Pancreatic organoids and uses thereof

Abstract

Disclosed herein are pancreatic organoids, including a posterior foregut cell population, and a vascular mesenchyme recombinant cell population and / or a splanchnic mesenchyme recombinant cell population. In particular embodiments, the pancreatic organoids include a vascular mesenchyme recombinant cell population and a splanchnic mesenchyme recombinant cell population. Also disclosed are methods of making and using the pancreatic organoids of the disclosure.

Description

PANCREATIC ORGANOIDS AND USES THEREOFPRIORITY

[0001] The present application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 669,081, PANCREATIC ORGANOIDS AND USES THEREOF IN MODELING HUMAN DEVELOPMENT, filed on filed July 9, 2024, which is currently co-pending herewith and which is incorporated by reference in its entirety.FIELD OF THE INVENTION

[0002] Aspects of the present disclosure generally relate to pancreatic organoids, methods of preparation, and compositions including the same, as well as uses thereof.BACKGROUND

[0003] The organs of the gastrointestinal tract are assembled from cells that derive from the three primary germ layers. The human pancreas is a branching structure with endocrine, ductal and exocrine structures, all surrounded by supporting cells like blood vessels and mesenchymally-derived fibroblasts.

[0004] The development of the pancreas is a complex process that relies on sequential interactions between the pancreatic epithelium and a variety of mesodermal cell types, including splanchnic mesenchyme and vascular endothelial cells. The pancreas derives from a region of the developing gut tube just distal to the stomach and first emerges as two buds from the posterior foregut that undergoing extensive growth, branching and differentiation through islet delamination and peripheral acini formation. This complex morphogenesis process results in the pancreas, with a ductal network connected to acini that secrete digestive enzymes and endocrine islets that secrete hormones.

[0005] Fueled by the promise of cell therapy for diabetes, much progress has been made in directing the differentiation of human pluripotent stem cells into endocrine cell clusters that secrete insulin. In contrast, recapitulating the complex morphogenetic processes of ductal and exocrine development of the pancreas in vitro has not been achieved to date.SUMMARY

[0006] Various embodiments of the disclosure relate to in vitro methods of preparing pancreatic organoids (PO). In some embodiments, the methods include: a) dissociating posterior splanchnic mesenchyme, vascular mesenchyme, and posterior foregut cells to respective single cell suspensions; b) recombining and aggregating the single cell suspensions of posterior splanchnic mesenchyme and / or vascular’ mesenchyme with the single cell suspension of posterior foregut cells; and c) culturing the recombined and aggregated single cell suspensions of posterior splanchnic mesenchyme and / or vascular mesenchyme with the single cell suspension of posterior foregut cells, while inhibiting a ROCK pathway, for a first period of time, to provide a precursor pancreatic organoid.

[0007] In some embodiments, the methods further include: d) culturing the precursor pancreatic organoid while activating and / or inhibiting one or more signaling pathways, for a second period of time, to provide an pancreatic organoid with committed lineage. In some embodiments, the methods further include: d) culturing the precursor pancreatic organoid without activating and / or inhibiting one or more signaling pathways, for a second period of time, to provide a pancreatic organoid with variably committed lineage. In some embodiments, the methods further include: e) culturing the pancreatic organoid without activating and / or inhibiting one or more signaling pathways, for a third period of time, to provide a cultured pancreatic organoid.

[0008] In some embodiments, the precursor pancreatic organoid is cultured in an inductive medium including growth factors and / or signaling pathway activators and / or inhibitors, to provide a pancreatic organoid with committed lineage. In some embodiments, the precursor pancreatic organoid is cultured in an inductive medium, including posterior foregut medium, classical gut medium, and / or basal mesenchyme differentiation medium; optionally wherein the inductive medium includes SANT-1, retinoic acid, TPB, LDN and / or KGF. In some embodiments, the precursor pancreatic organoid is cultured in a permissive medium, in the absence of growth factors and / or signaling pathway activators and / or inhibitors, to provide a pancreatic organoid with variably committed lineage.

[0009] In some embodiments, the single cell suspension of posterior foregut cells is cultured with single cell suspensions of both posterior splanchnic mesenchyme and vascular mesenchyme. In some embodiments, the single cell suspension of posterior foregut cells is cultured with a single cell suspension of vascular mesenchyme, and the pancreatic organoid includes a majority of pancreatic-like cell population. In some embodiments, the single cellsuspension of posterior foregut cells is cultured with a single cell suspension of posterior splanchnic mesenchyme, and the PO includes one or more stomach-pancrcas boundary cell population.

[0010] In some embodiments, the single cell suspensions of posterior splanchnic mesenchyme and vascular’ mesenchyme are recombined with the single cell suspension of posterior foregut cells in a ratio of at least about 1:1 to 10:1, or greater, mesenchymal cells to posterior foregut cells; optionally at least about 2:1 to 6:1; optionally about 4:1. In some embodiments, the posterior splanchnic mesenchyme and vascular mesenchyme are recombined with the single cell suspension of posterior foregut cells in a ratio of at least about 1:10 to 10:1, splanchnic mesenchymal cells to vascular mesenchymal cells; optionally at least about 5:1 to 1:5; optionally at least about 2:1 to 1:2; optionally about 1:1. In some embodiments, the single cell suspensions of posterior splanchnic mesenchyme and vascular mesenchyme are recombined with the single cell suspension of posterior foregut cells to provide at least about 1,000 to 100,000 cells / organoid; optionally at least about 2,000 to 50,000 cells / organoid; optionally about 10,000 cells / organoid.

[0011] In some embodiments, the first period of time is at least about 1 day, or longer; optionally at least about 1 day to 3 days, 1 day to 5 days, or longer; optionally at least about 1 day. In some embodiments, the second period of time is selected from at least about 1 day to 10 days, or longer; optionally at least about 1 day to 7 days; optionally at least about 7 days. In some embodiments, the third period of time is at least about 1 day, or longer; optionally 1 day to to 7 days, 1 day to 14 days, 1 day to 60 days, 1 day to 70 days, or longer; optionally at least about 1 day, 7 days, 60 days, 70 days, or longer.

[0012] In some embodiments, the posterior foregut cells are derived from definitive endoderm, and the posterior splanchnic mesenchyme and vascular mesenchyme are derived from pluripotent stem cells. In some embodiments, the posterior foregut cells are derived from definitive endoderm, which has been derived from pluripotent stem cells. In some embodiments, the pluripotent stem cells comprise embryonic stem cells or induced pluripotent stem cells. In some embodiments, the posterior splanchnic mesenchyme, vascular mesenchyme, and / or posterior foregut cells are derived from pluripotent stem cells; optionally embryonic stem cells or induced pluripotent stem cells; optionally human induced pluripotent stem cells.

[0013] In some embodiments, the posterior foregut cells express PDX1; and / or the splanchnic mesenchyme expresses vimentin and / or FOXF1; and / or the vascular mesenchymeexpresses F0XF1 , S0X17, and / or PECAM. In some embodiments, the precursor pancreatic organoid is cultured in an inductive medium, and the pancreatic organoid includes a region including one or more pancreatic-like cell population. In some embodiments, the precursor pancreatic organoid is cultured in an inductive medium, and the pancreatic organoid expresses cytokeratin-19, glucagon, c-peptide, neurogenin3, chromogranin A, claudin-18, E-cadherin, vimentin, PECAM, GATA4, PDX1, NKX6.1, FOXF1, WT-1, Ki67, and / or SOX9. In some embodiments, the precursor pancreatic organoid is cultured in a permissive medium, and wherein the pancreatic organoid includes a region including one or more pancreatic-like cell population, and one or more intestinal-like, duodenum-like, and / or stomach-like cell population; and / or and the pancreatic organoid expresses claudin-18, MUC5AC, SOX2, CDX2, and / or CDH17.

[0014] In some embodiments, the cells self-sort, or self-assemble, into two or more regions. In some embodiments, the cells self-sort, or self-assemble, into an inner region including pancreatic epithelium, or into an outer region including mesenchyme and / or vascular endothelium.

[0015] In some embodiments, dissociation includes enzymatic dissociation and / or mechanical dissociation. In some embodiments, enzymatic dissociation includes dissociating the organoid with trypsin, chymotrypsin, collagenase, papain, hyaluronidase, elastase, thermolysin, neutral protease, or any combination thereof; and / or mechanical dissociation includes manual dissociation and / or passing the organoid through successively narrower bore channels. In some embodiments, epithelial cells and / or mesenchymal cells are dissociated from the organoid by using an extracellular matrix depolymerization solution.

[0016] In some embodiments of the methods, steps a) through c) are performed in suspension culture. In some embodiments, steps d) and / or e) are performed in suspension culture. In some embodiments, the pancreatic organoid and / or precursor pancreatic organoid is in suspension culture. In some embodiments, the pancreatic organoid and / or precursor pancreatic organoid is free of extracellular matrix. In some embodiments, the pancreatic organoid is embedded in a basement membrane matrix.

[0017] Some embodiments of the methods further include providing posterior splanchnic mesenchyme, including: a) activating a TGF-b signaling pathway, activating a BMP signaling pathway, activating a Wnt signaling pathway, activating an FGF signaling pathway, and activating a PI3K signaling pathway, in dissociated pluripotent stem cells, to generate mid primitive streak cells; b) activating a TGF-b signaling pathway, activating a BMP signalingpathway, activating a retinoic acid signaling pathway, and activating a Wnt signaling pathway, in the mid primitive streak cells of step a), to generate posterior foregut lateral plate mesoderm; and c) activating a TGF-b signaling pathway, activating a BMP signaling pathway, activating a retinoic acid signaling pathway, activating a Wnt signaling pathway, and activating an FGF signaling pathway, in the posterior foregut lateral plate mesoderm of step b), to generate posterior splanchnic mesenchyme. Some embodiments of the methods include providing posterior splanchnic mesenchyme, including: a) contacting dissociated pluripotent stem cells with one or more TGF-b signaling pathway activator, BMP signaling pathway activator, Wnt signaling pathway activator, FGF signaling pathway activator, and PI3K signaling pathway activator, to generate mid primitive streak cells; b) contacting the mid primitive streak cells of step a) with one or more TGF-b signaling pathway activator, BMP signaling pathway activator, retinoic acid signaling pathway activator, and Wnt signaling pathway activator, to generate posterior foregut lateral plate mesoderm; and c) contacting the posterior foregut lateral plate mesoderm of step b) with one or more TGF-b signaling pathway activator, BMP signaling pathway activator, retinoic acid signaling pathway activator, Wnt signaling pathway activator, and FGF signaling pathway activator, to generate posterior splanchnic mesenchyme.

[0018] Some embodiments of the methods further include providing vascular mesenchyme, including: a) inhibiting a ROCK signaling pathway in dissociated pluripotent stem cells in aggregation media, to generate aggregates; b) activating a Wnt signaling pathway, and activating a BMP signaling pathway, in the aggregates of step a), to induce mesoderm formation; and c) activating a VEGF signaling pathway, and activating a cAMP signaling pathway, in the mesoderm of step b), to induce vascular mesenchyme formation. In some embodiments, the methods include providing vascular mesenchyme, including: a) contacting dissociated pluripotent stem cells in aggregation media with one or more ROCK signaling pathway inhibitor, to generate aggregates; b) contacting the aggregates of step a) with one or more Wnt signaling pathway activator, and BMP signaling pathway activator, to induce mesoderm formation; and c) contacting the mesoderm of step b) with one or more VEGF signaling pathway activator, and cAMP signaling pathway activator, to induce vascular mesenchyme formation.

[0019] Some embodiments of the methods further include providing posterior foregut cells, including: a) activating a TGF-b signaling pathway activator and activating a Wnt signaling pathway, for a first period of time, in definitive endoderm cells (DE); b) activating a TGF-b signaling pathway activator without activating a Wnt signaling pathway, for a secondperiod of time, in the cells of step a); and c) activating an FGF signaling pathway, activating an Shh signaling pathway, and activating a retinoic acid (RA) signaling pathway, and inhibiting a BMP signaling pathway, and inhibiting a CK2 signaling pathway, in the cells of step b), thereby differentiating the DE to posterior foregut cells. Some embodiments of the methods include providing posterior foregut cells, including: a) contacting definitive endoderm cells (DE) with one or more TGF-b signaling pathway activator and Wnt signaling pathway activator, for a first period of time; b) contacting the cells of step a) with a TGF-b signaling pathway activator for a second period of time; and c) contacting the cells of step b) with one or more FGF signaling pathway activator, Shh signaling pathway activator, retinoic acid (RA) signaling pathway activator, BMP signaling pathway inhibitor, and CK2 signaling pathway inhibitor, for a third period of time, thereby differentiating the DE to posterior foregut cells.

[0020] Some embodiments of the methods further include providing posterior foregut cells, including: a) activating an FGF signaling pathway activator and activating a Wnt signaling pathway for a first period of time, with or without inhibiting a BMP signaling pathway, in definitive endoderm cells (DE); and b) activating an FGF signaling pathway, activating a Wnt signaling pathway, and activating a retinoic acid (RA) signaling pathway for a second period of time, with or without inhibiting a BMP signaling pathway, in the cells of step a), thereby differentiating the DE to posterior foregut cells. Some embodiments of the methods include: a) contacting definitive endoderm cells (DE) with one or more FGF signaling pathway activator and Wnt signaling pathway activator for a first period of time, with or without one or more BMP inhibitor; and b) contacting the cells of step a) with one or more FGF signaling pathway activator, Wnt signaling pathway activator, and retinoic acid (RA) signaling pathway activator for a second period of time, with or without one or more BMP inhibitor, thereby differentiating the DE to posterior foregut cells.

[0021] Further embodiments of the disclosure include pancreatic organoids (POs), such as in vitro pancreatic organoids, including three-dimensional pancreatic organoids. In some embodiments, the POs are obtained by in vitro expansion, and the POs include a posterior foregut cell population; and a vascular mesenchyme recombinant cell population (VMPO) and / or a splanchnic mesenchyme recombinant cell population (SMPO). Embodiments of the disclosure also include in vitro pancreatic organoids prepared by the methods described herein, where the pancreatic organoids include a posterior foregut cell population; and a vascular mesenchymerecombinant cell population (VMPO) and / or a splanchnic mesenchyme recombinant cell population (SMPO).

[0022] In some embodiments, the PO includes a posterior foregut cell population, a VMPO, and a SMPO. In some embodiments, the PO includes endocrine cells, islets or islet-like structures, and / or ductal and / or exocrine structures. In some embodiments, the PO includes endocrine pancreas and / or stratified epithelial pancreatic progenitors. In some embodiments, the PO includes an epithelial cavity or network, and one or more lumen and / or microlumen. In some embodiments, the one or more lumen and / or microlumen is adjacent to the epithelial cavity or network. In some embodiments, one or more lumen and / or microlumen is polarized. In some embodiments, the PO includes one or more branched or interconnected ductal networks and / or acing structures. In some embodiments, the acing structures comprise acinar cells and / or acinar tissue localized at one or more ductal network terminus. In some embodiments, the acinar cells and / or acinar tissue are polarized. In some embodiments, the PO lacks nerve cells and / or immune cells. In some embodiments, the PO includes immune cells. In some embodiments, the PO does not undergo anisotropic growth and / or wherein the PO lacks hierarchical vascularization.

[0023] In some embodiments, the PO expresses one or more posterior foregut and / or pancreatic progenitor marker. In some embodiments, the PO expresses cytokeratin-19, glucagon, c-peptide, neurogenin3, chromogranin A, claudin-18, E-cadherin, vimentin, PECAM, GATA4, PDX1, NKX6.1, FOXF1, WT-1, Ki67, SOX2, and / or SOX9. In some embodiments, the posterior foregut cell population expresses NKX6.1; and / or wherein the VMPO expresses PDX1 and / or E-cadherin; and / or wherein the SMPO expresses SOX2, E-cadherin, and / or claudin-18. In some embodiments, the VMPO expresses PDX1. In some embodiments, the VMPO expresses claudin-18 only in a cystic lumen region. In some embodiments, the PO includes a posterior foregut cell population and a VMPO, and the posterior foregut cell population expresses NKX6.1, SOX9, and / or PDX1. In some embodiments, the VMPO and / or SMPO expresses FOXF1 and / or WT-1. In some embodiments, the PO expresses Ki67 after culturing for 60 days, or longer. In some embodiments, the PO includes endocrine cells expressing neurogenin3 and / or chromogranin A; islets or islet-like structures expressing glucagon and / or c-peptide; and / or an epithelial network expressing cytokeratin-19. In some embodiments, the PO includes acinar cells and / or acinar tissue expressing GATA4, and downregulated expression of SOX9 and / or PDX1. In some embodiments, the PO includes a ductal network expressing SOX9 and downregulatedexpression of GATA4. In some embodiments, the PO includes a lumen expressing SOX2, surrounded by or adjacent to an epithelium expressing PDX1.

[0024] In some embodiments, the PO secretes one or more pancreatic enzymes in acinar cells, acinar tissue, lumen, and / or microlumen. In some embodiments, the PO secretes zymogen granules, CPA1, mucin-1, and / or amylase in acinar cells, acinar tissue, lumen, and / or microlumen. In some embodiments, the PO secretes CPA1 and amylase, and wherein the CPA1 and amylase secretion is co-localized. In some embodiments, the PO includes a lumen adjacent to the epithelial network, wherein the lumen secretes mucin- 1.

[0025] In some embodiments, the posterior foregut cells, mesenchymal cells, and vascular cells are derived from pluripotent stem cells. In some embodiments, the pluripotent stem cells include embryonic stem cells or induced pluripotent stem cells. In some embodiments, the posterior foregut cells, SMPO, and / or VMPO are derived from human pluripotent stem cells in vitro.

[0026] In some embodiments, the PO is embedded in a basement membrane matrix. In some embodiments, the PO is in suspension culture.

[0027] In some embodiments, the PO is an artificial pancreatic organoid and / or is generated in vitro. In some embodiments, the PO is three-dimensional. In some embodiments, the PO is a mature pancreatic organoid and / or includes pancreatic architecture and hormones.

[0028] Further embodiments of the disclosure include in vitro compositions including POs as described above. Further embodiments of the disclosure include POs as described above, or compositions including the same, for use in methods of treating a pancreas-related disease or disorder and / or a disease or disorder involving endocrinogenesis, methods of scaling up in bioprocess manufacturing, methods of screening for therapeutic efficacy, method of modeling human pancreatic development and / or disease, a method of diagnosing pancreatic-related diseases or disorders, and / or the manufacture of medicaments for treating pancreas-related diseases or disorders and / or diseases or disorders involving endocrinogenesis. Further embodiments of the disclosure include uses of POs as described above, or compositions including the same, in methods of treating pancreas-related diseases or disorders and / or diseases or disorders involving endocrinogenesis, methods of scaling up in bioprocess manufacturing, methods of screening for therapeutic efficacy, methods of modeling human pancreatic development and / or disease, methods of diagnosing pancreatic-related diseases or disorders and / or diseases or disorders involving endocrinogenesis, or the manufacture of medicaments fortreating pancreas-related diseases or disorders and / or diseases or disorders involving cndocrinogcncsis .

[0029] Further embodiments of the disclosure include methods of administering POs as described above, or compositions including the same, to a subject in need thereof. In some embodiments, the methods include administering the POs as described above, or compositions including the same, to the subject. In some embodiments, administering includes transplanting POs as described above, or compositions including the same, into the subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a mouse or a human.

[0030] In some embodiments, the PO is transplanted after culturing for 7 days to 60 days, or longer, in vitro. In some embodiments, the PO, following transplant, engrafts under the kidney capsule of the subject. In some embodiments, transplanting the PO to the subject includes organoid engraftment, tissue growth, and / or improved tissue and / or organ function. In some embodiments, the PO, following transplant, matures in vivo. In some embodiments, the PO, following transplant, generates branched epithelial tissue. In some embodiments, the PO, following transplant, includes one or more branched or interconnected ductal networks and / or differentiated acing structures. In some embodiments, the PO, following transplant, secretes pancreatic enzymes in the acinar cells, acinar tissue, ducts, lumen, and / or microlumen. In some embodiments, the PO secretes zymogen granules, CPA1, and / or amylase in the acinar cells, acinar tissue, ducts, lumen, and / or microlumen. In some embodiments, the CPA1 and amylase secretion is co-localized.

[0031] In some embodiments, the subject has a pancreas-related disease or disorder and / or a disease or disorder involving endocrinogenesis. In some embodiments, the pancreas- related disease or disorder includes a type of cancer, cystic fibrosis, pancreatitis, exocrine pancreatic insufficiency (EPI), pancreatic cysts, bile duct cysts, and / or diabetes. In some embodiments, the type of cancer includes pancreatic ductal adenocarcinoma (PDAC), a pancreatic neuroendocrine tumor, and / or bile duct cancer (cholangiocarcinoma). In some embodiments, the subject has an increased survival rate following transplantation. In some embodiments, the PO is produced from pluripotent stem cells derived from the subject.

[0032] Further embodiments of the disclosure include methods for screening a candidate compound or composition, wherein the candidate compound or composition to be screened includes one or more exogenous agent, the method including: contacting POs asdescribed above, or compositions including the same, with the candidate compound or composition; culturing the PO with the candidate compound or composition for a period of time; and assessing one or more effects of the compound or composition on the PO, thereby screening the compound or composition. In some embodiments, the assessed effect includes therapeutic efficacy and / or toxicity of the compound or composition.

[0033] Further embodiments of the disclosure include kits including means for preparing POs as described above, or compositions including the same, or for performing any of the methods as described above. Further embodiments of the disclosure include kits including POs as described above, or compositions including the same. In some embodiments, one or more of the kit components are provided in separate vials. In some embodiments, one or more of the kit components are pre-loaded onto one or more assay platform. In some embodiments, one or more of the kit components are pre-frozen.

[0034] Further embodiments of the disclosure include methods of providing in vitro pancreatic mesenchyme, including: providing an PO according to the method of any of claims 1- 42; and dissociating the pancreatic organoid into respective mesenchymal and epithelial components. Further embodiments of the disclosure include pancreatic mesenchyme, including a vascular mesenchyme recombinant cell population (VMPO) and / or a splanchnic mesenchyme recombinant cell population (SMPO). Further embodiments of the disclosure include pancreatic mesenchyme, including a vascular mesenchyme recombinant cell population (VMPO) and / or a splanchnic mesenchyme recombinant cell population (SMPO), wherein the pancreatic mesenchyme is produced by the methods described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0035] 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.

[0036] FIGURE 1. Bioengineering approach to generate human pancreatic organoid (HPO).

