encoding ACTD 17, ACTD 18, ACTD 19, ACTD 20, ACTD 21, and ACTD 22. Supernatants were obtained, and analyzed by Western blot; the membrane was probed with an anti-activin A antibody (Mab 3381—left hand side), or an anti-precursor antibody (Mab 1203—right hand side).

FIG. 12 shows a representative IMAC purification profile for ACTD 20. After loading, the column was washed and protein eluted with a linear gradient of imidazole (0-500 mM) over 20 column volumes.

FIG. 13 shows the Western blot elution profiles for Imidazole fractions for ACTD 17, ACTD 18, ACTD 19, ACTD 20, ACTD 21, and ACTD 22.

FIG. 14 shows a representative Western blot for follistatin variant expression from the supernatants of HEK293-F cells transfected with vectors containing the follistatin genes ACTA 1, ACTA 2 and ACTA 3. The membrane was probed with the antibodies indicated.

FIG. 15 shows a representative IMAC purification profile for ACTA 3 (Panel A). After loading, the column was washed and protein eluted with a step gradient of Imidazole (10 mM, 50 mM, 150 mM, 250 mM and 500 mM). Panel B shows a silver stain gel of the elution profile for the IMAC purification.

FIG. 16 shows a Western blot (Panels A and B) of a representative purification of peptide variant ACTN 1 using an ACTA 3 affinity column. The membranes were probed with the antibodies indicated. Panel C shows a silver stain gel a representative purification of peptide variant ACTN 1 using an ACTA 3 affinity column.

FIG. 17 shows the differentiation of human embryonic stem cells into cells expressing markers characteristic of the definitive endoderm lineage. Differentiation was determined by measuring cell number (Panel A) and SOX17 intensity (Panel B) using an IN Cell Analyzer 1000 (GE Healthcare). Human embryonic stem cells were treated for four days with medium containing 20 ng/ml Wnt3a plus activin A at the concentrations indicated (black bars) or medium lacking Wnt3a but with activin A at the concentrations indicated (white bars).

FIG. 18 shows the ability of ACTN 1 (white bars) and a control activin A (hatched bars and solid bars) to differentiate human embryonic stem cells into cells expressing markers characteristic of the definitive endoderm lineage. Supernatants from HEK293-E cells transfected with ACTN 1 (white bars) and a control activin A (hatched bars), cloned into pcDNA3.1(−) were obtained and concentrated, then added to human embryonic stem cells at the dilutions shown. Differentiation was determined by measuring SOX17 intensity.

FIG. 19 shows the differentiation of human embryonic stem cells into cells expressing markers characteristic of the definitive endoderm lineage using activin A. Panel A shows a standard curve for human embryonic stem cell differentiation using commercial recombinant human activin A and measuring SOX17 intensity. Cells were treated with activin A at the concentrations indicated for four days. Data shown are mean expression levels of SOX17, as detected using an IN Cell Analyzer 1000 (GE Healthcare). Panel B shows the ability of ACTN 1 to differentiate human embryonic stem cells into cells expressing markers characteristic of the definitive endoderm lineage. Supernatants from HEK293-F cells (white bars) and CHO-S cells (black bars) transfected with ACTN 1 cloned into pUNDER (pUNDER), and a wildtype activin A control (OriGene) cloned into pCMV6-XL4 were added to human embryonic stem cells at the concentrations indicated, and SOX 17 expression levels were determined four days later.

FIG. 20 shows the standard curve of recombinant human activin A as supplied by the manufacturer of an activin A ELISA (Panel A). Panel B compares the standard curves of two commercial recombinant human activin A standards in an activin A ELISA, where open squares (□) indicate the activin A standard supplied by the manufacturer (R&D Systems) and closed triangles (▴) indicate activin A purchased from Peprotech.

FIG. 21 shows results using flow cytometric analysis for CXCR4 expression after various treatments during the first step of differentiation. Histograms with percentages of CXCR4 positive cells are shown for treatment with activin A, or no activin A or two variant histidine peptides (ACTD3 and ACTD8), tested as unpurified supernatant stocks or IMAC purified material.

FIG. 22 panels A through I, show relative percent intensity for SOX17 expression versus a dose titration of given peptide concentrations, where peptide concentrations were previously calculated from ELISA results. In each panel, representative curves compare wildtype activin A peptide (ACTN1) to a variant peptide. Relative fit for each of the curves is shown by representative R2 values.

