Patent Application
20210180026
Organogenesis is a complex and inter-connected process, orchestrated by multiple boundary tissue interactions1-7. However, to date, it has been unclear how individual, neighboring components coordinate to establish an integral multi-organ structure. Thus, multi-organ integration in stem cell culture has been a critical unmet challenge. Specifically, obtaining structurally and functionally integrated organoids having more than one tissue type is an unmet need in the art. More particularly, the patterning and balanced organogenesis of the hepato-biliary-pancreatic (HBP) system has not been successfully modelled in tissue culture due to technical complexities, hindering detailed mechanistic studies16,17. The instant disclosure seeks to address one or more of the aforementioned needs in the art.
Disclosed herein are hepato-biliary-pancreatic organoid (“HBPO” or “HBP organoid”) compositions, and methods of making and using hepato-biliary-pancreatic organoid compositions. The disclosed compositions may have two or more functions selected from hepatic tissue function, biliary tissue function, exocrine pancreatic function, and endocrine pancreatic tissue function. Methods of treating individuals using the hepato-biliary-pancreatic organoid compositions is also disclosed.
Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.
Definitions
Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.
As used herein and in the appended claims, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a method” includes a plurality of such methods and reference to “a dose” includes reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.
The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” may mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” may mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.
As used herein, the term “definitive endoderm (DE) cell” means one of the three primary germ layers produced by the process of gastrulation.
As used herein the term “wnt signalling pathway” means the wnt/beta-catenin pathway and is a signal transduction pathway that is mediated by Wnt ligands and frizzled cell surface receptors that acts through the beta-catenin protein.
As used herein the term “activator” with respect to a pathway, such as a “wnt pathway” means a substance that activates the Wnt/beta-catenin pathway such that Wnt/beta-catenin targets are increased.
As used herein, the term “FGF signaling pathway activator” means a substance that activates the FGF pathway such that FGF targets are increased.
As used herein, the term “BMP signaling pathway inhibitor” a substance that interferes with the BMP pathway and causes BMP targets to be decreased.
As used herein, the term “growth factor” means a substance capable of stimulating cellular processes including but not limited to growth, proliferation, morphogenesis or differentiation.
As used herein, the term “stable expression” of a marker means expression that does not change upon modification of the growth environment.
As used herein, the term “totipotent stem cells” (also known as omnipotent stem cells) are stem cells that can differentiate into embryonic and extra-embryonic cell types. Such cells can construct a complete, viable, organism. These cells are produced from the fusion of an egg and sperm cell. Cells produced by the first few divisions of the fertilized egg are also totipotent.
As used herein, the term “pluripotent stem cells (PSCs),” also commonly known as PS cells, encompasses any cells that can differentiate into nearly all cells, i.e., cells derived from any of the three germ layers (germinal epithelium), including endoderm (interior stomach lining, gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood, urogenital), and ectoderm (epidermal tissues and nervous system). PSCs can be the descendants of totipotent cells, derived from embryos (including embryonic germ cells) or obtained through induction of a non-pluripotent cell, such as an adult somatic cell, by forcing the expression of certain genes.
As used herein, the term “induced pluripotent stem cells (iPSCs),” also commonly abbreviated as iPS cells, refers to a type of pluripotent stem cells artificially derived from a normally non-pluripotent cell, such as an adult somatic cell, by inducing a “forced” expression of certain genes.
As used herein, the term “precursor cell” encompasses any cells that can be used in methods described herein, through which one or more precursor cells acquire the ability to renew itself or differentiate into one or more specialized cell types. In some aspects, a precursor cell is pluripotent or has the capacity to becoming pluripotent. In some aspects, the precursor cells are subjected to the treatment of external factors (e.g., growth factors) to acquire pluripotency. In some aspects, a precursor cell can be a totipotent stem cell; a pluripotent stem cell (induced or non-induced); a multipotent stem cell; and a unipotent stem cell. In some aspects, a precursor cell can be from an embryo, an infant, a child, or an adult. In some aspects, a precursor cell can be a somatic cell subject to treatment such that pluripotency is conferred via genetic manipulation or protein/peptide treatment.
In developmental biology, cellular differentiation is the process by which a less specialized cell becomes a more specialized cell type. As used herein, the term “directed differentiation” describes a process through which a less specialized cell becomes a particular specialized target cell type. The particularity of the specialized target cell type can be determined by any applicable methods that can be used to define or alter the destiny of the initial cell. Exemplary methods include but are not limited to genetic manipulation, chemical treatment, protein treatment, and nucleic acid treatment.
Disclosed herein are hepato-biliary-pancreatic organoid (“HBPO” or “HBP organoid”) compositions. The disclosed HBPO compositions may have, in some aspects, two or more functions selected from hepatic tissue function, biliary tissue function, exocrine pancreatic function, and endocrine pancreatic tissue function. In one aspect, the HBPO disclosed herein may be referred to interchangeably as a “multi-organ three-dimensional organoid” and may comprise an anterior region, a posterior region and a boundary region. In one aspect, the boundary region may expresse Pancreatic and Duodenal Homeobox 1 (PDX1) and Hematopoietically-Expressed Homeobox Protein (HHEX). In one aspect, the HBPO may comprise bile duct tissue and pancreatic tissue. In one aspect, the bile duct tissue and pancreatic tissue may be connected in the HBPO. In one aspect, the HBPO may comprise liver cells, pancreas cells, bile duct cells, and intestinal cells.
In one aspect, the HBPO may comprise endothelial cells, mesenchymal cells, or both endothelial cells and mesenchymal cells. The HBPO may comprise, in certain aspects, endoderm and mesoderm.
The disclosed HBPOs may be characterized by a branched structure, as shown, for example, in the corresponding figures herein.
In one aspect, the HBPOs disclosed herein may be characterized by expression of a functional exocrine marker. In one aspect, the marker may be amylase. In one aspect, the marker may be an endocrine marker, for example, insulin.