[0037] FIG. 1A) Illustration of the protocol with the derivation of splanchnic mesenchyme (SM), posterior foregut (PFG) and vascular mesenchyme (VM). FIG. IB) Flow cytometry analysis of PDX1 expression of PFG cells prior to recombination. Mean and standard deviation are shown for n=3 biologically independent experiments. FIG. 1C) Fluorescenceconfocal images of splanchnic mesenchyme before recombination. FIG. ID) Fluorescence confocal images of vascular’ mesenchyme before recombination. All images arc representative of n = 3 biologically independent experiments. Scale bar’s, 200 pm. DE, Definitive Endoderm; PGT, Primitive Gut Tube; Meso, mesoderm; LPM, Lateral Plate Mesoderm.

[0038] FIGURE 2. Mesenchyme source bias differentiation.

[0039] FIG. 2A) Brightfield and fluorescence images overlay showing evolution of the organoids after recombination. Posterior foregut cells are constitutively labelled with mCherry and vascular mesenchyme with gfp. Scale bars, 500 pm. FIG. 2B) Fluorescence confocal images of the organoids stained with Dapi (left) and corresponding NKX6.1 staining (right). Scale bar’s, 500 pm. FIG. 2C) Fluorescence confocal imaging of the organoids at day 22. Scale bars, 200 pm. FIG. 2D and FIG. 2E) Fluorescence confocal imaging of the organoids at day 60. Scale bars, 500 pm. FIG. 2F) Hematoxylin and Eosin staining of the organoids after 60 days of culture. Scale bars, 500 pm. FIG. 2G) Macroscopic images of the organoids after 10 weeks of maturation in vivo. Pie charts of the ratio of engraftment and complexity of the resulting epithelium is shown on the left. Ruler with 1 mm increment is shown as a scale. FIG. 2H) Hematoxylin and Eosin staining of the explanted organoids. Scale bar’s, 500 pm (left) and 200 pm (zoom, right). All images are representative of n = 3 biologically independent experiments.

[0040] FIGURE 3. Recombination with vascular mesenchyme results in robust pancreatic organoid formation.

[0041] FIG. 3A) Illustration of the protocol for growth of the pancreatic organoids. FIG. 3B) Fluorescent spinning disk images showing the timeline of organoid growth, with PFG self-sorting and growing inside the VM. Scale bars, 500 pm. FIG. 3C) Brightfield images showing the morphology of organoids cultured in inductive or permissive medium. Scale bars, 500 pm. FIG. 3D) Fluorescence confocal images of the organoids stained for PDX1, SOX2 and dapi. Scale bars, 200 pm. FIG. 3E, FIG. 3F, FIG. 3G, FIG. 3H, and FIG. 31) Fluorescence confocal images of the organoids showing differences in morphology, endocrine commitment and stomach markers when grown in different mediums. Scale bars, 200 pm. FIG. 3 J) Fluorescence images of organoids grown in permissive medium showing intestinal marker expression. Scale bars, 200 pm. All images are representative of a minimum of n = 3 biologically independent experiments.

[0042] FIGURE 4. Pancreatic organoids undergo ductal branching and acinar differentiation without additional induction.

[0043] FIG. 4A) Brightfield image showing organoids grown for 60 days. Scale bars, 500 |am. FIG. 4B) Hematoxylin and Eosin staining of day 60 organoids. Scale bars, 500 m (left) and 200 pm (zoom, right). FIG. 4C, FIG. 4D, FIG. 4E, FIG. 4F, FIG. 4G, FIG. 4H) Fluorescence confocal images of organoids cultured for either 40 or 60 days. Scale bars, 200 pm. All images are representative of a minimum of n = 3 biologically independent experiments.

[0044] FIGURE 5. Pancreatic organoid can mature in vivo.

[0045] FIG. 5A) Macroscopic images of the organoids after 10 weeks of maturation in vivo. Ruler with 1 mm increment is shown as a scale. FIG. 5B) Hematoxylin and Eosin staining of the explanted organoids, and compared with section of a 15-20 week human fetal pancreas. Scale bars, 500 pm (left) and 200 pm (zoom, right). FIG. 5C) Fluorescence confocal images showing nucleus (blue), CPA1 (green) and amylase (pink) for the explanted organoids and fetal pancreas. Scale bars, 100 pm. All images are representative of a minimum of n = 3 biologically independent experiments.

[0046] FIGURE 6. Protocol robustness across cell lines.

[0047] FIG. 6A) Flow cytometry quantification showing the percentage of endodermal cell expressing PDX1 (left), PDX+ / NKX6.1+ (middle), or the percentage of total cell coming from the endoderm differentiation (right) at different stages. FIG. 6B) Flow cytometry quantification of the percentage of cells expressing region specific markers during differentiation in iPSC 72.3 (left) or Hl (right).

[0048] FIGURE 7. Differentiation of acinar cells within hPOs.

[0049] FIG. 7A) Hematoxilin and eosin stain showing acini formation within the organoids. FIG. 7B) Immunostaining images showing zymogen granules present in the apical side of the acinar cells. FIG. 7C) Electron micrograph showing granules within the hPO.

[0050] FIGURE 8. Single cell atlas of organoid differentiation.

[0051] FIG. 8 A) UMAP showing that all the key pancreatic cell types are present during organoid differentiation. FIG. 8B) Bar graph showing the different population proportion during organoid differentiation. FIG. 8C) UMAP showing clustering of the different stages of organoid differentiation.

[0052] FIGURE 9. Single cell exocrine recluster.

[0053] FIG. 9A) UMAP showing key exocrine differentiation population and their representation at different stages of organoid development. FIG. 9B) Overlay of the different stages on the UMAP. FIG. 9C) Pseudotime analysis of the exocrine differentiation in the organoids. FIG. 9D) Expression of specific markers in the population.

[0054] FIGURE 10. Single cell endocrine recluster.

[0055] FIG. 10A) UMAP showing key endocrine differentiation population. FIG. 10B) Pseudotime analysis of the endocrine differentiation in the organoids. FIG. 10C) Expression of specific markers in the population.

[0056] FIGURE 11. Single cell mesenchyme recluster.

[0057] FIG. 11A) UMAP showing key mesenchymal population. FIG. 11B) Pseudotime analysis of the mesenchyme differentiation in the organoids. FIG. 11C) Expression of specific markers in the population.

[0058] FIGURE 12. Mesenchymal type can bias posterior foregut fate.

[0059] FIG. 12A) Flow cytometry quantification of the endodermal population 1 week post-combination in function of mesenchyme type. FIG. 12B) PCA plot of bulk RNA sequencing showing clustering of different mesenchyme before and after recombination. FIG. 12C) Volcano plot showing differentially expressed genes of Mes 1 vs Mes 3 showing upregulation of posterior genes in Mes 1. FIG. 12D) Bulk RNA sequencing showing gene signature of the mesenchyme before and 1 week after combination.

[0060] FIGURE 13. Endocrinogenesis can be recapitulatd in hPO.

[0061] Immuno staining showing islet-like cluster morphology and expression of specific endocrine, ductal and acinar markers within the same organoids, demonstrating endocrinogenesis .

[0062] FIGURE 14. Immune cells can survive and differentiate inside the hPOs.

[0063] FIG. 14A) Immunostaining showing immune cell incorporation and survival within the hPOs within disrupting exocrine differentiation. FIG. 14B) Immuno staining showing differentiation of immune cells towards macrophage lineage. FIG. 14C) Single cell UMAP of hPO with immune cells.DETAILED DESCRIPTION

[0064] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar’ symbols typically identify similarcomponents, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims arc not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.

[0065] The following description of various embodiments is exemplary and explanatory only and is not to be construed as limiting or restrictive in any way. Other embodiments, features, objects, and advantages of the present teachings will be apparent from the description and accompanying drawings, and from the claims.

[0066] The disclosure herein uses affirmative language to describe the numerous embodiments. The disclosure also includes embodiments in which subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, or procedures.

[0067] It should be understood that any use of subheadings herein are for organizational purposes, and should not be read to limit the application of those subheaded features to the various embodiments herein. Each and every feature described herein is applicable and usable in all the various embodiments discussed herein and that all features described herein can be used in any contemplated combination, regardless of the specific example embodiments that are described herein. It should further be noted that exemplary description of specific features are used, largely for informational purposes, and not in any way to limit the design, subfeature, and functionality of the specifically described feature.Overview

[0068] Due to the complex nature of pancreas development, in vitro recapitulation of morphogenetic processes, such as pancreatic budding, ductal plexus formation and regionalized acinar differentiation, has proven challenging to date. Pancreatic ductal organoids have been derived from fetal mouse tissues, as well as human adult and pluripotent stem cells, and they have been used to study ductal carcinoma and to model cystic fibrosis in human patients. However, although these organoids have contributed to deepening an understanding of pancreas biology, they are generally growing as spheroids or are characterized by limited budding, and do not recapitulate the native morphological processes and tissue architecture.

[0069] Given that mouse embryonic tissues have demonstrated growth and branching on recombination of early pancreatic epithelium with mesenchyme, ccll-ccll interactions between epithelium and cells found in mesenchyme are considered to be important for pancreas morphogenesis. Subsequent studies have further demonstrated the importance of mesenchyme, vascular cells, etc., in pancreas development. Thus, new approaches that incorporate mesodermal cell types, such as mesenchyme and vascular cells, can greatly facilitate efforts at generating pancreatic organoids that undergo morphogenesis more similar to normal development.

[0070] As described herein, human pancreatic organoids (HPOs) containing various pancreatic cell populations have been generated, including endocrine, ductal and exocrine structures. First, splanchnic mesenchyme and vascular organoids were generated, and these cell populations were used to drive morphogenetic development of pancreatic progenitors in a manner that is highly similar to what is seen in vivo. In some embodiments, the HPOs are entirely derived from pluripotent stem cells in vitro. This is the first description of an HPO with tissue complexity and architecture that is highly similar to human pancreas. In accordance with various embodiments, the HPOs are complex and branching, and contain ductal and acing structures, as well as endocrine pancreas. This new pancreatic organoid system mimics many characteristics of human pancreatic development, including epithelial stratification of pancreatic progenitors, ductal branching and generation of acinar cells localized at the tip of the ductal networks.

[0071] Pancreatic morphogenesis was achieved by combining mesenchymal and vascular endothelial cells with posterior foregut cells, all derived from pluripotent stem cells. In this process, after initial aggregation, the cells self-sort, or self-assemble, into two compartments, with the developing pancreatic epithelium on the inside, surrounded by mesenchyme and vascular endothelium. As seen during pancreas development in vivo, pancreatic epithelial growth in the organoid is dependent on mesenchyme and recapitulates many aspects of pancreatic development, starting with budding of multipotent progenitors (PDX1+ / NKX6.1+ / SOX9+) and subsequent formation of polarized microlumens. After 2 months of suspension culture, the pancreatic organoids are characterized by an interconnected ductal network in the center and regionalized differentiation of acinar tissue (CPA1+) at the periphery.

[0072] In addition, pancreatic organoids transplanted under the kidney capsule of mice were able to mature in vivo and produce similar ductal branching and acinar differentiation.Together, these findings represent an exciting new platform to study human pancreas development and disease in vitro.Definitions of Terms

[0073] Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. For purposes of the present disclosure, the following terms are explained below.

[0074] As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one. Some embodiments of the disclosure may consist of or consist essentially of one or more elements, method steps, and / or methods of the disclosure. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein and that different embodiments may be combined.

[0075] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” For example, “x, y, and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment. As used herein “another” may mean at least a second or more.

[0076] The term “ones” means more than one.

[0077] As used herein, the term “plurality” may be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

[0078] As used herein, the term “set of’ means one or more. For example, a set of items includes one or more items.

[0079] As used herein, the phrase “at least one of,” when used with a list of items, means different combinations of one or more of the listed items may be used and only one of the items in the list may be needed. The item may be a particular object, thing, step, operation, process, or category. In other words, “at least one of’ means any combination of items or number of items may be used from the list, but not all of the items in the list may be required. For example, without limitation, “at least one of item A, item B, or item C” means item A; item A and item B; item B; item A, item B, and item C; item B and item C; or item A and C. In some cases, “at least one of item A, item B, or item C” means, but is not limited to, two of item A, oneof item B, and ten of item C; four of item B and seven of item C; or some other suitable combination.

[0080] As used herein, “substantially” means sufficient to work for the intended purpose. The term “substantially” thus allows for minor, insignificant variations from an absolute or perfect state, dimension, measurement, result, or the like such as would be expected by a person of ordinary skill in the field but that do not appreciably affect overall performance. When used with respect to numerical values or parameters or characteristics that can be expressed as numerical values, “substantially” means within ten percent.

[0081] Throughout this specification, unless the context requires otherwise, the words “comprise”, “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that no other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0082] Reference throughout this specification to “one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” or “a further embodiment” or combinations thereof means that a particular' feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular- features, structures, or characteristics may be combined in any suitable manner in various embodiments.

[0083] As used herein, the terms “treatment,” “treating,” “treat,” and the like, with respect to a disease or condition, can refer to obtaining a desired pharmacologic and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or can be therapeutic in terms of a partial or complete cure fora disease and / or adverse effect attributable to the disease. For example, a treatment can include executing a protocol, which may include administering one or more drugs to a patient, in an effort to alleviate signs or symptoms of the disease. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. Alleviation can occur prior to signs or symptoms of the disease or condition appearing, as well as after their appearance. Thus, ‘‘treating” or “treatment” may include “preventing” or “prevention” of disease or undesirable condition. In addition, “treating” or “treatment” does not require complete alleviation of signs or symptoms, does not require a cure, and specifically includes protocols that have only a marginal effect on the patient.

[0084] “Treatment,” as used herein, thus can cover any treatment of a disease in a subject, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease and / or relieving one or more disease symptoms. “Treatment” can also encompass delivery of an agent or administration of a therapy in order to provide for a pharmacologic effect, even in the absence of a disease or condition.

[0085] The term “therapeutically effective” or “therapeutically effective amount” as used throughout this application can refer to an amount effective to achieve a desired and / or beneficial effect, and / or anything that promotes or enhances the well-being of the subject with respect to the medical treatment of a condition. This includes, but is not limited to, a reduction in the frequency or severity of one or more signs or symptoms of a disease. An effective amount can be administered in one or more administrations. In the methods, a therapeutically effective amount is an amount appropriate to treat an indication. By treating an indication is meant achieving any desirable effect, such as one or more of palliate, ameliorate, stabilize, reverse, slow, or delay disease progression, increase the quality of life, or to prolong life. Such achievement can be measured by any suitable method, such as measurement of tumor size or blood cell count, or any other suitable measurement.

[0086] The terms “effective amount” or “effective dose” as used herein have their plain and ordinary meaning as understood in light of the specification, and can refer to that amount of a recited composition or compound that, results in an observable effect. Actual dosage levels of active ingredients in an active composition of the presently disclosed subject matter can be varied so as to administer an amount of the active composition or compound that, is effectiveto achieve the desired response for a particular subject and / or application. The selected dosage level wall depend upon a variety of factors including, but not limited to, the activity of the composition, formulation, route of administration, combination with other drugs or treatments, severity of the condition being treated, and the physical condition and prior medical history of the subject being treated. In some embodiments, a minimal dose is administered, and dose is escalated in the absence of dose-limiting toxicity to a minimally effective amount. Determination and adjustment of an effective dose, as well as evaluation of when and how to make such adjustments, are contemplated herein.

[0087] The term “disease state” as used herein, can generally refer to a condition that affects the structure or function of an organism. Disease states can include, for example, stages of a disease progression.

[0088] As used herein, the term “assessing” can include any form of measurement, and includes determining if an element is present or not. The terms “determining,” “measuring,” “evaluating,” “assessing” and “assaying” can be used interchangeably and can include quantitative and / or qualitative determinations.

[0089] As used herein, the terms “modulated” or “modulation,” or “regulated” or “regulation” and “differentially regulated” can refer to both up regulation (z.e., activation or stimulation, e.g., by agonizing or potentiating) and down regulation (z.e., inhibition or suppression, e.g., by antagonizing, decreasing or inhibiting), unless otherwise specified or clear from the context of a specific usage.

[0090] As used herein, the term “subject” can refer to any member of the animal kingdom. In some embodiments, a subject is a human patient.

[0091] As used herein, the term “marker” or “biomarker” can refer to any measurable substance taken as a sample from a subject whose presence is indicative of some phenomenon. Non-limiting examples of such phenomenon can include a disease state, a condition, or exposure to a compound or environmental condition. In various embodiments described herein, biomarkers may be used for diagnostic purposes (e.g., to diagnose a disease state, a health state, an asymptomatic state, a symptomatic state, etc.). The term “biomarker” may be used interchangeably with the term “marker”. The term “marker” or “biomarker” can include a biological molecule, such as, for example, a nucleic acid, peptide, protein, hormone, and the like, whose presence or concentration can be detected and correlated with a known condition, such as a disease state. It can also be used to refer to a differentially expressed gene whose expressionpattem can be utilized as part of a predictive, prognostic or diagnostic process in healthy conditions or a disease state, or which, alternatively, can be used in methods for identifying a useful treatment or prevention therapy.

[0092] As used herein, the term “cellular phenotype” can refer to any determinable, observable, and / or measurable characteristic associated with a cell population.

[0093] As used herein, a “model” can include one or more in vitro or in vivo disease models; a model can also include algorithms, one or more mathematical techniques, one or more machine learning algorithms, or a combination thereof. A model can be used in a process and / or applied to an assay, in accordance with various embodiments as disclosed herein.

[0094] As used herein, a “process” can include one or more steps involving one or more features of one or more model as disclosed herein.

[0095] The terms “function” and “functional” as used herein have their plain and ordinary meaning as understood in light of the specification, and can refer to a biological, enzymatic, or therapeutic function.

[0096] The term “inhibit” as used herein has its plain and ordinary meaning as understood in light of the specification, and can refer to the reduction or prevention of a biological activity. The reduction can be by a percentage that is, is about, is at least, is at least about, is not more than, or is not more than about, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or an amount that is within a range defined by any two of the aforementioned values. As used herein, the term “delay” has its plain and ordinary meaning as understood in light of the specification, and refers to a slowing, postponement, or deferment of a biological event, to a time which is later than would otherwise be expected. The delay can be a delay of a percentage that is, is about, is at least, is at least about, is not more than, or is not more than about, 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or an amount within a range defined by any two of the aforementioned values. The terms inhibit and delay may not necessarily indicate a 100% inhibition or delay, A partial inhibition or delay may be realized.

[0097] As used herein, the term “isolated” has its plain and ordinary meaning as understood in light of the specification, and can refer to a substance and / or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and / or in an experimental setting), and / or (2) produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities may be separated from equal to, about, at least, at least about, not more than, or not more than about, 10%, about20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or 100% of the other components with which they were initially associated (or ranges including and / or spanning the aforementioned values). In some embodiments, isolated agents are, are about, are at least, are at least about, are not more than, or are not more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or 100% pure (or ranges including and / or spanning the aforementioned values). As used herein, a substance that is “isolated” may be “pure” (e.g., substantially free of other components). As used herein, the term “isolated cell” can refer to a cell not contained in a multi - cellular organism or tissue.

[0098] As used herein, “zz? vivo” is given its plain and ordinary meaning as understood in light of the specification and can refer to the performance of a method inside living organisms, usually animals, mammals, including humans, and plants, as opposed to a tissue extract or dead organism.

[0099] As used herein, “ev vivo” is given its plain and ordinary meaning as understood in light of the specification and can refer to the performance of a method outside a living organism with little alteration of natural conditions.

[0100] As used herein, “zn vitro” is given its plain and ordinary' meaning as understood in light of the specification and can refer to the performance of a method outside of biological conditions, e.g., in a petri dish or test tube.

[0101] The terms “nucleic acid” or “nucleic acid molecule” as used herein have their plain and ordinary meaning as understood in light of the specification, and can refer to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, those that appear in a cell naturally, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally -occurring nucleotides (such as DNA and RNA), or analogs of naturally-occurring nucleotides (e.g., enantiomeric forms of naturally-occurring nucleotides), or a combination of both. Modified nucleotides can have alterations in sugar moieties and / or in pyrimidine or purine base moieties. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters. Moreover, the entire sugar moiety can be replaced with statically andelectronically similar structures, such as aza- sugars and carbocyclic sugar analogs. Examples of modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoramlidate, or phosphoramidate. The term “nucleic acid molecule” also includes so-called “peptide nucleic acids,” which comprise naturally-occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either single stranded or double stranded. “Oligonucleotide” can be used interchangeable with nucleic acid and can refer to either double stranded or single stranded DNA or RNA. A nucleic acid or nucleic acids can be contained in a nucleic acid vector or nucleic acid construct (e.g. plasmid, virus, retrovirus, lentivirus, bacteriophage, cosmid, fosmid, phagemid, bacterial artificial chromosome (BAG), yeast artificial chromosome (YAC), or human artificial chromosome (HAG)) that can be used for amplification and / or expression of the nucleic acid or nucleic acids in various biological systems. Typically, the vector or construct will also contain elements including but not limited to promoters, enhancers, terminators, inducers, ribosome binding sites, translation initiation sites, start codons, stop codons, polyadenylation signals, origins of replication, cloning sites, multiple cloning sites, restriction enzyme sites, epitopes, reporter genes, selection markers, antibiotic selection markers, targeting sequences, peptide purification tags, or accessory genes, or any combination thereof.

[0102] A nucleic acid or nucleic acid molecule can comprise one or more sequences encoding different peptides, polypeptides, or proteins. These one or more sequences can be joined in the same nucleic acid or nucleic acid molecule adjacently, or with extra nucleic acids in between, e.g. linkers, repeats or restriction enzyme sites, or any other sequence that is, is about, is at least, is at least about, is not more than, or is not more than about, I, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases long, or any length in a range defined by any two of the aforementioned lengths. The term “downstream” on a nucleic acid as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a sequence being after the 3 ’-end of a previous sequence, on the strand containing the encoding sequence (sense strand) if the nucleic acid is double stranded. The term “upstream” on a nucleic acid as used herein has its plain and ordinary meaning as understood in light of the specification and refers toa sequence being before the 5’- end of a subsequent sequence, on the strand containing the encoding sequence (sense strand) if the nucleic acid is double stranded. The term “grouped” on a nucleic acid as used herein has its plain and ordinary meaning as understood in light of the specification and refers to two or more sequences that occur in proximity either directly or with extra nucleic acids in between, e.g. linkers, repeats, or restriction enzyme sites, or any other sequence that is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases long, or any length in a range defined by any two of the aforementioned lengths, but generally not with a sequence in between that encodes for a functioning or catalytic polypeptide, protein, or protein domain.