FIG. 23 shows results at the conclusion of the first step of differentiation using flow cytometric, PCR, and high content measure for multiple markers representative of definitive endoderm. Panel A shows FACS analysis for CXCR4 expression using a commercial source of activin A or wild type ACTN1 peptide during differentiation treatment. Panel B shows CXCR4 expression for two variant peptides (ACTN4 and ACTN48) compared to the wild type ACTN1 peptide. Panels C through F show high content analysis for cell number and SOX17 expression at the end of the first step of differentiation after treatment with wildtype activin A or individual variant peptides. Panels G and H show RT-PCR results for SOX17 and FOXA2 gene expression at the conclusion of the first step of differentiation after treatment with wildtype ACTN1 or variant peptides ACTN4 or ACTN48. The inset box shows CT values for each of the gene markers.

FIG. 24 shows results at the conclusion of the third step of differentiation after treatment with wildtype ACTN1 or variant peptides ACTN4 or ACTN48 during the first step of differentiation. Results depict high content analysis for cell number (panels A and B), PDX1 protein expression (panels C and D), CDX2 protein expression (panels E and F), or RT-PCR results for PDX1 or CDX2 (panels G and H). The inset box shows CT values for each of the gene markers.

FIG. 25 shows RT-PCR results at the conclusion of step four of differentiation after treatment with wildtype ACTN1 or. variant peptides ACTN4 or ACTN48 during the first step of differentiation. The inset box shows CT values for each of the gene markers.

DETAILED DESCRIPTION

For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the following subsections that describe or illustrate certain features, embodiments or applications of the present invention.

DEFINITIONS

Stem cells are undifferentiated cells defined by their ability at the single cell level to both self-renew and differentiate to produce progeny cells, including self-renewing progenitors, non-renewing progenitors, and terminally differentiated cells. Stem cells are also characterized by their ability to differentiate in vitro into functional cells of various cell lineages from multiple germ layers (endoderm, mesoderm and ectoderm), as well as to give rise to tissues of multiple germ layers following transplantation and to contribute substantially to most, if not all, tissues following injection into blastocysts.

Stem cells are classified by their developmental potential as: (1) totipotent, meaning able to give rise to all embryonic and extraembryonic cell types; (2) pluripotent, meaning able to give rise to all embryonic cell types; (3) multipotent, meaning able to give rise to a subset of cell lineages but all within a particular tissue, organ, or physiological system (for example, hematopoietic stem cells (HSC) can produce progeny that include HSC (self-renewal), blood cell restricted oligopotent progenitors, and all cell types and elements (e.g., platelets) that are normal components of the blood); (4) oligopotent, meaning able to give rise to a more restricted subset of cell lineages than multipotent stem cells; and (5) unipotent, meaning able to give rise to a single cell lineage (e.g., spermatogenic stem cells).

Differentiation is the process by which an unspecialized (“uncommitted”) or less specialized cell acquires the features of a specialized cell such as, for example, a nerve cell or a muscle cell. A differentiated or differentiation-induced cell is one that has taken on a more specialized (“committed”) position within the lineage of a cell. The term “committed”, when applied to the process of differentiation, refers to a cell that has proceeded in the differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type. De-differentiation refers to the process by which a cell reverts to a less specialized (or committed) position within the lineage of a cell. As used herein, the lineage of a cell defines the heredity of the cell, i.e., which cells it came from and what cells it can give rise to. The lineage of a cell places the cell within a hereditary scheme of development and differentiation. A lineage-specific marker refers to a characteristic specifically associated with the phenotype of cells of a lineage of interest and can be used to assess the differentiation of an uncommitted cell to the lineage of interest.

“β-cell lineage” refers to cells with positive gene expression for the transcription factor

PDX1 and at least one of the following transcription factors: NGN3, NKX2.2, NKX6.1, NEUROD, ISL1, HNF-3 beta, MAFA, PAX4, or PAX6. Cells expressing markers characteristic of the β cell lineage include β cells.