In certain aspects, the HBPOs disclosed herein may be characterized by a functional response to an exogenously administered agent. For example, in one aspect, the HBPO may secrete amylase in response to contact with cholecystokinin (CCK).
In certain aspects, the disclosed HBPOs may be characterized in that the HBPO may be substantially free of one or more of submucosal glands, transition zones, vasculature, immune cells, or submucosal layers. Such features distinguish the three-dimensional organoid structure from that of native tissue found endogenously in a human, or other mammal.
In one aspect, the HBPO is obtained by expansion of one or more precursor cells as defined above, for example, an iPSC obtained from an individual.
Also disclosed herein are methods of making a hepato-biliary-pancreatic organoid (“HBPO” or “HBP organoid”). In this aspect, the method may comprise contacting a first definitive endoderm with a Wnt signaling pathway activator, an FGF signaling pathway activator, and a BMP signaling pathway inhibitor to form an anterior gut spheroid; contacting a second definitive endoderm with a Wnt signaling pathway activator and an FGF signaling pathway activator to form a posterior gut spheroid; contacting the anterior gut spheroid with the posterior gut spheroid until the anterior gut spheroid and the posterior gut spheroid are fused to form a boundary organoid having a foregut-midgut boundary; and culturing the boundary organoid having the foregut-midgut boundary to form the HBPO; wherein the HBPO comprises biliary tissue and pancreatic tissue. (See, for example,
As used herein, the term hepato-biliary-pancreatic organoid (“HBPO” or “HBP organoid”) generally refers to the organoid at the stage in which multi-organ domains (hepatic, biliary and pancreatic organ domains) are segregated, which usually occurs around D30. With regard to the term “boundary organoid,” this may include an organoid after D7, which includes organoid compositions that do not yet have tissue specification.
In one aspect, the anterior gut spheroids may comprise cells expressing SRY-box 2 (SOX2). In one aspect, the anterior gut spheroid may be characterized by SRY-box 2 (SOX2) expression. In one aspect, the anterior gut spheroids may be substantially free of Pancreatic and Duodenal Homeobox 1 (PDX1) expressing cells. In one aspect, the posterior gut spheroids comprise cells expressing Pancreatic and Duodenal Homeobox 1 (PDX1). In one aspect, the posterior gut spheroids may express CDX2. In one aspect, the posterior gut spheroid may be characterized by Caudal Type Homeobox 2 (CDX2) expression. In one aspect, the posterior gut spheroids may comprise cells expressing Caudal type homeobox 2 (CDX2). In one aspect, the HBPO may comprise cells expressing Pancreatic and Duodenal Homeobox 1 (PDX1), cells expressing Hematopoietically-expressed homeobox protein (HHEX), and cells expressing Prospero-Related Homeobox 1 (PROX1). In one aspect, the fused anterior gut spheroid and posterior gut spheroid may comprise a biliary-pancreatic primordium characterized by expression of SOX2, CDX2, HHEX, and PDX1, wherein PDX1 expression is localized to the boundary region of the fused anterior gut spheroid and the posterior gut spheroid.
In one aspect, the HBPO may be embedded into a morphogenic factor, preferably a basement membrane matrix, such as, for example, Matrigel. The method may further comprise excising PROX1 positive regions from the HBPO and culturing the excised HBPO to form invaginating epithelium and a branching structure.
In one aspect, the HBPO may be cultured with rotation, for example, using the methods disclosed herein.
In one aspect, the disclosed methods may be used to obtain an HBPO as described herein, in particular, an HBPO comprising endoderm and mesoderm.
In one aspect, the definitive endoderm may be derived from a precursor cell selected from an embryonic stem cell, an embryonic germ cell, an induced pluripotent stem cell, a mesoderm cell, a definitive endoderm cell, a posterior endoderm cell, a posterior endoderm cell, and a hindgut cell, preferably a definitive endoderm derived from a pluripotent stem cell, more preferably a definitive endoderm derived from a pluripotent stem cell selected from an embryonic stem cell, an adult stem cell, or an induced pluripotent stem cell.
In one aspect, the definitive endoderm may be derived from contacting a pluripotent stem cell with one or more molecules selected from Activin, the BMP subgroups of the TGF-beta superfamily of growth factors; Nodal, Activin A, Activin B, BMP4, Wnt3a, and combinations thereof.
In one aspect, the WNT signaling pathway activator may be selected from one or more molecules selected from the group consisting of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, a GSKβ inhibitor (e.g., CHIR99021, i.e. “CHIRON”), BIO, LY2090314, SB-216763, lithium, porcupine inhibitors IWP, LGK974, C59, SFRP inhibitor WAY-316606, beta-catenin activator DCA.
In one aspect, the FGF signaling pathway activator may be selected from one or more molecules selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, and combinations thereof, preferably FGF4 or FGF10, or a combination thereof.
In one aspect, the BMP signaling pathway inhibitor may be selected from Noggin, Dorsomorphin, LDN189, DMH-1, and combinations thereof, preferably wherein the BMP signaling pathway inhibitor is Noggin.
In one aspect, the disclosed methods may be conducted in vitro. In certain aspect, at least one step of the method may be carried out on a solid support. For example, the solid support may be selected from collagen, basement membrane matrix (Matrigel), or a combination thereof.