[0103] The nucleic acids described herein comprise nucleobases. Primary, canonical, natural, or unmodified bases are adenine, cytosine, guanine, thymine, and uracil. Other nucleobases include but are not limited to purines, pyrimidines, modified nucleobases, 5- methylcytosine, pseudouridine, dihydrouridine, inosine, 7-methylguanosine, hypoxanthine, xanthine, 5,6-dihydrouracil, 5-hydroxymethylcytosine, 5 -bromouracil, isoguanine, isocytosine, aminoallyl bases, dye-labeled bases, fluorescent bases, or biotin-labeled bases.

[0104] The terms “peptide”, “polypeptide”, and “protein” as used herein have their plain and ordinary meaning as understood in light of the specification and refer to macromolecules comprised of amino acids linked by peptide bonds. The numerous functions of peptides, polypeptides, and proteins are known in the art, and include but are not limited to enzymes, structure, transport, defense, hormones, or signaling. Peptides, polypeptides, and proteins are often, but not always, produced biologically by a ribosomal complex using a nucleic acid template, although chemical syntheses are also available. By manipulating the nucleic acid template, peptide, polypeptide, and protein mutations such as substitutions, deletions, truncations, additions, duplications, or fusions of more than one peptide, polypeptide, or protein can be performed. These fusions of more than one peptide, polypeptide, or protein can be joined in the same molecule adjacently, or with extra amino acids in between, e.g. linkers, repeats, epitopes, or tags, or any other sequence that is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases long, or any length in a range defined by any two of the aforementioned lengths. The term “downstream” on a polypeptide as used herein has its plain and ordinary meaning as understood in light of thespecification and refers to a sequence being after the C-terminus of a previous sequence. The term “upstream” on a polypeptide as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a sequence being before the N- terminus of a subsequent sequence.

[0105] The term “purity” of any given substance, compound, or material as used herein has its plain and ordinary meaning as understood in light of the specification and can refer to the actual abundance of the substance, compound, or material relative to the expected abundance. For example, the substance, compound, or material may be at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure, including all decimals in between. Purity may be affected by unwanted impurities, including but not limited to nucleic acids, DNA, RNA, nucleotides, proteins, polypeptides, peptides, amino acids, lipids, cell membrane, cell debris, small molecules, degradation products, solvent, carrier, vehicle, or contaminants, or any combination thereof. In some embodiments, the substance, compound, or material is substantially free of host cell proteins, host cell nucleic acids, plasmid DNA, contaminating viruses, proteasomes, host cell culture components, process related components, mycoplasma, pyrogens, bacterial endotoxins, and adventitious agents. Purity' can be measured using technologies including but not limited to electrophoresis, SDS-PAGE, capillary electrophoresis, PCR, rtPCR, qPCR, chromatography, liquid chromatography, gas chromatography, thin layer chromatography, enzyme-linked immunosorbent assay (ELISA), spectroscopy, UV-visible spectrometry, infrared spectrometry, mass spectrometry, nuclear magnetic resonance, gravimetry, or titration, or any combination thereof.

[0106] The term “yield” of any given substance, compound, or material as used herein has its plain and ordinary meaning as understood in light of the specification and can refer to the actual overall amount of the substance, compound, or material relative to the expected overall amount. For example, the yield of the substance, compound, or material is, is about, is at least, is at least about, is not more than, or is not more than about 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the expected overall amount, including ail decimals in between. Yield may be affected by the efficiency of a reaction or process, unwanted side reactions, degradation, quality of the input substances, compounds, or materials, or loss of the desired substance, compound, or material during any step of the production.

[0107] The term “% w / w” or “% wt / wt” as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a percentage expressed in termsof the weight of the ingredient or agent over the total weight of the composition multiplied by 100. The term “% v / v” or “% vol / vol” as used herein has its plain and ordinary meaning as understood in the light of the specification and refers to a percentage expressed in terms of the liquid volume of the compound, substance, ingredient, or agent over the total liquid volume of the composition multiplied by 100.

[0108] The term “basement membrane matrix” or “extracellular matrix” as used herein has its plain and ordinary meaning m light of the specification and refers to any biological or synthetic compound, substance, or composition that enhances cell attachment and / or growth. Any extracellular matrix, as well as any mimetic or derivative thereof, known in the art can be used for the methods disclosed herein. Some examples of extracellular matrices, or numerics or derivative thereof, include but are not limited to cell-based feeder layers, polymers, proteins, polypeptides, nucleic acids, sugars, lipids, poly-lysine, poly-ornithine, collagen, gelatin, fibronectin, vitronectin, laminin, elastin, tenascin, heparan sulfate, entactin, osteopontin, basement membrane, Matrigel, hydrogel, PEI, WGA, or hyaluronic acid, or any combination thereof. hPSC-Derived Pancreatic Organoids

[0109] While development of the exocrine pancreas has historically been understudied compared to the endocrine pancreas and its role in diabetes, some work has focused on recapitulating whole pancreas development or modeled diseases of the exocrine compartment, such as pancreatic adenocarcinomas and other forms of cancer. Ductal and acinar cells can be involved development and disease, with increasing evidence pointing to an important role for the exocrine pancreas in islet dysfunction and some forms of diabetes. Importantly, endocrine cells are derived from the bipotent ductal progenitors, and subsequent delamination and islet formation likely respond to signals from the exocrine compartment and other supporting cells of neural, vascular, and mesenchymal origin.

[0110] By expanding progenitors isolated from fetal murine pancreas or adult human pancreas, pancreatic organoids can be propagated in vitro. However, such organoids differentiate with limited efficiency towards acinar or endocrine cells. Pluripotent stem cell-derived pancreatic organoids have been generated, resulting in ductal organoids that can be cultured as hollow spheres (Hohwieler et al., Gut 66, 473-486 (2017)), although organoids prepared according to this protocol are limited to epithelium. The Hohweiler differentiation protocol has been adapted to model different diseases, such as cystic fibrosis and pancreatic ductaladenocarcinoma (PDAC). However, the limited morphogenesis and differentiation potential of these systems has hindered the study of human development and exocrine disease in vitro.

[0111] In contrast, the pancreatic organoids of the present disclosure have mesenchyme and vascular endothelium which are both normal and essential components of the developing pancreas. These additional cell types incorporated into the pancreatic organoids of the present disclosure result in formation of tissue that is more similar to actual pancreas. The organoids of the disclosure thus undergo developmental processes that are much more similar to the developing human pancreas and form structures that are not observed in the Hohwieler organoids in culture. The additional cell types (mesenchyme and vascular) allow recapitulation of many aspects of pancreatic development, such as extensive branching with spatially relevant differentiation and cellular phenotype. These features are not seen in other pancreatic organoids described to date (including that of Hohwieler et al.). The level of organization of the cells and structures within the pancreatic organoids of the present disclosure have heretofore never been described, and are closer to fetal pancreas than to published organoids.

[0112] As compared to naturally occurring human pancreas, the pancreatic organoids of the present disclosure lack various types of cells present in fetal pancreas, such as nerve cells and immune cells. In addition to missing some more cell types, the pancreatic organoids of the present disclosure lack the macroscopic scale properties of the fetal pancreas, such as the anisotropic growth, hierarchical vascularization, and organ-level features that depend on interactions with blood or other organs during development.

[0113] Evidence of the importance of the mesenchyme in pancreas morphogenesis emerged in the 1960s, when it was demonstrated that isolated pancreatic epithelium adopted a round morphology and failed to grow in culture. When recombined with the mesenchyme from which it had been separated, the epithelium could resume growth and formed a branching network reminiscent of in vivo development. More recently, strong evidence has confirmed the important role of the mesenchyme in proliferation of the pancreatic bud as well as induction of branching morphogenesis. Since then, some of the mechanisms involved have been elucidated, including secretion of FGF7 and FGF10 by the pancreatic mesenchyme to promote progenitor expansion, as well as the secretion of basement membrane laminin by the mesenchyme to promote ductal and acinar differentiation. Localized gradients formed by the evolving interaction between the epithelium and the mesenchyme can drive branching morphogenesis, as has been shown for other organs.

[0114] In accordance with various embodiments of the present disclosure, pancreatic organoids have been engineered by recombining posterior foregut cells with mesenchymal cells, both derived from pluripotent stem cells. Posterior foregut cells were chosen based on their potential to differentiate into both endocrine and exocrine lineages. By contrast, there is limited information on the nature of human pancreatic mesenchyme, and a protocol for its differentiation from pluripotent stem cells has heretofore been lacking.

[0115] To maximize cell-cell communication and potential for co-development with the pancreatic epithelium, two different sources of mesenchyme were selected: posterior foregut mesenchyme and vascular mesenchyme. The former is the precursor to pancreatic mesenchyme, whereas the mesenchyme derived from a vascular organoid protocol was used to take advantage of the important role of vasculature on early pancreas morphogenesis. As shown herein, the source of mesenchyme strongly impacts lineage commitment of the organoids, with vascular mesenchyme either repressing stomach fate or promoting pancreatic fate.

[0116] Using the bioengineering approach described herein, pancreatic organoids can be cultured in suspension, facilitating potential downstream applications, such as, for example, bioprocess scaling up, drug screening, disease modelling, etc. Recombination with mesenchymal cells also provides a system free of external extracellular matrix (ECM) that allows for the investigation of the importance of natural ECM production and its regulation by mesenchyme- epithelial interactions.

[0117] The study described herein presents a novel in vitro model of human pancreatic development, recapitulating many aspects of pancreas organogenesis. First, pancreatic epithelial cells bud from a main lumen before rapidly expanding as a highly stratified epithelium that express the same markers as human multipotent pancreatic progenitor cells (e.g. GATA4, PDX1, SOX9, NKX6.1, and the like). Subsequently, multiple microlumens are formed in the pancreatic domain, slowly coalescing to form a ductal network with secretion of pancreatic specific mucin in the lumen. Importantly, maturation of the ductal network robustly recapitulates the compartmentalization of a distinct ductal network with the terminal acinar differentiation seen in human and mammalian development. After 7 weeks of culture post-recombination, the ducts maintain high expression of SOX9 with downregulation of GATA4 expression. In contrast, the peripheral acinar compartment keeps high expression of GATA4 while decreasing SOX9 and PDX1 expression, recapitulating the transcription factor patterns seen in vivo. Maturing acinar cells arc also strongly polarized, with formation of an apical lumen with robust deposition oflaminin on the basal side. Pancreatic enzymes are also found both inside the acinar cells as well as in the lumen, confirming functional secretion of the zymogen granules in the lumen of the acini.

[0118] This represents the first pancreatic organoid that recapitulates human exocrine fetal development. This organoid system thus opens many avenues of research in various areas, including, for example, but not limited to, development, disease modelling, and study of exocrine biology.

[0119] Thus, various embodiments of the disclosure relate to in vitro or ex vivo pancreatic organoids (POs), including a posterior foregut cell population, and a vascular mesenchyme recombinant cell population (VMPO) and / or a splanchnic mesenchyme recombinant cell population (SMPO), as well as methods of making and using the same. POs, such as human pancreatic organoids (HPOs), as described herein, can include a posterior foregut cell population, a VMPO, and a SMPO.

[0120] Pancreatic organoids, such as HPOs, can be derived from progenitor cells. These include, for example, induced pluripotent stem cells (iPSCs). In some embodiments, the iPSCs can be subject-derived, where the subject can be healthy or having a diseased condition, and are identical in genetic content to the respective patient. They express pancreatic markers that are expressed in the pre-natal stages of development. Furthermore, they are clonal and therefore react similarly to external stimuli and biochemical perturbations. These POs and HPOs can be scalable and tractable, allowing screening approaches to test a vast array of drugs and small molecules.

[0121] POs and HPOs can have various uses, such as, for example, methods of treating a pancreas-related disease or disorder and / or a disease or disorder involving endocrinogenesis, methods of scaling up in bioprocess manufacturing, methods of screening compounds, compositions, or potential treatments, for therapeutic efficacy, methods of modeling human pancreatic development and / or disease, methods of diagnosing a pancreatic-related disease or disorder, the manufacture of a medicament for treating a pancreas-related disease or disorder, and the like.

[0122] Further detail is provided in Examples 1-6 and the sections that follow.Methods of Producing Pancreatic Organoids

[0123] Generating various types of organoids from pluripotent stem cells, and precursors thereof, such as definitive endoderm, gut endoderm, foregut endoderm, mid / hindgutendoderm, lateral plate mesoderm, splanchnic mesoderm, splanchnic mesenchyme, vascularized mesoderm, vascularized mesenchyme, and the like, arc generally known in the ail. Exemplary methods may be found in PCT publications WO 2011 / 140441, WO 2015 / 183920, WO2016 / 061464, WO 2017 / 192997, WO 2018 / 085622, WO 2018 / 085623, WO 2018 / 106628, WO2018 / 200481, WO 2018 / 226267, WO 2019 / 074793, WO 2020 / 023245, WO 2020 / 160371, WO2020 / 243633, WO 2021 / 030373, WO 2021 / 041443, WO 2022 / 072553, WO 2023 / 102133, WO2023 / 023180, WO 2023 / 137467, and WO 2023 / 278676, each of which is hereby expressly incorporated by reference in its entirety. Any methods for producing definitive endoderm, gut endoderm, foregut endoderm, mid / hindgut endoderm, lateral plate mesoderm, splanchnic mesoderm, splanchnic mesenchyme, vascularized mesoderm, vascularized mesenchyme, and the like, from pluripotent stem cells disclosed herein or otherwise known in the ail are applicable to the methods described herein.

[0124] Embodiments of methods for producing pancreatic organoids are provided herein. In some embodiments, the methods include dissociating splanchnic mesenchyme, vascular mesenchyme, and posterior foregut cells to respective single cell suspensions; recombining and aggregating the single cell suspensions of posterior splanchnic mesenchyme and / or vascular mesenchyme with the single cell suspension of posterior foregut cells; and culturing the recombined and aggregated single cell suspensions of posterior splanchnic mesenchyme and / or vascular mesenchyme with the single cell suspension of posterior foregut cells, while inhibiting a ROCK pathway, for a first period of time, to provide a precursor pancreatic organoid.

[0125] The methods can optionally include an additional step of culturing the precursor pancreatic organoid with or without activating and / or inhibiting one or more signaling pathways, for a second period of time, to provide an induced pancreatic organoid. In some embodiments, the methods can further optionally include an additional step of culturing the induced pancreatic organoid with activating and / or inhibiting one or more signaling pathways, for a second period of time, to provide a pancreatic organoid. In some embodiments, culturing the precursor pancreatic organoids is in an inductive medium including growth factors and / or signaling pathway activators and / or inhibitors, such as, for example, posterior foregut medium, classical gut medium, and / or basal mesenchyme differentiation medium, to provide an induced pancreatic organoid with committed lineage. In some embodiments, the inductive medium can include, for example, SANT-1, retinoic acid, TPB, LDN and / or KGF. In some embodiments, themethods can further optionally include an additional step of culturing the induced or non-induced pancreatic organoid without activating and / or inhibiting one or more signaling pathways, for a third period of time, to provide a pancreatic organoid. In some embodiments, the pancreatic organoid cultured following a pulse (e.g., the second period of time as referenced above) with inductive medium includes a region including one or more pancreatic-like cell population; optionally wherein the region including one or more pancreatic-like cell population includes a majority of the cells of the pancreatic organoid; optionally wherein the region including one or more pancreatic-like cell population includes at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the cells of the pancreatic organoid. In some embodiments, the pancreatic organoid cultured following a pulse (e.g., the second period of time as referenced above) with inductive medium expresses one or more pancreatic markers, such as, for example, cytokeratin-19, glucagon, c-peptide, neurogenin3, chromogranin A, claudin-18, E- cadherin, vimentin, PECAM, GATA4, PDX1, NKX6.1, FOXF1, WT-1, Ki67, and / or SOX9.

[0126] In some embodiments, the precursor pancreatic organoid can be cultured in a permissive medium in the absence of growth factors and / or signaling pathway activators and / or inhibitors, to provide a cultured pancreatic organoid with variably committed lineage. In some embodiments, the methods can further optionally include an additional step of culturing the pancreatic organoid without activating and / or inhibiting one or more signaling pathways, for a third period of time, to provide a cultured pancreatic organoid. In some embodiments, the pancreatic organoid cultured following a pulse (e.g., the second period of time as referenced above) with permissive medium, and without any pulse of inductive medium, includes a region including one or more pancreatic-like cell population, as well as one or more intestinal-like, duodenum-like, and / or stomach-like cell population; optionally wherein the region including one or more pancreatic-like cell population includes a majority of the cells of the pancreatic organoid. In some embodiments, the pancreatic organoid cultured following a pulse (e.g., the second period of time as referenced above) with inductive medium expresses one or more gastrointestinal markers, such as, for example, claudin-18, MUC5AC, SOX2, CDX2, and / or CDH17.

[0127] In some embodiments, the cells of the posterior foregut, splanchnic mesenchyme, and vascular mesenchyme self-sort, or self-assemble, into two or more regions, following any of steps c), d), or e). For example, the cells can self-sort, or self-assemble, into aninner region including pancreatic epithelium, or into an outer region including mesenchyme and / or vascular endothelium.

[0128] In some embodiments, the first period of time is at least about 1 day, or longer; optionally at least about 1 day to 3 days, 1 day to 5 days, or longer; optionally at least about 1 day. In some embodiments, the second period of time is selected from at least about 1 day to 10 days, or longer; optionally at least about 1 day to 7 days; optionally at least about 7 days. In some embodiments, the third period of time is at least about 1 day, or longer; optionally 1 day to to 7 days, 1 day to 14 days, 1 day to 60 days, 1 day to 70 days, or longer; optionally at least about 1 day, 7 days, 60 days, 70 days, or longer.

[0129] In some embodiments, the single cell suspension of posterior foregut cells can be cultured with single cell suspensions of both posterior splanchnic mesenchyme and vascular mesenchyme. In some embodiments, the single cell suspension of posterior foregut cells is cultured with a single cell suspension of vascular mesenchyme, and the pancreatic organoid includes a majority of pancreatic-like cell population. In some embodiments, the single cell suspension of posterior foregut cells is cultured with a single cell suspension of posterior splanchnic mesenchyme, and wherein the PO comprises one or more stomach-pancreas boundary cell population.

[0130] In some embodiments, the single cell suspensions of posterior splanchnic mesenchyme and vascular mesenchyme are recombined with the single cell suspension of posterior foregut cells in a ratio of at least about 1:1 to 10:1, or greater, mesenchymal cells to posterior foregut cells; optionally at least about 2:1 to 6:1; optionally about 4:1. In some embodiments, the single cell suspensions of posterior splanchnic mesenchyme and vascular mesenchyme are recombined with the single cell suspension of posterior foregut cells to provide at least about 1,000 to 100,000 cells / organoid; optionally at least about 2,000 to 50,000 cells / organoid; optionally about 10,000 cells / organoid. In some embodiments, the posterior splanchnic mesenchyme and vascular mesenchyme are recombined with the single cell suspension of posterior foregut cells in a ratio of at least about 1:10 to 10:1, splanchnic mesenchymal cells to vascular mesenchymal cells; optionally at least about 5:1 to 1:5; optionally at least about 2:1 to 1:2; optionally about 1:1.

[0131] In some embodiments, the posterior foregut cells are derived from definitive endoderm, optionally wherein the definitive endoderm has been derived from pluripotent stem cells, and the posterior splanchnic mesenchyme and vascular mesenchyme arc derived frompluripotent stem cells, such as, for example, embryonic stem cells or induced pluripotent stem cells. In some embodiments, the posterior splanchnic mesenchyme, vascular' mesenchyme, and / or posterior foregut cells are derived from pluripotent stem cells, such as, for example, embryonic stem cells or induced pluripotent stem cells; optionally human pluripotent stem cells.

[0132] In some embodiments, the splanchnic mesenchyme, vascular' mesenchyme, and / or posterior foregut cells can be dissociated into single cell suspensions via enzymatic dissociation, such as, for example, via dissociating the organoid with trypsin, chymotrypsin, collagenase, papain, hyaluronidase, elastase, thermolysin, neutral protease, or any combination thereof. In some embodiments, the splanchnic mesenchyme, vascular mesenchyme, and / or posterior foregut cells can be dissociated into single cell suspensions via enzymatic dissociation, such as, for example, via manual dissociation and / or passing the organoid through successively narrower bore channels. In some embodiments, the splanchnic mesenchyme, vascular mesenchyme, and / or posterior foregut cells can be dissociated into single cell suspensions via use of an extracellular matrix depolymerization solution.

[0133] The methods of forming pancreatic organoids as described herein can be performed in suspension culture and can be free of extracellular' matrix. Alternatively, the methods of forming pancreatic organoids as described herein can be performed in a basement membrane matrix, such as Matrigel.

[0134] Some embodiments of the methods can include methods of producing the splanchnic mesenchyme, vascular mesenchyme, and / or posterior foregut cells.

[0135] In some embodiments, splanchnic mesenchyme is produced by a method including: a) activating a TGF-b signaling pathway, activating a BMP signaling pathway, activating a Wnt signaling pathway, activating an FGF signaling pathway, and activating a PI3K signaling pathway, in dissociated pluripotent stem cells, for a first period of time, to generate mid primitive streak cells; b) activating a TGF-b signaling pathway, activating a BMP signaling pathway, activating a retinoic acid signaling pathway, and activating a Wnt signaling pathway, in the mid primitive streak cells of step a), for a second period of time, to generate posterior foregut lateral plate mesoderm; and c) activating a TGF-b signaling pathway, activating a BMP signaling pathway, activating a retinoic acid signaling pathway, activating a Wnt signaling pathway, and activating an FGF signaling pathway, in the posterior foregut lateral plate mesoderm of step b), for a third period of time, to generate posterior splanchnic mesenchyme. In some embodiments, splanchnic mesenchyme is produced by a method including: a) contacting dissociated pluripotentstem cells with a TGF-b signaling pathway activator, a BMP signaling pathway activator, a Wnt signaling pathway activator, an FGF signaling pathway activator, and a PI3K signaling pathway activator, for a first period of time, to generate mid primitive streak cells; b) contacting the mid primitive streak cells of step a) with a TGF-b signaling pathway activator, a BMP signaling pathway activator, a retinoic acid signaling pathway activator, and a Wnt signaling pathway activator, for a second period of time, to generate posterior foregut lateral plate mesoderm; and c) contacting the posterior foregut lateral plate mesoderm of step b) with a TGF-b signaling pathway activator, a BMP signaling pathway activator, a retinoic acid signaling pathway activator, a Wnt signaling pathway activator, and an FGF signaling pathway activator, for a third period of time, to generate posterior splanchnic mesenchyme. In some embodiments, the first period of time is, is about, is at least, is at least about, is not more than, or is not more than about, 0.5, 1, 2, 3, or 4 days, or a range defined by any two of the preceding values, for example 0.5-4, 1-4, 0.5-2, or 3-4 days. In some embodiments, the second period of time is, is about, is at least, is at least about, is not more than, or is not more than about 0.5, 1, or 2 days, or a range defined by any two of the preceding values, for example 0.5-4, 1-4, 0.5-2, or 3-4 days. In some embodiments, the third period of time is, is about, is at least, is at least about, is not more than, or is not more than about, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or a range defined by any two of the preceding values, for example 1-3, 1-5, 4-30, 10-30, 20-30, 4-17, 4-12, or 10-25 days.