“Cells expressing markers characteristic of the definitive endoderm lineage”, or “Stage 1 cells”, or “Stage I”, as used herein, refers to cells expressing at least one of the following markers: SOX17, GATA4, HNF-3 beta, GSC, CERT, Nodal, FGF8, Brachyury, Mix-like homeobox protein, FGF4 CD48, eomesodermin (EOMES), DKK4, FGF17, GATA6, CXCR4, C-Kit, CD99, or OTX2. Cells expressing markers characteristic of the definitive endoderm lineage include primitive streak precursor cells, primitive streak cells, mesendoderm cells and definitive endoderm cells.

“Cells expressing markers characteristic of the pancreatic endoderm lineage”, as used herein, refers to cells expressing at least one of the following markers: PDX1, HNF-1 beta, PTF-1 alpha, HNF6, or HB9. Cells expressing markers characteristic of the pancreatic endoderm lineage include pancreatic endoderm cells, primitive gut tube cells, and posterior foregut cells.

“Cells expressing markers characteristic of the pancreatic endocrine lineage”, or “Stage 5 cells”, or “Stage 5”, as used herein, refers to cells expressing at least one of the following markers: NGN3, NEUROD, ISL1, PDX1, NKX6.1, PAX4, or PTF-1 alpha. Cells expressing markers characteristic of the pancreatic endocrine lineage include pancreatic endocrine cells, pancreatic hormone expressing cells, and pancreatic hormone secreting cells, and cells of the β-cell lineage.

“Definitive endoderm”, as used herein, refers to cells which bear the characteristics of cells arising from the epiblast during gastrulation and which form the gastrointestinal tract and its derivatives. Definitive endoderm cells express the following markers: HNF-3 beta, GATA4, SOX17, Cerberus, OTX2, goosecoid, C-Kit, CD99, and MIXL1.

“Extraembryonic endoderm”, as used herein, refers to a population of cells expressing at least one of the following markers: SOX7, AFP, or SPARC.

“Markers”, as used herein, are nucleic acid or polypeptide molecules that are differentially expressed in a cell of interest. In this context, differential expression means an increased level for a positive marker and a decreased level for a negative marker. The detectable level of the marker nucleic acid or polypeptide is sufficiently higher or lower in the cells of interest compared to other cells, such that the cell of interest can be identified and distinguished from other cells using any of a variety of methods known in the art.

“Mesendoderm cell”, as used herein, refers to a cell expressing at least one of the following markers: CD48, eomesodermin (EOMES), SOX17, DKK4, HNF-3 beta, GSC, FGF17, or GATA6.

“Pancreatic endocrine cell”, or “pancreatic hormone expressing cell”, as used herein, refers to a cell capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin, or pancreatic polypeptide.

“Pancreatic endoderm cell”, or “Stage 4 cells”, or “Stage 4”, as used herein, refers to a cell capable of expressing at least one of the following markers: NGN3, NEUROD, ISL1, PDX1, PAX4, or NKX2.2.

“Pancreatic hormone producing cell”, as used herein, refers to a cell capable of producing at least one of the following hormones: insulin, glucagon, somatostatin, or pancreatic polypeptide.

“Pancreatic hormone secreting cell”, as used herein, refers to a cell capable of secreting at least one of the following hormones: insulin, glucagon, somatostatin, or pancreatic polypeptide.

“Posterior foregut cell” or “Stage 3 cells”, or “Stage 3”, as used herein, refers to a cell capable of secreting at least one of the following markers: PDX1, HNF1, PTF1 alpha, HB9, or PROX1.

“Pre-primitive streak cell”, as used herein, refers to a cell expressing at least one of the following markers: Nodal, or FGF8.

“Primitive gut tube cell” or “Stage 2 cells”, or “Stage2”, as used herein, refers to a cell capable of secreting at least one of the following markers: HNF1, or HNF4 alpha.

“Primitive streak cell”, as used herein, refers to a cell expressing at least one of the following markers: Brachyury, Mix-like homeobox protein, or FGF4.

The Peptides of the Present Invention

The present invention provides peptides capable of differentiating pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage. In one embodiment, the peptides of the present invention are peptides comprising the amino acid sequence of activin A containing at least one point mutation. The at least one point mutation may be within the region of activin A that facilitates binding to the receptor. Alternatively, the at least one point mutation may be within the region of activin A that is within the homo-dimer interface.