In one aspect, a method for making an HBPO having an endothelium is disclosed. In this aspect, the method may comprise the step of culturing an HBPO according to the methods disclosed herein, with endothelial cells derived from induced pluripotent stem cells. The endothelial cells may be formed into an endothelial cell (EC) spheroid prior to culturing with an HBPO. For example, for endothelial cell (EC) differentiation, human iPSCs may be dissociated using Accutase (Thermo Fisher Scientific Inc., Waltham, Mass., USA) and plated on Laminin 511 E8 fragment (iMatrix-511™, provided by Nippi, Inc.) at varying optimal density (depending on the cell line) in StemFit® (Ajinomoto Co., Inc.) with Rho-associated kinase inhibitor (Y-27632). From the next day, they may first be differentiated into mesoderm using the Priming Medium having B27 medium (1:1 mixture of DMEM:F12 (1:1) with 1% Glutamax and 1% B27 (all Life Technologies) with Wnt activator and bone morphogenetic protein 4 (BMP4) for three days. The priming medium may then be replaced with EC induction medium, StemPro-34 SFM medium (Life Technologies) supplemented with VEGF and forskolin. The induction medium may be renewed daily. On day seven of differentiation, ECs are dissociated and subjected to FACS analysis. iPSC derived ECs should display typical endothelial morphology with junctional localization of CD144 and CD31. ECs may be re-plated on Fibronectin coated dishes at a density of 50,000 cells cm−2 in EC Expansion Medium consisting of StemPro-34 SFM supplemented with VEGF-A. EC Expansion Medium may be replaced every other day. The differentiated EC may be dissociated to single cells and formed into spheroids using the spheroid formation protocol of the anterior gut spheroids and posterior gut spheroids as described herein. The ECs may then be mixed with the fused anterior gut spheroid and the posterior gut spheroid on 96 well round bottom ultra-low attachment plate in gut growth medium for 24 hours to form fused EC spheroid, the anterior gut spheroid and the posterior gut spheroid.
In one aspect, a method for making an HBPO having a mesenchyme is disclosed. In this aspect, the method may comprise the step of culturing an HBPO according to a method as disclosed herein with mesenchymal cells derived from induced pluripotent stem cells. The mesenchyme cells may be formed into a mesenchyme cell (MC) spheroid prior to culturing with the HBPO. The disclosed multi-organ, three-dimensional organoid and mesenchymal cells or a mesenchymal spheroid may be cultured together to improve the growth and maturation of the multi-organ, three-dimensional organoid. For example, for mesenchyme (MSC) differentiation, human iPSCs may be differentiated to mesoderm. The mesoderm cells may then be exposed to 3 days of additional Activin A and PDGFBB. Differentiated MSC may then be dissociated to single cells and formed into spheroids using the spheroid formation protocol of the anterior gut spheroid and posterior gut spheroid. The MSC spheroid may then be mixed with the fused anterior gut spheroid and posterior gut spheroid on 96 well round bottom ultra-low attachment plate in gut growth medium for 24 hours to form a fused MSC spheroid, anterior gut spheroid and posterior gut spheroid.
In one aspect, a method of making a hepato-biliary-pancreatic organoid (“HBPO”) is disclosed, wherein the method may comprise deriving an anterior gut spheroid from anterior gut cells and a deriving a posterior gut spheroid from posterior gut cells to make a boundary organoid; and culturing the boundary organoid in gut growth medium to make the HBPO. In one aspect, a method of making a hepato-biliary-pancreatic organoid (“HBPO”), may comprise deriving an anterior gut spheroid and deriving a posterior gut spheroid from anterior gut cells and posterior gut cells to make the HBPO in the absence of inducing factors; and culturing the boundary organoids in a gut growth medium to make HBPO.
By “inducing factors,” it is meant a factor used to direct differentiation, for example, retinoic acid (RA; Sigma, MO, USA), hepatocyte growth factor (HGF; PeproTech, NJ, USA), 0.1 μM Dexamethasone (Dex; Sigma) and 20 ng/mL Oncostatin M (OSM; R&D Systems) for hepatocyte differentiation. Many other inducing factors are known for pancreas and biliary also, and would be readily understood by one of skill in the art.
In one aspect, a of method of making a hepato-biliary-pancreatic organoid (“HBPO”) is disclosed, wherein the method may comprise culturing a definitive endoderm (DE) differentiated from a pluripotent stem cell (PSC) in gut growth medium to make anterior gut cells and posterior gut cells, making an anterior gut spheroid from anterior gut cells and a posterior gut spheroid from posterior gut cells to make a boundary organoid in the absence of inducing factors; and (iii) culturing boundary organoids in gut growth medium to make an HBPO. In one aspect, the gut growth medium may be a medium comprising a ROCK inhibitor. In one aspect, the ROCK inhibitor may be Y-27632. In one aspect, the growth medium is Advanced DMEM/F-12 (Dulbecco's Modified Eagle Medium/Ham's F-12), a widely used basal medium that allows the culture of mammalian cells with reduced Fetal Bovine Serum (FBS) supplementation, available at https://www.thermofisher.com/order/catalog/product/12634010, with addition of Glutamine, HEPES, N2 and B27. In one aspect, the media may include addition of the following cytokines to Advanced DMEM: Noggin (BMP inhibitor), CHIR (WNT activator), FGF4 for anterior gut cells, CHIR and FGF4 (without Noggin) for posterior gut cells.
In one aspect, a machine for producing an HBPO is disclosed herein. The machine may comprise a gut growth medium containing a Wnt signaling pathway activator and an FGF signaling pathway activator, in the presence or absence of a BMP signaling pathway inhibitor; and a gut growth medium containing a ROCK inhibitor.
In one aspect, use of a plurality of culture mediums for the production of an HBPO, is disclosed, wherein the plurality of culture mediums comprise a gut growth medium containing a Wnt signaling pathway activator and an FGF signaling pathway activator, optionally comprising a BMP signaling pathway inhibitor; and an intestinal growth medium containing a ROCK inhibitor.
In one aspect, the HBPO may be transplanted into a mammal, such as a non-human mammal, for a period of time to increase growth and maturation of the HBPO. In one aspect, the HBPO may be transplanted to rescue organ dysfunction, for example, in an individual in need thereof. In one example, In vitro generated HBPOs may be collected and transplanted into the subscapular site of kidney or mesentery of the small intestine in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. An increase in growth of the organoid may be observed at 8 weeks after the transplant. The viability and growth can be monitored based on blood tests such as human-Alb, human-C-peptide levels.