[0136] In some embodiments, vascular mesenchyme is produced by a method including: a) inhibiting a ROCK signaling pathway in dissociated pluripotent stem cells in aggregation media, for a first period of time, to generate aggregates; b) activating a Wnt signaling pathway, and activating a BMP signaling pathway, in the aggregates of step a), for a second period of time, to induce mesoderm formation; and c) activating a VEGF signaling pathway, and activating a cAMP signaling pathway, in the mesoderm of step b), for a third period of time, to induce vascular mesenchyme formation. In some embodiments, vascular mesenchyme is produced by a method including: a) contacting dissociated pluripotent stem cells in aggregation media with a ROCK signaling pathway inhibitor, for a first period of time, to generate aggregates; b) contacting the aggregates of step a) with a Wnt signaling pathway activator, and a BMP signaling pathway activator, for a second period of time, to induce mesoderm formation; and c) contacting the mesoderm of step b) with a VEGF signaling pathway activator, and a cAMP signaling pathway activator, for a third period of time, to induce vascularmesenchyme formation. In some embodiments, the first period of time is, is about, is at least, is at least about, is not more than, or is not more than about, 0.5, 1, 2, 3, or 4 days, or a range defined by any two of the preceding values, for example 0.5-4, 1-4, 0.5-2, or 3-4 days. In some embodiments, the second period of time is, is about, is at least, is at least about, is not more than, or is not more than about 0.5, 1, 2, 3, or 4 days, or a range defined by any two of the preceding values, for example 0.5-4, 1-4, 0.5-3, or 1-4 days. In some embodiments, the third period of time is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or a range defined by any two of the preceding values, for example 1-3, 1-5, 4-30, 10-30, 20-30, 4- 17, 4-12, or 10-25 days.

[0137] In some embodiments, posterior foregut cells are produced by a method including: a) activating an FGF signaling pathway activator and activating a Wnt signaling pathway for a first period of time, with or without inhibiting a BMP signaling pathway, definitive endoderm cells (DE); and b) activating an FGF signaling pathway, activating a Wnt signaling pathway, and activating a retinoic acid (RA) signaling pathway for a second period of time, with or without inhibiting a BMP signaling pathway, in the cells of step a), thereby differentiating the DE to posterior foregut cells. In some embodiments, posterior foregut cells are produced by a method including: a) contacting DE with an FGF signaling pathway activator and a Wnt signaling pathway activator for a first period of time, with or without a BMP inhibitor; b) contacting the cells of step a) with the FGF signaling pathway activator, the Wnt signaling pathway activator, and a retinoic acid (RA) signaling pathway activator for a second period of time, with or without a BMP inhibitor, thereby differentiating the DE to posterior foregut cells. In some embodiments, the DE has been derived from pluripotent stem cells. In some embodiments, the pluripotent stem cells are embryonic stem cells and / or induced pluripotent stem cells. In some embodiments, the first period of time is, is about, is at least, is at least about, is not more than, or is not more than about, 0.5, 1, 2, 3, or 4 days, or a range defined by any two of the preceding values, for example 0.5-4, 1-4, 0.5-2, or 3-4 days. In some embodiments, the second period of time is, is about, is at least, is at least about, is not more than, or is not more than about 0.5, 1, or 2 days, or a range defined by any two of the preceding values, for example 0.5-4, 1-4, 0.5-2, or 3-4 days. In some embodiments, the third period of time is, is about, is at least, is at least about, is not more than, or is not more than about, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or a range defined by anytwo of the preceding values, for example 4-30, 10-30, 20-30, 4-17, 4-12, or 10-25 days. In some embodiments, the basement membrane matrix is Matrigcl. In some embodiments, the pancreatic organoid, DE, and / or pluripotent stem cells are derived from a subject.

[0138] In some embodiments of the methods of making pancreatic organoids, the FGF signaling pathway activator is selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF4, FGF 5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF 10, FGF 11, FGF 12, FGF13, FGF 14, FGF 15, FGF 16, FGF 17, FGF 18, FGF 19, FGF20, FGF21 , FGF22, and FGF23. in some embodiments, the FGF signaling pathway activator is FGF4. In some embodiments, the FGF signaling pathway activator is contacted at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 ng / mE, or any concentration within a range defined by any two of the aforementioned concentrations, including 100-1000 ng / mE, 100-500 ng / mL, 500-1000 ng / mL, 250-750 ng / mL, or 400-600 ng / mL, In some embodiments, the FGF signaling pathway activator is contacted at a concentration of 500 ng / mL or about 500 ng / mL.

[0139] In some embodiments of the methods of making pancreatic organoids, the Wnt signaling pathway activator is selected from the group consisting of Wntl, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, WntlOa, WntlOb, Wntl l, Wntl6, BML 284, IQ-1, WAY 262611, CHIR99021, CHIR98014, AZD2858, BIO, AR-A014418, SB 216763, SB 415286, aloisine, indirubin, alsterpaullone, kenpaullone, lithium chloride, TDZD 8, and TWS119. In some embodiments, the Wnt signaling pathway activator is CHIR99021. In some embodiments, the Wnt signaling pathway activator is contacted at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, or 3.5 mM, or any concentration within a range defined by any two of the aforementioned concentrations, including 0.5-3.5 mM, 0.5-2 mM, 2-3.5 mM, 1-3 mM, or 1.5-2.5 mM. In some embodiments, the Wnt signaling pathway activator is contacted at a concentration of 2 mM or about 2 mM.

[0140] In some embodiments of the methods of making pancreatic liver organoids, the RA signaling pathway activator is selected from the group consisting of retinoic acid, alL trans retinoic acid, 9-eis retinoic acid, CD437, EC23, BS 493, TTNPB, and AMS 80. In some embodiments, the RA signaling pathway activator is RA. In some embodiments, the RA signaling pathway activator is contacted at a concentration that is, is about, is at least, is at leastabout, is not more than, or is not more than about, 1 , 1.1 , 1.2, 1.3, 1 .4, 1 .5, 1 .6, 1 .7, 1 .8, 1 .9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.9, or 3 mM, or any concentration within a range defined by any two of the aforementioned concentrations, including 1-3 mM, 1-2 mM, 2-3 mM, or 1.5-2.5 mM. In some embodiments, the RA signaling pathway activator is contacted at a concentration of 2 mM or about 2 mM.

[0141] Also disclosed herein are the pancreatic organoids produced by any of the methods described herein.Characteristics of Pancreatic OrganoidsPancreatic organoids according to the present disclosure include a posterior foregut cell population, and a vascular mesenchyme recombinant cell population and / or a splanchnic mesenchyme recombinant cell population, and can have various structural and functional characteristics, which recapitulate one or more aspects of in vivo pancreatic development, particularly human pancreatic development. For example, in some embodiments, pancreatic organoids according to the disclosure can include one or more pancreas-like cellular populations, such as endocrine cells, islets, ductal and / or exocrine structures, and / or endocrine pancreas and / or stratified epithelial pancreatic progenitors. For example, in some embodiments, pancreatic organoids according to the disclosure can include one or more pancreas-like structural features, such as an epithelial cavity or network, and one or more lumen and / or microlumen; optionally wherein the one or more lumen and / or microlumen is polarized. In some embodiments, the one or more lumen and / or microlumen can be adjacent to the epithelial cavity or network. In some embodiments, pancreatic organoids according to the disclosure can include one or more branched or interconnected ductal networks and / or acing structures; optionally wherein the acing structures comprise acinar cells and / or acinar tissue localized at one or more ductal network terminus, and / or wherein the acinar cells and / or acinar tissue are polarized. In some embodiments, pancreatic organoids according to the disclosure lack nerve cells and / or immune cells. In some embodiments, pancreatic organoids according to the disclosure do not undergo anisotropic growth and / or lack hierarchical vascularization

[0142] In some embodiments, pancreatic organoids according to the disclosure can express one or more pancreas-associated markers or biomarkers. For example, in some embodiments, pancreatic organoids according to the disclosure can include one or more posterior foregut and / or pancreatic progenitor marker. In some embodiments, pancreatic organoidsaccording to the disclosure can express cytokeratin-19, glucagon, c-peptide, neurogenin3, chromogranin A, claudin-18, E-cadhcrin, vimcntin, PECAM, GATA4, PDX1, NKX6.1, FOXF1, WT-1, Ki67, SOX2, and / or SOX9. In some embodiments, the posterior foregut cell population of the pancreatic organoids according to the disclosure can express NKX6.1, and / or the vascular mesenchymal cell population can express PDX1 and / or E-cadherin; and / or the splanchnic mesenchymal cell population can express SOX2, E-cadherin, and / or claudin-18. In some embodiments, the vascular mesenchymal cell population of the pancreatic organoids according to the disclosure can express claudin-18 only in a cystic lumen region. In some embodiments, pancreatic organoids according to the disclosure can include a posterior foregut cell population and a vascular mesenchymal cell population, and wherein the posterior foregut cell population expresses NKX6.1, SOX9, and / or PDX1. In some embodiments, pancreatic organoids according to the disclosure can include a splanchnic mesenchymal cell population and a vascular mesenchymal cell population, and wherein the splanchnic mesenchymal cell populand / or vascular mesenchymal cell population express FOXF1 and / or WT-1. In some embodiments, cultured pancreatic organoids according to the disclosure can express Ki67 after culturing for 60 days, or longer.

[0143] . In some embodiments, pancreatic organoids according to the disclosure can include endocrine cells expressing neurogenin3 and / or chromogranin A; islets or islet-like structures expressing glucagon and / or c-peptide; and / or an epithelial network expressing cytokeratin-19. In some embodiments, pancreatic organoids according to the disclosure can include acinar cells and / or acinar tissue expressing GATA4, and downregulated expression of SOX9 and / or PDX1. In some embodiments, pancreatic organoids according to the disclosure can include a ductal network expressing SOX9 and downregulated expression of GATA4. In some embodiments, pancreatic organoids according to the disclosure can include a lumen expressing SOX2, surrounded by or adjacent to an epithelium expressing PDX1.

[0144] Pancreatic organoids according to the disclosure have been shown to secrete various pancreatic enzymes, as with normal, in vivo pancreatic development and function. For example, in some embodiments, pancreatic organoids according to the disclosure can secrete one or more pancreatic enzymes in the acinar cells, acinar tissue, lumen, and / or microlumen. In some embodiments, pancreatic organoids according to the disclosure can secrete zymogen granules, CPA1, mucin- 1, and / or amylase in the acinar cells, acinar tissue, lumen, and / or microlumen. In some embodiments, pancreatic organoids according to the disclosure can secrete CPA1 andamylase, wherein the CPA1 and amylase secretion is co-localized. In some embodiments, pancreatic organoids according to the disclosure can include a lumen adjacent to the epithelial network, wherein the lumen secretes mucin- 1.Pancreatic-Related Diseases and Disorders

[0145] The pancreatic organoids of the disclosure can be used in treatment and / or studying or modeling pancreatic-related diseases and disorders, for which their ability to recapitulate one or more aspects of in vivo pancreatic development, particularly human pancreatic development, is particularly advantageous and renders them applicable to a wide range of conditions. In some embodiments, the methods include administering any of the pancreatic organoids or pancreatic cell compositions disclosed herein. Also disclosed herein are the pancreatic organoids or pancreatic cell compositions disclosed herein for use in the manufacture of a medicament for the treatment of a pancreatic-related disease or disorder. Also disclosed herein are the pancreatic organoids or pancreatic cell compositions disclosed herein for use in the treatment of a pancreatic-related disease or disorder in a subject in need thereof.

[0146] Pancreatic -related diseases and disorders relevant to the pancreatic organoids of the disclosure can include conditions such as cancer (e.g. pancreatic ductal adenocarcinoma (PDAC), pancreatic neuroendocrine tumor, bile duct cancer (cholangiocarcinoma), etc.), cystic fibrosis, pancreatitis, exocrine pancreatic insufficiency (EPI), pancreatic cysts, bile duct cysts, diabetes, and the like. One skilled in the art will appreciate other pancreatic-related diseases and conditions for which the pancreatic organoids or pancreatic cell compositions disclosed herein could have relevance.

[0147] For example, the pancreatic organoid can be transplanted into a subject having pancreatic dysfunction and / or failure, where the transplanted pancreatic organoids engraft onto the pancreas of the subject. Following transplantation, the subject can have increased survival rate and or improved symptoms.

[0148] For example, these pancreatic organoids can be used an in vitro human model system for studying pancreatic cell function and developmental divergence, studying pancreatic- related disease, identifying and / or screening for therapeutic targets, and / or identifying therapeutic compounds and / or compositions effective in treating a pancreatic-related disease or disorder. Accordingly, the pancreatic organoids of the disclosure can allow for new developments in pancreatic disease treatment and study.Stem Cells

[0149] The term “totipotent stem cells” (also known as omnipotent stem cells) as used herein has its plain and ordinary meaning as understood in light of the specification and 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.

[0150] The term “embryonic stem cells (ESCs),” also commonly abbreviated as ES cells, as used herein has its plain and ordinary meaning as understood in light of the specification and refers to cells that are pluripotent and derived from the inner cell mass of the blastocyst, an early- stage embryo. For purpose of the present disclosure, the term "ESCs" is used broadly sometimes to encompass the embryonic germ cells as well.

[0151] The term “pluripotent stem cells (PSCs)” as used herein has its plain and ordinary' meaning as understood in light of the specification and encompasses any cells that can differentiate into nearly all cell types of the body, 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 inner cell mass cells of the preimplantation blastocyst or obtained through induction of a non-pluripotent cell, such as an adult somatic cell, by forcing the expression of certain genes. Pluripotent stem cells can be derived from any suitable source. Examples of sources of pluripotent stem cells include mammalian sources, including human, rodent, porcine, and bovine.

[0152] The term “induced pluripotent stem cells (iPSCs),” also commonly abbreviated as iPS cells, as used herein has its plain and ordinary meaning as understood in light of the specification and 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. hiPSC refers to human iPSCs. In some methods known in the ail, iPSCs may be derived by transfection of certain stem cell-associated genes into non-pluripotent cells, such as adult fibroblasts. Transfection may be achieved through viral transduction using viruses such as retroviruses or lentiviruses. Transfected genes may include the master transcriptional regulators Oct-3 / 4 (PUU5F1) and Sox2, although other genes may 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 arc typically isolated through morphologicalselection, doubling time, or through a reporter gene and antibiotic selection. As used herein, iPSCs include first generation iPSCs, second generation iPSCs in mice, and human induced pluripotent stem cells. In some methods, 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 other methods, a lentiviral system is used to transform somatic cells with GCT4, SOX2, NANOG, and LIN28. Genes whose expression are induced in iPSCs include but are not limited to Oct-3 / 4 (POU5F1); certain members of the Sox gene family (e.g., Soxl, Sox2, Sox3, and Soxl5); certain members of the Klf family (e.g., KID, Klf2, Klf4, and Klf5), certain members of the Mye family (e.g., C-myc, L-myc, and N- myc), Nanog, LIN28, Tert, Fbxl5, ERas, EC ATI 5- 1, ECAT15-2, Tell, b-Catenm, EC ATI, Esgi, Dnmt3L, EC ATS, Gdf3, Fthll7, Sall4, Rexl, UTF1, Stella, Stat3, Grb2, Prdml4, Nr5al, Nr5a2, or E-cadherin, or any combination thereof.

[0153] The term “precursor cell” as used herein has its plain and ordinary meaning as understood in light of the specification and 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 embodiments, a precursor cell is pluripotent or has the capacity to becoming pluripotent. In some embodiments, the precursor cells are subjected to the treatment of external factors (e.g., growth factors) to acquire pluripotency. In some embodiments, a precursor cell can be a totipotent (or omnipotent) stem cell; a pluripotent stem cell (induced or non-induced); a multipotent stem cell; an oligopotent stem cells and a unipotent stem cell. In some embodiments, a precursor cell can be from an embryo, an infant, a child, or an adult. In some embodiments, a precursor cell can be a somatic cell subject to treatment such that pluripotency is conferred via genetic manipulation or protein / peptide treatment. Precursor cells include embryonic stem cells (ESC), embryonic carcinoma cells (ECs), and epiblast stem cells (EpiSC).

[0154] In some embodiments, one step can include obtaining stem cells that are pluripotent or can be induced to become pluripotent. In some embodiments, 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. It would be understood by one of skill in the art that the methods and systems described herein arc applicable to any stem cells.

[0155] Additional stem cells that can be used in embodiments in accordance with the present disclosure include but arc 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 (IJW- SCRMC); Novocell, Inc. (San Diego, Calif.); Cellartis AB (Goteborg, Sweden); ES Cell International Pte Etd (Singapore); Techmon 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 disclosure include but are not limited to SA01 (SA001); SA02 (SA002); ESDI (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 (HI); WA07 (H7); WA09 (H9); WA13 (HI 3); WA14 (HI 4). Exemplary human pluripotent cell lines include but are not limited to TkDA3-4, 1231 A3, 317-D6, 317-A4, CDH1, 5-T-3, 3-34- 1, NAFLD27, NAFLD77, NAFLD150, WD90, WD91, WD92, 1.20012. C213, 1383D6, FF, or 317-12 cells.

[0156] 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.

[0157] In some embodiments, an adenovirus can be used to transport the requisite four genes, resulting in iPSCs substantially 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 embodiments, non-viral based technologies are employed to generate iPSCs. In some embodiments, reprogramming can be accomplished via plasmid without any virus transfection system at all, although at very low efficiencies. In other embodiments, direct deliver}' 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 thecells via poly-arginine anchors was sufficient to induce pluripotency. In some embodiments, the expression of pluripotency induction genes can also be increased by treating somatic cells with FGF2 under low oxygen conditions.

[0158] The term “feeder cell” as used herein has its plain and ordinary meaning as understood in light of the specification and refers to cells that support the growth of pluripotent stem cells, such as by secreting growth factors into the medium or displaying on the cell surface. Feeder cells are generally adherent cells and may be growth arrested. For example, feeder cells are growth-arrested by irradiation (e.g. gamma rays), mitomycin-C treatment, electric pulses, or mild chemical fixation (e.g. with formaldehyde or glutaraldehyde). However, feeder cells do not necessarily have to be growth arrested. Feeder cells may serve purposes such as secreting growth factors, displaying growth factors on the cell surface, detoxifying the culture medium, or synthesizing extracellular matrix proteins. In some embodiments, the feeder cells are allogeneic or xenogeneic to the supported target stem cell, which may have implications m downstream applications. In some embodiments, the feeder cells are mouse cells. In some embodiments, the feeder cells are human cells. In some embodiments, the feeder cells are mouse fibroblasts, mouse embryonic fibroblasts, mouse STO cells, mouse 3T3 cells, mouse SNL 76 / 7 cells, human fibroblasts, human foreskin fibroblasts, human dermal fibroblasts, human adipose mesenchymal cells, human bone marrow mesenchymal cells, human amniotic mesenchymal cells, human amniotic epithelial cells, human umbilical cord mesenchymal cells, human fetal muscle cells, human fetal fibroblasts, or human adult fallopian tube epithelial cells. In some embodiments, conditioned medium prepared from feeder cells is used in lieu of feeder cell co-culture or in combination with feeder cell co-culture. In some embodiments, feeder cells are not used during the proliferation of the target stem cells.Differentiation of PSCs

[0159] Known methods for producing definitive endoderm from pluripotent cells (e.g., iPSCs or ESCs) are applicable to the methods described herein. In some embodiments, pluripotent cells are derived from a morula. In some embodiments, pluripotent stem cells are stem cells. Stem cells used in these methods can include, but are not limited to, embryonic stem cells or induced pluripotent 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 embodiments, human embryonic stem cells are used toproduce definitive endoderm. In some embodiments, human embryonic germ cells are used to produce definitive endoderm. In some embodiments, iPSCs arc used to produce definitive endoderm, in some embodiments, human iPSCs (hiPSCs) are used to produce definitive endoderm.

[0160] In some embodiments, PSCs, such as ESCs and iPSCs, undergo directed differentiation into embryonic germ layer cells, organ tissue progenitor cells, and then into tissue such as liver tissue or any other biological tissue. In some embodiments, the directed differentiation is done in a stepwise manner to obtain each of the differentiated cell types where molecules (e.g. growth factors, ligands, agonists, antagonists) are added sequentially as differentiation progresses. In some embodiments, the directed differentiation is done in a nonstepwise manner where molecules (e.g. growth factors, ligands, agonists, antagonists) are added at the same time. In some embodiments, directed differentiation is achieved by selectively activating certain signaling pathways in the PSCs or any downstream cells.

[0161] In some embodiments, human embryonic stem cells are used to produce pluripotent stem cells, definitive endoderm, mid primitive streak cells, plate mesoderm, lateral plate mesoderm, posterior foregut lateral plate mesoderm, posterior foregut, foregut spheroids, mid / hindgut spheroids, splanchnic mesenchyme, vascular mesenchyme, intestinal organoids, esophageal organoids, gastric organoids, colonic organoids, vascularized intestinal organoids, vascularized esophageal organoids, vascularized gastric, and / or vascularized colonic organoids, or any combination thereof. In some embodiments, iPSCs are used to produce pluripotent stem cells, definitive endoderm, mid primitive streak cells, plate mesoderm, lateral plate mesoderm, posterior foregut lateral plate mesoderm, posterior foregut, foregut spheroids, mid / hindgut spheroids, splanchnic mesenchyme, vascular mesenchyme, intestinal organoids, esophageal organoids, gastric organoids, colonic organoids, vascularized intestinal organoids, vascularized esophageal organoids, vascularized gastric, and / or vascularized colonic organoids, or any combination thereof. In some embodiments, human iPSCs (hiPSCs) are used to produce pluripotent stem cells, definitive endoderm, mid primitive streak cells, plate mesoderm, lateral plate mesoderm, posterior foregut lateral plate mesoderm, posterior foregut, foregut spheroids, mid / hindgut spheroids, splanchnic mesenchyme, vascular mesenchyme, intestinal organoids, esophageal organoids, gastric organoids, colonic organoids, vascularized intestinal organoids, vascularized esophageal organoids, vascularized gastric, and / or vascularized colonic organoids, or any combination thereof.