The peptides of the present invention may contain one point mutation. Alternatively, the peptides of the present invention may contain multiple point mutations. In one embodiment, the at least one point mutation is determined by analyzing the crystallographic structure of activin A, wherein specific amino acid residues are chosen for mutation. The at least one point mutation may be in the form of an insertion of at least one amino acid residue. Alternatively, the at least one point mutation may be in the form of a deletion of at least one amino acid residue. Alternatively, the at least one point mutation may be in the form of a substitution of at least one amino acid residue.

The substitution of the at least one amino acid may be in the form of a substitution of at least one random amino acid at the specific location. Alternatively, the substitution of the at least one amino acid may be in the form of a substitution of at least one specific amino acid at the specific location. In one embodiment, the at least one specific amino acid used to substitute is chosen using a computational prediction that the at least one specific amino acid would have on the resulting homo-dimer formation.

In one embodiment, at least one point mutation was introduced into the amino acid sequence of activin A at least one amino acid residue selected from the group consisting of: 10I, 16F, 39Y, 41E, 43E, 74F, 75A, 76N, 77L, 78K, 79S, and 82V.

In one embodiment, at least one point mutation was introduced into the amino acid sequence of activin A at least one amino acid residue selected from the group consisting of: 16F, 18V, 19S, 20F, 37A, 38N, 39Y, 41E, 74F, 82V, 107N, 109I, 110V, and 116S.

The amino acid sequences of the peptides of the present invention may be found in Table 1.

In one embodiment, the amino acid sequences of the peptides of the present invention are back-translated into a nucleic acid sequence. The nucleic acid sequence may be synthesized and inserted into an expression vector to allow expression in mammalian cells. The nucleic acid sequence may be inserted into the expression vector pcDNA3.1(−). Alternatively, the nucleic acid sequence may be inserted into a variant of the pcDNA3.1(−) vector, wherein the vector has been altered to enhance the expression of the inserted nucleic acid sequence in mammalian cells. In one embodiment, the variant of the pcDNA3.1(−) vector is known as pUNDER.

The nucleic acid sequences of the peptides of the present invention may be found in Table 2.

The expression vector, containing a nucleic acid sequence of a peptide of the present invention may be transiently transfected into a mammalian cell. Alternatively, the expression vector, containing a nucleic acid sequence of a peptide of the present invention may be stably transfected into a mammalian cell. Any transfection method is suitable for the present invention. Such transfection method may be, for example, CaCl₂-mediated transfection, or LIPOFECTAMINE™-mediated transfection. See Example 2, for an example of a suitable transfection method.

The mammalian cell may be cultured in suspension, or, alternatively, as a monolayer. An example of a mammalian cell that may be employed for the present invention may be found in Example 2, and an alternative mammalian cell that may be employed for the present invention may be found in Example 3.

In an alternate embodiment, the peptides of the present invention may be expressed in an insect cell expression system, such as, for example, the system described in Kron, R et al (Journal of Virological Methods 72 (1998) 9-14).

Purification of the Peptides of the Present Invention

The peptides of the present invention may be isolated from the mammalian cells wherein they are expressed. In one embodiment, the mammalian cells are fractionated, and the supernatants containing the peptides of the present invention are removed. The peptides may be purified from the supernatants. Alternatively, the supernatants may be used directly. In the case where the supernatants are used directly, the supernatant is applied directly to human pluripotent stem cells. In one embodiment, the supernatant is concentrated prior to application to human pluripotent stem cells.

In the case where the peptides of the present invention are purified from the supernatant, the peptides may be purified using any suitable protein purification technique, such as, for example, size exclusion chromatography. In one embodiment, the peptides of the present invention are purified by affinity chromatography.

In one embodiment, the peptides of the present invention are purified by affinity chromatography by a method comprising the steps of:

- a. Transfecting cells with a vector encoding a peptide of the present invention,
- b. Allowing the expression of the peptide in the cells,
- c. Fractionating the cells and collecting the supernatant containing the peptide,
- d. Passing the supernatant through an affinity purification column, that is packed with a solid matrix containing a ligand that is capable of specifically binding the peptide, and
- e. Eluting the bound peptide off the solid matrix, therein obtaining a purified preparation of the peptide.

In one embodiment, the ligand that is capable of specifically binding the peptides of the present invention is follistatin.

