MICROENVIRONMENTS FOR SELF-ASSEMBLY OF ISLET ORGANOIDS FROM STEM CELLS DIFFERENTIATION

Abstract

Human pluripotent stem cells (hPSCs) are promising cell source to produce therapeutic endocrine cells for diabetes treatment. A gel solution made by decellularized tissue-specific extracellular matrix (dpECM) significantly promotes three-dimensional (3D) islet-like organogenesis during induced hPSC differentiation into endocrine lineages. Islet organoids are self-organized even in a two-dimensional (2D) culture mode. Cells derived from hPSCs differentiated on such ECM coated substrates exhibit similar cellular composition to native pancreatic islets. These cells express islet signature markers insulin, PDX-1, C-peptide, MafA, glucagon, somatostatin, and pancreatic polypeptide, and secrete more insulin in response to glucose level compared to a traditional matrix substrate (Matrigel). The dpECM facilitates generating more C-peptide+/glucagon cells rather than C-peptide+/glucagon+ cells. Remarkably, dpECM also facilitated intra-organoid vascularity by generating endothelial cells and pericytes. Furthermore, dpECM niches also induced intra-organoid microvascularization during pancreatic differentiation.

Claims

1. A method of creating a synthetic organoid, comprising: extracting tissue-specific matrix from a mammalian organ; coating a growth surface with cell growth factors comprising collagen and mucopolysaccharides, and the extracted tissue-specific matrix; culturing stem cells on the surface; and inducing the stem cells to differentiate on the surface.

2. The method according to claim 1, wherein said inducing the stem cells to differentiate on the surface further comprises differentiating the step cells to produce at least four coexisting distinct cell types which are differentiated with respect to the stem cells.

3. The method according to claim 1, wherein the cell growth factors comprise a solubilized basement membrane preparation extracted from a cell culture, comprising laminin, collagen IV, heparin sulfate proteoglycans, and entactin/nidogen.

4. The method according to claim 1, further comprising cross linking the collagen after commencing culturing of the stem cells on the surface.

5. The method according to claim 1, wherein: the tissue-specific matrix comprises pancreas tissue-specific matrix from a mammal; and the stem cells are pluripotent stem cells; wherein said inducing the stem cells to differentiate on the surface comprises inducing the pluripotent stem cells to differentiate through at least four stages of differentiation on the surface into differentiated pancreatic cells.

6. The method according to claim 5, wherein the pluripotent stem cells differentiate into at least pancreatic beta cells; pancreatic alpha cells; pancreatic delta cells; and pancreatic polypeptide cells, and the differentiated pluripotent stem cells comprise: about 50-70% pancreatic beta cells; about 20-30% pancreatic alpha cells; about 10% pancreatic delta cells; and about <5% pancreatic polypeptide cells.

7. The method according to claim 1, wherein the mammalian organ is a pancreas, the stems cells are human pluripotent stem cells, and said inducing comprises inducing the stem cells to differentiate through at least four stages of differentiation, further comprising producing human insulin and human somatostatin from the synthetic organoid.

8. The method according to claim 7, further comprising testing the synthetic organoid for a modulation of a response to glucose selectively dependent on at least one drug.

9. The method according to claim 1, wherein said inducing the stem cells to differentiate comprises sequentially incubating the stem cells in: Media in Stage 1 (S1): RPMI 1640 containing B27, 50 ng/ml activin A and 0.5-1 mM sodium butyrate; Media in Stage 2 (S2): RPMI 1640 containing B27, 250 M ascorbic acid, 50 ng/ml keratinocyte growth factor, 50 ng/ml Noggin, 1 M retinoic acid, 300 nM ()-indolactam V, and 100 nM LDN193189; Media in Stage 3 (S3): DME/F12 containing B27, 1 M RA, 200 nM LDN, 300 nM ILV, 1 M 3,3,5-Triiodo-L-thyronine sodium salt, 10 M ALK5 inhibitor II, and 10 g/ml heparin, supplemented with glucose to a final concentration of 20 mM; Media in Stage 4 (S4): RPMI 1640 containing B27, 1 M T3, 10 M ALKi, 1 mM N-acetyl cysteine, 0.5 M R428, 10 M trolox, 100 nM -secretase inhibitor XX, 10 M zinc sulfate, 10 mM nicotinamide, and 10 g/ml HP, supplemented with glucose to a final concentration of 20 mM glucose; and Media in Stage 5 (S5): CMRL supplement containing 10% fetal bovine serum, 1 M T3, 10 M ALKi, 0.5 M R428, and 10 mM Nic.

10. The method according to claim 9, wherein the synthetic organoid comprises pancreatic alpha cells, pancreatic beta cells, pancreatic delta cells, and pancreatic polypeptide cells, further comprising implanting the synthetic organoid into a mammal.

11. The method according to claim 1, wherein the synthetic organoid is maintained within an extracorporeal blood circulation loop of a mammal.

12. A synthetic pancreatic islet organoid, comprising: a pancreas tissue-specific matrix; a collagen support material; about 50-70% pancreatic beta cells; about 20-30% pancreatic alpha cells; about 10% pancreatic delta cells; and about <5% pancreatic polypeptide cells, the pancreatic beta cells, pancreatic alpha cells, pancreatic delta cells, and pancreatic polypeptide cells being derived from mammalian pluripotent stem cells, and the pancreas tissue-specific matrix being derived from a mammal.

13. The synthetic pancreatic islet organoid according to claim 12, wherein the pancreas tissue specific matrix is processed for cellular removal, substantially without detergent treatment.

14. The synthetic pancreatic islet organoid according to claim 13, wherein the pancreas tissue-specific matrix is processed for cellular removal by cycles of high and low osmotic tension.

15. The synthetic pancreatic islet organoid according to claim 12, wherein the pancreas tissue-specific matrix has a residual DNA content of less than about 1% of the DNA of the mammalian tissue from which it is derived.

16. The synthetic pancreatic islet organoid according to claim 12, wherein the collagen support material is cross linked.

17. The synthetic pancreatic islet organoid according to claim 12, further comprising a bioreactor containing the synthetic organoid, configured expose the synthetic organoid to an extracorporeal blood flow of a living mammal.

18. A method of screening a drug, comprising: providing a synthetic organoid, comprising at least one cross-linked collagen-containing surface, coated with osmotically processed acellular tissue-specific matrix extracted from a mammalian organ substantially without use of detergent, and cell growth factors comprising collagen, mucopolysaccharides, and basement membrane factors, and pluripotent stem cells cultured and induced into differentiation on the surface, to at least two organ-specific differentiated cell types; determining a modulation of response of the synthetic organoid to glucose by a drug.

19. The method according to claim 18, wherein the pluripotent stem cells are human pluripotent stem cells induced into four stages of differentiation into at least pancreatic beta cells; pancreatic alpha cells; pancreatic delta cells; and pancreatic polypeptide cells, and the differentiated pluripotent stem cells comprise: about 50-70% pancreatic beta cells; about 20-30% pancreatic alpha cells; about 10% pancreatic delta cells; and about <5% pancreatic polypeptide cells.

20. The method according to claim 18, wherein the synthetic organoid is maintained within an extracorporeal blood circulation loop of a mammal.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0109] FIGS. 1A-1D shows dpECM preparation. FIG. 1A shows Gel electrophoresis of DNA isolated from two dpECM replicates. Equal amount of native rat pancreatic tissues was used as control (rPan). FIG. 1B shows DNA quantitation (meanSD) of dpECM in comparison to native pancreatic tissues, n>3. Values were expressed as meanSD. *, p<0.05. FIG. 1C shows live-and-dead cell staining of iPSCs cultured on MG or dpECM coated dishes for 24 h. Bars, 50 m. FIG. 1D shows live/dead cell dual-staining of iPSCs cultured on either MG or dpECM coated dishes for 24 h after seeding. Scale bars, 200 m.

[0110] FIGS. 2A-2E shows a schematic summary of the 4-stage, 21-day differentiation protocol and characterization of marker genes during the differentiation. FIG. 2A shows in vitro differentiation protocol of iPSCs to islet tissue. Basal media as well as key molecules used are shown for each stage of differentiation. FIGS. 2B-2E show the profile of marker gene expression of hPSC-derived cells during 51 to S4 of differentiation. Cells were 2D cultured on MG- or MG-/dpECM-coated substrates using indicated concentrations. Expression levels of mRNA were assessed for DE (FIG. 2B), posterior foregut (FIG. 2C), pancreatic progenitor (FIG. 2D), and -cell markers (FIG. 2E). Gene expression was relative to that in cells grown on MG-coated dishes. Results were from three or more experiments and shown as meanSD. *, p<0.05; **, p<0.01; ***, p<0.001, compared to MG group. Cells were 2D cultured on MG or MG/dpECM (M+d) coated plates as described in Methods. The MG-dpECM ratios (w:w) of prepared MG/dpECM mixtures were 4:1, 2:1, and 1:1, respectively.

[0111] FIGS. 3A-3C shows microscopic examination of self-assembly of islet-like organoids at the end of Stage 2, 3, and 4 of iPSC differentiation. FIG. 3A show micrographs of cell clusters formed on MG/dpECM coated plates. Black scale bars, 1,000 m; white scale bars, 200 m. FIG. 3B shows number of cell clusters formed on MG/dpECM coated plates. Tiled images covering an area of 0.53 cm2 were randomly selected and analyzed by ImageJ software. Data are shown as meanSD (n=8). *, p<0.05; **, p<0.01; ***, p<0.001. FIG. 3C show diameters of cell clusters formed on MG/dpECM coated plates. Gray bars indicate average diameter of aggregates.

[0112] FIGS. 4A-4E show organoids generated from iPSCs grown on dpECM coated substrates showed similar composition to native islets. FIG. 4A shows representative immunofluorescent staining of S4 cells labeled for C-peptide (C-PEP, green) and glucagon (GCG; red). 4,6-diamidino-2-phenylindole (DAPI, blue) was used for counterstaining nuclei. Scale bars, 50 m. FIG. 4B shows representative immunofluorescent staining of S4 cells labeled for somatostatin (SST, green), pancreatic polypeptide (PPY; red), and DAPI. Scale bars, 50 m. (FIGS. 4C-4E) Representative flow cytometric results of S4 cells stained for C-PEP and GCG (FIG. 4C), SST (FIG. 4D), and PPY (FIG. 4E). Numbers in quadrants represent the percentage of total counted cells.

[0113] FIGS. 4F-4L show cell compositions of S4 cells during iPSC pancreatic differentiation on MG/dpECM coated plates. S4 cells were immunofluorescently labeled for (FIG. 4F) C-peptide (C-PEP, green) and glucagon (GCG; red); (FIG. 4G) somatostatin (SST, green) and pancreatic polypeptide (PPY; red). 4,6-diamidino-2-phenylindole (DAPI, blue) was used for counterstaining cell nuclei. Scale bars, 50 m. (FIGS. 4H-4I) Representative flow cytometric analysis of C-PEP (FIG. 4H), GCG (FIG. 4I), SST (FIG. 4J), and PPY (FIG. 4K) expressing cells in S4 cells. SSC: side scatter. (FIG. 4L) Representative flow cytometric analysis of S4 cells dual-stained for C-PEP and GCG. Numbers in quadrants represent the percentage of total counted cells.

[0114] FIGS. 5A-5C show characterization of S4 cells. (FIG. 5A) Flow cytometric analysis of insulin (INS) and Nkx6.1 expression in S4 cells. Numbers in quadrants represent the percentage of total counted cells, illustrating the typical population of both NKX6.1+ and insulin+ cells generated at S4. (FIG. 5B and FIG. 5C) Representative immunofluorescent staining of S4 cells cultured on various conditions. (B) pancreatic and duodenal homeobox 1 (PDX-1, green) and C-peptide (C-PEP, red). (FIG. 5C) MAF bZIP transcription factor A (MAFA, green) and glucagon (GCG, red). 4,6-diamidino-2-phenylindole (DAPI, blue) was used for counterstaining nuclei. Bars, 50 m.