[0162] In some embodiments, the embryonic stem cells or germ cells or iPSCs are treated with one or more small molecule compounds, activators, inhibitors, or growth factors for a time that is, is about, is at least, is at least about, is not more than, or is not more than about, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 120 hours, 150 hours, 180 hours, 240 hours, 300 hours or any time within a range defined by any two of the aforementioned times, for example 6 hours to 300 hours, 24 hours to 120 hours, 48 hours to 96 hours, 6 hours to 72 hours, or 24 hours to 300 hours, in some embodiments, more than one small molecule compounds, activators, inhibitors, or growth factors are added. In these cases, the more than one small molecule compounds, activators, inhibitors, or growth factors can be added simultaneously or separately.

[0163] In some embodiments, the embryonic stem cells, germ cells, PSCs, iPSCs, or definitive endoderm, mid primitive streak cells, plate mesoderm, lateral plate mesoderm, posterior foregut lateral plate mesoderm, posterior foregut, foregut spheroids, mid / hindgut spheroids, splanchnic mesenchyme, vascular mesenchyme, intestinal organoids, esophageal organoids, gastric organoids, colonic organoids, vascularized intestinal organoids, vascularized esophageal organoids, vascularized gastric, and / or vascularized colonic organoids, are treated with one or more small molecule compounds, activators, inhibitors, or growth factors at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 10 ng / mL, 20 ng / mL, 50 ng / mL, 75 ng / mL, 100 ng / mL, 120 ng / mL, 150 ng / mL, 200 ng / mL, 500 ng / mL, 1000 ng / mL, 1200 ng / mL, 1500 ng / mL, 2000 ng / mL, 5000 ng / mL, 7000 ng / niL, 10000 ng / mL, or 15000 ng / mL, or any concentration that is within a range defined by any two of the aforementioned concentrations, for example, 10 ng / mL to 15000 ng / mL, 100 ng / mL to 5000 ng / mL, 500 ng / mL to 2000 ng / mL, 10 ng / mL to 2000 ng / mL, or 1000 ng / mL to 15000 ng / mL. In some embodiments, concentration of the one or more small molecule compounds, activators, inhibitors, or growth factors is maintained at a constant level throughout the treatment. In some embodiments, concentration of the one or more small molecule compounds, activators, inhibitors, or growth factors is varied during the course of the treatment. In some embodiments, more than one small molecule compounds, activators, inhibitors, or growth factors are added. In these cases, the more than one small molecule compounds, activators, inhibitors, or growth factors can differ in concentrations.

[0164] In some embodiments, the ESCs or iPSCs, or the ESCs, germ cells, or iPSCs arc cultured in growth media that supports the growth of stem cells. In some embodiments, theESCs or iPSCs, or the ESCs, germ cells, or iPSCs, are cultured in stem cell growth media. In some embodiments, the stem cell growth media is RPMI 1640, DMEM, DMEM / F12, or Advanced DMEM / F12. In some embodiments, the stem cell growth media comprises fetal bovine serum (FBS). In some embodiments, the stem cell growth media comprises FBS at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0,6%, 0.7%, 0.8%, 0,9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or any percentage within a range defined by any two of the aforementioned concentrations, for example 0% to 20%, 0.2% to 10%, 2% to 5%, 0% to 5%, or 2% to 20%. In some embodiments, the stem cell growth media does not contain xenogeneic components. In some embodiments, the growth media comprises one or more small molecule compounds, activators, inhibitors, or growth factors.

[0165] In some embodiments, populations of cells enriched in definitive endoderm cells are used. In some embodiments, the definitive endoderm cells are isolated or substantially purified. In some embodiments, pluripotent stem cells are prepared from somatic cells the isolated or substantially purified definitive endoderm cells express one or more (e.g. at least 1, 3) of SOX 17, FOXA2, or CXRC4 markers to a greater extent than one or more (e.g. at least 1, 3, 5) of GCT4, AFP, I'M, SPARC, or SGX7 markers.

[0166] In some embodiments, pluripotent stem cells are prepared from somatic cells. In some embodiments, pluripotent stem cells are prepared from biological tissue obtained from a biopsy. In some embodiments, the pluripotent stem cells are cryopreserved. In some embodiments, the somatic cells are cryopreserved. In some embodiments, pluripotent stem cells are prepared from PBMCs. In some embodiments, human PSCs are prepared from human PBMCs. In some embodiments, pluripotent stem cells are prepared from cryopreserved PBMCs. In some embodiments, pluripotent stem cells are prepared from PBMCs by viral transduction. In some embodiments, PBMCs are transduced with Sendai virus, lentivirus, adenovirus, or adeno- associated virus, or any combination thereof. In some embodiments, PBMCs are transduced with Sendai virus comprising expression vectors for Oct3 / 4, Sox2, Klf4, or L-Myc, or any combination thereof. In some embodiments, PBMCs are grown on a feeder cell substrate. In some embodiments, PBMCs are grown on a mouse embryonic fibroblast (MEF) feeder cell substrate. In some embodiments, PBMCs are grown on an irradiated MEF feeder cell substrate. In some embodiments, iPSCs arc grown on an irradiated MEF feeder cell substrate. In someembodiments, iPSCs are grown in RPMI 1640, DMEM, DMEM / F12, mTeSR 1 , or mTeSR Plus media.

[0167] In some embodiments, iPSCs are expanded in cell culture. In some embodiments, iPSCs are expanded in an extracellular matrix, or mimetic or derivative thereof. In some embodiments, the extracellular matrix, or mimetic or derivative thereof, comprises polymers, proteins, polypeptides, nucleic acids, sugars, lipids, poly-lysine, poly-omithine, collagen, gelatin, fibronectin, vitronectin, laminin, elastin, tenascin, heparan sulfate, entactin, nidogen, osteopontin, basement membrane, Matrigel, Geltrex, hydrogel, PEI, WGA, or hyaluronic acid, or any combination thereof. In some embodiments, PSCs are expanded in Matrigel, Geltrex, or 1% gelatin, or any combination thereof. In some embodiments, iPSCs are expanded in Matrigel. In some embodiments, the iPSCs are expanded in cell culture while inhibiting a ROCK signaling pathway. In some embodiments, the iPSCs are expanded in cell culture comprising a ROCK inhibitor (e.g. Y-27632).

[0168] In some embodiments, inhibiting one or more signaling pathways, e.g. via providing proteins, activators, or inhibitors of the FGF, Wnt, BMP, TGF-b, EGF, PI3K, ROCK, cAMP, VEGF, Notch, HH, and / or retinoic acid (RA) pathways, or any combination thereof, can be used to mimic development in culture to obtain various cell types used herein that are differentiated from pluripotent stem cells. In some embodiments, cellular constituents associated with the FGF, Wnt, BMP, TGF-b, EGF, PI3K, ROCK, cAMP, VEGF, Notch, HH, and / or retinoic acid (RA) signaling pathways, for example, natural inhibitors, antagonists, activators, or agonists of the pathways can be used to result in inhibition or activation of the FGF, Wnt, BMP, TGF-b, EGF, PI3K, ROCK, cAMP, VEGF, Notch, HH, and / or retinoic acid (RA) signaling pathways. In some embodiments, siRNA and / or shRNA targeting cellular constituents associated with the FGF, Wnt, BMP, TGF-b, EGF, PI3K, ROCK, cAMP, VEGF, Notch, HH, and / or retinoic acid (RA) signaling pathways are used to inhibit or activate these pathways. Furthermore, the methods disclosed herein may also involve the use of an EGF pathway activator acting as a mitogen, which promotes proliferation and growth of desired cell populations.

[0169] In some embodiments, stem cells are treated with one or more growth factors to differentiate to definitive endoderm cells. Such growth factors can include growth factors from the TGF-beta superfamily. In some embodiments, the one or more growth factors comprise the Nodal / Activin and / or the BMP subgroups of the TGF-beta superfamily of growth factors. Insome embodiments, the one or more growth factors are selected from the group consisting of Nodal, Activin A, Activin B, A8301, BMP4, Wnt3a or combinations of any of these growth factors. In some embodiments, the stem cells are contacted with Activin A and / or A8301. In some embodiments, the stem cells are contacted with Activin A and BMP4.

[0170] In some embodiments, pluripotent stem cells, pluripotent stem cells, definitive endoderm, mid primitive streak cells, plate mesoderm, lateral plate mesoderm, posterior foregut lateral plate mesoderm, posterior foregut, foregut spheroids, mid / hindgut spheroids, splanchnic mesenchyme, vascular mesenchyme, intestinal organoids, esophageal organoids, gastric organoids, colonic organoids, vascularized intestinal organoids, vascularized esophageal organoids, vascularized gastric, and / or vascularized colonic organoids, or any combination thereof, are contacted with an FGF pathway activator or inhibitor. In some embodiments, the FGF pathway activator comprises an FGF protein. In some embodiments, the FGF protein comprises a recombinant FGF protein. In some embodiments, the FGF pathway activator comprises one or more of FGF1, FGF2, FGF3, FGF4, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15 (FGF19, FGF15 / FGF19), FGF16, FGF17, FGF18, FGF20, FGF21, FGF22, or FGF23. In some embodiments, the cells are not treated with an FGF pathway activator or inhibitor. FGF pathway activation and / or inhibition as used with embodiments provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein. The FGF pathway activator or inhibitor provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0171] In some embodiments, pluripotent stem cells, definitive endoderm, mid primitive streak cells, plate mesoderm, lateral plate mesoderm, posterior foregut lateral plate mesoderm, posterior foregut, foregut spheroids, mid / hindgut spheroids, splanchnic mesenchyme, vascular- mesenchyme, intestinal organoids, esophageal organoids, gastric organoids, colonic organoids, vascularized intestinal organoids, vascularized esophageal organoids, vascularized gastric, and / or vascularized colonic organoids, or any combination thereof, are contacted with a Wnt pathway activator or Wnt pathway inhibitor. In some embodiments, the Wnt pathway activator comprises a Wnt protein. In some embodiments, the Wnt protein comprises a recombinant Wnt protein. In some embodiments, the Wnt pathway activator comprises Wntl, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, WntlOa, WntlOb, Wntll, Wntl6, BML 284, IQ-1, WAY 262611, CHIR99021,CHIR 98014, AZD2858, BIO, AR-A014418, SB 216763, SB 415286, aloisine, indirubin, alstcrpaullonc, kcnpaullonc, lithium chloride, TDZD 8, TWS119, or any combination thereof. In some embodiments, the Wnt pathway activator comprises a GSK3 pathway inhibitor. In some embodiments, the Wnt pathway activator comprises CHIR99021, CHIR 98014, AZD2858, BIO, AR-A014418, SB 216763, SB 415286, aloisine, indirubin, alsterpaullone, kenpaullone, lithium chloride, TDZD 8, or TWS119, or any combination thereof. In some embodiments, the Wnt pathway inhibitor comprises C59, PNU 74654, KY-02111, PRI-724, FH-535, DIF-1, or XAV939, or any combination thereof. In some embodiments, the cells are not treated with a Wnt pathway activator or Wnt pathway inhibitor. Wnt pathway activation and / or inhibition as used with embodiments provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein. The Wnt pathway activator or Wnt pathway inhibitor provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0172] In some embodiments, pluripotent stem cells, pluripotent stem cells, definitive endoderm, mid primitive streak cells, plate mesoderm, lateral plate mesoderm, posterior foregut lateral plate mesoderm, posterior foregut, foregut spheroids, mid / hindgut spheroids, splanchnic mesenchyme, vascular mesenchyme, intestinal organoids, esophageal organoids, gastric organoids, colonic organoids, vascularized intestinal organoids, vascularized esophageal organoids, vascularized gastric, and / or vascularized colonic organoids, or any combination thereof, are contacted with a BMP pathway activator or BMP pathway inhibitor. In some embodiments, the BMP pathway activator comprises a BMP protein. In some embodiments, the BMP protein is a recombinant BMP protein. In some embodiments, the BMP pathway activator comprises BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP11, BMP15, IDE1, or IDE2, or any combination thereof. In some embodiments, the BMP pathway inhibitor comprises Noggin, Dorsomorphin, RepSox, LY364947, LDN193189, SB431542, or any combination thereof. In some embodiments, the cells are not treated with a BMP pathway activator or BMP pathway inhibitor. BMP pathway activation and / or inhibition as used with embodiments provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein. The BMP pathway activator or BMP pathway inhibitor provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0173] In some embodiments, pluripotent stem cells, pluripotent stem cells, definitive endoderm, mid primitive streak cells, plate mesoderm, lateral plate mesoderm, posterior foregut lateral plate mesoderm, posterior foregut, foregut spheroids, mid / hindgut spheroids, splanchnic mesenchyme, vascular mesenchyme, intestinal organoids, esophageal organoids, gastric organoids, colonic organoids, vascularized intestinal organoids, vascularized esophageal organoids, vascularized gastric, and / or vascularized colonic organoids, or any combination thereof, are contacted with a TGF-beta (TGF-b) pathway activator or TGF-b pathway inhibitor. In some embodiments, the TGF-b family comprises bone morphogenetic protein (BMP), growth and differentiation factor (GDF), anti-Miillerian hormone, Activin, and Nodal pathways. In some embodiments, the TGF-b pathway activator comprises TGF-b 1, TGF-b 2, TGF-b 3, Activin A, Activin B, Nodal, a BMP, IDE1, IDE2, or any combination thereof. In some embodiments, the TGF-b pathway inhibitor comprises A83O1, RepSox, LY365947, SB431542, or any combination thereof. In some embodiments, the cells are not treated with a TGF-b pathway activator or TGF- b pathway inhibitor. TGF-b pathway activation and / or inhibition as used with embodiments provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein. The TGF-b pathway activator or TGF-b pathway inhibitor provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0174] In some embodiments, pluripotent stem cells, pluripotent stem cells, definitive endoderm, mid primitive streak cells, plate mesoderm, lateral plate mesoderm, posterior foregut lateral plate mesoderm, posterior foregut, foregut spheroids, mid / hindgut spheroids, splanchnic mesenchyme, vascular mesenchyme, intestinal organoids, esophageal organoids, gastric organoids, colonic organoids, vascularized intestinal organoids, vascularized esophageal organoids, vascularized gastric, and / or vascularized colonic organoids, or any combination thereof, are contacted with an EGF pathway activator or inhibitor. In some embodiments, the EGF pathway activator is EGF. In some embodiments, the cells are not treated with an EGF pathway activator or inhibitor. EGF pathway activation and / or inhibition as used with embodiments provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein. The EGF pathway activator provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0175] In some embodiments, pluripotent stem cells, pluripotent stem cells, definitive endoderm, mid primitive streak cells, plate mesoderm, lateral plate mesoderm, posterior foregut lateral plate mesoderm, posterior foregut, foregut spheroids, mid / hindgut spheroids, splanchnic mesenchyme, vascular mesenchyme, intestinal organoids, esophageal organoids, gastric organoids, colonic organoids, vascularized intestinal organoids, vascularized esophageal organoids, vascularized gastric, and / or vascularized colonic organoids, or any combination thereof, are contacted with a PI3K pathway activator or inhibitor. In some embodiments, the PI3K pathway activator comprises 740 Y-P, or erucic acid, or any combination thereof. In some embodiments, the PI3K pathway inhibitor comprises wortmannin, LY294002, hibiscone C, PI- 103, IC-87114, ZSTK474, AS-605240, PIK-75, PIK-90, PIK-294, PIK-293, AZD6482, PF- 04691502, GSK1059615, quercetin, pluripotin, flurbiprofen, GDC-0941, dactolisib, pictilisib, idelalisib, buparlisib, rigosertib, copanlisib, duvelisib, alpelisib, or any combination thereof. In some embodiments, the cells are not treated with a PI3K pathway activator. PI3K pathway activation and / or inhibition as used with embodiments provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein. The PI3K pathway activator or inhibitor provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0176] In some embodiments, pluripotent stem cells, pluripotent stem cells, definitive endoderm, mid primitive streak cells, plate mesoderm, lateral plate mesoderm, posterior foregut lateral plate mesoderm, posterior foregut, foregut spheroids, mid / hindgut spheroids, splanchnic mesenchyme, vascular mesenchyme, intestinal organoids, esophageal organoids, gastric organoids, colonic organoids, vascularized intestinal organoids, vascularized esophageal organoids, vascularized gastric, and / or vascularized colonic organoids, or any combination thereof, are contacted with a ROCK pathway activator or inhibitor. In some embodiments, the ROCK pathway activator or inhibitor comprises Y -27632. In some embodiments, the cells are not treated with a ROCK pathway activator. ROCK pathway activation and / or inhibition as used with embodiments provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein. The ROCK pathway activator or inhibitor provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0177] In some embodiments, pluripotent stem cells, pluripotent stem cells, definitive endoderm, mid primitive streak cells, plate mesoderm, lateral plate mesoderm, posterior foregut lateral plate mesoderm, posterior foregut, foregut spheroids, mid / hindgut spheroids, splanchnic mesenchyme, vascular mesenchyme, intestinal organoids, esophageal organoids, gastric organoids, colonic organoids, vascularized intestinal organoids, vascularized esophageal organoids, vascularized gastric, and / or vascularized colonic organoids, or any combination thereof, are contacted with a cAMP pathway activator. In some embodiments, the cAMP pathway activator or inhibitor comprises forskolin. In some embodiments, the cells arc not treated with a cAMP pathway activator. cAMP pathway activation and / or inhibition as used with embodiments provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein. The cAMP pathway activator provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0178] In some embodiments, pluripotent stem cells, pluripotent stem cells, definitive endoderm, mid primitive streak cells, plate mesoderm, lateral plate mesoderm, posterior foregut lateral plate mesoderm, posterior foregut, foregut spheroids, mid / hindgut spheroids, splanchnic mesenchyme, vascular mesenchyme, intestinal organoids, esophageal organoids, gastric organoids, colonic organoids, vascularized intestinal organoids, vascularized esophageal organoids, vascularized gastric, and / or vascularized colonic organoids, or any combination thereof, are contacted with a VEGF pathway activator. In some embodiments, the VEGF pathway activator or inhibitor comprises VEGF-A. In some embodiments, the cells are not treated with a VEGF pathway activator or inhibitor. VEGF pathway activation and / or inhibition as used with embodiments provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein. The VEGF pathway activator provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0179] In some embodiments, pluripotent stem cells, pluripotent stem cells, definitive endoderm, mid primitive streak cells, plate mesoderm, lateral plate mesoderm, posterior foregut lateral plate mesoderm, posterior foregut, foregut spheroids, mid / hindgut spheroids, splanchnic mesenchyme, vascular mesenchyme, intestinal organoids, esophageal organoids, gastric organoids, colonic organoids, vascularized intestinal organoids, vascularized esophageal organoids, vascularized gastric, and / or vascularized colonic organoids, or any combinationthereof, are contacted with a Shh pathway activator. Tn some embodiments, the Shh pathway activator or inhibitor comprises SANT-1. In some embodiments, the cells arc not treated with a Shh pathway activator or inhibitor. Shh pathway activation and / or inhibition as used with embodiments provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein. The Shh pathway activator provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0180] In some embodiments, pluripotent stem cells, pluripotent stem cells, definitive endoderm, mid primitive streak cells, plate mesoderm, lateral plate mesoderm, posterior foregut lateral plate mesoderm, posterior foregut, foregut spheroids, mid / hindgut spheroids, splanchnic mesenchyme, vascular mesenchyme, intestinal organoids, esophageal organoids, gastric organoids, colonic organoids, vascularized intestinal organoids, vascularized esophageal organoids, vascularized gastric, and / or vascularized colonic organoids, or any combination thereof, are contacted with a CK2 pathway activator. In some embodiments, the CK2 pathway activator or inhibitor comprises TPB. In some embodiments, the cells are not treated with a CK2 pathway activator or inhibitor. CK2 pathway activation and / or inhibition as used with embodiments provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein. The CK2 pathway activator provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0181] In some embodiments, pluripotent stem cells, pluripotent stem cells, definitive endoderm, mid primitive streak cells, plate mesoderm, lateral plate mesoderm, posterior foregut lateral plate mesoderm, posterior foregut, foregut spheroids, mid / hindgut spheroids, splanchnic mesenchyme, vascular mesenchyme, intestinal organoids, esophageal organoids, gastric organoids, colonic organoids, vascularized intestinal organoids, vascularized esophageal organoids, vascularized gastric, and / or vascularized colonic organoids, or any combination thereof, are contacted with a Notch pathway activator or Notch signaling pathway inhibitor. In some embodiments, the Notch signaling pathway activator comprises a Notch protein. In some embodiments, the Notch protein is a recombinant Notch protein. In some embodiments, the Notch pathway activator comprises JAG1, JAG2, Notch 1, Notch 2, Notch 3, or Notch 4, or any combination thereof. In some embodiments, the Notch pathway inhibitor comprises Compound E, LY411575, DBZ, or DAPT, or any combination thereof. In some embodiments, the cells arcnot treated with a Notch pathway activator or inhibitor. Notch pathway activation and / or inhibition as used with embodiments provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein. The Notch pathway activator provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0182] In some embodiments, pluripotent stem cells, pluripotent stem cells, definitive endoderm, mid primitive streak cells, plate mesoderm, lateral plate mesoderm, posterior foregut lateral plate mesoderm, posterior foregut, foregut spheroids, mid / hindgut spheroids, splanchnic mesenchyme, vascular mesenchyme, intestinal organoids, esophageal organoids, gastric organoids, colonic organoids, vascularized intestinal organoids, vascularized esophageal organoids, vascularized gastric, and / or vascularized colonic organoids, or any combination thereof, are contacted with a HH pathway activator or HH signaling pathway inhibitor. In some embodiments, the HH signaling pathway activator comprises a HH protein. In some embodiments, the HH protein is a recombinant HH protein. In some embodiments, the HH signaling pathway activator comprises SHH, IHH, DHH, purmorphamine (PMA), GSA 10, SAG, or any combination thereof. In some embodiments, the HH signaling pathway inhibitor comprises HPI-1, cyclopamine, GANT 58, or GANT61, or any combination thereof. In some embodiments, the cells are not treated with a HH pathway activator or inhibitor. HH pathway activation and / or inhibition as used with embodiments provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein. The HH pathway activator provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0183] In some embodiments, pluripotent stem cells, pluripotent stem cells, definitive endoderm, mid primitive streak cells, plate mesoderm, lateral plate mesoderm, posterior foregut lateral plate mesoderm, posterior foregut, foregut spheroids, mid / hindgut spheroids, splanchnic mesenchyme, vascular mesenchyme, intestinal organoids, esophageal organoids, gastric organoids, colonic organoids, vascularized intestinal organoids, vascularized esophageal organoids, vascularized gastric, and / or vascularized colonic organoids, or any combination thereof, are contacted with a retinoic acid pathway activator. In some embodiments, the retinoic acid pathway activator comprises retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS 493, TTNPB, or AM580, or any combination thereof. In some embodiments, the cells arc not treated with a retinoic acid pathway activator or inhibitor. Retinoic acid pathwayactivation and / or inhibition as used with embodiments provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein. The retinoic acid pathway activator provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0184] In some embodiments, for any of the small molecule compounds, pathway activators, pathway inhibitors, or growth factors, the cells are contacted for a time that is, is about, is at least, is at least about, is not more than, or is not more than about, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 120 hours, 150 hours, 180 hours, 240 hours, 300 hours or any time within a range defined by any two of the aforementioned times, for example 1 hour to 300 hours, 24 hours to 120 hours, 48 hours to 96 hours, 6 hours to 72 hours, or 24 hours to 300 hours. In some embodiments, more than one small molecule compounds, activators, inhibitors, or growth factors are added. In these cases, the more than one small molecule compounds, activators, inhibitors, or growth factors can be added simultaneously or separately.