In one embodiment, the peptides of the present invention are further modified to contain at least one region that is capable of specifically binding to the ligand on the solid substrate in the affinity purification column. In one embodiment, the peptides of the present invention are further modified to contain at least one metal binding site within their amino acid sequence. The further modification may consist of deleting amino acid resides to form the region that is capable of specifically binding to the ligand on the solid substrate in the affinity purification column. Alternatively, the further modification may consist of inserting amino acid resides to form the region that is capable of specifically binding to the ligand on the solid substrate in the affinity purification column. Alternatively, the further modification may consist of substituting amino acid resides to form the region that is capable of specifically binding to the ligand on the solid substrate in the affinity purification column. In one embodiment, the at least one metal binding site consists of two histidine residues. In one embodiment, the histidine residues are substituted into the amino acid sequence of the peptide comprising the amino acid sequence of activin A containing at least one point mutation. Table 3 lists peptides of the present invention that have been further modified to contain metal binding sites. In these embodiments, the ligand that is capable of specifically binding the peptide is nickel.

In an alternate embodiment, the peptides of the present invention are purified according to the methods described in Pangas, S. A. and Woodruff (J. Endocrinol. 172 (2002) 199-210).

In an alternate embodiment, the peptides of the present invention are purified according to the methods described in Arai, K. Y. et al (Protein Expression and Purification 49 (2006) 78-82).

Isolation, Expansion and Culture of Pluripotent Stem Cells
Characterization of Pluripotent Stem Cells

The pluripotency of pluripotent stem cells can be confirmed, for example, by injecting cells into severe combined immunodeficient (SCID) mice, fixing the teratomas that form using 4% paraformaldehyde, and then examining them histologically for evidence of cell types from the three germ layers. Alternatively, pluripotency may be determined by the creation of embryoid bodies and assessing the embryoid bodies for the presence of markers associated with the three germinal layers.

Propagated pluripotent stem cell lines may be karyotyped using a standard G-banding technique and compared to published karyotypes of the corresponding primate species. It is desirable to obtain cells that have a “normal karyotype,” which means that the cells are euploid, wherein all human chromosomes are present and not noticeably altered.

Sources of Pluripotent Stem Cells

The types of pluripotent stem cells that may be used include established lines of pluripotent cells derived from tissue formed after gestation, including pre-embryonic tissue (such as, for example, a blastocyst), embryonic tissue, or fetal tissue taken any time during gestation, typically but not necessarily before approximately 10 to 12 weeks gestation. Non-limiting examples are established lines of human embryonic stem cells or human embryonic germ cells, such as, for example, the human embryonic stem cell lines H1, H7, and H9 (WiCell). Also contemplated is use of the compositions of this disclosure during the initial establishment or stabilization of such cells, in which case the source cells would be primary pluripotent cells taken directly from the source tissues. Also suitable are cells taken from a pluripotent stem cell population already cultured in the absence of feeder cells. Also suitable are mutant human embryonic stem cell lines, such as, for example, BG01v (BresaGen, Athens, Ga.).

In one embodiment, human embryonic stem cells are prepared as described by Thomson et al. (U.S. Pat. No. 5,843,780; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133 ff., 1998; Proc. Natl. Acad. Sci. U.S.A. 92:7844, 1995).

In one embodiment, pluripotent stem cells are prepared as, described by Takahashi et al. (Cell 131: 1-12, 2007).

Culture of Pluripotent Stem Cells

In one embodiment, pluripotent stem cells are typically cultured on a layer of feeder cells that support the pluripotent stem cells in various ways. Alternatively, pluripotent stem cells are cultured in a culture system that is essentially free of feeder cells but nonetheless supports proliferation of pluripotent stem cells without undergoing substantial differentiation. The growth of pluripotent stem cells in feeder-free culture without differentiation is supported using a medium conditioned by culturing previously, with another cell type. Alternatively, the growth of pluripotent stem cells in feeder-free culture without differentiation is supported using a chemically defined medium.

The pluripotent stem cells may be plated onto a suitable culture substrate. In one embodiment, the suitable culture substrate is an extracellular matrix component, such as, for example, those derived from basement membrane or that may form part of adhesion molecule receptor-ligand couplings. In one embodiment, the suitable culture substrate is MATRIGEL® (Becton Dickenson). MATRIGEL® is a soluble preparation from Engelbreth-Holm-Swarm tumor cells that gels at room temperature to form a reconstituted basement membrane.