[0115] FIGS. 6A-6E show that dpECM promotes Glucose-responsive insulin-secretion of islet organoids matured in 3D cultures. (FIG. 6A) A five-stage differentiation strategy. After S3, cells were either continuously cultured on MG/dpECM substrates (2D cultures) or transferred into ultra-low attachment plates for suspension cultures (3D culture). (FIG. 6B) DTZ staining of cell clusters at S5. Undifferentiated iPSCs cultured in ultra-low attachment plates for 24 hrs served as a control. Scale bars, 200 m. Glucose-stimulated insulin secretion (GSIS) analysis was performed by challenging cells differentiated in 2D (FIG. 6C) or 3D (FIG. 6D) conditions with 2 mM and 20 mM glucose for 30 min at each step. The insulin release in response to secretagogue such as 30 mM KCl was also measured. 2 mM glucose was used along with KCl when determining insulin release in response to a secretogogue. Insulin secretion was determined as IU insulin per g cellular DNA (n=4). (FIG. 6E) Insulin secretion from ESCs-derived cells at S5 of suspension culture (n=3). Data are shown as meanSD. *, p<0.05; **, p<0.01.

[0116] FIG. 6F-6I. IMR90 and H9 aggregates, 2D vs, 3D. (FIGS. 6F-H) insulin secretion of IMR90 S5 2D, IMR S5 Aggregates, and H9 SW5 2D, with respect to glucose concentration (2 mM, 20 mM, 30 mM KCl), on MG, M+d 4:1, and M+d 2:1. (FIG. 6I) insulin secretion of IMR90 S5 2D vs. 3D, with respect to glucose concentration (2 mM, 20 mM, 30 mM KCl) and condition (MG, M+d 4:1, and M+d 2:1).

[0117] FIG. 7A-7E show representative flow cytometric analysis of cell compositions of islet organoid at Stage 5. Representative flow cytometric analysis of C-PEP (FIG. 7A), GCG (FIG. 7A), SST (FIG. 7C), and PPY (FIG. 7D) expressing cells in S5 aggregates. SSC: side scatter. (FIG. 7E) Representative flow cytometric analysis of S5 cells dual-stained for C-PEP and GCG. Numbers in quadrants represent the percentage of total counted cells.

[0118] FIG. 8A shows iPSCs (IMR90) cultured on MG- or dpECM-coated substrates. Twenty-four hours after seeding, phase contrast images were captured using a Nikon microscope. Bars, 200 m.

[0119] FIG. 8B shows cell clusters formed on MG and MG/dpECM substrates. Phase contrast micrographs taken at 24 h after seeding. Scale bars, 200 m.

[0120] FIG. 9 shows flow cytometry analysis of SOX17 expression in DE stage. SOX17-positive cells were gated using isotype control (Black square). Red square indicates the percentage of cells expressing high level of SOX17.

[0121] FIG. 10 shows a comparison of iPSC DE differentiation on MG with those on MG/dpECM substrates. TaqMan qPCR analysis of SOX17 and FOXA2 expression in cells at the end of DE stage. Cells cultured on MG-coated (MG) or two-fold increased MG ECM coated substrates (2MG). Expression levels were normalized to that in cells cultured MG-coated dish. Values are shown as meanSD (n=3). NS: not statistically significant.

[0122] FIG. 11 shows cell clusters observed at day 7 of S5 in 2D cultures. (A) Micrographs of cell clusters formed on MG/dpECM coated plates. Black scale bars, 1,000 m; white scale bars, 200 m. Tiled images covering an area of 0.53 cm2 were randomly selected and analyzed by ImageJ software. (B) Number of cell clusters formed on MG/dpECM coated plates. Tiled images covering an area of 0.53 cm2 were randomly selected and analyzed by ImageJ software. Data are shown as meanSD (n=8). *, p<0.05. (C) Diameters of cell clusters formed on MG/dpECM coated plates. Gray bars indicate average diameter of aggregates.

[0123] FIGS. 12A-12C show fine tuning of mechanical properties of collagen scaffolds by treating with different molecular weights of PEGDA. The shear storage modulus (FIG. 12A), the shear loss modulus (FIG. 12BB), and the Young's modulus (FIG. 12CC) of the PEGDA treated collagen scaffolds were determined using a rheometer. All experiments were performed in triplicate. Error bars indicate standard deviation. *, p<0.02.

[0124] FIGS. 13A-13E show Vasculogenesis of islet organoid at S5. FIG. 13A shows cryosectioned islet organoids stained with H&E. Black scale bars, 50 m; white scale bars, 10 m. White arrowheads indicate capillary-like structures. FIG. 13B shows capillary density in S5 organoids as analyzed by Image-Pro Plus (Version 6.0). *, p<0.05. FIGS. 13C and 13D show representative images of cryosectioned islet organoids stained by antibodies against (FIG. 13C) CD31, or (FIG. 13D) C-peptide (CP) and CD31. White arrowheads indicate direct contact between CD31+ cells and CP+ cells. FIG. 13E shows representative flow cytometric analysis of CD31 and NG2 expressing cells in S5 organoids. SSC: side scatter. Numbers in quadrants represent the percentage of total counted cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Example 1

[0125] Rat pancreas were cut into 3 mm thick slices, and treated repeatedly with deionized water and sodium chloride-ammonia hydroxide solution for four days. After rinse and lyophilization, total DNA content of dpECM was examined. dpECM was milled and reconstituted by pepsin-containing acetic acid and neutralized. One hour before seeding human induced pluripotent stem cells (iPSCs), six-well plates were coated with matrigel (MG) and varied concentrations of dpECM. MG coated plates serve as a control for comparison. To differentiate iPSCs into islet organoids, a novel stepwise differentiation protocol developed was used. Expression of pancreatic marker genes and proteins were examined by quantitative real-time PCR and flow cytometric analyses at the end of each stage of differentiation.

[0126] The dpECM procedure developed in this study enables the removal of approximately 99% of DNA from animal pancreas. At the end of stage I of differentiation, the expression of definitive endoderm marker genes SOX17 and FOXA2 were increased 3.8 and 2 folds, respectively, when the cells were cultured on dpECM plus MG coated surfaces compared to cells cultured on MG-coated surfaces. At stage II of differentiation, the expression of pancreatic progenitor markers ISL-1 and PDX1 mRNA increased 2.5 folds in cells cultured on dpECM and MG coated surfaces. At stage III of differentiation, the pancreatic endoderm markers Nkx6.1 and PDX1 mRNA increased 4.7 and 3 folds in cells cultured on dpECM and MG coated surfaces. Notably, the expression of insulin increased 9 folds in cells cultured on dpECM and MG coated surfaces. Importantly, the gene expression levels of PDX1, Nkx6.1, glucagon, and insulin from iPSC-derived cells are comparable to those in human pancreas. The experimental results indicate that dpECM facilities the differentiation of hPSCs into functional islet organoids. Experimental data obtained from flow cytometry confirmed that more than 60 percent of cells expressed insulin at the end of differentiation using dpECM as additional substrates.

[0127] dpECM gel offers an excellent tissue niche for hPSC islet differentiation.

[0128] Materials and Methods

[0129] Preparation of Rat dpECM Gel

[0130] Rat pancreata were obtained from the Laboratory Animal Resources at the Binghamton University. Briefly, male and female Sprague Dawley rats (Charles River) were euthanized by CO2 asphyxiation according to the American Veterinary Medical Association (AVMA) guidelines. Pancreata were isolated and rinsed with cold PBS twice then stored at 80 C. until use. The frozen pancreata were cut into 1.5 mm sections using a deli-style slicer (Chef's choice 632, Edge Craft Corporation). The slices were rinsed with deionized water for 5 times on a tube rotator (Boekel Industries) at 4 C. with a speed of 20 rpm. Then the tissues were processed 4 cycles with hyper/hypotonic washes: gently shaken in hypertonic solution containing 100 g/l sodium chloride and 0.1% ammonium hydroxide at 4 C. for 12 hours. The materials were then transferred into deionized water and shaken at 4 C. for 12 hours. The resulting tissues were extensively rinsed with deionized water to remove any residue chemicals. The decellularized pancreatic tissues were lyophilized using Freezone freeze dry system (LABCONCO) and comminuted using a Wiley Mini Mill (Thomas Scientific). To solubilize the dpECM powder, 100 mg of lyophilized dpECM powder was digested by 10 mg of pepsin (Sigma) in 5 ml of 0.02 N acetic acid for 48 h at room temperature with continuous stirring. The resultant dpECM solution was aliquoted and stored at 80 C. until use.

[0131] Characterization of dpECM

[0132] Decellularization efficiency was evaluated by extracting DNA from lyophilized dpECM using a DNeasy Blood and Tissue Kit (QIAGEN) according to manufacturer's instructions. DNA content in dpECM was quantified using Synergy H1 Microplate Reader (BioTek) and also examined by 1% agarose gel electrophoresis.

[0133] Cytotoxicity of dpECM was examined using a viability/cytotoxicity kit (Life Technologies), according to manufacturer's instructions. Briefly, 24 h after seeding onto Matrigel- (Corning Life Science) or Matrigel- and dpECM-coated 6-well plate, the cultured iPSCs were rinsed twice with PBS and incubated with 2 M Calcein A M and 2 M ethidium homodimer reagents at 37 C. for 10 min. After rinsing again with PBS, the cell viability was evaluated using a Nikon fluorescence microscope.

[0134] Cell Culture

[0135] Undifferentiated iPSC line IMR90 and hESC line H9 (WiCell Research Institute) were maintained in mTeSR1 medium (Stemcell Technologies). Cells were passaged every 4 days at ratios of 1:3 to 1:5 as reported previously [Nair G, Hebrok M. Islet formation in mice and men: lessons for the generation of functional insulin-producing beta-cells from human pluripotent stem cells. Curr Opin Genet Dev 32, 171-180 (2015).; Seymour P A, Sander M. Historical perspective: beginnings of the beta-cell: current perspectives in beta-cell development. Diabetes 60, 364-376 (2011)]. For induced differentiation into endocrine tissue, cells were dissociated by Accutase (Stemcell Technologies) and seeded onto 80 g/ml of Matrigel (MG) or MG with various amount of dpECM-coated 6-well plate with a density of 110.sup.6 cells/well and cultured in mTeSR1 medium. The dpECM concentrations used were: 20 g/ml indicated as M+d 25%, 40 g/ml (M+d 50%), or 80 g/ml (M+d 100%). The ratios (w/w) of dpECM vs MG in the mixed ECM gel (M+d) were 1:4, 1:2, and 1:1. A five-stage differentiation protocol as dictated in (FIGS. 2A and 6A) was adopted to differentiate iPSCs and ESCs into islet organoids at 24 h post seeding. Twenty-four hours after seeding, cells were cultured in varied differentiation media following the timeframe shown in FIG. 2A.

[0136] Media in Stage 1 (S1) included RPMI 1640 (Corning), B27 (Gibco), 50 ng/ml activin A (PeproTech) and 1 mM sodium butyrate (NaB, Sigma-Aldrich) for the first 24 h. The NaB was reduced into 0.5 mM from day 2- to day 4 as described elsewhere [Seymour P A, Sander M. Historical perspective: beginnings of the beta-cell: current perspectives in beta-cell development. Diabetes 60, 364-376 (2011).; Steiner D J, Kim A, Miller K, Hara M. Pancreatic islet plasticity: interspecies comparison of islet architecture and composition. Islets 2, 135-145 (2010)].