[0185] In some embodiments, the PSCs are differentiated into definitive endoderm cells. In some embodiments, the PSCs are differentiated into posterior foregut cells, splanchnic mesenchyme, and / or vascular mesenchyme. In some embodiments, the PSCs are differentiated into lateral plate mesoderm. In some embodiments, the PSCs are differentiated into posterior foregut cells. In some embodiments, the PSCs are differentiated into splanchnic mesenchyme. In some embodiments, the PSCs are differentiated into vascular mesenchyme. In some embodiments, the PSCs are differentiated into a pancreatic organoid or a pancreatic organoid precursor. In some embodiments, the PSCs are differentiated into a mature pancreatic organoid. In some embodiments, the differentiation is in vitro. In some embodiments, the PSCs are differentiated into vascularized gastrointestinal organoids. In some embodiments, the PSCs are differentiated into vascularized intestinal, vascularized esophageal, vascularized colonic, and / or vascularized gastric organoids, and / or a precursor or immature version of said organoids.

[0186] In some embodiments, any of the cells disclosed herein may be cryopreserved for later use. In some embodiments, the cells can be cryoprcscrvcd according to methods generally known in the art, optionally including one or more cryoprotectants.

[0187] Cryoprotectants are cell composition additives to improve efficiency and yield of low temperature cryoprcscrvation by preventing formation of large ice crystals.Cryoprotectants include but are not limited to DMSO, ethylene glycol, glycerol, propylene glycol, trehalose, formamide, mcthyl-formamidc, dimcthyl-formamidc, glycerol 3 -phosphate, proline, sorbitol, diethyl glycol, sucrose, triethylene glycol, polyvinyl alcohol, polyethylene glycol, or hydroxy ethyl starch. Cryoprotectants can be used as part of a cryopreservation medium, which include other components such as nutrients (e.g. albumin, serum, bovine serum, fetal calf serum (FCS)) to enhance post-thawing survivability of the cells, in these cry opreservation media, at least one cryoprotectant may be found at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or any percentage within a range defined by any two of the aforementioned numbers.Gene Editing

[0188] Embodiments of the disclosure can include PSCs, iPSCs, definitive endoderm cells, posterior foregut spheroids, or organoids which have been or which can be genetically modified or edited according to methods known in the ait. For example, gene editing using CRISPR nucleases such as Cas9 are explored in PCT Publications WO 2013 / 176772, WO 2014 / 093595, WO 2014 / 093622, WO 2014 / 093655, WO 2014 / 093712, WO 2014 / 093661, WO 2014 / 204728, WO 2014 / 204729, WO 2015 / 071474, WO 2016 / 115326, WO 2016 / 141224, WO 2017 / 023803, and WO 2017 / 070633, each of which is hereby expressly incorporated by reference in its entirety.Methods of Using Pancreatic Organoids

[0189] The pancreatic organoids as described herein can be used in various methods. For example, in some embodiments, the pancreatic organoids disclosed herein can be used in a method of treating a pancreas-related disease or disorder and / or a disease or disorder involving endocrinogenesis, a method of scaling up in bioprocess manufacturing, a method of screening for therapeutic efficacy, a method of modeling human pancreatic development and / or disease, a method of diagnosing a pancreatic-related disease or disorder, and / or the manufacture of a medicament for treating a pancreas-related disease or disorder and / or and / or a disease or disorder involving endocrinogenesis.

[0190] In some embodiments, the pancreatic organoids disclosed herein can be administered to a subject, e.g. via transplantation. In some embodiments, the pancreaticorganoids disclosed herein are transplanted into a mammal, such as a mouse, such as an immunocompromised mouse. In some embodiments, the subject has a pancrcas-rclatcd disease or disorder and / or a disease or disorder involving endocrinogenesis. In some embodiments, the subject has a pancreas-related disease or disorder and / or a disease or disorder involving endocrinogenesis, and the subject has an increased survival rate following transplantation. In some embodiments, the organoid is produced from pluripotent stem cells derived from the subject

[0191] In some embodiments, the organoid is transplanted to and engrafts under the kidney capsule of the mammal. In some embodiments, the transplantation results in organoid engraftment, tissue growth, and / or improved tissue and / or organ function. In some embodiments, the transplanted organoid grows about 50x, 150x, 200x, 250x, 300x, 400x, 500x, 600x, 700x, 800x, 900x, lOOOx, l lOOx, 1200x, 1300x, 1400x, or 1500x or at least 50x, 150x, 200x, 250x, 300x, 400x, 500x, 600x, 700x, 800x, 900x, lOOOx, llOOx, 1200x, 1300x, 1400x, or 1500x in volume. In some embodiments, the transplanted organoid matures in vivo.

[0192] In some embodiments, the transplanted organoid generates branched epithelial tissue. In some embodiments, the transplanted organoid generates one or more branched or interconnected ductal networks and / or differentiated acing structures. In some embodiments, the transplanted organoid secretes pancreatic enzymes (e.g. zymogen granules, CPA1, and / or amylase) in the acinar cells, acinar tissue, ducts, lumen, and / or microlumen.

[0193] Also disclosed herein are methods of screening. In some embodiments, the methods comprise contacting any one of the pancreatic organoids disclosed herein with a compound or composition of interest and assessing an effect of the compound or composition. In some embodiments, assessing an effect includes determining a change in phenotype in the cell population or tissue. In some embodiments, the pancreatic organoids disclosed herein are derived from stem cells obtained from a subject. In some embodiments, the subject comprises a disease and the change in phenotype in response to the compound of interest is associated with an improvement of the disease. In some embodiments, the assessed effect includes determining therapeutic efficacy and / or toxicity of the compound or composition. Also disclosed herein are methods of using the pancreatic organoids as described herein in diagnostic screening, e.g. as a companion diagnostic.Pharmaceutical Compositions

[0194] Embodiments of the disclosure can include pharmaceutical compositions. Such pharmaceutical compositions can include one or more additional pharmaceutically acceptable components, which can include carriers, excipients, and / or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed or that have an acceptable level of toxicity. A “pharmaceutically acceptable” “diluent,” “excipient,” and / or “carrier” as used herein have their plain and ordinary meaning as understood in light of the specification and are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with administration to humans, cats, dogs, or other vertebrate hosts. Typically, a pharmaceutically acceptable diluent, excipient, and / or carrier is a diluent, excipient, and / or earner approved by a regulatory agency of a Federal, a state government, or other regulatory agency, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans as well as non-human mammals, such as cats and dogs. The term diluent, excipient, and / or “carrier” can refer to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Such pharmaceutical diluent, excipient, and / or earners can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water, saline solutions and aqueous dextrose and glycerol solutions can be employed as liquid diluents, excipients, and / or carriers, particularly for injectable solutions. Suitable pharmaceutical diluents and / or excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. A non-limiting example of a physiologically acceptable carrier is an aqueous pH buffered solution. The physiologically acceptable carrier may also comprise one or more of the following: antioxidants, such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin, gelatin, immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, ammo acids, carbohydrates such as glucose, mannose, or dextrins, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, saltforming counterions such as sodium, and nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®. The composition, if desired, can also contain minor amounts of wetting, bulking, emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, sustained release formulations and the like. The formulation should suit the mode of administration.

[0195] Additional excipients with desirable properties include but are not limited to preservatives, adjuvants, stabilizers, solvents, buffers, diluents, solubilizing agents, detergents, surfactants, chelating agents, antioxidants, alcohols, ketones, aldehydes, ethylenediaminetetraacetic acid (EDTA), citric acid, salts, sodium chloride, sodium bicarbonate, sodium phosphate, sodium borate, sodium citrate, potassium chloride, potassium phosphate, magnesium sulfate sugars, dextrose, fructose, mannose, lactose, galactose, sucrose, sorbitol, cellulose, serum, amino acids, polysorbate 20, polysorbate 80, sodium deoxycholate, sodium taurodeoxycholate, magnesium stearate, octylphenol ethoxylate, benzethonium chloride, thimerosal, gelatin, esters, ethers, 2-phenoxyethanol, urea, or vitamins, or any combination thereof. Some excipients may be in residual amounts or contaminants from the process of manufacturing, including but not limited to serum, albumin, ovalbumin, antibiotics, inactivating agents, formaldehyde, glutaraldehyde, b-propiolactone, gelatin, cell debris, nucleic acids, peptides, ammo acids, or growth medium components or any combination thereof. The amount of the excipient may be found in composition at a percentage that is, is about, is at least, is at least about, is not more than, or is not more than about, 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% w / w or any percentage by weight in a range defined by any two of the aforementioned numbers.

[0196] Pharmaceutical compositions can include one or more “pharmaceutically acceptable salts”, which can include relatively non-toxic, inorganic and organic acid, or base addition salts of compositions or excipients, including without limitation, analgesic agents, therapeutic agents, other materials, and the like. Examples of pharmaceutically acceptable salts include those derived from mineral acids, such as hydrochloric acid and sulfuric acid, and those derived from organic acids, such as ethanesulfonic acid, benzenesulfonic acid, p- toluenesulfonic acid, and the like. Examples of suitable inorganic bases for the formation of salts include the hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, zinc, and the like. Salts may also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts. For example, the class of such organic bases may include but are not limited to mono-, di-, and trialkylamines, including methylamine, dimethylamine, and triethylamine; mono-, di-, or trihydroxyalkylamines including mono-, di-, and triethanolamine; ammo acids, including glycine, arginine and lysine; guanidine;N-methylglucosamine; N-methylglucamine; L-glutamine; N-methylpiperazine; morpholine; cthylcncdiaminc; N-bcnzylphcncthylaminc; trihydroxymethyl ammocthanc.

[0197] Proper formulation is dependent upon the route of administration chosen. Techniques for formulation and administration of the compounds described herein are known to those skilled in the ait. Multiple techniques of administering a compound exist in the ait including, but not limited to, enteral, oral, rectal, topical, sublingual, buccal, intraaural, epidural, epicutaneous, aerosol, parenteral delivery, including intramuscular, subcutaneous, intra-arterial, intravenous, intraportal, intra-articular, intradermal, peritoneal, intramedullary injections, intrathecal, direct intraventricular, intraperitoneal, intranasal or intraocular injections. Pharmaceutical compositions will generally be tailored to the specific intended route of administration.

[0198] As used herein, a “carrier” has its plain and ordinary meaning as understood in light of the specification and can refer to a compound, particle, solid, semi- solid, liquid, or diluent that facilitates the passage, delivery and / or incorporation of a compound to cells, tissues and / or bodily organs.

[0199] As used herein, a “diluent” has its plain and ordinary meaning as understood in light of the specification and can refer to an ingredient in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically necessary or desirable. For example, a diluent may be used to increase the bulk of a potent drug whose mass is too small for manufacture and / or administration. It may also be a liquid for the dissolution of a drug to be administered by injection, ingestion or inhalation. A common form of diluent in the ail is a buffered aqueous solution such as, without limitation, phosphate buffered saline that mimics the composition of human blood.Dosage and Administration Routes

[0200] Embodiments of the disclosure can include methods of administering or treating an animal, which can involve administering an amount of at least one treatment, that is effective to treat the disease, condition, or disorder that the organism has, or is suspected of having, or is susceptible to, or to bring about a desired physiological effect. In some embodiments, the disease, condition, or disorder can be a liver-related disease or disorder.

[0201] In some embodiments, at least one treatment can include a composition or pharmaceutical composition, which can be administered to an animal (e.g., mammals, primates, monkeys, or humans) in an amount of about 0.005 to about 50 mg / kg body weight, about 0.01 toabout 15 mg / kg body weight, about 0.1 to about 10 mg / kg body weight, about 0.5 to about 7 mg / kg body weight, about 0.005 mg / kg, about 0.01 mg / kg, about 0.05 mg / kg, about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 3 mg / kg, about 5 mg / kg, about 5.5 mg / kg, about 6 mg / kg, about 6.5 mg / kg, about 7 mg / kg, about 7.5 mg / kg, about 8 mg / kg, about 10 mg / kg, about 12 mg / kg, or about 15 mg / kg. In regal'd to some conditions, the dosage can be about 0.5 mg / kg human body weight or about 6.5 mg / kg human body weight. In some instances, some subjects (e.g., mammals, mice, rabbits, feline, porcine, or canine) can be administered a dosage of about 0.005 to about 50 mg / kg body weight, about 0.01 to about 15 mg / kg body weight, about 0.1 to about 10 mg / kg body weight, about 0.5 to about 7 mg / kg body weight, about 0.005 mg / kg, about 0.01 mg / kg, about 0.05 mg / kg, about 0.1 mg / kg, about 1 mg / kg, about 5 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 80 mg / kg, about 100 mg / kg, or about 150 mg / kg. Of course, those skilled in the art will appreciate that it is possible to employ many concentrations in the methods of the present disclosure, and using, in part, the guidance provided herein, will be able to adjust and test any number of concentrations in order to find one that achieves the desired result in a given circumstance. In some embodiments, a dose or a therapeutically effective dose of a compound disclosed herein will be that which is sufficient to achieve a plasma concentration of the compound or its active metabolite(s) within a range set forth herein, e.g., about 1-10 nM, 10-100 nM, 0.1-1 pM, 1-10 pM, 10-100 pM, 100-200 pM, 200-500 pM, or even 500-1000 pM, preferably about 1-10 nM, 10-100 nM, or 0.1-1 pM.

[0202] In other embodiments, a treatment can be administered in combination with one or more other therapeutic agents for a given disease, condition, or disorder.

[0203] The compounds and pharmaceutical compositions are preferably prepared and administered in dose units. Solid dose units are tablets, capsules and suppositories. For treatment of a subject, depending on activity of the compound, manner of administration, nature and severity of the disease or disorder, age and body weight of the subject, different daily doses can be used.

[0204] Under certain circumstances, however, higher or lower daily doses can be appropriate. The administration of the daily dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administrations of subdivided doses at specific intervals.

[0205] A treatment can be administered locally or systemically in a therapeutically effective dose. Amounts effective for this use will, of course, depend on the severity of thedisease or disorder and the weight and general state of the subject. Typically, dosages used in vitro can provide useful guidance in the amounts useful for in situ administration of the pharmaceutical composition, and animal models can be used to determine effective dosages for treatment of particular disorders.

[0206] Various considerations are described, e. g. , in Langer, 1990, Science, 249: 1527; Goodman and Gilman's (eds.), 1990, Id., each of which is herein incorporated by reference and for all purposes. Dosages for parenteral administration of active pharmaceutical agents can be converted into corresponding dosages for oral administration by multiplying parenteral dosages by appropriate conversion factors. As to general applications, the parenteral dosage in mg / mL times 1.8 = the corresponding oral dosage in milligrams (“mg”). As to oncology applications, the parenteral dosage in mg / mL times 1.6 = the corresponding oral dosage in mg. An average adult weighs about 70 kg. See e.g., Miller-Keane, 1992, Encyclopedia & Dictionary of Medicine, Nursing & Allied Health, 5th Ed., (W. B. Saunders Co.), pp.1708 and 1651.

[0207] It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

[0208] In some embodiments, the administration can include a unit dose of one or more treatments in combination with a pharmaceutically acceptable carrier and, in addition, can include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, and excipients. In certain embodiments, the carrier, vehicle or excipient can facilitate administration, delivery and / or improve preservation of the composition. In other embodiments, the one or more carriers, include but are not limited to, saline solutions such as normal saline, Ringer's solution, PBS (phosphate-buffered saline), and generally mixtures of various salts including potassium and phosphate salts with or without sugar additives such as glucose. Carriers can include aqueous and non-aqueous sterile injection solutions that can contain antioxidants, buffers, bacteriostats, bactericidal antibiotics, and solutes that render the formulation isotonic with the bodily fluids of the intended recipient; and aqueous and non-aqueous sterile suspensions, which can include suspending agents and thickening agents. In other embodiments, the one or more excipients can include, but are not limited to water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. Nontoxic auxiliary substances, such as wetting agents, buffers, oremulsifiers may also be added to the composition. Oral formulations can include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate.

[0275] The quantity of active component in a unit dose preparation can be varied or adjusted from 0.1 mg to 10000 mg, more typically 1.0 mg to 1000 mg, most typically 10 mg to 500 mg, according to the particular application and the potency of the active component. The composition can, if desired, also contain other compatible therapeutic agents.

[0209] A treatment can be administered to subjects by any number of suitable administration routes or formulations. The treatment, such as an immunotherapy, can also be used to treat subjects for a variety of diseases. Subjects include but are not limited to mammals, primates, monkeys (e.g., macaque, rhesus macaque, or pig tail macaque), humans, canine, feline, bovine, porcine, avian (e.g., chicken), mice, rabbits, and rats. In particular embodiments described herein, the subject is a human.

[0210] The route of administration of the compounds of the treatments described herein can be of any suitable route. Administration routes can be, but are not limited to the oral route, the parenteral route, the cutaneous route, the nasal route, the rectal route, the vaginal route, and the ocular route. In other embodiments, administration routes can be parenteral administration, a mucosal administration, intravenous administration, subcutaneous administration, topical administration, intradermal administration, oral administration, sublingual administration, intranasal administration, or intramuscular administration. The choice of administration route can depend on the compound identity (e.g., the physical and chemical properties of the compound) as well as the age and weight of the animal, the particular disease (e.g., type of cancer), and the severity of the disease (e.g., stage or severity of cancer). Of course, combinations of administration routes can be administered, as desired.

[0211] Some embodiments of the disclosure include a method for providing a subject with a treatment which comprises one or more administrations of one or more compositions; the compositions may be the same or different if there is more than one administration.Toxicity

[0212] The ratio between toxicity and therapeutic effect for a particular treatment is its therapeutic index and can be expressed as the ratio between LD50 (the amount of compound lethal in 50% of the population) and ED50 (the amount of compound effective in 50% of the population). Compounds that exhibit high therapeutic indices are preferred. Therapeutic indexdata obtained from in vitro assays, cell culture assays and / or animal studies can be used in formulating a range of dosages for use in humans. The dosage of such compounds preferably lies within a range of plasma concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. See, e.g. Fingl et al., In: THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, Ch.l, p.l, 1975. The exact formulation, route of administration, and dosage can be chosen by the individual practitioner in view of the patient’s condition and the particular method in which the compound is used. For in vitro formulations, the exact formulation and dosage can be chosen by the individual practitioner in view of the patient’s condition and the particular method in which the compound is used.