Other extracellular matrix components and component mixtures are suitable as an alternative. Depending on the cell type being proliferated, this may include laminin, fibronectin, proteoglycan, entactin, heparan sulfate, and the like, alone or in various combinations.

The pluripotent stem cells may be plated onto the substrate in a suitable distribution and in the presence of a medium that promotes cell survival, propagation, and retention of the desirable characteristics. All these characteristics benefit from careful attention to the seeding distribution and can readily be determined by one of skill in the art.

Suitable culture media may be made from the following components, such as, for example, Dulbecco's modified Eagle's medium (DMEM), Gibco #11965-092; Knockout Dulbecco's modified Eagle's medium (KO DMEM), Gibco #10829-018; Ham's F12/50% DMEM basal medium; 200 mM L-glutamine, Gibco #15039-027; non-essential amino acid solution, Gibco 11140-050; β-mercaptoethanol, Sigma #M7522; human recombinant basic fibroblast growth factor (bFGF), Gibco #13256-029.

Formation of Pancreatic Hormone Producing Cells from Pluripotent Stem Cells

In one embodiment, the present invention provides a method for producing pancreatic hormone producing cells from pluripotent stem cells, comprising the steps of:

- a. Culturing pluripotent stem cells,
- b. Differentiating the pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage,
- c. Differentiating the cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the pancreatic endoderm lineage, and
- d. Differentiating the cells expressing markers characteristic of the pancreatic endoderm lineage into cells expressing markers characteristic of the pancreatic endocrine lineage.

In one aspect of the present invention, the pancreatic endocrine cell is a pancreatic hormone producing cell. In an alternate aspect, the pancreatic endocrine cell is a cell expressing markers characteristic of the β-cell lineage. A cell expressing markers characteristic of the β-cell lineage expresses PDX1 and at least one of the following transcription factors: NGN3, NKX2.2, NKX6.1, NEUROD, ISL1, HNF-3 beta, MAFA, PAX4, or PAX6. In one aspect of the present invention, a cell expressing markers characteristic of the β-cell lineage is a β-cell.

Pluripotent stem cells suitable for use in the present invention include, for example, the human embryonic stem cell line H9 (NIH code: WA09), the human embryonic stem cell line H1 (NIH code: WA01), the human embryonic stem cell line H7 (NIH code: WA07), and the human embryonic stem cell line SA002 (Cellartis, Sweden). Also suitable for use in the present invention are cells that express at least one of the following markers characteristic of pluripotent cells: ABCG2, cripto, CD9, FOXD3, Connexin43, Connexin45, OCT4, SOX2, Nanog, hTERT, UTF-1, ZFP42, SSEA-3, SSEA-4, Tra1-60, or Tra1-81.

Markers characteristic of the definitive endoderm lineage are selected from the group consisting of SOX17, GATA4, HNF-3beta, GSC, CER1, Nodal, FGF8, Brachyury, Mix-like homeobox protein, FGF4 CD48, eomesodermin (EOMES), DKK4, FGF17, GATA6, CXCR4, C-Kit, CD99, and OTX2. Suitable for use in the present invention is a cell that expresses at least one of the markers characteristic of the definitive endoderm lineage. In one aspect of the present invention, a cell expressing markers characteristic of the definitive endoderm lineage is a primitive streak precursor cell. In an alternate aspect, a cell expressing markers characteristic of the definitive endoderm lineage is a mesendoderm cell. In an alternate aspect, a cell expressing markers characteristic of the definitive endoderm lineage is a definitive endoderm cell.

Markers characteristic of the pancreatic endoderm lineage are selected from the group consisting of PDX1, HNF-1beta, PTF1 alpha, HNF6, HB9 and PROX1. Suitable for use in the present invention is a cell that expresses at least one of the markers characteristic of the pancreatic endoderm lineage. In one aspect of the present invention, a cell expressing markers characteristic of the pancreatic endoderm lineage is a pancreatic endoderm cell.