[0137] Media in Stage 2 (S2) consisted of RPMI 1640, B27, 250 M ascorbic acid (Vc, Sigma-Aldrich), 50 ng/ml keratinocyte growth factor (KGF, PeproTech), 50 ng/ml Noggin (PeproTech), 1 M retinoic acid (RA, Sigma-Aldrich), 300 nM ()-indolactam V (ILV, AdipoGen), and 100 nM LDN193189 (LDN, Sigma-Aldrich).

[0138] Media in Stage 3 (S3), cells were cultured in DME/F12 (HyClone) containing B27, 1 M RA, 200 nM LDN, 300 nM ILV, 1 M 3,3,5-Triiodo-L-thyronine sodium salt (T3, Sigma-Aldrich), 10 M ALK5 inhibitor II (ALKi, Enzo Life Sciences), 10 g/ml heparin (HP, Sigma-Aldrich), and supplemented glucose to a final concentration of 20 mM.

[0139] Media in Stage 4 (S4), the differentiation media contained RPMI 1640, B27, 1 M T3, 10 M ALKi, 1 mM N-acetyl cysteine (N-Cys, Sigma-Aldrich), 0.5 M R428 (SelleckChem), 10 M trolox (Enzo Life Sciences), 100 nM -secretase inhibitor XX (Millipore), 10 M zinc sulfate (Sigma-Aldrich), 10 mM nicotinamide (Nic, Sigma-Aldrich), 10 g/ml HP, and 20 mM glucose. [Otonkoski, Timo, Gillian M. Beattie, Martin I. Mally, Camillo Ricordi, and Alberto Hayek. Nicotinamide is a potent inducer of endocrine differentiation in cultured human fetal pancreatic cells. Journal of Clinical Investigation 92, no. 3 (1993): 1459.]

[0140] Media in Stage 5 (S5), the S4 medium was replaced with CMRL supplement containing 10% fetal bovine serum (ATCC), 1 M T3, 10 M ALKi, 0.5 M R428, and 10 mM Nic for 7 days (FIG. 6A).

[0141] All differentiation media were changed every two days, unless otherwise specified.

[0142] For aggregate culture, differentiated cells at the end of stage 3 were dissociated with Dispase (STEMCELL Technologies) and further cultured in 24-well ultra-low attachment plate (Corning) with stage 4 differentiation medium for 7 days and stage 5 differentiation medium for another 7 days in a 24-well ultra-low attachment plate (Corning Life Science).

[0143] Quantitative Real-Time Polymerase Chain Reaction (qPCR)

Gene expression was evaluated by TaqMan qRT-PCR analysis as described in previous work [Nair G, Hebrok M. Islet formation in mice and men: lessons for the generation of functional insulin-producing beta-cells from human pluripotent stem cells. Curr Opin Genet Dev 32, 171-180 (2015).; Seymour P A, Sander M. Historical perspective: beginnings of the beta-cell: current perspectives in beta-cell development. Diabetes 60, 364-376 (2011)]. Briefly, total RNA was isolated from cells using an RNeasy Mini Kit (Qiagen) and 200 g of RNA from each sample was subjected to a Multiplex PCR Kit (QIAGEN) using CFX Connect Real-Time PCR system (BIO-RAD). Data were normalized to an internal housekeeping gene (Cyclophilin A) and then calculated as fold change relative to differentiated cells cultured on MG using C method. Human pancreatic RNA (Clontech) was used as positive control. For each sample, at least three independent experiments were performed. Primers are listed in Table 1.

TABLE-US-00001 TABLE1 PrimersandprobesforqRT-PCR Sequencesofprimersandprobes(5to3) Genes orAssayIDsfromAppliedBiosystems SOX17 Forward:CAGCAGAATCCAGACCTGCASEQIDNO.001 Reverse:GTCAGCGCCTTCCACGACTSEQIDNO.002 Probe:FAM-ACGCCGAGTTGAGCAAGATGCTGG-BHQSEQIDNO.003 FOXA2 Forward:CCGACTGGAGCAGCTACTATGSEQIDNO.004 Reverse:TACGTGTTCATGCCGTTCATSEQIDNO.005 Probe:FAM-CAGAGCCCGAGGGCTACTCCTCC-BHQSEQIDNO.006 PDX-1 Forward:CCTTTCCCATGGATGAAGTCSEQIDNO.007 Reverse:CGTCCGCTTGTTCTCCTCSEQIDNO.008 Probe:FAM-AAGCTCACGCGTGGAAAGGCC-BHQSEQIDNO.009 ISL-1 Hs00158126_m1 NKx6.1 Hs00232355_m1 PTF1A Forward:CAGGCCCAGAAGGTCATCSEQIDNO.010 Reverse:GGGAGGGAGGCCATAATCSEQIDNO.011 Probe:FAM-ATCTGCCATCGGGGCACCC-BHQSEQIDNO.012 Insulin Forward:GGGAGGCAGAGGACCTGSEQIDNO.013 Reverse:CCACAATGCCACGCTTCTSEQIDNO.014 Probe:FAM-AGGTGGGGCAGGTGGAGCTG-BHQSEQIDNO.015 Glucagon Forward:GCTGCCAAGGAATTCATTGCSEQIDNO.016 Reverse:CTTCAACAATGGCGACCTCTTCSEQIDNO.017 Probe:FAM-TGAAAGGCCGAGGAAGGCGAGATT-BHQSEQIDNO.018 MAFA Hs01651425_s1 Somatostatin Hs00356144_m1 Pancreatic Hs00237001_m1 polypeptide CyclophilinA 4310883E

[0144] Flow Cytometry

[0145] Cells were treated with Accutase for 7 min to acquire single cell suspension. After washing with PBS, the cells were fixed in 4% paraformaldehyde (PFA) on ice for 15 min, permeablized with 0.25% Triton X-100 in PBS for 15 min, and blocked with 1% BSA in PBS-Tween at room temperature for 30 min. The following primary and secondary antibodies were used for intracellular staining: anti-insulin (Alexa Fluor 647-conjugated, 1:50, Cell Signaling), anti-Nkx6.1 (PE-conjugated, 1:20, BD Biosciences), anti-SOX17 (APC-conjugated, 1:10, R&D Systems), anti-C-peptide (1:20, DSHB at University of Iowa), anti-glucagon (1:80, R&D Systems), anti-somatostatin (1:100, Millipore), anti-pancreatic polypeptide (1:30, R&D SYSTEMS), mouse IgG (Alexa Fluor 647-conjugated, 1:20, BD Biosciences), mouse IgG (PE-conjugated, 1:40, BD Biosciences), goat IgG (APC-conjugated, 1:10, R&D SYSTEMS), goat anti-rat IgG-FITC (1:50, R&D SYSTEMS), donkey anti-mouse IgG-NL557 (1:200, R&D SYSTEMS), goat anti-rabbit-Alexa Fluor 488 (1:2000, Abcam). Flow cytometric analysis was performed on a FACS Aria II flow cytometer (Becton Dickinson) using FlowJo software (Version 10, FlowJo, LLC). Antibodies used in this study are summarized in Table 2.

TABLE-US-00002 TABLE 2 Antibodies used in flow cytometry Antibodies Species Manufacturer Category Dilution Insulin Rabbit Cell Signaling Primary 1:50 antibody Nkx6.1 Mouse BD Biosciences Primary 1:20 antibody SOX17 Goat R&D SYSTEMS Primary 1:10 antibody C-peptide Rat DSHB at University Primary 1:20 of Iowa antibody Mouse IgG Mouse BD Biosciences Isotype 1:40 control Mouse IgG Mouse BD Biosciences Isotype 1:20 control Goat IgG Goat R&D SYSTEMS Isotype 1:10 control Rat IgG Goat R&D SYSTEMS Secondary 1:50 antibody Mouse IgG Donkey R&D SYSTEMS Secondary 1:20 antibody Rabbit IgG Goat Abcam Secondary 1:2000 antibody

[0146] Immunofluorescence Microscopy

[0147] Immunofluorescent staining was carried out as described previously [Nair G, Hebrok M. Islet formation in mice and men: lessons for the generation of functional insulin-producing beta-cells from human pluripotent stem cells. Curr Opin Genet Dev 32, 171-180 (2015).]. In brief, cells were rinsed with PBS for three times and fixed in 4% PFA for 15 min on ice. The cells were then permeablized with 0.25% Triton X-100 in PBS and blocked with 1% BSA in PBST. Marker protein expression was labelled by incubating overnight at 4 C. with primary and secondary antibodies, anti-C-peptide (1:20, DSHB at University of Iowa), anti-glucagon (1:50, R&D Systems), anti-PDX1 (1:100, BD Biosciences), anti-MAFA (1:400, Abcam), anti-somatostatin (1:100, Millipore), anti-pancreatic polypeptide (1:50, R&D Systems), goat anti-rat IgG-FITC (1:50, R&D SYSTEMS), donkey anti-mouse IgG-NL557 (1:200, R&D Systems), goat anti-rabbit-Alexa Fluor 488 (1:1000, Abcam), goat anti-mouse-Alexa Fluor 488 (1:1000, Abcam). Nuclei were counterstained with Vectashield Mounting Medium containing 4,6-diamidino-2-phenylindole (Vector Laboratories). Images were captured using Nikon Eclipse Ti microscope. Antibodies used in immunofluorescent staining are listed in Table 3.

TABLE-US-00003 TABLE 3 Antibodies used in immunofluorescent staining Antibodies Species Manufacturer Category Dilution C-peptide Rat DSHB at University Primary 1:20 of Iowa antibody Glucagon Mouse R&D Systems Primary 1:50 antibody PDX1 Rabbit Abcam Primary 1:100 antibody MAFA Rabbit Abcam Primary 1:400 antibody Somatostatin Rat Millipore Primary 1:100 antibody Pancreatic Mouse R&D Systems Primary 1:50 polypeptide antibody Rat IgG Goat R&D Systems Secondary 1:50 antibody Mouse IgG Donkey R&D Systems Secondary 1:200 antibody Rabbit IgG Goat Abcam Secondary 1:1000 antibody Mouse IgG Goat Abcam Secondary 1:1000 antibody

[0148] DTZ Staining

[0149] Dithizone (DTZ) (Sigma) was performed by first dissolving 5 mg of DTZ in 1 ml of dimethyl sulfoxide (DMSO), then diluted at 1:5 with PBS. The resultant solution was filtered through 0.2 m nylon filter. Cellular aggregates were incubated in the DTZ solution at 37 C. for 2 to 3 minutes. Images were captured under an inverted phase contrast microscope.

[0150] Insulin Enzyme-Link Immunosorbent Assay (ELISA)

[0151] hPSC-derived cells at the end of differentiation (S5) were washed twice with PBS and preincubated in Krebs-Ringer buffer (KRB, Boston BioProducts) containing 120 mM sodium chloride, 5 mM potassium chloride, 2 mM calcium chloride, 1 mM magnesium chloride, 5.5 mM HEPES, and 1 mM D-glucose for 4 h to remove residual insulin. After rinsing twice with KRB, the cells were sequentially incubated with KRB containing 2 mM or 20 mM D-glucose or 30 mM KCl with 2 mM glucose at 37 C. for 30 min, unless otherwise specified. The respective supernatants were collected and human insulin level was measured using a human insulin ELISA kit (ALPCO Diagnostics) according to manufacturer's instructions. Total DNA content from each sample was determined by a DNeasy Blood and Tissue Kit (QIAGEN) and Synergy H1 Microplate Reader (BioTek).

[0152] Aggregates Analysis

[0153] At indicated time points, 2D cultured cells in 6-well plate were placed onto stage of a Nikon Eclipse Ti microscope and tiled bright field images were taken using 40 objective lens, covering an area of 0.53 cm2. The resulting images were analyzed by ImageJ software (National Institutes of Health, Version 1.50b). Thresholds for each were set as 0 to 200 in black and white mode. Particles whose sizes were between 10,000 to 250,000 m.sup.2 and circularity between 0.18 to 1.00 were counted. Diameters of aggregates were calculated from identified areas based on the circular forms of aggregates (area=(d/2).sup.2) where is circumference, and d is diameter.