[0213] Having described the various technical aspects in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the scope of the disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.EXAMPLES

[0214] The following non-limiting examples are provided to further illustrate embodiments of the disclosure. 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 disclosure, and thus can 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 can 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 disclosure.EXAMPLE 1Materials and MethodsAnimals

[0215] Mice used for organoid transplantation were housed in the animal facility at Cincinnati Children’s Hospital Medical Center (CCHMC) in accordance with NIH Guidelines for the Care and Use of Laboratory animals. Animals were maintained on a 12-hour light-dark cycle with access to water and standard chow ad libitum. Healthy male and female immune-deficient NSG (N0D.Cg-PrkdcscidI12rgtmlWjl / SzJ) mice, aged between 8 and 16 weeks old, were used for all transplantation. All experiments were performed with the approval of the Institutional Animal Care and Use Committee (IACUC) of CCHMC.Human ESC / iPSC lines and maintenance

[0216] Human embryonic stem cell (hESC) lines Hl (WA-01) were purchased from WiCell (NIH approval number NIHhESC-10-0043). Human induced pluripotent stem cell (iPSC) line 72.3- GFP and 72.3-mCherry were generated and obtained from the CCHMC Pluripotent Stem Cell Facility (PSCF) and approved by the institutional review board (IRB) at CCHMC. All hPSC lines were screened for pluripotency and the absence of karyotypic abnormalities and mycoplasma contamination by the CCHMC PSCF. All human hPSCs were maintained in an undifferentiated state as colonies in feeder-free conditions. They were plated on human- ES- cell- qualified Matrigel (BD Biosciences) and maintained at 37°C with 5% CO2 with daily replacement of mTeSRl media (STEMCELL Technologies). Cells were routinely passaged with 0.5 mM EDTA in PBS (Life Technologies) when colony confluency reached 75-90%.Differentiation of hPSCs into splanchnic mesenchyme

[0217] Posterior splanchnic mesenchyme was obtained using a previously published protocol (Kishimoto, et al., Nat Protoc 17, 2699-2719 (2022)). Briefly, 90% confluent hPSCs colonies were dissociated into single cells using 0.5 mM EDTA in PBS, resuspended in mTesRl with 1 pM thiazovivin (Tocris), and passaged 1 :20 onto new Geltrex-coated 12-well plates. To generate mid primitive streak cells, hPSCs were exposed to 30 ng / ml Activin A (R&D), 40 ng / ml BMP4 (R&D Systems), 6mM CHIR99021 (ReproCell), 20 ng / ml FGF2 (ThermoFisher Scientific), and lOOnM PIK90 (EMD Millipore) for 24 hours. A basal media composed of Advanced DMEM / F12 (ThermoFisher Scientific) supplemented with B27 supplement (IX, ThermoFisher Scientific), N2 supplement (IX, ThermoFisher Scientific), HEPES (13 mM, ThermoFisher Scientific), L-Glutamine (2 mM ThermoFisher Scientific), and penicillinstreptomycin (IX, ThermoFisher Scientifics) was used for this and all subsequent differentiation steps. To generate posterior foregut lateral plate mesoderm, cells were then exposed to A83O1 (1 mM, Tocris), BMP4 (30 ng / ml), Retinoic acid (RA, 2mM, Sigma), and C59 (1 mM, Cellagen Technology) for 24 hours. For splanchnic mesoderm generation, cells were cultured in A8301 (1 mM), BMP4 (30 ng / ml), C59 (1 mM), FGF2 (20 ng / ml), and RA (2 mM, Sigma- Aldrich) from Day 2 to Day 4.Differentiation of hPSCs into vascular mesenchyme

[0218] Vascular mesenchyme was obtained using a previously published protocol to generate vascular organoids (Wimmer, et ah, Nat Protoc 14, 3082-3100 (2019)). Briefly, human pluripotent stem cells were dissociated into single cells with 0.5 mM EDTA and resuspend in aggregation media supplemented with 50uM ROCK inhibitor Y -27632. Aggregation media was prepared by mixing 40ml knockOut DMEM / F12, 10ml knockOut serum replacement, 0.5ml GlutaMAX, 0.5ml NEAA, 35pl 1:100 b-Mercaptoethanol in PBS, 0.5ml Antibiotic- Antimycotic. Stem cell aggregates containing 1000 cells per aggregates were produced using overnight culture in AggreWell400 (Stem Cell Technologies). Basal medium for the two-step differentiation protocol was produced as follows: 500ml media included 250ml DMEM / F12, 250ml Neurobasal medium, 10ml B27 supplement, 5ml N2 supplement, 2.5ml GlutaMAX, 350ul b- Mercaptoethanol diluted 1:100 in PBS, 5ml Antibiotic- Antimycotic. Aggregates were transferred to ultralow attachment 6-well plate (Coming) with 3 ml of differentiation medium and cultured on an orbital shaker at 100 rpm. For mesodermal induction (stage 1 - 3 days), basal medium was supplemented with 12 pM CHIR99021 and 30 ng / mL BMP-4. The medium for vascular specification step (stage 2 - 2 days) was supplemented with 100 ng / mL VEGF-A and 2 pM forskolin.Differentiation of hPSCs into posterior foregut cells

[0219] Posterior foregut (PFG) cells were generated following previously published protocols to generate pancreatic progenitors (Balboa, et al., Nat Biotechnol 40, 1042-1055 (2022)). Briefly, confluent hPSC cultures were dissociated using trypLE (Life Technologies) and resuspended as single cells in mTeSRl supplemented with 10 pM of ROCK inhibitor Y-27632 (Tocris). Cells were seeded at a density of 1.35 xlO5cells / cm2on a 6-well plate coated with growth factor reduced Matrigel (Corning) and fed mTeSRl the following day. Definitive endoderm differentiation was initiated 48 hours after seeding by exposing the cells to Activin A (100 ng / ml) and CH1R99021 (3pM) for 24h and Activin A (lOOng / ml) only for two more days. Basal medium for definitive endoderm was as follows: MCDB 131 medium (Life Technologies) supplemented with 1.5 g / 1 sodium bicarbonate (Sigma), Glutamax IX (Life Technologies), 10 mM glucose (Sigma) and 0.5% BSA (fatty acid free BSA, Proliant). Primitive gut tube fate was specified for 3 days using the same basal medium supplemented with 50ng / ml KGF (R&D) and 0.25 mM Ascorbic acid. Posterior foregut cells were then differentiated for 2 days using 0.25 mM ascorbic acid, 50 ng / ml of FGF7, 0.25 mM SANT-1 (Sigma), 1 mM retinoic acid (Sigma),100 nM LDN193189 (Stemgent), 1 :200 ITS-X (Life technologies), and 200 nM TPB (Tocris). Basal medium for posterior gut tube induction was as follows: MCDB 131 medium supplemented with 1.5 g / 1 sodium bicarbonate, Glutamax IX, 15 mM glucose and 2% BSA.Recombination and spheroid patterning

[0220] Single cell suspensions of mesenchymal cells and posterior foregut cells were generated using trypLE and recombined in a 4:1 ratio to make 10,000 cells / organoids (8000 mesenchyme and 2000 PFG) in Aggrewell 800 (StemCell Technologies). Cells were recombined in posterior foregut medium supplemented with 20 pM of ROCK inhibitor and 2% fetal bovine serum (FBS). The following day, pancreatic organoids were harvested and transferred to an ultralow adhesion plate with 3ml of either posterior foregut medium or classical gut medium (same as basal mesenchyme differentiation medium), both supplemented with 2% FBS. The pancreatic specification stage lasts for 1 week, with daily medium changes. Medium was then switched to permissive medium (gut medium) for all conditions to allow for uncontrolled morphogenesis. Medium was changed every two days, and organoids were split between multiple wells if the medium became yellow at the time of feeding.In vivo transplantation

[0221] Progenitor aggregates and human pancreatic organoids were ectopically transplanted into the kidney capsule of NSG mice (Eicher, et al., Cell Stem Cell 29, 36-5 l.e6 (2022)). Briefly, a small incision was made to mice under anesthesia to access the kidney and organoids were transplanted into the kidney subcapsular space using a small needle. Engrafted organoids were harvested 6-12 weeks after transplantation and analyzed using immunofluorescence and histological coloration.Tissue Processing, IHC, and Microscopy

[0222] Organoids were washed with lx phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde (PFA) at room temperature (RT) for 1 hour, washed, and stored in PBS at 4°C. Organoids were then put in 30% sucrose in PBS overnight, or 70% ethanol at 4°C overnight for downstream cryogenic or paraffin processing, respectively. Tissues were then embedded in either O.C.T. Compound (Tissue-Tek) or paraffin and were serially sectioned at a thickness of 8 pm onto Superfrost Plus glass slides (Fisherbrand). Cryosection slides and paraffin slides were stored at -80°C and room temperature, respectively. Routine Hematoxylin & Eosin (H&E) staining was performed by the Research Pathology Core at CCHMC. Frozen slides were thawedto room temperature (RT) and rehydrated in PBS, while paraffin slides were deparaffinized, rchydratcd, and subjected to heat- and pressure-induced antigen retrieval in citrate buffer (0.192% citric acid and 0.0005% Tween 20 in dH20 of pH 6.0 with NaOH) for 30 minutes and brought to RT on ice. All slides and cells were washed with PBS, permeabilized with 0.5% Triton X-100 in PBS (PBST) for 15 min at RT and then blocked with 5% normal donkey serum (NDS, Jackson ImmunoResearch) in PBS for one hour at RT. Tissue was incubated at 4°C overnight in primary antibodies diluted in 5% NDS in PBST. The following day, tissue was washed and incubated with secondary antibodies at RT for one hour, thoroughly washed, and mounted with Fluoromount-G (Southern Biotech). Brightfield and fluorescence images of live tissue samples were captured using either a Leica DMC5400 or a Nikon Ti-E inverted Al confocal microscope. Images were processed and quantified using Nikon NIS Elements.EXAMPLE 2Mesenchymal type biases pancreatic organoid morphogenesis and diff erentiation

[0223] As described herein, a developmentally inspired tissue engineering approach was developed, where developmentally relevant cells are differentiated from pluripotent stem cells before being recombined for further development. Directed differentiation of pluripotent stem cells through endoderm, posterior foregut and pancreatic progenitors has been shown herein to generate both ductal and endocrine cells, depending on the induction of different pathways aimed at mimicking in vivo pancreatic development.

[0224] Using this approach, a population of posterior foregut cells was derived with the competence to become pancreatic progenitors. These posterior foregut cells were then recombined with different mesenchymal populations that could provide support for growth and differentiation (FIG. 1A). The endoderm-derived population uniformly express the posterior foregut marker pancreatic and duodenal homeobox 1 (PDXl) (FIG. IB), a transcription factor essential for pancreas development that is also expressed in the duodenum and the antral stomach.

[0225] In vivo, the splanchnic mesenchyme is a precursor to the mesenchymal tissue surrounding the posterior foregut, and eventually gives rise to much of the pancreatic mesenchyme. To present knowledge, however, differentiation of mesenchymal cells specific to pancreas development has not been achieved in vitro, and the ideal population for recombination with pancreatic endoderm is heretofore unknown.

[0226] Accordingly, two populations of mesenchyme were evaluated. The first mesenchymal population follows a 3-stcp protocol mimicking the development of mesodermal lineage during the primitive streak, followed by differentiation to lateral plate mesoderm and a foregut specific splanchnic mesenchyme (Kishimoto, et al., Nat Protoc 17, 2699-2719 (2022)). This results in a population expressing vimentin and the splanchnic mesenchyme marker FOXF1 (FIG. 1C). Since vascularization and proximity to major vessels is an important part of pancreas development, the second mesenchyme population, herein called vascular’ mesenchyme, was derived from precursors of vascular organoids that had gone through mesodermal commitment and vascular specification (Wimmer, et al., Nature 565, 505-510 (2019)). After vascular specification, vascular mesenchyme organoids expressed the splanchnic mesenchyme marker FOXF1 in the middle and vascular markers such as SOX17 and PECAM in the periphery (FIG. ID).

[0227] Posterior foregut cells and the mesenchymal cells were then dissociated into single cells, recombined into aggregates, and cultured in suspension. Whether recombined with splanchnic or vascular- or both mesenchyme, the posterior foregut cells rapidly self-sort into an internal compartment surrounded by mesenchymal cells (FIG. 2A). Interestingly, in organoids containing both mesenchymal cell types, the vascular mesenchyme was less strongly associated with the endoderm compartment, with most of the endodermal surface interacting with the splanchnic mesenchyme.

[0228] After one week of pancreatic induction (stage 4), the organoids started to express the pancreatic progenitor marker NKX6.1 (FIG. 2B). Most of the posterior foregut cells (constitutivly labelled with mCherry) expressed multipotent pancreatic progenitor markers (NKX6.1, SOX9, PDX1) when recombined with vascular mesenchyme, whereas a large domain negative for these markers was formed when splanchnic mesenchyme was used (FIG. 2C). This non-pancreatic domain showed strong SOX2 expression combined with convoluted epithelial formation around a central lumen filled with dead cells. In contrast, the putative pancreatic domain, making most of the vascular mesenchyme recombinant (VMPO) and a portion of the splanchnic mesenchyme recombinant (SMPO), was characterized by a cauliflower-like, rapidly growing epithelium (E-cadherin positive). Culturing the organoids for an extra 6 weeks in permissive medium (no added growth factors) resulted in expansion of the SOX2 domain in SMPO, potentially due to increased proliferation in the non-pancreatic compartments (FIG. 2D).

[0229] Considering the spatial proximity of the developing pancreas with the stomach, and the key role of SOX2 in gastric development, the study investigated if the SMPO would express gastric specific markers. Indeed, SMPO showed strong expression of claudin-18, a tight junction protein uniquely expressed in the stomach (FIG. 2E). In contrast, claudin-18 expression was restricted to a cystic lumen in VMPO, with most of the organoid strongly expressing PDX1. Together, these data demonstrate that the type of mesenchyme can greatly affect lineage commitment of the posterior foregut cells, with splanchnic mesenchyme recombinant resulting in stomach-pancreas boundary organoids whereas vascular- mesenchyme recombinant yielded highly pure pancreatic organoids (FIG. 2F).

[0230] Additionally, the study showed that transplantation of organoids right after recombination results in divergent outcomes based on the mesenchyme used. Here, the splanchnic mesenchyme was unable to efficiently support growth of the posterior foregut cells in vivo, with only one transplant out of four generating epithelial tissue after 10 weeks of transplantation. Transplantation with vascular mesenchyme not only resulted in more vascularized transplant (FIG. 2G), but generated complex epithelial structures in most mice (FIG. 2H). Since the vascular mesenchyme is a precursor for vascular organoids that can form vascular network both in vitro and in vivo, the outcome of the transplantation can reflect more the ability of the early organoid to engraft in the kidney capsule than their potential for supporting posterior foregut growth and differentiation. Nevertheless, it shows that pancreatic organoid can engraft under the kidney capsule and can be used to study organoid development in vivo.EXAMPLE 3Recombination with vascular mesenchyme generates highly pure pancreatic organoids

[0231] Given the current knowledge on the in vitro differentiation of posterior foregut towards pancreatic progenitors, ductal organoids, and endocrine cells, the effect of vascular mesenchyme on posterior foregut differentiation in vitro was investigated. To do this, freshly recombined VMPOs were cultured in either in inductive medium containing the classical molecules for pancreatic progenitor differentiation (SANT-1, retinoic acid, TPB, LDN and KGF), or in permissive medium without growth factors (FIG. 3A). In inductive medium, pancreatic organoids formed an epithelial cavity with many folds inside the mesenchyme (FIG. 3B), with most of the epithelium transitioning to rapidly proliferating multipotent progenitorsafter the initial pancreatic induction week (FIG. 3C). Further culture resulted in pancreatic organoids forming a main SOX2 positive lumen surrounded by highly stratified PDX1 positive epithelium becoming progressively more lobular (FIG. 3D), even after removal of the pancreatic induction cocktail. In contrast, pancreatic organoid directly cultured in permissive medium after recombination exhibits increased proliferation of the mesenchyme compartments and a less organized boundary delimited by the transcription factors SOX2 and PDX1. This results in absence or very rare instance of the stereotypical “cauliflower” structure typically seen in organoid cultured in inductive medium.

[0232] In addition, the use of pro-pancreatic factors in the medium during the first week of culture, resulted in increased endocrine cells in the organoids, as demonstrated by an increased number of cells expressing the endocrine markers neurogenin3 and chromogranin A (FIG. 3E, FIG. 3F). Islet- specific markers glucagon and c-peptide were also elevated, with most positive cells located in the middle of the organoids where progenitors competent for endocrine differentiation would be expected (FIG. 3G). More diverse cell fates were achieved when permissive medium was used, with the organoids showing a bigger stomach-like compartment characterized by expression of tight junction protein claudin-18 and stomach-enriched mucin MUC5AC (FIG. 31), as well as frequent differentiation towards a CDX2 and CDH17 positive intestinal-like domain (FIG. 3J).

[0233] These results confirm the competency of the PDX1+ posterior foregut cells to give rise to organ rudiment of pancreatic, duodenum and stomach fate. More importantly, it shows that organoid fate can be controlled using an initial pulse of pancreatic specification to generate organoids with a strong pancreatic lineage commitment.EXAMPLE 4Pancreatic organoids undergo ductal branching and acinar differentiation without further induction

[0234] During pancreatic development, the luminal plexus formed within the stratified pancreatic epithelium is remodeled into a ductal network with acinar differentiation occurring at the end of terminal ducts. Organoids cultured for several weeks in vitro showed extensive cellular rearrangement and cleft formation at the periphery of the pancreatic domain (FIG. 4A). Hematoxylin and eosin staining confirm that the organoids contained an interconnected tubular network with acini-like formation located at the periphery of theorganoids (FIG. 4B). Immunostaining at day 40 and day 60 confirmed the pancreatic nature of the tubular network, with pancreas-specific mucin- 1 expressed in the lumen of the cytokcratin-19 positive epithelial network (FIG. 4C).

[0235] Importantly, SOX9 is shown to be still highly expressed in the ducts, whereas it is downregulated in the surrounding acinar tissue (FIG. 4D), recapitulating the restriction of SOX9 expression to the ductal compartment. In addition, the organoids recapitulate the pattern of protein expression in the developing acinar compartment as well (FIG. 4E), with GATA4 being progressively restricted to the acini at the end terminal of the ductal network. Overall, pancreatic organoids also show downregulated expression of PDX1 in the acinar compartment, also consistent with what is known of acinar development in human development. Carboxypeptidase Al (CPA1) is also expressed throughout the outer epithelial layer of the pancreatic epithelium, and was found abundantly in the lumen of the acini and downstream ducts, confirming acinar differentiation into functional secretory cells (FIG. 4F).

[0236] At day 60, amylase, another enzyme typically secreted by the exocrine pancreas, was faintly detected in some acinar cells and their apical lumen. Immunofluorescence for basement membrane laminin and the tight junction protein zo-1 also showed proper polarization of the acinar region, with deposition of extracellular matrix on their basal side and formation of a connected lumen on their apical side (FIG. 4G). Of note, FOXF1+ mesenchymal cells were localized close to the developing duct and acinar region, which may develop into pancreatic mesenchymal cells. WT-1 was also expressed in the outer layer of the mesenchyme, which can correspond to serosa differentiation in response to suspension culture without external addition of ECM. Many cells were still positive for the proliferation marker Ki67 after 60 days in culture, indicating that the organoid can further grow and differentiate to reach a more mature state. However, although the organoids are still growing beyond 60 days of culture, the stomachlike lumens do not appeal- to undergo complex morphogenetic events and still maintain a cystic growth without folding or crypt formation (FIG. 4H).

[0237] To investigate the potential of recombined aggregates and organoids to generate pancreatic tissue in vivo, they were transplanted under the kidney capsule of immune deficient NSG mice. Here, organoids transplanted right after recombination were compared with organoids grown in vitro for 10 weeks to evaluate the potential of posterior foregut cells as well as a committed pancreatic endoderm that was specified towards pancreatic fate in vitro. Organoids transplanted right after recombination were grown in vivo for 10 weeks, whereas theorganoids differentiated in vitro were implanted for 6 weeks, bringing the total maturation time after recombination to 10 and 16 weeks respectively.

[0238] Both transplantation timepoints resulted in robust engraftment under the kidney capsule and growth of an epithelium compartment (FIG. 5A). After in vivo maturation, the organoids were organized into a branching epithelial compartment reminiscent of the human fetal pancreas branching ducts around 15 weeks post conception (FIG. 5B). In both cases, the endoderm mostly differentiates into cells of pancreatic lineage, with cells adopting an acinar fate with high CPA1 expression (FIG. 5C). Interestingly, based on both histology and enzyme localization, 15 weeks fetal pancreas were more similar to organoids transplanted right after recombination and cultured for 10 weeks. Indeed, amylase was found mostly in the ducts and did not colocalized with CPA1. In contrast, organoids cultured in vitro after recombination showed much higher colocalization of the two enzymes, a feature expected of more mature pancreatic tissue.EXAMPLE 5Pancreatic organoid protocol characterization

[0239] Further experimentation was conducted on differing starting materials in order to demonstrate whether the pancreatic organoid protocol described herein can be recapitulated across cell lines. The protocol was conducted in both induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs).

[0240] The protocol was demonstrated to be robust across lines (FIGs. 6A-B). The percentage of endodermal cell expressing PDX1 and / or PDX+ / NKX6.1+, the percentage of total cells coming from endoderm differentiation, and the relevant marker expression, were all consistent regardless of whether iPSCs vs ESCs were used as stalling material. Accordingly, the pancreatic organoid generation protocol was found to be robust across different cell lines.

[0241] In addition, differentiation of acinar cells was observed within the hPOs, as shown by hematoxilin and eosin staining (FIG. 7A), immunostaning (FIG. 7B), and electron micrograph (FIG. 7C). Single cell analysis was conducted as well, showing that all the key pancreatic cell types are present during organoid differentiation (FIGs. 8A-8C), including acinar ductal precursors, acinar cells, gastrointestinal-like cells, ductal cells, endocrine cells, stromal cells, stromal precursor cells, endothelial cells, and mitotic cells. Single cell exocrine, endocrine, and mesenchyme recluster analyses additionally were conducted to show key exocrine,endocrine, and mesenchymal differentiation populations and their representation at different stages of organoid development, as well as expression of specific markers in the populations (FIGs. 9A-9D, FIGs. 10A-10C, FIGs. 11A-11C). In particular, it was shown that mesenchymal type can bias posterior foregut fate (FIGs. 12A-12D).EXAMPLE 6Endocrinogenesis can be achieved, and immune cells can differentiate, in pancreatic organoids

[0242] Endocrinogenesis can be recapitulatd in hPOs (FIG. 13). The hPOs generate endocrine cells, and this can further be enhanced by using endocrine differentiation cocktail, composed of XXi, T3, LDN and Sant-1. Differentiation with endocrine cocktail results in isletlike structures forming inside hPO, with the relevant pancreatic architecture and hormones. To date, there are no known iPSC-derived organoids which have been able to generate islet-like structures within a ductal network comprising exocrine tissue. Further, immune cells were shown to have the ability to survive and differentiate inside the hPOs (FIGs. 14A-14C).

[0243] The demonstration of endocrinogenesis, by making islet like clusters within the pancreatic organoid, can have important implications. For example, the pancreatic organoid can thus be used to model or treat diabetes-related conditions and can also broaden the relevance of the pancreatic organoid and associated model to both endocrine and exocrine pancreatic diseases.

[0244] Together, these results demonstrate that this new organoid system recapitulates many aspects of human pancreatic development in vitro and that organoids transplanted in vivo can result in highly mature tissues resembling human fetal pancreas.

[0245] The various methods and techniques described above provide a number of ways to cany out the disclosure. Of course, it is to be understood that not necessarily all objectives or advantages described can be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as taught or suggested herein. A variety of alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another, or several features, while others specifically exclude one, another, or several features, while still others mitigate a particular' feature by inclusion of one, another, or several advantageous features.

[0246] Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be employed in various combinations by one of ordinary skill in this art to perform methods in accordance with the principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in diverse embodiments.

[0247] Although the application has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the disclosure extend beyond the specifically disclosed embodiments to other alternative embodiments and / or uses and modifications and equivalents thereof.

[0248] In some embodiments, the numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the application are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the application are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.

[0249] In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment of the application (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (for example, “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the application anddoes not pose a limitation on the scope of the application otherwise claimed. No language in the specification should be construed as indicating any non-claimcd element essential to the practice of the application.

[0250] Preferred embodiments of this application are described herein. Variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the application can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this application include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the application unless otherwise indicated herein or otherwise clearly contradicted by context.

[0251] All patents, patent applications, publications of patent applications, and other material, such as articles, books, specifications, publications, documents, things, and / or the like, referenced herein are hereby incorporated herein by this reference in their entirety for all purposes, excepting any prosecution file history associated with same, any of same that is inconsistent with or in conflict with the present document, or any of same that may have a limiting affect as to the broadest scope of the claims now or later associated with the present document. By way of example, should there be any inconsistency or conflict between the description, definition, and / or the use of a term associated with any of the incorporated material and that associated with the present document, the description, definition, and / or the use of the term in the present document shall prevail.

[0252] In closing, it is to be understood that the embodiments of the application disclosed herein are illustrative of the principles of the embodiments of the disclosure. Other modifications that can be employed can be within the scope of the application. Thus, by way of example, but not of limitation, alternative configurations of the embodiments of the application can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present application are not limited to that precisely as shown and described.