In one aspect, a method of treating an individual, comprising transplanting an HBPO as described herein is disclosed. In this aspect, an organoid produced according to any of the methods used herein, may be transplanted into an individual in need thereof, using standard surgical procedures as known in the art. In one aspect, the individual may have a disease state selected from hepatic disease, such as liver failure and congenital liver diseases, pancreatic disease, such as diabetes, or biliary disease.
In one aspect, methods of identifying a treatment for one or more disease states selected from a biliary inflammatory disease and/or a pancreatic inflammatory disease such as pancreatitis is disclosed. In this aspect, the disclosed organoid compositions may be contacted with a potential therapeutic agent of interest with an organoid composition as disclosed herein. A measure of organ activity may then be detected in the organoid. Based on the output of this detection, it may be determined whether the potential therapeutic agent of interest improves the measure of the disease state, and may thereby be used to identify therapeutic agents likely to be useful in treating a disease state which involves a malfunction of any of the tissues embodied in the HBPO.
Pluripotent Stem Cells Derived from Embryonic Cells
In some aspects, one step may include obtaining stem cells that are pluripotent or that can be induced to become pluripotent. In some aspects, pluripotent stem cells are derived from embryonic stem cells, which are in turn derived from totipotent cells of the early mammalian embryo and are capable of unlimited, undifferentiated proliferation in vitro. Embryonic stem cells are pluripotent stem cells derived from the inner cell mass of the blastocyst, an early-stage embryo. Methods for deriving embryonic stem cells from blastocytes are well known in the art. Human embryonic stem cells H9 (H9-hESCs) are used in the exemplary embodiments described in the present application, but it would be understood by one of skill in the art that the methods and systems described herein are applicable to any stem cells.
Additional stem cells that can be used in embodiments in accordance with the present invention include but are not limited to those provided by or described in the database hosted by the National Stem Cell Bank (NSCB), Human Embryonic Stem Cell Research Center at the University of California, San Francisco (UCSF); WISC cell Bank at the Wi Cell Research Institute; the University of Wisconsin Stem Cell and Regenerative Medicine Center (UW-SCRMC); Novocell, Inc. (San Diego, Calif.); Cellartis AB (Goteborg, Sweden); ES Cell International Pte Ltd (Singapore); Technion at the Israel Institute of Technology (Haifa, Israel); and the Stem Cell Database hosted by Princeton University and the University of Pennsylvania. Exemplary embryonic stem cells that can be used in embodiments in accordance with the present invention include but are not limited to SA01 (SA001); SA02 (SA002); ES01 (HES-1); ES02 (HES-2); ES03 (HES-3); ES04 (HES-4); ES05 (HES-5); ES06 (HES-6); BG01 (BGN-01); BG02 (BGN-02); BG03 (BGN-03); TE03 (13); TE04 (14); TE06 (16); UCO1 (HSF1); UC06 (HSF6); WA01 (H1); WA07 (H7); WA09 (H9); WA13 (H13); WA14 (H14). More details on embryonic stem cells can be found in, for example, Thomson et al., 1998, “Embryonic Stem Cell Lines Derived from Human Blastocysts,” Science 282 (5391):1145-1147; Andrews et al., 2005, “Embryonic stem (ES) cells and embryonal carcinoma (EC) cells: opposite sides of the same coin,” Biochem Soc Trans 33:1526-1530; Martin 1980, “Teratocarcinomas and mammalian embryogenesis,”. Science 209 (4458):768-776; Evans and Kaufman, 1981, “Establishment in culture of pluripotent cells from mouse embryos,” Nature 292(5819): 154-156; Klimanskaya et al., 2005, “Human embryonic stem cells derived without feeder cells,” Lancet 365 (9471): 1636-1641; each of which is hereby incorporated herein in its entirety.
Induced Pluripotent Stem Cells (iPSCs)
In some aspects, iPSCs are derived by transfection of certain stem cell-associated genes into non-pluripotent cells, such as adult fibroblasts. Transfection may be achieved through viral vectors, such as retroviruses. Transfected genes include the master transcriptional regulators Oct-3/4 (Pouf51) and Sox2, although it is suggested that other genes enhance the efficiency of induction. After 3-4 weeks, small numbers of transfected cells begin to become morphologically and biochemically similar to pluripotent stem cells, and are typically isolated through morphological selection, doubling time, or through a reporter gene and antibiotic selection. As used herein, iPSCs include but are not limited to first generation iPSCs, second generation iPSCs in mice, and human induced pluripotent stem cells. In some aspects, a retroviral system is used to transform human fibroblasts into pluripotent stem cells using four pivotal genes: Oct3/4, Sox2, Klf4, and c-Myc. In alternative aspects, a lentiviral system is used to transform somatic cells with OCT4, SOX2, NANOG, and LIN28. Genes whose expression are induced in iPSCs include but are not limited to Oct-3/4 (e.g., Pou5fl); certain members of the Sox gene family (e.g., Sox1, Sox2, Sox3, and Sox15); certain members of the Klf family (e.g., Klf1, Klf2, Klf4, and Klf5), certain members of the Myc family (e.g., C-myc, L-myc, and N-myc), Nanog, and LIN28.
In some aspects, non-viral based technologies are employed to generate iPSCs. In some aspects, an adenovirus can be used to transport the requisite four genes into the DNA of skin and liver cells of mice, resulting in cells identical to embryonic stem cells. Since the adenovirus does not combine any of its own genes with the targeted host, the danger of creating tumors is eliminated. In some aspects, reprogramming can be accomplished via plasmid without any virus transfection system at all, although at very low efficiencies. In other aspects, direct delivery of proteins is used to generate iPSCs, thus eliminating the need for viruses or genetic modification. In some embodiment, generation of mouse iPSCs is possible using a similar methodology: a repeated treatment of the cells with certain proteins channeled into the cells via poly-arginine anchors was sufficient to induce pluripotency. In some aspects, the expression of pluripotency induction genes can also be increased by treating somatic cells with FGF2 under low oxygen conditions.