[0154] Statistical Analysis

[0155] Data are presented as meansstandard deviation (SD) of at least three independent experiments. Statistical analysis was calculated by Student's t-test, difference between groups were considered significant with p values <0.05.

[0156] Preparation of dpECM

[0157] After washing by hyper/hypotonic solutions, the appearance of rat pancreata changed from bright red color to a mostly white and translucent. To assess the efficacy of decellularization, total DNAs were extracted and examined by electrophoresis. The result showed that no residual presence of cellular DNA in dpECM (FIG. 1A). Furthermore, it was confirmed that DNA content decreased from 6.262.31 g/mg dry weight in normal rat pancreas to 0.060.05 g/mg dry weight after decellularization (P<0.01) (FIG. 1B), indicating removal of 96% of the DNA from the tissues. By solubilizing in acetic acid with pepsin, a homogeneous and translucent dpECM gel solution was obtained. The biocompatibility of the dpECM gel was analyzed by live-and-dead cell assay. As shown in FIG. 1C, cells seeded on dpECM-coated surface revealed similar cell viability when compared to MG-coated surface. Notably, although cell culture plates coated with dpECM showed a concentration-dependent reduction in iPSCs attachment within 24 h after seeding (FIG. 8A), the cells cultured on dpECM-coated surface were able to reach 100% confluence during the 28-day differentiation protocol (data not shown). These results suggest that the decellularization process successfully removed pancreatic cellular components, the resulting dpECM solution exhibited excellent biocompatibility to support iPSCs in vitro. FIG. 1D shows live/dead cell dual-staining of iPSCs cultured on either MG or dpECM coated dishes for 24 h after seeding. Scale bars, 200 m.

[0158] The ECM prepared using this protocol preserve growth factors and tissue niches that are essential to islet development from hPSCs. To prepare rat dpECMs, rat pancreata were treated with hyper/hypotonic solutions designed to completely remove cells from pancreatic tissues. Pancreatic tissue samples turned white and translucent after washing with hyper/hypotonic solutions. Total DNAs were extracted and examined to assess efficiency of the decellularization. Residual cellular DNA was not detected by electrophoresis in these dpECMs (FIG. 1A). The DNA content decreased from 6.262.31 g/mg dry weight in normal rat pancreas to 0.060.05 g/mg dry weight after decellularization (p<0.01) (FIG. 1B), suggesting successful removal of 99% of DNAs from the tissues. A homogeneous and translucent dpECM gel solution was obtained by solubilizing the dpECMs in acetic acid in the presence of pepsin.

[0159] The biocompatibility of the dpECMs was characterized through a live/dead cell assay. As shown in FIGS. 1C and 1D, cells seeded on dpECM-coated substrates revealed a cell viability similar to those grown on the MG-coated substrates, albeit cells were less attached to dpECM coated substrates (FIG. 1C). Mixing dpECM with MG improved cell attachment (FIG. 1D). Further increase of dpECM in MG appeared to suppress cell attachment within 24 hours after seeding. Nevertheless, cells were able to reach 100% confluence after culturing several days (data not shown). These experimental results suggested that dpECMs support cell growth. The mixing of dpECM with MG seems to improve cell attachment during seeding.

[0160] During experiments, it was discovered that dpECM promotes remarkably the formation of cell clusters during iPSC pancreatic differentiation in 2D cultures. The growth factors and tissue niches preserved in dpECM during decellularization appear to offer tissue-inspired niches for pancreatic endocrine development. These effects were systematically characterized, to investigate whether cell clusters formed in the presence of dpECM are actually islet organoids that are physiologically functional.

[0161] To determine the instructive effect of dpECM on hPSC pancreatic differentiation, iPSCs were differentiated on MG/dpECM coated dishes using a four-stage differentiation protocol as shown in FIG. 2A, i.e. differentiating cells stepwise toward definitive endoderm (DE) (S1), posterior foregut (S2), pancreatic progenitor (S3), and hormone-expressing endocrine cells (S4). To interrogate whether the instructive effect of dpECM on iPSC pancreatic differentiation is dose-dependent, culture plates were coated with MG/dpECM mixtures at different ratios (4:1, 2:1, and 1:1). It is clear that the addition of dpECM to MG substrates enhanced directed differentiation of iPSCs toward pancreatic lineages. Key pancreatic endocrine marker genes, including PDX-1, insulin, Ptf1, Nkx6.1, and MafA, expressed at much higher levels in cells differentiated on MG/dpECM coated plates (FIG. 2B-E), suggesting an instructive effect of dpECMs on iPSC pancreatic lineage specification. It seemed that an increase in dpECM concentration in the MG/dpECM mixture stimulated higher degree of pancreatic lineage specification at Stage 3, a key stage of pancreatic endocrine development. The expression levels of PDX1 and NKX6.1 in cells differentiated on 2:1 mixed MG/dpECM coated plates were much higher than those differentiated on 4:1 mixed MG/dpECM coated plates. Further increase in dpECM concentrations does not appear to enhance iPSC pancreatic differentiation. Accordingly, a 2:1 mixed MG/dpECM matrix was used for the subsequent experiments. The flow cytometric analysis confirmed these observations. While SOX17.sup.+ DE cells reached 98% at the end of Stage 1 in all conditions, 84.1% cells expressed a high level of SOX17 when differentiated on MG/dpECM coated plates, whereas only 64.6% cells reached a high level of SOX17 expression in cells differentiated on MG coated plates (FIG. 8B).

[0162] Further investigation measured whether increase in the amount of MG coated on culture plates has a similar effect on iPSC pancreatic differentiation. As shown in FIG. 10, the expression of SOX17 and FOXA2, two DE marker genes, were at almost the same level in cells differentiated on MG or 2MG (doubling the amount of MG used for coating) coated plates. It is clear that the increase in coating matrix does not contribute to the enhancement of iPSC pancreatic lineage specification. The preferential factors entailed in dpECM promoted the iPSC pancreatic differentiation.

[0163] Starting from S2, significant amount of self-assembled clusters were constantly observed to appear in dpECM/MG-coated plates compared with that of MG-coated plates (FIG. 3A). Clusters larger than 100 m in diameter were considered as organoids and evaluated them by quantitative image analysis. The results showed that higher number of organoids were found on 2:1 mixed MG/dpECM substrates than 4:1 mixed MG/dpECM substrates, while MG only coating failed to generate organoids, albeit smaller condensed colonies ultimately appeared (FIG. 3B). To determine the size distribution of the organoids, the diameter of each identified organoid from S2 to S4 was monitored. The organoids formed on dpECM coated condition have large variance in size ranging from 100 m to 430 m, while no aggregate larger than 200 m was found on MG only coating (FIG. 3C). These experimental data confirm the presence of dpECM induces self-assembly of organoids during pancreatic differentiation. The same phenomenon has been confirmed as well during differentiation process from human ESC line H9 cells (data not shown).

[0164] The organoids formed on dpECM coated condition have large variance in size.

[0165] dpECM Enhances the Expression of Pancreatic Marker Genes During hPSC Islet Tissue Differentiation

[0166] FIGS. 2A-2E shows a schematic summary of the 4-stage, 21-day differentiation protocol and characterization of marker genes during the differentiation. FIG. 2A shows in vitro differentiation protocol of iPSCs to islet tissue. Basal media as well as key molecules used are shown for each stage of differentiation. FIGS. 2B-2E show the profile of marker gene expression of hPSC-derived cells during 51 to S4 of differentiation. Cells were 2D cultured on MG- or MG-/dpECM-coated substrates using indicated concentrations. Expression levels of mRNA were assessed for DE (FIG. 2B), posterior foregut (FIG. 2C), pancreatic progenitor (FIG. 2D), and -cell markers (FIG. 2E). Gene expression was relative to that in cells grown on MG-coated dishes. Results were from three or more experiments and shown as meanSD. *, p<0.05; **, p<0.01; ***, p<0.001, compared to MG group. Cells were 2D cultured on MG or MG/dpECM (M+d) coated plates as described in Methods. The MG-dpECM ratios (w:w) of prepared MG/dpECM mixtures were 4:1, 2:1, and 1:1, respectively.

[0167] To determine the regulative role of dpECM in pancreatic differentiation, iPSCs cultured on various concentrations of dpECM coating were differentiated by a four-stage differentiation protocol through definitive endoderm (S1), posterior foregut (S2), pancreatic progenitor (S3), and hormone-expressing endocrine cells (S4) (FIG. 2A). Cells grown on dpECM/MG-coated substrates demonstrated higher levels of gene expression for most of the key markers compared with cells cultured on MG-coated substrates (FIGS. 2B-E). Particularly, mRNAs of PDX-1, insulin, Ptf1, Nkx6.1, and MafA from cells grown in dpECM/MG-coated substrates showed multiple folds' enhancement at each stage of differentiation.

[0168] Differentiation efficiency was also assessed by flow cytometric analysis. Cells cultured on either coating condition tested possessed more than 98% of SOX17+ cells at the end of definitive endoderm (DE) stage (S1) (FIG. 9). There were 84.1% of DE cells possessing higher level of SOX17 expression from cells cultured on dpECM/MG-coated substrates as compared to 64.6% from cells cultured on MG-coated substrates (FIG. 9). This result is consistent with quantitative analysis of gene expression (FIG. 2B).

[0169] To rule out the possibility of the increased gene and protein expression in dpECM-containing groups was caused by higher amount of protein coated on culture plates compared with MG group, the expression of DE marker genes was evaluated in cells cultured on MG-coated with the same amount of dpECM/MG proteins as described in Materials and Methods (FIG. 10). Experimental results indicate that cells cultured on increased ECM amount of MG-coated substrates expressed similar levels of both SOX17 and FOXA2 genes compared with cells cultured on regular MG-coated plate. This experimental result suggests that, first of all, the amount of MG coating is sufficient for stem cell attachment, proliferation and differentiation. Further increase in the amount of MG for plate coating has no influence on stem cell attachment and differentiation. Second, the augmentation of efficiency of hPSC-derived endocrine differentiation is indeed contributed by dpECM.

[0170] dpECM Triggers the Self-Assembly of Islet-Like Organoids During hPSC Pancreatic Differentiation

[0171] FIGS. 3A-3C show microscopic examination of self-assembly of islet-like organoids at the end of stage 2, 3, and 4 of iPSC differentiation. Black bars, 1000 m; white bars, 200 m. Tiled images covering an area of 0.53 cm.sup.2 were randomly selected and analyzed by ImageJ software. Data are shown as meanSD (n=8). *, p<0.05; **, p<0.01; ***, p<0.001. FIG. 3A show micrographs of cell clusters formed on MG/dpECM coated plates. Black scale bars, 1,000 m; white scale bars, 200 m. FIG. 3B shows number of cell clusters formed on MG/dpECM coated plates. Tiled images covering an area of 0.53 cm2 were randomly selected and analyzed by ImageJ software. Data are shown as meanSD (n=8). *, p<0.05; **, p<0.01; ***, p<0.001. FIG. 3C show diameters of cell clusters formed on MG/dpECM coated plates. Gray bars indicate average diameter of aggregates.

[0172] It was observed that cells grown on dpECM/MG-coated substrates started forming aggregates from Stage 2 of differentiation and these aggregates grew to the end of the entire differentiation (FIGS. 3A-3C). Significant number of aggregates self-assembled in dpECM/MG-substrates compared with that of MG-coated substrates. Clusters larger than 100 m in diameter were considered as organoids and then quantitatively evaluated them by quantitative image analysis. The results showed that dpECM triggered e the formation of organoids in a dose-dependent manner, while MG only coating failed to generate organoids, albeit smaller condensed colonies ultimately appeared.