Claims

CLAIMSWhat is claimed is:

1. An in vitro method of preparing a pancreatic organoid (PO), the method comprising: a) dissociating posterior splanchnic mesenchyme, vascular mesenchyme, and posterior foregut cells to respective single cell suspensions; b) recombining and aggregating the single cell suspensions of posterior splanchnic mesenchyme and / or vascular mesenchyme with the single cell suspension of posterior foregut cells; and c) culturing the recombined and aggregated single cell suspensions of posterior splanchnic mesenchyme and / or vascular mesenchyme with the single cell suspension of posterior foregut cells, while inhibiting a ROCK pathway, for a first period of time, to provide a precursor pancreatic organoid.

2. The method of claim 1, the method further comprising: d) culturing the precursor pancreatic organoid while activating and / or inhibiting one or more signaling pathways, for a second period of time, to provide an pancreatic organoid with committed lineage.

3. The method of claim 1, the method further comprising: d) culturing the precursor pancreatic organoid without activating and / or inhibiting one or more signaling pathways, for a second period of time, to provide a pancreatic organoid with variably committed lineage.

4. The method of any of claims 2-3, the method further comprising: e) culturing the pancreatic organoid without activating and / or inhibiting one or more signaling pathways, for a third period of time, to provide a cultured pancreatic organoid.

5. The method of claim 2, wherein, in step d), the precursor pancreatic organoid is cultured in an inductive medium comprising growth factors and / or signaling pathway activators and / or inhibitors, to provide a pancreatic organoid with committed lineage.

6. The method of claim 5, wherein, in step d), the precursor pancreatic organoid is cultured in an inductive medium, comprising posterior foregut medium, classical gut medium,and / or basal mesenchyme differentiation medium; optionally wherein the inductive medium comprises SANT-1, retinoic acid, TPB, LDN and / or KGF.

7. The method of claim 3, wherein, in step d), the precursor pancreatic organoid is cultured in a permissive medium, in the absence of growth factors and / or signaling pathway activators and / or inhibitors, to provide a pancreatic organoid with variably committed lineage.

8. The method of any preceding claim, wherein the single cell suspension of posterior foregut cells is cultured with single cell suspensions of both posterior splanchnic mesenchyme and vascular mesenchyme.

9. The method of any preceding claim, wherein the single cell suspension of posterior forcgut cells is cultured with a single cell suspension of vascular mesenchyme, and wherein the pancreatic organoid comprises a majority of pancreatic-like cell population.

10. The method of any preceding claim, wherein the single cell suspension of posterior foregut cells is cultured with a single cell suspension of posterior splanchnic mesenchyme, and wherein the PO comprises one or more stomach-pancreas boundary cell population.

11. The method of any preceding claim, wherein the single cell suspensions of posterior splanchnic mesenchyme and vascular’ mesenchyme are recombined with the single cell suspension of posterior foregut cells in a ratio of at least about 1:1 to 10:1, or greater, mesenchymal cells to posterior foregut cells; optionally at least about 2:1 to 6:1; optionally about 4:1.

12. The method of any preceding claim, wherein the posterior splanchnic mesenchyme and vascular- mesenchyme are recombined with the single cell suspension of posterior foregut cells in a ratio of at least about 1:10 to 10:1, splanchnic mesenchymal cells to vascular mesenchymal cells; optionally at least about 5:1 to 1:5; optionally at least about 2:1 to 1:2; optionally about 1:1.

13. The method of any preceding claim, wherein the single cell suspensions of posterior splanchnic mesenchyme and vascular- mesenchyme are recombined with the single cell suspension of posterior foregut cells to provide at least about 1,000 to 100,000 cells / organoid; optionally at least about 2,000 to 50,000 cells / organoid; optionally about 10,000 cells / organoid.

14. The method of any preceding claim, wherein the first period of time is at least about 1 day, or longer; optionally at least about 1 day to 3 days, 1 day to 5 days, or longer; optionally at least about 1 day.

15. The method of any of claims 2-14, wherein the second period of time is selected from at least about 1 day to 10 days, or longer; optionally at least about 1 day to 7 days; optionally at least about 7 days.

16. The method of any of claims 3-15, wherein the third period of time is at least about 1 day, or longer; optionally 1 day to to 7 days, 1 day to 14 days, 1 day to 60 days, 1 day to 70 days, or longer; optionally at least about 1 day, 7 days, 60 days, 70 days, or longer.

17. The method of any preceding claim, wherein the posterior foregut cells arc derived from definitive endoderm, and wherein the posterior splanchnic mesenchyme and vascular mesenchyme are derived from pluripotent stem cells.

18. The method of any preceding claim, wherein the posterior foregut cells are derived from definitive endoderm, which has been derived from pluripotent stem cells.

19. The method of any of claims 17-18, wherein the pluripotent stem cells comprise embryonic stem cells or induced pluripotent stem cells.

20. The method of any preceding claim, wherein the posterior splanchnic mesenchyme, vascular mesenchyme, and / or posterior foregut cells arc derived from pluripotent stem cells; optionally embryonic stem cells or induced pluripotent stem cells; optionally human induced pluripotent stem cells.

21. The method of any preceding claim, wherein the posterior foregut cells express PDX1; and / or wherein the splanchnic mesenchyme expresses vimentin and / or FOXF1; and / or wherein the vascular mesenchyme expresses FOXF1, SOX17, and / or PECAM.

22. The method of any of claims 2-21, wherein, in step d), the precursor pancreatic organoid is cultured in an inductive medium, and wherein the pancreatic organoid comprises a region comprising one or more pancreatic-like cell population.

23. The method of any of claims 2-22, wherein, in step d), the precursor pancreatic organoid is cultured in an inductive medium, and wherein the pancreatic organoid expressescytokeratin- 19, glucagon, c-peptide, neurogenin3, chromogranin A, claudin-18, E-cadherin, vimcntin, PECAM, GATA4, PDX1, NKX6.1, FOXF1, WT-1, Ki67, and / or SOX9.

24. The method of any of claims 2-22, wherein, in step d), the precursor pancreatic organoid is cultured in a permissive medium, and wherein the pancreatic organoid comprises a region comprising one or more pancreatic-like cell population, and one or more intestinal-like, duodenum-like, and / or stomach-like cell population; and / or and wherein the pancreatic organoid expresses claudin-18, MUC5AC, SOX2, CDX2, and / or CDH17.

25. The method of any preceding claim, wherein, in the method, the cells self-sort into two or more regions.

26. The method of claim xx, wherein the cells self-sort into an inner region comprising pancreatic epithelium, or into an outer region comprising mesenchyme and / or vascular endothelium.

27. The method of any preceding claim, wherein dissociation comprises enzymatic dissociation and / or mechanical dissociation.

28. The method of claim 27, wherein enzymatic dissociation comprises dissociating the organoid with trypsin, chymotrypsin, collagenase, papain, hyaluronidase, elastase, thermolysin, neutral protease, or any combination thereof; and / or wherein mechanical dissociation comprises manual dissociation and / or passing the organoid through successively narrower bore channels.

29. The method of any preceding claim, wherein epithelial cells and / or mesenchymal cells are dissociated from the organoid by using an extracellular matrix depolymerization solution.

30. The method of any preceding claim, wherein steps a) through c) are performed in suspension culture.

31. The method of any of claims 2-30, wherein steps d) and / or e) are performed in suspension culture.

32. The method of any preceding claim, wherein the precursor pancreatic organoid and / or pancreatic organoid is in suspension culture.

33. The method of any preceding claim, wherein the precursor pancreatic organoid and / or pancreatic organoid is free of extracellular matrix.

34. The method of any preceding claim, wherein the pancreatic organoid is embedded in a basement membrane matrix.

35. The method of any preceding claim, the method further comprising providing posterior splanchnic mesenchyme, comprising: a) activating a TGF-b signaling pathway, activating a BMP signaling pathway, activating a Wnt signaling pathway, activating an FGF signaling pathway, and activating a PI3K signaling pathway, in dissociated pluripotent stem cells, to generate mid primitive streak cells; b) activating a TGF-b signaling pathway, activating a BMP signaling pathway, activating a retinoic acid signaling pathway, and activating a Wnt signaling pathway, in the mid primitive streak cells of step a), to generate posterior foregut lateral plate mesoderm; and c) activating a TGF-b signaling pathway, activating a BMP signaling pathway, activating a retinoic acid signaling pathway, activating a Wnt signaling pathway, and activating an FGF signaling pathway, in the posterior foregut lateral plate mesoderm of step b), to generate posterior splanchnic mesenchyme.

36. The method of any preceding claim, the method further comprising providing posterior splanchnic mesenchyme, comprising: a) contacting dissociated pluripotent stem cells with one or more TGF-b signaling pathway activator, BMP signaling pathway activator, Wnt signaling pathway activator, FGF signaling pathway activator, and PI3K signaling pathway activator, to generate mid primitive streak cells; b) contacting the mid primitive streak cells of step a) with one or more TGF-b signaling pathway activator, BMP signaling pathway activator, retinoic acid signaling pathway activator, and Wnt signaling pathway activator, to generate posterior foregut lateral plate mesoderm; and c) contacting the posterior foregut lateral plate mesoderm of step b) with one or more TGF-b signaling pathway activator, BMP signaling pathway activator, retinoic acid signaling pathway activator, Wnt signaling pathway activator, and FGF signaling pathway activator, to generate posterior splanchnic mesenchyme.

37. The method of any preceding claim, the method further comprising providing vascular mesenchyme, comprising: a) inhibiting a ROCK signaling pathway in dissociated pluripotent stem cells in aggregation media, to generate aggregates; b) activating a Wnt signaling pathway, and activating a BMP signaling pathway, in the aggregates of step a), to induce mesoderm formation; and c) activating a VEGF signaling pathway, and activating a cAMP signaling pathway, in the mesoderm of step b), to induce vascular mesenchyme formation.

38. The method of any preceding claim, the method further comprising providing vascular mesenchyme, comprising: a) contacting dissociated pluripotent stem cells in aggregation media with one or more ROCK signaling pathway inhibitor, to generate aggregates; b) contacting the aggregates of step a) with one or more Wnt signaling pathway activator, and BMP signaling pathway activator, to induce mesoderm formation; and c) contacting the mesoderm of step b) with one or more VEGF signaling pathway activator, and cAMP signaling pathway activator, to induce vascular mesenchyme formation.

39. The method of any preceding claim, the method further comprising providing posterior foregut cells, comprising: a) activating a TGF-b signaling pathway activator and activating a Wnt signaling pathway, for a first period of time, in definitive endoderm cells (DE); b) activating a TGF-b signaling pathway activator without activating a Wnt signaling pathway, for a second period of time, in the cells of step a); and c) activating an FGF signaling pathway, activating an Shh signaling pathway, and activating a retinoic acid (RA) signaling pathway, and inhibiting a BMP signaling pathway, and inhibiting a CK2 signaling pathway, in the cells of step b), thereby differentiating the DE to posterior foregut cells.

40. The method of any preceding claim, the method further comprising providing posterior foregut cells, comprising:a) contacting definitive endoderm cells (DE) with one or more TGF-b signaling pathway activator and Wnt signaling pathway activator, for a first period of time; b) contacting the cells of step a) with a TGF-b signaling pathway activator for a second period of time; and c) contacting the cells of step b) with one or more FGF signaling pathway activator, Shh signaling pathway activator, retinoic acid (RA) signaling pathway activator, BMP signaling pathway inhibitor, and CK2 signaling pathway inhibitor, for a third period of time, thereby differentiating the DE to posterior foregut cells.

41. The method of any preceding claim, the method further comprising providing posterior foregut cells, comprising: a) activating an FGF signaling pathway activator and activating a Wnt signaling pathway for a first period of time, with or without inhibiting a BMP signaling pathway, in definitive endoderm cells (DE); and b) activating an FGF signaling pathway, activating a Wnt signaling pathway, and activating a retinoic acid (RA) signaling pathway for a second period of time, with or without inhibiting a BMP signaling pathway, in the cells of step a), thereby differentiating the DE to posterior foregut cells.

42. The method of any of claims xx-xx, the method further comprising providing posterior foregut cells, comprising: a) contacting definitive endoderm cells (DE) with one or more FGF signaling pathway activator and Wnt signaling pathway activator for a first period of time, with or without one or more BMP inhibitor; and b) contacting the cells of step a) with one or more FGF signaling pathway activator, Wnt signaling pathway activator, and retinoic acid (RA) signaling pathway activator for a second period of time, with or without one or more BMP inhibitor, thereby differentiating the DE to posterior foregut cells.

43. A pancreatic organoid (PO) obtained by in vitro expansion, comprising: a posterior foregut cell population; anda vascular mesenchyme recombinant cell population (VMPO) and / or a splanchnic mesenchyme recombinant cell population (SMPO).

44. A pancreatic organoid (PO) organoid obtained by in vitro expansion and prepared by the method of any of claims 1-42, comprising: a posterior foregut cell population; and a vascular mesenchyme recombinant cell population (VMPO) and / or a splanchnic mesenchyme recombinant cell population (SMPO).

45. The PO of any of claims 43-44, wherein the PO comprises a posterior foregut cell population, a VMPO, and a SMPO.

46. The PO of any of claims 43-45, wherein the PO comprises endocrine cells, islets or islet-like structures, and / or ductal and / or exocrine structures.

47. The PO of any of claims 43-46, wherein the PO comprises endocrine pancreas and / or stratified epithelial pancreatic progenitors.

48. The PO of any of claims 43-47, wherein the PO comprises an epithelial cavity or network, and one or more lumen and / or microlumen.

49. The PO of claim 48, wherein the one or more lumen and / or microlumen is adjacent to the epithelial cavity or network.

50. The PO of claim 48, wherein one or more lumen and / or microlumen is polarized.

51. The PO of any of claims 43-50, wherein the PO comprises one or more branched or interconnected ductal networks and / or acing structures.

52. The PO of claim 51, wherein the acing structures comprise acinar cells and / or acinar tissue localized at one or more ductal network terminus.

53. The PO of claim 52, wherein the acinar cells and / or acinar tissue are polarized.

54. The PO of any of claims 43-53, wherein the PO lacks nerve cells and / or immune cells.

55. The PO of any of claims 43-53, wherein the PO comprises immune cells.

56. The PO of any of claims 43-54, wherein the PO does not undergo anisotropic growth and / or wherein the PO lacks hierarchical vascularization.

57. The PO of any of claims 43-56, wherein the PO expresses one or more posterior forcgut and / or pancreatic progenitor marker.

58. The PO of any of claims 43-57, wherein the PO expresses cytokeratin-19, glucagon, c-peptide, neurogenin3, chromogranin A, claudin-18, E-cadherin, vimentin, PECAM, GATA4, PDX1, NKX6.1, FOXF1, WT-1, Ki67, SOX2, and / or SOX9.

59. The PO of claim 58, wherein the posterior foregut cell population expresses NKX6.1; and / or wherein the VMPO expresses PDX1 and / or E-cadherin; and / or wherein the SMPO expresses SOX2, E-cadherin, and / or claudin-18; and / or wherein the VMPO expresses PDX1.

60. The PO of claim 59, wherein the VMPO expresses claudin-18 only in a cystic lumen region.

61. The PO of any of claims 43-60, wherein the PO comprises a posterior foregut cell population and a VMPO, and wherein the posterior foregut cell population expresses NKX6.1, SOX9, and / or PDX1.

62. The PO of any of claims 43-61, wherein the VMPO and / or SMPO expresses FOXF1 and / or WT-1.

63. The PO of any of claims 43-62, wherein the PO expresses Ki67 after culturing for 60 days, or longer.

64. The PO of any of claims 43-63, wherein the PO comprises endocrine cells expressing neurogenin3 and / or chromogranin A; islets or islet-like structures expressing glucagon and / or c-peptide; and / or an epithelial network expressing cytokeratin-19.

65. The PO of any of claims 43-64, wherein the PO comprises acinar cells and / or acinar- tissue expressing GATA4, and downregulated expression of SOX9 and / or PDX1.

66. The PO of any of claims 43-65, wherein the PO comprises a ductal network expressing SOX9 and downregulated expression of GATA4.

67. The PO of any of claims 43-66, wherein the PO comprises a lumen expressing SOX2, surrounded by or adjacent to an epithelium expressing PDX1.

68. The PO of any of claims 43-67, wherein the PO secretes one or more pancreatic enzymes in acinar cells, acinar tissue, lumen, and / or microlumen.

69. The PO of any of claims 43-68, wherein the PO secretes zymogen granules, CPA1, mucin-1, and / or amylase in acinar cells, acinar tissue, lumen, and / or microlumcn.

70. The PO of any of claims 43-69, wherein the PO secretes CPA1 and amylase, and wherein the CPA1 and amylase secretion is co-localized.

71. The PO of any of claims 43-70, wherein the PO comprises a lumen adjacent to the epithelial network, wherein the lumen secretes mucin- 1.

72. The PO of any of claims 43-71, wherein the posterior foregut cells, mesenchymal cells, and vascular cells are derived from pluripotent stem cells.

73. The PO of claim 72, wherein the pluripotent stem cells comprise embryonic stem cells or induced pluripotent stem cells.

74. The PO of any of claims 43-73, wherein the posterior foregut cells, SMPO, and / or VMPO are derived from human pluripotent stem cells in vitro.

75. The PO of any of claims 43-74, wherein the PO is embedded in a basement membrane matrix.

76. The PO of any of claims 43-75, wherein the PO is in suspension culture.

77. The PO of any of claims 43-76, wherein the PO is an artificial pancreatic organoid and / or is generated in vitro.

78. The PO of any of claims 43-77, wherein the PO is three-dimensional.

79. The PO of any of claims 43-78, wherein the PO is a mature pancreatic organoid and / or comprises pancreatic architecture and hormones.

80. An in vitro composition comprising the PO of any of claims 43-79.

81. The PO of any of claims 43-79, or the composition of claim 80, for use in a method of treating a pancreas-related disease or disorder and / or a disease or disorder involving endocrinogenesis, a method of scaling up in bioprocess manufacturing, a method of screening for therapeutic efficacy, a method of modeling human pancreatic development and / or disease, a method of diagnosing a pancreatic -related disease or disorder and / or a disease or disorder involving endocrinogenesis, and / or the manufacture of a medicament for treating a pancreas- related disease or disorder and / or a disease or disorder involving endocrinogenesis.

82. Use of the PO of any of claims 43-79, or the composition of claim 80, a method of treating a pancrcas-rclatcd disease or disorder and / or a disease or disorder involving endocrinogenesis, a method of scaling up in bioprocess manufacturing, a method of screening for therapeutic efficacy, a method of modeling human pancreatic development and / or disease, a method of diagnosing a pancreatic-related disease or disorder, or the manufacture of a medicament for treating a pancreas-related disease or disorder and / or a disease or disorder involving endocrinogenesis.

83. A method comprising administering the PO of any of claims 43-79, or the composition of claim 80, to a subject in need thereof.

84. A method of treating a pancreas-related disease or disorder and / or a disease or disorder involving endocrinogenesis in a subject in need thereof, the method comprising administering the PO of any one of claims 43-79, or the composition of claim 80, to the subject.

85. The method of any one of claims 83-84, wherein administering comprises transplanting the PO of any one of claims 43-79, or the composition of claim 80, into the subject.

86. The method of any of claims 83-85, wherein the subject is a mammal.

87. The method of any of claims 83-86, wherein the subject is a mouse or a human.

88. The method of any of claims 85-87, wherein the PO is transplanted after culturing for 7 days to 60 days, or longer, in vitro.

89. The method of any of claims 85-88, wherein the PO, following transplant, engrafts under the kidney capsule of the subject.

90. The method of any of claims 85-89, wherein transplanting the PO to the subject comprises organoid engraftment, tissue growth, and / or improved tissue and / or organ function.

91. The method of any of claims 85-90, wherein the PO, following transplant, matures in vivo.

92. The method of any of claims 85-91, wherein the PO, following transplant, generates branched epithelial tissue.

93. The method of any of claims 85-92, wherein the PO, following transplant, comprises one or more branched or interconnected ductal networks and / or differentiated acing structures.

94. The method of any of claims 85-93, wherein the PO, following transplant, secretes pancreatic enzymes in the acinar cells, acinar tissue, ducts, lumen, and / or microlumen.

95. The method of any of claims 85-94, wherein the PO secretes zymogen granules, CPA1, and / or amylase in the acinar cells, acinar- tissue, ducts, lumen, and / or microlumen.

96. The method of claim 95, wherein the CPA1 and amylase secretion is colocalized.

97. The method of any of claims 83-96, wherein the subject has a pancreas-related disease or disorder, and / or a disease or disorder involving endocrinogenesis.

98. The method of claim 97, wherein the pancreas-related disease or disorder comprises a type of cancer, cystic fibrosis, pancreatitis, exocrine pancreatic insufficiency (EPI) or other exocrine pancreatic condition, endocrine pancreatic condition, pancreatic cysts, bile duct cysts, and / or diabetes.

99. The method of claim 98, wherein the type of cancer comprises pancreatic ductal adenocarcinoma (PDAC), a pancreatic neuroendocrine tumor, and / or bile duct cancer (cholangiocarcinoma) .

100. The method of any of claims 85-99, wherein the subject has an increased survival rate following transplantation.

101. The method of any of claims 81-100, wherein the PO is produced from pluripotent stem cells derived from the subject.

102. A method for screening a candidate compound or composition, wherein the candidate compound or composition to be screened comprises one or more exogenous agent, the method comprising: contacting the PO of any of claims 43-78, or the composition of claim 79, with the candidate compound or composition; culturing the PO with the candidate compound or composition for a period of time; andassessing one or more effects of the compound or composition on the PO, thereby screening the compound or composition.

103. The method of claim 102, wherein the assessed effect comprises therapeutic efficacy and / or toxicity of the compound or composition.

104. A kit comprising means for preparing an PO according to any one of claims 43- 78, or the composition according to claim 79, or for performing the method according to any one of claims 1-42 or 83-103.

105. A kit comprising the PO according to any one of claims 43-78, or the composition according to claim 79.

106. The kit of any of claims 104-105, wherein one or more of the kit components are provided in separate vials.

107. The kit of any of claims 104-106, wherein one or more of the kit components are pre-loaded onto one or more assay platform.

108. The kit of any of claims 104-107, wherein one or more of the kit components are pre-frozen.

109. A method of providing an in vitro pancreatic mesenchyme, comprising: providing an PO according to the method of any of claims 1-42; and dissociating the pancreatic organoid into respective mesenchymal and epithelial components.

110. A pancreatic mesenchyme, comprising a vascular mesenchyme recombinant cell population (VMPO) and / or a splanchnic mesenchyme recombinant cell population (SMPO).

111. A pancreatic mesenchyme, comprising a vascular mesenchyme recombinant cell population (VMPO) and / or a splanchnic mesenchyme recombinant cell population (SMPO), wherein the pancreatic mesenchyme is produced by the method of claim 109.

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