More details on embryonic stem cells can be found in, for example, Kaji et al., 2009, “Virus free induction of pluripotency and subsequent excision of reprogramming factors,” Nature 458:771-775; Woltjen et al., 2009, “piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells,” Nature 458:766-770; Okita et al., 2008, “Generation of Mouse Induced Pluripotent Stem Cells Without Viral Vectors,” Science 322(5903):949-953; Stadtfeld et al., 2008, “Induced Pluripotent Stem Cells Generated without Viral Integration,” Science 322(5903):945-949; and Zhou et al., 2009, “Generation of Induced Pluripotent Stem Cells Using Recombinant Proteins,” Cell Stem Cell 4(5):381-384; each of which is hereby incorporated herein in its entirety.
In some aspects, exemplary iPS cell lines include but not limited to iPS-DF19-9; iPS-DF19-9; iPS-DF4-3; iPS-DF6-9; iPS(Foreskin); iPS(IMR90); and iPS(IMR90).
More details on the functions of signaling pathways relating to DE development can be found in, for example, Zorn and Wells, 2009, “Vertebrate endoderm development and organ formation,” Annu Rev Cell Dev Biol 25:221-251; Dessimoz et al., 2006, “FGF signaling is necessary for establishing gut tube domains along the anterior-posterior axis in vivo,” Mech Dev 123:42-55; McLin et al., 2007, “Repression of Wnt/β-catenin signaling in the anterior endoderm is essential for liver and pancreas development. Development,” 134:2207-2217; Wells and Melton, 2000, Development 127:1563-1572; de Santa Barbara et al., 2003, “Development and differentiation of the intestinal epithelium,” Cell Mol Life Sci 60(7): 1322-1332; each of which is hereby incorporated herein in its entirety.
Any methods for producing definitive endoderm from pluripotent cells (e.g., iPSCs or ESCs) are applicable to the methods described herein. In some aspects, pluripotent cells are derived from a morula. In some aspects, pluripotent stem cells are stem cells. Stem cells used in these methods can include, but are not limited to, embryonic stem cells. Embryonic stem cells can be derived from the embryonic inner cell mass or from the embryonic gonadal ridges. Embryonic stem cells or germ cells can originate from a variety of animal species including, but not limited to, various mammalian species including humans. In some aspects, human embryonic stem cells are used to produce definitive endoderm. In some aspects, human embryonic germ cells are used to produce definitive endoderm. In some aspects, iPSCs are used to produce definitive endoderm.
In one aspect, the definitive endoderm may be derived from a precursor cell selected from an embryonic stem cell, an embryonic germ cell, an induced pluripotent stem cell, a mesoderm cell, a definitive endoderm cell, a posterior endoderm cell, a posterior endoderm cell, and a hindgut cell. In one aspect, the definitive endoderm may be derived from a pluripotent stem cell. In one aspect, the definitive endoderm may be derived from a pluripotent stem cell selected from an embryonic stem cell, an adult stem cell, or an induced pluripotent stem cell. In one aspect, the DE may be a DE monolayer, wherein greater than 90% of the cells in the DE monolayer co-express FOXA2 and SOX17.
In one aspect, the definitive endoderm may be derived from contacting a pluripotent stem cell with one or more molecules selected from Activin, the BMP subgroups of the TGF-beta superfamily of growth factors; Nodal, Activin A, Activin B, BMP4, Wnt3a, and combinations thereof.
In one aspect, the BMP signaling pathway inhibitor may be selected from Noggin, Dorsomorphin, LDN193189, DMH-1, and combinations thereof. In one aspect, the BMP signaling pathway inhibitor is Noggin. The BMP inhibitor may be present at a concentration of between from about 50 to about 1500 ng/ml.
In one aspect, the WNT signaling pathway activator may be selected from one or more molecules selected from the group consisting of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, a GSKβ inhibitor (e.g., CHIR99021, i.e. “CHIRON”), BIO, LY2090314, SB-216763, lithium, SFRP inhibitor WAY-316606, beta-catenin activator DCA. The concentration of the Wnt pathway activator may be, for example, used at a concentration between about 50 to about 1500 ng/ml. There are many ways to activate the Wnt/beta-catenin pathway (see http://web.stanford.edu/group/nusselab/cgi-bin/wnt/). Suitable Some existing wnt signaling pathway activators include but are not limited to protein-based activators, which may include Wnt ligands including but not limited to Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt8, et al; modifiers of Wnt ligand activity including but not limited to activated Wnt frizzled receptors, (LRP) co-receptors, R-spondin proteins, Dkk proteins, regulators of Wnt ligand secretion and trafficking (Wntless, Porcupine), inhibiting beta-catenin degredation APC and GSK3beta inhibition, activated beta-catenin, constitutively active TCF/Lef proteins and chemical activators, which may include over 28 known chemicals that either activate or inhibit Wnt/beta-catenin signaling. Some activators include but are not limited to GSK3-beta inhibitors CHIR99021 (CHIRON), BIO, LY2090314, SB-216763, lithium, SFRP inhibitor WAY-316606, beta-catenin activator DCA.
In one aspect, the FGF signaling pathway activator may be one or more molecules selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, and combinations thereof, preferably FGF4 or FGF10, or a combination thereof. In one aspect, the concentration of the FGF pathway activator may be used at a concentration between about 50 to about 1500 ng/ml, though it will be understood by one of ordinary skill in the art that various concentrations may be used. Proteins and chemicals that stimulate the FGF receptor and signaling components downstream of the receptors including MAPK, MEK, ERK proteins and chemicals that modulate their activity. FGF signaling can be activated by inhibiting inhibitors of FGF signaling pathways including but not limited to Sprouty protein family members.