[0173] Organoids formed by self-organizing stem cells recapitulate complex cell-cell interactions during de novo generation of 3D islet-like clusters. As such, they harbor geometric constraints and environmental cues that are essential for islet neogenesis [Greggio, Chiara, Filippo De Franceschi, and Anne Grapin-Botton. Concise Reviews: In Vitro-Produced Pancreas Organogenesis Models in Three Dimensions: Self-Organization From Few Stem Cells or Progenitors. Stem Cells 33, no. 1 (2015): 8-14]. To determine the size distribution of the organoids, the diameter of each identified organoid was monitored from S2 to S4. The organoids formed with large variance in size, and with increasing numbers of large organoids (diameter >200 m) on dpECM coated condition, confirming that the presence of dpECM induces self-assembly of organoids during pancreatic differentiation.

[0174] iPSCs Differentiated on dpECM Exhibit Similar Cellular Composition to Pancreatic Islets

[0175] The organoids shown in FIGS. 3A-3C were hypothesized to be islet organoids having fetal status. To characterize the identity of these organoids formed on dpECM-coated substrates, the organoids at the end of S4 were investigated regarding expression of islet signature composition through immunofluorescence staining and flow cytometric analyses.

[0176] FIGS. 4A-4E show organoids generated from iPSCs grown on dpECM coated substrates showed similar composition to native islets. FIG. 4A shows representative immunofluorescent staining of S4 cells labeled for C-peptide (C-PEP, green) and glucagon (GCG; red). 4,6-diamidino-2-phenylindole (DAPI, blue) was used for counterstaining nuclei. Scale bars, 50 m. FIG. 4B shows representative immunofluorescent staining of S4 cells labeled for somatostatin (SST, green), pancreatic polypeptide (PPY; red), and DAPI. Scale bars, 50 m. (FIGS. 4C-4E) Representative flow cytometric results of S4 cells stained for C-PEP and GCG (FIG. 4C), SST (FIG. 4D), and PPY (FIG. 4E). Numbers in quadrants represent the percentage of total counted cells.

[0177] FIGS. 4F-4L show cell compositions of S4 cells during iPSC pancreatic differentiation on MG/dpECM coated plates. S4 cells were immunofluorescently labeled for (FIG. 4F) C-peptide (C-PEP, green) and glucagon (GCG; red); (FIG. 4G) somatostatin (SST, green) and pancreatic polypeptide (PPY; red). 4,6-diamidino-2-phenylindole (DAPI, blue) was used for counterstaining cell nuclei. Scale bars, 50 m. (FIGS. 4H-4I) Representative flow cytometric analysis of C-PEP (FIG. 4H), GCG (FIG. 4I), SST (FIG. 4J), and PPY (FIG. 4K) expressing cells in S4 cells. SSC: side scatter. (FIG. 4L) Representative flow cytometric analysis of S4 cells dual-stained for C-PEP and GCG. Numbers in quadrants represent the percentage of total counted cells.

[0178] As revealed by FIG. 4A, more C-peptide (C-PEP)-positive cells are detected from cells grown on dpECM/MG-coated substrates compared to those on MG-coated surface. Interestingly, the C-PEP+ cells identified on high dpECM/MG coating showed a clustered pattern, while they were scattered randomly through the entire cells cultured on MG coating (FIG. 4A). Remarkably, although C-PEP and glucagon (GCG)-dual positive cells were identified in all culture conditions (FIG. 4A), the percentage of polyhormonal cells decreased in a dpECM dose-dependent manner., In contrast, C-PEP+/GCG-cells significantly increased from approximately 10% on MG to 35% on dpECM substrates (FIG. 4C), suggesting dpECM helps maturation of islet tissues during hPSC-derived islet development. Notably, the overall C-PEP+ cell population was higher than 60% (FIG. 4C), indicating an effective development of islet-like organoids.

[0179] Similar to INS+ and GCG+ cells, the somatostatin (SST)-positive and pancreatic polypeptide (PPY)-positive population showed a more notable increment on dpECM coating condition, although they constituted a minority in differentiated cells (FIG. 4B). In contrast to the other three cell types, PPY+ cells were hardly detectable on MG coating condition. Flow cytometry analysis enumerated that on dpECM/MG-coated substrates higher proportion of SST+ cells and PPY+ cells, which contributed to 9.35% and 1.01% of the total cell population, respectively, could be achieved (FIGS. 4D and 4E). These data illustrated a comparative ratio to that of human islet, in which 50-70% of the cells are -cells, 20-30% are -cells, 10% are cells, and <5% are PP cells [Shih H P, Wang A, Sander M. Pancreas organogenesis: from lineage determination to morphogenesis. Annu Rev Cell Dev Biol 29, 81-105 (2013).; Jennings R E, et al. Development of the human pancreas from foregut to endocrine commitment. Diabetes 62, 3514-3522 (2013).; Rose S D, Swift G H, Peyton M J, Hammer R E, MacDonald RJ. The role of PTF1-P48 in pancreatic acinar gene expression. J Biol Chem 276, 44018-44026 (2001).]. Taken together, the hPSCs differentiated on dpECM niches demonstrated similar cellular arrangement to an islet, suggesting dpECM provides necessary microenvironmental context that is required for the development of islet organoids. It implies that dpECM niches permit self-organizing an islet tissue during hPSC differentiation even in a 2D culture condition.

[0180] To determine whether cell clusters formed from iPSCs in the presence of dpECM are islets or islet organoids, cell compositions and architectures of cells collected were characterized at end of S4. The expression of pancreatic endocrine hormones such as c-peptide (C-PEP, a peptide released from the pancreatic beta-cells during cleavage of insulin from proinsulin), glucagon (GCG), somatostatin (SST), and pancreatic polypeptide (PPY) in cell clusters were detected using immunostaining.

[0181] As shown in FIG. 4F-4G, GCG, SST, and PPY were expressed in small subsets of cells that were mixed with insulin-secreting cells. Flow cytometry data showed that more than 60% of S4 cells expressed C-PEP among all differentiation conditions (FIG. 4H), where GCG.sup.+ cells decreased significantly from 53.4312.40% of cells differentiated on MG to 39.918.61% and 31.366.31% of cells differentiated on 4:1 and 2:1 mixed MG/dpECM substrates, respectively (FIG. 4I). SST.sup.+ cells were increased from 4.850.81% on MG to 5.812.39% on 4:1 mixed MG/dpECM and further upraised to 10.131.10% on 2:1 mixed MG/dpECM substrate (FIG. 4J). Similarly, the population of PPY.sup.+ cells increased from 0.500.07% on MG to 0.720.28% and 1.060.07% on 4:1 and 2:1 mixed MG/dpECM substrates, respectively (FIG. 4K). These results suggested cells differentiated on 2:1 mixed MG/dpECM substrates showed similar cellular arrangement to islet, which possess comparative cellular proportions to that of human islet, in which 50-70% of the cells are -cells, 20-30% are -cells, 10% are cells, and <5% are PP cells [Stefan Y, Orci L, Malaisse-Lagae F, Perrelet A, Patel Y, Unger R H. Quantitation of endocrine cell content in the pancreas of nondiabetic and diabetic humans. Diabetes 31, 694-700 (1982).; Ichii H, et al. A novel method for the assessment of cellular composition and beta-cell viability in human islet preparations. Am J Transplant 5, 1635-1645 (2005).; Brissova M, et al. Assessment of human pancreatic islet architecture and composition by laser scanning confocal microscopy. J Histochem Cytochem 53, 1087-1097 (2005)].

[0182] Nevertheless, some insulin-secreting cells co-expressed glucagon as shown in FIG. 4L, 53.8013.01% of cells differentiated on MG coated plates co-expressed C-PEP and GCG, suggesting their low maturity. In contrast, the percentage of C-PEP and GCG polyhormonal cells was reduced in cell clusters formed on MG/dpECM coated plates: 40.409.48% of cells cultured on 4:1 mixed MG/dpECM substrates, whereas only 31.655.87% of cells cultured on 2:1 mixed MG/dpECM substrate. Together, these results suggest using dpECM as a culture substrate can improve islet organogenesis and maturation during in vitro differentiation of hPSCs.

[0183] To further characterize the iPSCs-derived organoids under MG/dpECM substrates, co-expression of insulin (INS) and NKX6.1 was detected in cells collected at the end of S4 by flow cytometric analysis. About 29.0%, 26.9% and 21.3% of cells co-expressed INS and NKX6.1 in cells differentiated on 2:1 and 4:1 mixed MG/dpECM, and MG substrates, respectively (FIG. 4F). The Insulin-secreting cells were 40.4%, 41.4% and 37.7% in cells differentiated on 2:1 and 4:1 mixed MG/dpECM, and MG substrates, respectively. These experimental results supported the hypothesis that dpECM encourages self-assembly of cell clusters that are similar to pancreatic endocrine islets. The co-staining of cells with antibodies against PDX-1 and C-PEP revealed that almost all of the cells were PDX-1.sup.+, while PDX-1.sup.+/C-PEP.sup.+ cells were mostly found in cells differentiated on MG/dpECM substrates (FIG. 5B).

[0184] To determine whether these cell clusters are capable of secreting insulin in response to glucose stimulation, glucose-stimulated insulin secretion (GSIS) analysis was carried out, which showed that either with or without the presence of dpECM, the S4 cells did not show glucose-responsive insulin secretion. However, the intracellular insulin could be purged out when depolarized by KCl solution, suggesting these cells had acquired exocytosis capability (data not shown).

[0185] Clearly, these islet-like organoids developed from iPSCs on MG/dpECM coated substrates were immature. The detection of significant number of multi-hormone expressing endocrine cells in these cell clusters suggested that their cell compositions and structure are very similar to fetal islets.

[0186] As demonstrated by optical sections through the 3D cultured organoids, the core of the mouse islets was almost exclusively composed of C-PEP.sup.+ and GCG.sup.+ cells were dispersed throughout the organoids. Flow cytometry revealed that 3D culture further increased C-PEP+ cells (FIG. 7A) with sharp decline of the GCG+ population (FIG. 7B) as compared to S4 cells (FIGS. 4H and 4I). Cells transferred from 2:1 mixed MG/dpECM substrate produced 9.610.56% of SST.sup.+ cells (FIG. 7C) and 1.190.21% PPY.sup.+ cells (FIG. 7D), similar to the populations observed in S4 cells (FIGS. 4E and F) and adult human islets. Moreover, the population of CPEP.sup.+/GCG.sup.+ polyhormonal cells from 2:1 mixed MG/dpECM substrate decreased significantly in S5 organoids when compared to S4 cells (FIGS. 7E and 4G), indicating further maturation of organoids without losing physiological cellularity of non- cells at S5. Taken together, these demonstrate for the first time, that dpECM promotes the differentiation and maturation of islet tissue development from both iPSCs and ESCs.

[0187] dpECM Facilitates Assembly of Islet Cellularity During hPSC Pancreatic Differentiation

[0188] FIGS. 5A-5C show characterization of S4 cells. (FIG. 5A) Flow cytometric analysis of insulin (INS) and Nkx6.1 expression in S4 cells. Numbers in quadrants represent the percentage of total counted cells, illustrating the typical population of both NKX6.1+ and insulin+ cells generated at S4. (FIGS. 5B and 5C) Representative immunofluorescent staining of S4 cells cultured on various conditions. (FIG. 5B) pancreatic and duodenal homeobox 1 (PDX-1, green) and C-peptide (C-PEP, red). (FIG. 5C) MAF bZIP transcription factor A (MAFA, green) and glucagon (GCG, red). 4,6-diamidino-2-phenylindole (DAPI, blue) was used for counterstaining nuclei. Bars, 50 m.