Markers characteristic of the pancreatic endocrine lineage are selected from the group consisting of NGN3, NEUROD, ISL1, PDX1, NKX6.1, PAX4, and PTF-1 alpha. In one embodiment, a pancreatic endocrine cell is capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide. Suitable for use in the present invention is a cell that expresses at least one of the markers characteristic of the pancreatic endocrine lineage. In one aspect of the present invention, a cell expressing markers characteristic of the pancreatic endocrine lineage is a pancreatic endocrine cell. The pancreatic endocrine cell may be a pancreatic hormone expressing cell. Alternatively, the pancreatic endocrine cell may be a pancreatic hormone secreting cell.

Formation of Cells Expressing Markers Characteristic of the Definitive Endoderm Lineage

In one aspect of the present invention, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells with medium containing a peptide of the present invention, for an amount of time sufficient to enable the pluripotent stem cells to differentiate into cells expressing markers characteristic of the definitive endoderm lineage.

The pluripotent stem cells may be treated with medium containing a peptide of the present invention for about one day to about seven days. Alternatively, the pluripotent stem cells may be treated with medium containing a peptide of the present invention for about one day to about six days. Alternatively, the pluripotent stem cells may be treated with medium containing a peptide of the present invention for about one day to about five days. Alternatively, the pluripotent stem cells may be treated with medium containing a peptide of the present invention for about one day to about four days. Alternatively, the pluripotent stem cells may be treated with medium containing a peptide of the present invention for about one day to about three days. Alternatively, the pluripotent stem cells may be treated with medium containing a peptide of the present invention for about one day to about two days. In one embodiment, the pluripotent stem cells may be treated with medium containing a peptide of the present invention for about four days.

The pluripotent stem cells may be cultured on a feeder cell layer. Alternatively, the pluripotent stem cells may be cultured on an extracellular matrix.

In one aspect of the present invention, the pluripotent stem cells are cultured and differentiated on a tissue culture substrate coated with an extracellular matrix. The extracellular matrix may be a solubilized basement membrane preparation extracted from mouse sarcoma cells (as sold by BD Biosciences under the trade name MATRIGEL™). Alternatively, the extracellular matrix may be growth factor-reduced MATRIGEL™. Alternatively, the extracellular matrix may be fibronectin. In an alternate embodiment, the pluripotent stem cells are cultured and differentiated on tissue culture substrate coated with human serum.

The extracellular matrix may be diluted prior to coating the tissue culture substrate. Examples of suitable methods for diluting the extracellular matrix and for coating the tissue culture substrate may be found in Kleinman, H. K., et al., Biochemistry 25:312 (1986), or Hadley, M. A., et al., J. Cell. Biol. 101:1511 (1985).

In one embodiment, the extracellular matrix is MATRIGEL™. In one embodiment, the tissue culture substrate is coated with MATRIGEL™ at a 1:10 dilution. In an alternate embodiment, the tissue culture substrate is coated with MATRIGEL™ at a 1:15 dilution. In an alternate embodiment, the tissue culture substrate is coated with MATRIGEL™ at a 1:30 dilution. In an alternate embodiment, the tissue culture substrate is coated with MATRIGEL™ at a 1:60 dilution.

In one embodiment, the extracellular matrix is growth factor-reduced MATRIGEL™. In one embodiment, the tissue culture substrate is coated with growth factor-reduced MATRIGEL™ at a 1:10 dilution. In an alternate embodiment, the tissue culture substrate is coated with growth factor-reduced MATRIGEL™ at a 1:15 dilution. In an alternate embodiment, the tissue culture substrate is coated with growth factor-reduced MATRIGEL™ at a 1:30 dilution. In an alternate embodiment, the tissue culture substrate is coated with growth factor-reduced MATRIGEL™ at a 1:60 dilution.

The pluripotent stem cells may be treated with medium containing a peptide of the present invention that has been purified from the supernatant of the cell that expressed the peptide. Alternatively, the pluripotent stem cells may be treated with medium containing a peptide of the present invention that has been not purified from the supernatant of the cell that expressed the peptide.

In the case where the pluripotent stem cells are treated with medium containing a peptide of the present invention that has been not purified from the supernatant of the cell that expressed the peptide, the supernatant may be used at a final concentration of about 1:10 dilution to about 1:100. In one embodiment, supernatant may be used at a final concentration of about 1:10 dilution to about 1:50. In one embodiment, supernatant may be used at a final concentration of about 1:10 dilution to about 1:40. In one embodiment, supernatant may be used at a final concentration of about 1:20 dilution to about 1:50.