The following non-limiting examples are provided to further illustrate embodiments of the invention disclosed herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches that have been found to function well in the practice of the invention, and thus may be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes may be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Organogenesis is a complex and inter-connected process, orchestrated by multiple boundary tissue interactions1-7. However, it is currently unclear how individual, neighboring components coordinate to establish an integral multi-organ structure. Disclosed herein is the continuous patterning and dynamic morphogenesis of hepatic, biliary and pancreatic structures, invaginating from a three-dimensional culture of human pluripotent stem cell (PSC).
Applicant has found that the boundary interactions between anterior and posterior gut spheroids differentiated from human PSCs enables autonomous emergence of hepato-biliary-pancreatic (HBP) organ domains specified at the foregut-midgut boundary organoids in the absence of extrinsic factor supply. Whereas transplant-derived tissues were dominated by midgut derivatives, long-term culture of micro dissected HBP organoids develop into a segregated hepato-pancreato-biliary anlage, followed by the recapitulation of early morphogenetic events including the invagination and branching of three different and inter-connected organ structures, reminiscent of tissues derived from mouse explanted foregut-midgut culture. Mis-segregation of multi-organ domains incurred by a genetic mutation in HES1 abolishes the biliary specification potential in culture, as seen in vivo8,9. Applicant has demonstrated that the experimental multi-organ integrated model can be established by the juxta-positioning of foregut and midgut tissues, and which may serve as a tractable, manipulatable and easily accessible model for the study of complicated endoderm organogenesis in human.
The hepato-biliary-pancreatic (HBP) anlage, which is demarcated by HHEX (Hematopoietically-expressed homeobox protein) and PDX1 (Pancreatic and duodenal homeobox 1) expression is first specified at the boundary between the foregut-midgut10, and forms an epithelial vesicle invaginating ventrally from the primitive gut11-13. The disruption of boundary-defining genes around this area such as BMPR1A (Bone morphogenetic protein receptor, type 1A)1, HLX (H2.0-like homeobox)2, CDX2 (Caudal type homeobox 2)3, NKX3-2 (NK3 Homeobox 2)4, HHEX5, PDX1 and SOX9 (SRY-box 9)6 significantly alters balanced organogenesis along the stomach-HBP-intestine in vivo1-7. Subsequent diversification of HBP lineages is likely mediated by adjacent mesenchymal BMP at their boundary by indirectly repressing SOX9 in the posterior liver bud cells14. Thus, contiguous, dynamic organogenesis occurs in a complex environment and is likely driven by successive neighboring tissue interactions5,6,15. However, the patterning and balanced organogenesis of the HBP system has not been successfully modelled in tissue culture due to technical complexities, hindering detailed mechanistic studies16,17.
Here, Applicant used a three-dimensional differentiation approach using human pluripotent stem cells (PSCs) to specify gut spheroids with distinct regional identities comprised of both endoderm and mesoderm. Applicant demonstrated that antero-posterior interactions recapitulate the foregut (marked by SOX2, SRY-Box 2) and the midgut (marked by CDX2) boundary in vitro, modeling the inter-coordinated specification and invagination of the human hepato-biliary-pancreatic system.
To develop a foregut-midgut boundary model, Applicant first exposed definitive endoderm cells, which were patterned as previously described 18,19, to the recombinant protein FGF4, and the small molecule CHIR99021 (that modulates the WNT pathway) in the presence of a BMP antagonist Noggin for anterior gut and in the absence of Noggin for posterior gut identity (
To trace the source of HHEX and PDX1 expressing cells, the bAP organoids were established using non-labeled anterior and GFP-labeled posterior gut spheroids. Applicant found that both HHEX and PDX1 expression overlapped with GFP, suggesting that the HBP progenitors originate from the posterior gut (
Applicant further established a reporter human iPSC to track their fate by visualizing tdTomato under the common progenitor marker of the liver, bile duct, and pancreas, a prospero-related homeobox 1 (PROX1) using CRISPR/Cas9 genome editing (
To delineate the HBP progenitor self-inductive mechanism, Applicant evaluated the boundary specific expression profiles of known inductive signaling pathways. Amongst FGF, BMP, HH (hedgehog), NOTCH and RA (retinoic acid) signals, RNAseq identified that the signal downstream of RA were activated prominently at the boundary region, but not in the anterior or posterior regions at D11 (
In support, Applicant exposed the bAP organoids at D9 to various RA signaling agonists or antagonists. The RA receptor antagonist BMS493 strongly suppressed the gene expression of both HHEX and PDX1 (
It has been noted that stem cell-derived embryonic endodermal cells are highly plastic and usually generate intestinal tissues18,25. To examine whether there is an ability to form HBP tissues from progenitors in vivo, transplantations of human PROX1 expressing organoids into immunodeficient mice were performed. One month transplant derived tissues exhibited the small intestinal tissue markers Keratin 20 (CK20), CDX2, and EpCAM, but negligible expression of the other HBP markers (
Because the HBP organoids generated predominantly duodenum tissue in vivo, Applicant next excised PROX1 positive regions from the D13 boundary organoids and cultured them in different formats to effectively model HBP organogenesis (
To determine whether longer-term culture can produce more mature tissue, organoids were cultured until D90. The D90 organoids were morphologically similar to mouse E14.5 explanted organ culture (
HES1 (Hes family bHLH transcription factor 1) is a transcription factor that regulates the posterior foregut lineage15,27. In Hes1 knock-out rodents, conversion of the biliary system to pancreatic tissue occurs due to failed pancreato-biliary organ segregation8,9. To elucidate whether the HBP organoid recapitulates HES1 mediated developmental process, Applicant established HES1 KO on PROX1 reporter iPSCs by CRISPR/Cas9 system (
Multi-organ integration in stem cell culture is a critical unmet challenge. The instant disclosure demonstrates the generation of a human three-dimensional antero-posterior boundary system that leads to structurally and functionally integrated HBP organoids developed at foregut-midgut border.