[0189] To further characterize the iPSCs-derived organoids and other endocrine cells generated by the unique microenvironment of dpECM, flow cytometry was performed for detecting insulin (INS) and NKX6.1 expression in S4 cells. The results showed that cells cultured on dpECM possessed higher proportion of INS+ cells as well as INS+/NKX6.1+ cells compared to cells cultured on MG-coated substrates (FIG. 5A). This is consistent with the previous finding that dpECM allows generating more C-PEP+/GCG-cells, rather than C-PEP+/GCG+ cells (FIG. 4C). Co-staining of the PDX-1 and C-PEP revealed that almost all of the cells were PDX-1+ under each culture condition, while PDX-1+/C-PEP+ cells were mostly found from cells grown on dpECM-coated niches (FIG. 5B). Furthermore, immunostaining result also confirmed the expression of MAF bZIP transcription factor A (MAFA), a critical functional marker expressed in adult -cells [Pan F C, Brissova M. Pancreas development in humans. Curr Opin Endocrinol Diabetes Obes 21, 77-82 (2014).; Kim A, Miller K, Jo J, Kilimnik G, Wojcik P, Hara M. Islet architecture: A comparative study. Islets 1, 129-136 (2009).] in S4 cells cultured on dpECM-containing coating condition (FIG. 5C).

[0190] Next, to determine whether the iPSC-derived cells are able to secrete insulin in response to glucose stimulation, glucose-stimulated insulin secretion (GSIS) was carried out analysis. ELISA results unveiled that either with or without the presence of dpECM, the S4 cells did not show glucose-responsiveness. However, the intracellular insulin was purged out when depolarized by KCl solution, suggesting S4 cells had acquired exocytosis capability (data not shown). Notably, there was a dpECM dose-dependent increment in the level of insulin secretion when dpECM was introduced in coating plates, which implicates the promotive role of dpECM in -cell lineage decision.

[0191] dpECM Augments Maturation of Islet Organoids During hPSC Pancreatic Differentiation in 3D Cultures

[0192] In order to generate functional islet tissues that are glucose responsive insulin-secretion tissues, Stage 5 was introduced into the differentiation protocol based on the strategy reported by other group [Bosco D, et al. Unique arrangement of alpha- and beta-cells in human islets of Langerhans. Diabetes 59, 1202-1210 (2010). with slight modifications. 2D culture was compared with suspension-based 3D aggregate culture, the latter was achieved by transferring differentiated cells into ultra-low attachment plates for S4 and S5 differentiation (FIG. 6A). First, the existence of insulin secretion cells in 3D cultured organoids was confirmed by DTZ staining. DTZ selectively chelates zinc in the insulin-containing secretory granules existed in insulin-producing cells [Cabrera O, Berman D M, Kenyon N S, Ricordi C, Berggren P O, Caicedo A. The unique cytoarchitecture of human pancreatic islets has implications for islet cell function. Proc Natl Acad Sci USA 103, 2334-2339 (2006).]. Most of the organoids appeared crimson red, while undifferentiated hPSCs were not stained (FIG. 6B).

[0193] Further investigation by GSIS assay demonstrated that the S5 cells under 2D culture did not show glucose-responsiveness in either MG or dpECM groups (FIG. 6C), although more organoids were formed from cells cultured on dpECM coating condition (FIG. 11). In sharp contrast, considerable improvement of insulin secretion was found in cell aggregates comparing with 2D cultured cells (FIG. 6D).

[0194] As shown in FIG. 6D, aggregation differentiation condition at S4 and S5 permits insulin secretion correlated with glucose levels at all the substrate coating condition tested. Cells from MG group secreted insulin at low and high concentrations were 0.770.34 IU/g DNA and 1.400.61 IU/g DNA, respectively. In addition, with the stimulus of KCl the depolarized cells released 1.810.46 IU/g DNA. A more remarkable difference was found in cells from two dpECM groups showing 2.020.71 (M+d 25%) and 2.900.64 (M+d 50%) fold more insulin secreted when respond from low to high glucose concentrations (FIG. 6D). Especially, the overall amount of insulin released from dpECM-treated cells was 2 times more than that from control group, which further validated previous findings that dpECM enhances insulin expression and maturation (FIG. 2E and FIG. 4C).

[0195] Having characterized the unique role of the dpECM played in human iPSC differentiation towards islet tissue development, the dpECM-coating and differentiation procedures developed in were investigated to see if they are robust to other hPSC lines. hESC line H9 has been widely studied and reported by many research groups including us [Nair G, Hebrok M. Islet formation in mice and men: lessons for the generation of functional insulin-producing beta-cells from human pluripotent stem cells. Curr Opin Genet Dev 32, 171-180 (2015).; Seymour P A, Sander M. Historical perspective: beginnings of the beta-cell: current perspectives in beta-cell development. Diabetes 60, 364-376 (2011).; Steiner D J, Kim A, Miller K, Hara M. Pancreatic islet plasticity: interspecies comparison of islet architecture and composition. Islets 2, 135-145 (2010)]. Thus, H9 cells were induced into differentiation toward endocrine tissue using the same protocol shown in FIG. 6A. hESC-derived cells at the end of five-stage differentiation also demonstrated sugar level responsive insulin secretion. Likewise, cells after exposed on dpECM-coated environment and differentiated in 3D condition at later stage of differentiation, are able to produce more insulin compared to MG-alone environment (FIG. 6E). Taken together, dpECM promotes the differentiation and maturation of islet tissue development from both human iPSCs and hESCs.

[0196] FIGS. 6A-6E show that dpECM promotes insulin secretion in response to glucose level at the end of differentiation in 3D cultures. (FIG. 6A) Schematic outlining differentiation strategy for S5. For S4 and S5, 2D differentiated cells were either continuously differentiated under 2D condition (2D culture) or transferred into an ultra-low attachment plate for aggregate formation (3D culture). (FIG. 6B) DTZ staining of S5 aggregates. Undifferentiated iPSCs cultured in ultra-low attachment plate for 24 hrs were used as a control. (FIGS. 6C and 6D) Higher capacity of insulin secretion from cells grown on dpECM coated substrates during S1-S4 upon glucose challenges. Glucose-stimulated insulin secretion (GSIS) analysis was performed using cells cultured in 2D (FIG. 6C) or 3D (FIG. 6D) at S5 (n=4). Cells were challenged with 2 mM, 20 mM glucose, and 30 mM KCl with 2 mM glucose for 30 min at each step. (FIG. 6E) Insulin secretion from hESCs-derived cells at S5 (n=3). Data are shown as meanSD. *, p<0.05; **, p<0.01.

[0197] FIG. 8A shows iPSCs (IMR90) cultured on MG- or dpECM-coated substrates. Twenty-four hours after seeding, phase contrast images were captured using a Nikon microscope. Bars, 200 m.

[0198] FIG. 9 shows flow cytometry analysis of SOX17 expression in DE stage. SOX17-positive cells were gated using isotype control (Black square). Red square indicates the percentage of cells expressing high level of SOX17.

[0199] FIG. 10 shows TaqMan qPCR analysis of SOX17 and FOXA2 expression in cells at the end of DE stage. Cells cultured on MG-coated (MG) or two-fold increased MG ECM coated substrates (2MG). Expression levels were normalized to that in cells cultured MG-coated dish. Values are shown as meanSD (n=3). NS: not statistically significant.

[0200] FIG. 11 shows microscopic examination of cellular aggregates at day 7 of S5 under 2D culture. Black bars, 1000 m; white bars, 200 m. Tiled images covering an area of 0.53 cm2 were randomly selected and analyzed by ImageJ software. Data are shown as meanSD (n=8). *, p<0.05.

[0201] dpECM Promotes Intra-Organoid Vascularity in the Combination of 2D and 3D Culture

[0202] The important role of vascularization in differentiation and development of pancreas have been reported recently [Brissova M, Shostak A, Shiota M, Wiebe P O, Poffenberger G, Kantz J, et al. Pancreatic islet production of vascular endothelial growth factor-A is essential for islet vascularization, revascularization, and function. Diabetes 2006, 55(11): 2974-2985.; Ballian N, Brunicardi F C. Islet vasculature as a regulator of endocrine pancreas function. World J Surg 2007, 31(4): 705-714.]. During the process to characterize the ILOs generated in this work, lumen structures in dpECM-treated S5 samples were observed. Therefore, the effect of dpECM treatment on vascularization during islet organoid formation was investigated. The intra-organoid vasculature in S5 cells was investigated by H&E staining (FIG. 13A). Many lumens were observed in dpECM treated groups. Morphometric analysis of organoids from M+d 4:1 group showed 7-fold higher capillary density compared to MG group, and the density further increased to 10-fold in M+d 2:1 group (FIG. 13B). Furthermore, there was an increased number of CD31+ endothelial cells (ECs) in dpECM-treated organoids (FIG. 13C). Interestingly, some CD31+ cells located closely to CP+ cells (FIG. 13D, arrowheads), suggesting a physical interaction that is beneficial for the production and secretion of insulin. The flow cytometry results showed that M+d 2:1 group contained 4.431.23% of CD31+ cells, which is significantly higher than 1.400.09% in MG group (p<0.05) (FIG. 13E). Notably, pericyte marker neuron-glial antigen 2 (NG2) was also detectable in M+d 4:1 and M+d 2:1 treated organoids (2.200.16% and 1.420.12%, respectively) as compared to an imperceptible population in MG group (0.470.43%). These experimental results reveal that dpECM also induces vasculogenesis in addition to islet organogenesis during iPSC islet lineage progression.

[0203] While microcirculation is a critical requirement for survival and proper function of transplanted grafts, a successful prevascularization of organoid may reduce posttransplantation-mediated ischemia, thus improve the outcome [Brissova M, Powers A C. Revascularization of transplanted islets: can it be improved? Diabetes 2008, 57(9): 2269-2271]. A significant amount of capillaries formed in dpECM-treated organoids were observed (FIGS. 13A and 13B), which is accordance with the previously reported inductive role of decellularized ECM in vascularization [Moore M C, Pandolfi V, McFetridge P S. Novel human-derived extracellular matrix induces in vitro and in vivo vascularization and inhibits fibrosis. Biomaterials 2015, 49: 37-46.; Fercana G R, Yerneni S, Billaud M, Hill J C, VanRyzin P, Richards T D, et al. Perivascular extracellular matrix hydrogels mimic native matrix microarchitecture and promote angiogenesis via basic fibroblast growth factor. Biomaterials 2017, 123: 142-154]. dpECM treatment during hPSC pancreatic differentiation generated not only CD31+ endothelial cells, but also NG2+ pericytes (FIG. 13E), which are involved in supporting EC migration and morphogenesis during the early stages of neovascularization [Fukushi J, Makagiansar I T, Stallcup W B. NG2 proteoglycan promotes endothelial cell motility and angiogenesis via engagement of galectin-3 and alpha3beta1 integrin. Molecular biology of the cell 2004, 15(8): 3580-3590]. Intraislet ECs, apart from their conductive role in angiogenesis, also interact with -cells and enhance insulin production and secretion [Johansson A, Lau J, Sandberg M, Borg L A, Magnusson P U, Carlsson P O. Endothelial cell signalling supports pancreatic beta cell function in the rat. Diabetologia 2009, 52(11): 2385-2394.] by providing growth factors, basement membrane components, or direct-contact signaling [Penko D, Rojas-Canales D, Mohanasundaram D, Peiris H S, Sun W Y, Drogemuller C J, et al. Endothelial progenitor cells enhance islet engraftment, influence beta-cell function, and modulate islet connexin 36 expression. Cell Transplant 2015, 24(1): 37-48]. In the in vitro generated organoids, our data indicate that dpECM treatment leads to increased number of ECs along with -cells (FIGS. 13C-13E), which may partially contribute to the improved insulin production and secretory capacity of -cells in dpECM groups.