Methods
Maintenance of PSCs
Human PSC lines were maintained as described previously. Undifferentiated hPSCs were maintained on feeder-free conditions in mTeSR1 medium (StemCell technologies, Vancouver, Canada) or Stem Fit medium (Ajinomoto Co, Japan) on plates coated either with Matrigel Growth Factor Reduced (Corning Inc., New York, N.Y., USA) at 1/30 dilution or iMatrix-511 (Nippi, Japan) at 0.25 ug/cm2 in an incubator with 5% CO2/95% air at 37° C.
Differentiation of PSCs into Anterior and Posterior Gut Spheroid
Differentiation of hPSCs into definitive endoderm was induced using previously described methods18,19 with modifications. In brief, colonies of hiPSCs were isolated in Accutase (Thermo Fisher Scientific Inc., Waltham, Mass., USA) and 150,000 cells/mL were plated on Matrigel coated tissue culture plate (VWR Scientific Products, West Chester, Pa.). Medium was changed to RPMI 1640 medium (Life Technologies, Carlsbad, Calif.) containing 100 ng/mL Activin A (R&D Systems, Minneapolis, Minn.) and 50 ng/mL bone morphogenetic protein 4 (BMP4; R&D Systems) at Day 1, 100 ng/mL Activin A and 0.2% fetal calf serum (FCS; Thermo Fisher Scientific Inc.) at Day 2 and 100 ng/mL Activin A and 2% FCS at Day 3. For Day 4-7, cells were cultured in gut growth medium (Advanced DMEM/F12 (Thermo Fisher Scientific Inc.) with 15 mM HEPES, 2 mM L-glutamine, penicillin-streptomycin, B27 (Life Technologies) and N2 (Gibco, Rockville, Md.)) supplemented with 200 ng/mL noggin (NOG; R&D Systems), 500 ng/ml fibroblast growth factor 4 (FGF4; R&D Systems) and 2 μM CHIR99021 (Stemgent, Cambridge, Mass., USA) for anterior gut cell induction and supplemented with 500 ng/ml FGF 4 and 3 μM CHIR99021 for posterior gut cell induction. Cultures for cell differentiation were maintained at 37° C. in an atmosphere of 5% CO2/95% air and the medium was replaced every day.
Anterior-Posterior Boundary Spheroid Formation
On Day 7, anterior or posterior gut cells were dissociated to single cells by incubation with TrypLE Express (Life Technologies) at 37° C. Cells were centrifuged at 1000 rpm for 3 minutes and, after removing supernatant, the pellet was re-suspended in gut growth medium containing 10 uM of Y-27632 dihydrochloride (Tocris Bioscience, Bristol, United Kingdom). The anterior or posterior gut cell suspensions were plated on 96 well round bottom ultra-low attachment plate (Corning Inc) at density of 10,000 cells/well and incubated at 37° C. for 24 hours to form spheroid. On Day 8, generated single anterior gut spheroid and posterior gut spheroid were mixed on 96 well round bottom ultra-low attachment plate in gut growth medium for 24 hours to form fused boundary spheroids (A-P spheroids).
Hepato-Biliary-Pancreatic (HBP) Organoid Culture and Transplantation
On Day 9, A-P spheroids were embedded in Matrigel drop and were cultured in gut growth medium to generate multi-organ HBP organoids. For longer-term culture, HBP organoids were dissected and/or transferred to transwell for air-liquid interface culture at Day 13. Cultures for spheroid were maintained at 37° C. in an atmosphere of 5% CO2/95% air and the gut growth medium were replaced every 4 days. Single HBP organoid at Day 13 was transplanted into the subcapsule of the kidney in male immune deficient NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) mice, aged 12 weeks old. All experiments were performed under the approval of the Institutional Animal Care and Use Committee of CCHMC (protocols IACUC2018-0096).
H&E Staining and Immunohistochemistry
Spheroid and organoid were collected from Matrigel, fixed in 4% paraformaldehyde (PFA) and embedded in paraffin. Sections were subjected to H&E and immunohistochemical staining. The primary antibodies were listed in Table 1. Immunohistochemical staining was performed by using ultraView Universal DAB Detection Kit (Roche Diagnostics, Basel, Switzerland). The specimens were observed under a microscope.
For whole mount immunohistochemical staining, spheroid and organoid were collected from Matrigel and removed remaining Matrigel by treating with Cell recovery solution at 4° C. for 30 min. The tissues were washed by PBS and were fixed in 4% PFA at 4° C. for overnight. The fixed samples were treated by 4% PFA with 0.5% Triton X100 at room temperature for 15 min and permeabilized with 0.1% Tween 20 (Sigma) at room temperature for 15 min. The samples were treated with blocking solution (1% BSA, 0.3% Triton X100) at room temperature for 1 hour and were incubated overnight at 4° C. with the primary antibodies diluted in blocking solution. After washing, fluorescent dye-conjugated secondary antibodies were applied to the samples at room temperature for 2 hours. The primary and secondary antibodies are listed in Table 1. After the secondary antibody reaction, the samples were washed three times. Nuclei were stained with DAPI mounting solution.
The stained section and whole mount samples were observed under a Nikon A1Rsi inverted confocal microscope.
RNA Isolation, RT-qPCR
RNA was isolated using the RNeasy mini kit (Qiagen, Hilden, Germany). Reverse transcription was carried out using the SuperScript IV First-Strand Synthesis System for RT-PCR (Invitrogen, CA, USA) according to manufacturer's protocol. qPCR was carried out using TaqMan gene expression master mix (Applied Biosystems) on a QuantStudio 3 Real-Time PCR System (Thermo). All primers and probe information for each target gene was obtained from the Universal ProbeLibrary Assay Design Center (https://qper.probefinder.com/organism.jsp) and listed in Table 2.