[0204] Taken together, the capability of dpECM for representing the complex microenvironmental signals that facilitate pancreatic-lineage decision of hPSCs. Utilizing dpECM as an in vitro cell culture substrate, demonstrate an approach for generating islets from hPSCs. dpECM plays an instructive role in cell fate specification in early islet development, which gives rise to vascularized islet-like organoid with multicellular composition.

Conclusion

[0205] Taken together, the present technology provides an efficient and effective approach to create niches that permit self-assembly of islet organoids during stem cell differentiation. These organoids showed similar cellular composition to native pancreatic islets. The organoids consist of -cells, -cells, cells, and PP cells. They express islet signature markers insulin, PDX-1, C-peptide, MafA, glucagon, somatostatin, and pancreatic polypeptide. Furthermore, these cells secrete more insulin in response to glucose level compared to a traditional matrix substrate (Matrigel). Remarkably, the dpECM developed in this study facilitates generating more C-peptide+/glucagon cells rather than C-peptide+/glucagon+ cells. These findings provide the first evidence of a promotive role of the materials developed in recapitulating functional pancreatic islets during induced hPSC differentiation.

[0206] The microenvironments that allow self-organization of islet organoids have not been previously elucidated. The technology improves understanding of -cell maturation and islet organogenesis during hPSC pancreatic differentiation. It also provides an improved system for generating mature islet organoids for islet transplantation and/or for use in drug screening and pathological studies, leading to a cure to diabetes.

Example 2

[0207] The feasibility of generating islet-like clusters from mouse embryonic stem cells (mESCs) within a collagen scaffold is demonstrated above. These cell clusters consisted of , , and cells and exhibited a characteristic mouse islet architecture that has a (3 cell core surrounded by and cells. The clusters were capable of KATP channel dependent insulin secretion upon glucose challenge. No PP cells were detected in these cell clusters, distinct from adult islets.

[0208] Building upon this mESC work, full islet organoids (consisting of four subtypes of pancreatic endocrine cells) from human embryonic stem cells (hESCs) were developed within a biomimetic scaffold. The cytostructural analysis of these organoids revealed a typical architecture of human adult islets, comprising , , , and PP cells. Both cells and non- cells were mixed to form organoids that secrete insulin and C-peptide in response to glucose challenges. The insulin secretory granules were detected in these organoids, indicating the degree of maturation.

[0209] When working on human embryonic stem cells (hESCs), collagen scaffolds become weaker and partially collapse after 10-15 days of differentiation. Efforts to overcome this issue included mixing collagen with Matrigel during scaffolding. The incorporation of Matrigel in biomimetic collagen scaffolds improved not only mechanical strength, but also hESC definitive endoderm (DE) differentiation, as indicated by elevated expression of DE markers such as Sox17, Foxa2, and CXCR4.

[0210] The mechanical strength of collagen scaffolds was also enhanced by treating or mixing collagen with other chemicals such as polyethylene (glycol) diacrylate (PEGDA) to enhance their mechanical properties during crosslinking. By selecting different molecular weight and adjusting the ratio of PEGDA during treatment, the stiffness of the collagen scaffolds may be adjusted to be close to that of human pancreas.

[0211] These scaffolds are believed to be superior for islet development due to their improved mechanical properties. The collagen scaffolds are penetrated with-PEGDA to enhance the stiffness and mechanical stability of the scaffolds for islet organoid developed from iPSCs. PEGDA is a polymer approved by FDA for various clinical applications. The stiffness of the collagen scaffolds is finely tuned by treating with different molecular weights of PEGDA, as demonstrated in FIGS. 12A-12C, which shows fine tuning of mechanical properties of collagen scaffolds by treating with different molecular weights of PEGDA. The shear storage modulus (FIG. 12A), the shear loss modulus (FIG. 12B), and the Young's modulus (FIG. 12C) of the PEGDA treated collagen scaffolds were determined using a rheometer. All experiments were performed in triplicate. Error bars indicate standard deviation. *, p<0.02.

[0212] A stiffness of 7% 2 kDa PEGDA treated collagen scaffolds is 2.79 kPa, whereas the stiffness of human pancreases is in a range from 1.15 to 2.09 kPa.sup.71. Accordingly, these scaffolds offer better mechanical cues needed for islet development from iPSCs.

[0213] To treat the collagen scaffolds with PEGDA, the cell-laden scaffolds prepared as described above were immersed in a 7% 2 KDa PEGDA solution in the presence of a photoinitiator 2-Hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone (Sigma-Aldrich) at 0.3 or 0.15 w/v % for 45 min, followed by incubating in a 5% CO.sub.2 incubator at 37 C. for 24 hr. The scaffolds are rinsed with PBS twice and placed in a culture medium under a long wavelength UV lamp (6 Watt, 365 nm) for 6 min to crosslink collagen-PEGDA networks. After crosslinking, the scaffolds are ready for islet development as described above.

[0214] While it has been reported that a low-intensity long wavelength UV and a relatively short exposure time (365 nm, between 220 min) does not alter gene expression profiles of human MSCs, its effect on iPSCs remains unknown. Accordingly, other photoinitiators and light sources for crosslinking may be used, for example eosin Y photosensitizer enables PEGDA crosslinking under a visible light, which is more biocompatible to stem cells. Several iPSC lines including IMR90 and DF4 (WiCell) may be used.