RNA Sequencing
Sample preparation for RNA sequencing was performed using SMART-seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech Laboratories) according manufacture's user manual.
Briefly, First-strand cDNA synthesis was primed by the 3′ SMART-seq CDS Primer II A and uses the SMART-Seq v4 Oligonucleotide for template switching at the 5′ end of the transcript. PCR Primer II A amplified cDNA from the SMART sequences introduced by 3′ SMART-Seq CDS Primer II A and the SMART-Seq v4 Oligonucleotide by PCR. PCR-amplified cDNA was purified by immobilization on AMPure XP beads. The beads were then washed with 80% ethanol and cDNA was eluted with Elution Buffer. Amplified cDNA was validated using the Agilent 2100 Bioanalyzer and Agilent's High Sensitivity DNA Kit (Agilent) according Kit User Manual. The full-length cDNA output of the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing was processed with the Nextera XT DNA Library Preparation Kit (Illumina).
The RNA profiles were compiled with Kallisto software and expressed as transcripts per million (TPM). The data sets filtered by the threshold requiring greater than 0 in at least 1 sample were first subjected to gene functional classification based on gene sets related to FGF, BMP, Hedgehog, NOTCH and RA signaling pathway. The lists of gene sets were acquired from Molecular Signatures Database (ver 6.2). Unbiased cluster analysis for filtered data sets performed by using Cluster 3.0 software. Detail of gene sets included in each cluster were listed in Table 3.
Flow Cytometry.
For flow cytometry, Anterior gut cells were differentiated from GFP-Labeled iPSCs and posterior gut cells were differentiated from mCherry-Labeled iPSCs. At day 13, A-P spheroid were dissociated to single cells by the treatment of TrypLE Express for 10 min at 37° C. After PBS wash, the single cells were incubated with BV421-conjugated EpCAM antibody (BioLegend) at room temperature for 30 min. After PBS wash, cell sorting was performed by BD FACS AriaII (BD Biosciences). Analysis was performed by BD FACS DIVA software and FlowJo (FlowJo, LLC).
CRISPR Editing
The plasmid encoding Cas9-2A-GFP was acquired from addgene (#44719, doi: 10.1016/j.stem.2013.03.006). Guide RNA targeting the N-terminus of PROX1 or HES1 was synthesized by Integrated DNA Technologies, cloned into the pGL3-U6-sgRNA-PGK-puromycin vector (addgene #51133, doi: 10.1038/nmeth.2857) and sequenced using the RV3 universal primer. To construct the HDR template, homology arms flanking the PROX1 start codon were independently amplified from genomic DNA and then fused to tdTomato via overlap extension PCR using the high-fidelity taq polymerase iProof (Bio-Rad). The resulting PCR product was then cloned into the pCR-Blunt II-TOPO cloning vector (Invitrogen) and confirmed by Sanger sequencing.
Human iPSCs were transfected with 2 μg of each plasmid using the Lipofectamine 3000 following the manufacturer's instructions. Twenty-four hours after transfection, cells were sorted by GFP expression to select for positively transfected cells. Clonal cells were expanded for 2 weeks and screened for inserted PROX1-tdTomato or depleted HES1 exon1 sequence and karyotyped.
Amylase ELISA.
To measure Amylase secretion level of organoids, 200 pL of culture supernatant was collected from organoid embedded in Matrigel. The culture supernatants were collected at 48 hrs time points after the culture and stored at −80° C. until use. The supernatant was centrifuged at 1,500 rpm for 3 min and to pellet debris, and the resulting supernatant was assayed with Human Amylase ELISA Kit according to the manufacturer's instructions.
Statistics and Reproducibility.
Statistical analysis was performed using unpaired two-tailed Student's t-test, Dunn-Holland-Wolfe test, or Welch's t-test. Results were shown mean±s.d.; P values<0.05 were considered statistically significant. N-value refers to biologically independent replicates, unless noted otherwise. For comparisons between unpaired 2 groups, non-parametric Brunner-Munzel test was performed when 2 samples were independent, and the variances of the samples were unequal. For comparisons between more than 2 samples, non-parametric Kruskal-Wallis and post hoc Dunn-Holland-Wolf test were performed.
Mouse Whole Embryo Culture
The culturing of the multi-organ three-dimensional organoid may be carried out using a whole embryo culture system (Ikemoto Scientific Technology, Tokyo, Japan) with improved growth. The multi-organ three-dimensional organoid may be transferred into a sterile glass roller bottle containing culture media and the flask closed by silicon plug. The number of transferred organoids depends on how long they will be cultured. The culture bottles may be attached to a rotator drum and rotated at 20 rpm and 37° C. in the dark while being continuously supplied with a gas mixture (mainly 20% O2, 5% CO2 and 75% N2, but the O2 concentration can be modified with balanced with N2). The organoids can be cultured for a few weeks (with the longest trial being 10 weeks). The culture media is changed every 5-7 days and the viability and growth can be monitored based on the organoid length and media components such as Alb, Amy, C-peptide levels.
The Rotator-type Bottle Culture System (Ikemoto Scientific Technology Co., Ltd) was used for whole embryo culture. E9.0 Prox1-GFP mouse embryo was dissected and transfer to culture bottle with Advanced DMEM/F12 supplemented B27 and N2 supplements. The temperature inside the whole embryo culture system was kept at 37.0° C.
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All percentages and ratios are calculated by weight unless otherwise indicated.
All percentages and ratios are calculated based on the total composition unless otherwise indicated.
It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.
The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as “20 mm” is intended to mean “about 20 mm.”
Every document cited herein, including any cross referenced or related patent or application, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.
While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications may be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
This application claims priority to and benefit of 62/703,559, entitled “Modeling hepato-biliary-pancreatic organogenesis from the foregut-midgut boundary in humans,” filed Jul. 26, 2019, the contents of which are incorporated in their entirety for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/041985 | 7/16/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62703559 | Jul 2018 | US |