REFERENCES

[0215] Each of which is expressly incorporated herein by reference in its entirety: [0216] Agmon, Gillie, and Karen L. Christman. Controlling stem cell behavior with decellularized extracellular matrix scaffolds. Current Opinion in Solid State and Materials Science 20, no. 4:193-201 (2016). [0217] Alfonso, Andrea L., Jung Lee, E. Alice, M. S. Hitscherich, and M. S. Ma. Decellularized Pancreatic Tissue. Journal of Health Disparities Research and Practice 9, no. 5:60 (2016). [0218] An D, Ji Y, Chiu A, Lu Y C, Song W, Zhai L, Qi L, Luo D and Ma M. Developing robust, hydrogel-based, nanofiber-enabled encapsulation devices (NEEDs) for cell therapies. Biomaterials; 37:40-8 (2015). [0219] Badylak, Stephen F., Doris Taylor, and Korkut Uygun. Whole-organ tissue engineering: decellularization and recellularization of three-dimensional matrix scaffolds. Annual review of biomedical engineering 13:27-53 (2011). [0220] Bahney C S, Lujan T J, Hsu C W, Bottlang M, West J L and Johnstone B. Visible light photoinitiation of mesenchymal stem cell-laden bioresponsive hydrogels. Eur Cell Mater.; 22:43-55 (2011). [0221] Ballian N, Brunicardi F C. Islet vasculature as a regulator of endocrine pancreas function. World J Surg 2007, 31(4): 705-714. [0222] Barabaschi G D, Manoharan V, Li Q and Bertassoni L E. Engineering Pre-vascularized Scaffolds for Bone Regeneration. Adv Exp Med Biol.; 881:79-94 (2015). [0223] Basford C L, et al. The functional and molecular characterisation of human embryonic stem cell-derived insulin-positive cells compared with adult pancreatic beta cells. Diabetologia 55, 358-371 (2012). [0224] Benninger R K, Piston D W. Cellular communication and heterogeneity in pancreatic islet insulin secretion dynamics. Trends Endocrinol Metab 25, 399-406 (2014). [0225] Bersini S and Moretti M. 3D functional and perfusable microvascular networks for organotypic microfluidic models. J Mater Sci Mater Med.; 26:180 (2015). [0226] Bhatia S N, Ingber D E. Microfluidic organs-on-chips. Nat Biotechnol 32, 760-772 (2014). [0227] Bi H, Ming L, Cheng R, Luo H, Zhang Y, Jin Y. Liver extracellular matrix promotes B M-MSCs hepatic differentiation and reversal of liver fibrosis through activation of integrin pathway. J Tissue Eng Regen Med 2016. [0228] Bi H, Ye K and Jin S (2016). Engineering tissue substrates for generation of islet-like organoids from human pluripotent stem cell differentiation. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress, Montreal, Canada, 17 May-22 May 2016. doi: 10.3389/conf.FBIOE.2016.01.02293 (Published Online: 30 Mar. 2016). www.frontiersin.org/10.3389/conf.FBIOE.2016.01.02293/2893/10th_World_Biomaterials_Congress/all_events/event_abstract. [0229] Bogorad M I and Searson P C. Real-time imaging and quantitative analysis of doxorubicin transport in a perfusable microvessel platform. Integr Biol (Camb).; 8:976-84 (2016). [0230] Bogorad M I, DeStefano J, Karlsson J, Wong A D, Gerecht S and Searson P C. Review: in vitro microvessel models. Lab Chip; 15:4242-55 (2015). [0231] Bosco D, et al. Unique arrangement of alpha- and beta-cells in human islets of Langerhans. Diabetes 59, 1202-1210 (2010). [0232] Brereton H C, et al. Homotypic cell contact enhances insulin but not glucagon secretion. Biochem Bioph Res Co 344, 995-1000 (2006). [0233] Brissova M and Powers A C. Revascularization of transplanted islets: can it be improved? Diabetes; 57:2269-71 (2008). [0234] Brissova M, Aamodt K, Brahmachary P, Prasad N, Hong J Y, Dai C, Mellati M, Shostak A, Poffenberger G, Aramandla R, Levy S E and Powers A C. Islet microenvironment, modulated by vascular endothelial growth factor-A signaling, promotes beta cell regeneration. Cell Metab.; 19:498-511 (2014). [0235] Brissova M, Shostak A, Fligner C L, Revetta F L, Washington M K, Powers A C and Hull R L. Human Islets Have Fewer Blood Vessels than Mouse Islets and the Density of Islet Vascular Structures Is Increased in Type 2 Diabetes. J Histochem Cytochem.; 63:637-45 (2015). [0236] Brissova M, Shostak A, Shiota M, Wiebe P O, Poffenberger G, Kantz J, Chen Z, Carr C, Jerome W G, Chen J, Baldwin H S, Nicholson W, Bader D M, Jetton T, Gannon M and Powers A C. Pancreatic islet production of vascular endothelial growth factora is essential for islet vascularization, revascularization, and function. Diabetes; 55:2974-85 (2006). [0237] Brissova, M, M. J. Fowler, W. E. Nicholson, A. Chu, B. Hirshberg, D. M. Harlan, A. C. Powers, Assessment of human pancreatic islet architecture and composition by laser scanning confocal microscopy, J Histochem Cytochem 53(9):1087-97 (2005). [0238] Broutier L, et al. Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation. Nature protocols 11, 1724-1743 (2016). [0239] Bruin J E, et al. Characterization of polyhormonal insulin-producing cells derived in vitro from human embryonic stem cells. Stem cell research 12, 194-208 (2014). [0240] Bruin J E, Rezania A, Xu J, Narayan K, Fox J K, O'Neil J J and Kieffer T J. Maturation and function of human embryonic stem cell-derived pancreatic progenitors in macroencapsulation devices following transplant into mice. Diabetologia; 56:1987-98 (2013). [0241] Cabrera O, Berman D M, Kenyon N S, Ricordi C, Berggren P O, Caicedo A. The unique cytoarchitecture of human pancreatic islets has implications for islet cell function. Proc Natl Acad Sci USA 103, 2334-2339 (2006). [0242] Carvalho C P, Barbosa H C, Britan A, Santos-Silva J C, Boschero A C, Meda P and Collares-Buzato C B. Beta cell coupling and connexin expression change during the functional maturation of rat pancreatic islets. Diabetologia; 53:1428-37 (2010). [0243] Chao K C, Chao K F, Fu Y S, Liu S H. Islet-like clusters derived from mesenchymal stem cells in Wharton's Jelly of the human umbilical cord for transplantation to control type 1 diabetes. PLoS One 3, e1451 (2008). [0244] Chen S, Borowiak M, Fox J L, Maehr R, Osafune K, Davidow L, Lam K, Peng L F, Schreiber S L, Rubin L L and Melton D. A small molecule that directs differentiation of human ESCs into the pancreatic lineage. Nat Chem Biol.; 5:258-65 (2009). [0245] Chen, Wenhui, Yasuhiko Tabata, and Yen Wah Tong. Fabricating tissue engineering scaffolds for simultaneous cell growth and drug delivery. Current pharmaceutical design 16, no. 21:2388-2394 (2010). [0246] Cheng, Jennifer Y C, Michael Raghunath, John Whitelock, and Laura Poole-Warren. Matrix components and scaffolds for sustained islet function. Tissue Engineering Part B: Reviews 17, no. 4:235-247 (2011). [0247] Coronel, Maria M., and Cherie L. Stabler. Engineering a local microenvironment for pancreatic islet replacement. Current opinion in biotechnology 24, no. 5:900-908 (2013). [0248] Crapo, Peter M., Thomas W. Gilbert, and Stephen F. Badylak. An overview of tissue and whole organ decellularization processes. Biomaterials 32, no. 12:3233-3243 (2011). [0249] Cross V L, Zheng Y, Won Choi N, Verbridge S S, Sutermaster B A, Bonassar L J, Fischbach C and Stroock A D. Dense type I collagen matrices that support cellular remodeling and microfabrication for studies of tumor angiogenesis and vasculogenesis in vitro. Biomaterials; 31:8596-607 (2010). [0250] D'Amour K A, Agulnick A D, Eliazer S, Kelly O G, Kroon E and Baetge E E. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat Biotechnol.; 23:1534-41 (2005). [0251] D'Amour K A, Bang A G, Eliazer S, Kelly O G, Agulnick A D, Smart N G, Moorman M A, Kroon E, Carpenter M K and Baetge E E. Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat Biotechnol.; 24:1392-401 (2006). [0252] Davis, Nicolynn E., Liese N. Beenken-Rothkopf, Annie Mirsoian, Nikola Kojic, David L. Kaplan, Annelise E. Barron, and Magali J. Fontaine. Enhanced function of pancreatic islets co-encapsulated with ECM proteins and mesenchymal stromal cells in a silk hydrogel. Biomaterials 33, no. 28:6691-6697 (2012). [0253] De Carlo, E., S. Baiguera, M. T. Conconi, S. Vigolo, C. Grandi, S. Lora, C. Martini et al. Pancreatic acellular matrix supports islet survival and function in a synthetic tubular device: in vitro and in vivo studies. International journal of molecular medicine 25, no. 2:195-202 (2010). [0254] El-Gohary Y, Sims-Lucas S, Lath N, Tulachan S, Guo P, Xiao X, Welsh C, Paredes J, Wiersch J, Prasadan K, Shiota C and Gittes G K. Three-dimensional analysis of the islet vasculature. Anat Rec (Hoboken).; 295:1473-81 (2012). [0255] Faulk D M, et al. The effect of detergents on the basement membrane complex of a biologic scaffold material. Acta Biomater 10, 183-193 (2014). [0256] Forster N A, Penington A J, Hardikar A A, Palmer J A, Hussey A, Tai J, Morrison W A and Feeney S J. A prevascularized tissue engineering chamber supports growth and function of islets and progenitor cells in diabetic mice. Islets; 3:271-83 (2011). [0257] Fotino N, Fotino C, Pileggi A. Re-engineering islet cell transplantation. Pharmacol Res 98, 76-85 (2015). [0258] Fu J, Wiraja C, Muhammad H B, Xu C and Wang D A. Improvement of endothelial progenitor outgrowth cell (EPOC)-mediated vascularization in gelatin-based hydrogels through pore size manipulation. Acta Biomater. (2017). [0259] Fu, Ru-Huei, Yu-Chi Wang, Shih-Ping Liu, Ton-Ru Shih, Hsin-Lien Lin, Yue-Mi Chen, Jiun-Huei Sung et al. Decellularization and recellularization technologies in tissue engineering. Cell transplantation 23, no. 4-5:621-630 (2014). [0260] Gibot L, Galbraith T, Bourland J, Rogic A, Skobe M and Auger F A. Tissue-engineered 3D human lymphatic microvascular network for in vitro studies of lymphangiogenesis. Nat Protoc.; 12:1077-1088 (2017). [0261] Goh S K, et al. Perfusion-decellularized pancreas as a natural 3D scaffold for pancreatic tissue and whole organ engineering. Biomaterials 34, 6760-6772 (2013). [0262] Goh, Saik Kia. Bio-engineered pancreas with human embryonic stem cells and whole organ derived extracellular matrix scaffolds. PhD diss., University of Pittsburgh (2015). [0263] Graham M L, Janecek J L, Kittredge J A, Hering B J and Schuurman H J. The streptozotocin-induced diabetic nude mouse model: differences between animals from different sources. Comp Med.; 61:356-60 (2011). [0264] Greggio C, De Franceschi F, Grapin-Botton A. Concise reviews: In vitro-produced pancreas organogenesis models in three dimensions: self-organization from few stem cells or progenitors. Stem Cells 33, 8-14 (2015). [0265] Greggio C, et al. Artificial three-dimensional niches deconstruct pancreas development in vitro. Development 140, 4452-4462 (2013). [0266] Halban P A. Cellular sources of new pancreatic beta cells and therapeutic implications for regenerative medicine. Nat Cell Biol 6, 1021-1025 (2004). [0267] Haque M A, Nagaoka M, Hexig B, Akaike T. Artificial extracellular matrix for embryonic stem cell cultures: a new frontier of nanobiomaterials. Science and technology of advanced materials 11, 014106 (2010). [0268] Hoshiba, Takashi, Guoping Chen, Chiho Endo, Hiroka Maruyama, Miyuki Wakui, Eri Nemoto, Naoki Kawazoe, and Masaru Tanaka. Decellularized extracellular matrix as an in vitro model to study the comprehensive roles of the ECM in stem cell differentiation. Stem cells international 2016 (2015). [0269] Hrvatin S, et al. Differentiated human stem cells resemble fetal, not adult, beta cells. Proc Natl Acad Sci USA 111, 3038-3043 (2014). [0270] Hughes C S, Postovit L M, Lajoie G A. Matrigel: a complex protein mixture required for optimal growth of cell culture. Proteomics 10, 1886-1890 (2010). [0271] Hunter C S and Stein R W. Evidence for Loss in Identity, De-Differentiation, and Trans-Differentiation of Islet beta-Cells in Type 2 Diabetes. Front Genet.; 8:35 (2017). [0272] Hunter J D, Cannon J A. Biomaterials: So Many Choices, So Little Time. What Are the Differences? Clin Colon Rect Surg 27, 134-139 (2014). [0273] Ichii, H, L. Inverardi, A. Pileggi, R. D. Molano, O. Cabrera, A. Caicedo, S. Messinger, Y. Kuroda, P. O. Berggren, C. Ricordi, A novel method for the assessment of cellular composition and beta-cell viability in human islet preparations, Am J Transplant 5(7):1635-45 (2005). [0274] Ionescu-Tirgoviste C, Gagniuc P A, Gubceac E, Mardare L, Popescu I, Dima S and Militaru M. A 3D map of the islet routes throughout the healthy human pancreas. Sci Rep.; 5:14634 (2015). [0275] Islam M S. The islets of Langerhans. Preface. Adv Exp Med Biol.; 654:vii-viii (2010). [0276] Jain R, Lammert E. Cell-cell interactions in the endocrine pancreas. Diabetes, obesity & metabolism 11 Suppl 4, 159-167 (2009). [0277] Jang H K, Kim B S. Modulation of stem cell differentiation with biomaterials. International journal of stem cells 3, 80-84 (2010). [0278] Jansson L and Carlsson P O. Graft vascular function after transplantation of pancreatic islets. Diabetologia; 45:749-63 (2002). [0279] Jansson L, Barbu A, Bodin B, Drott C J, Espes D, Gao X, Grapensparr L, Kallskog O, Lau J, Liljeback H, Palm F, Quach M, Sandberg M, Stromberg V, Ullsten S and Carlsson P O. Pancreatic islet blood flow and its measurement. Ups J Med Sci.; 121:81-95 (2016). [0280] Jeffries E M, Nakamura S, Lee K W, Clampffer J, Ijima H and Wang Y. Micropatterning electrospun scaffolds to create intrinsic vascular networks. Macromol Biosci.; 14:1514-20 (2014). [0281] Jennings R E, Berry A A, Strutt J P, Gerrard D T and Hanley N A. Human pancreas development. Development; 142:3126-37 (2015). [0282] Jennings R E, et al. Development of the human pancreas from foregut to endocrine commitment. Diabetes 62, 3514-3522 (2013). [0283] Jiang J, Au M, Lu K, Eshpeter A, Korbutt G, Fisk G and Majumdar A S. Generation of insulin-producing islet-like clusters from human embryonic stem cells. Stem Cells; 25:1940-53 (2007). [0284] Jiang W, et al. In vitro derivation of functional insulin-producing cells from human embryonic stem cells. Cell Res 17, 333-344 (2007). [0285] Jin S, Wang X, Wang W and Ye K. Bioinspired 3D Scaffold for Inducing Differentiation of Embryonic Stem Cells. In: E. Jabbari, D. Kim, A. Ghaemmaghami and A. Khademhosseini, eds. Handbook of Biomimetics and Bioinspiration Singapore. World Scientific Publishing Co, Pte, Ltd Toh Tuck Link; 9(3):1241-1256 (2014). [0286] Jin S, Yao H, Krisanarungson P, Haukas A, Ye K. Porous membrane substrates offer better niches to enhance the Wnt signaling and promote human embryonic stem cell growth and differentiation. Tissue Eng Part A 18, 1419-1430 (2012). [0287] Jin S, Yao H, Weber J L, Melkoumian Z K, Ye K. A synthetic, xeno-free peptide surface for expansion and directed differentiation of human induced pluripotent stem cells. PLoS One 7, e50880 (2012). [0288] Jin S. Regeneration of Islet -cells from stem cells and progenitors. Journal of Stem Cell Research and Transplantation; 1(1):4-8 (2014). [0289] Justin A W, Brooks R A and Markaki A E. Multi-casting approach for vascular networks in cellularized hydrogels. J R Soc Interface; 13 (2016). [0290] Kang H W, Lee S J, Ko I K, Kengla C, Yoo J J and Atala A. A 3D bioprinting system to produce human-scale tissue constructs with structural integrity. Nat Biotechnol.; 34:312-9 (2016). [0291] Kasimir M T, et al. Comparison of different decellularization procedures of porcine heart valves. Int J Artif Organs 26, 421-427 (2003). [0292] Kaufman-Francis K, Koffler J, Weinberg N, Dor Y and Levenberg S. Engineered vascular beds provide key signals to pancreatic hormone-producing cells. PLoS One.; 7:e40741 (2012). [0293] Kibbey M C. Maintenance of the EHS sarcoma and Matrigel preparation. Journal of tissue culture methods 16, 227-230 (1994). [0294] Kim A, Miller K, Jo J, Kilimnik G, Wojcik P, Hara M. Islet architecture: A comparative study. Islets 1, 129-136 (2009). [0295] Kim Y, Kim H, Ko U H, Oh Y, Lim A, Sohn J W, Shin J H, Kim H and Han Y M. Islet-like organoids derived from human pluripotent stem cells efficiently function in the glucose responsiveness in vitro and in vivo. Sci Rep.; 6:35145 (2016). [0296] Kim, Youngjin, Hyeongseok Kim, Ung Hyun Ko, Youjin Oh, Ajin Lim, Jong-Woo Sohn, Jennifer H. Shin, Hail Kim, and Yong-Mahn Han. Islet-like organoids derived from human pluripotent stem cells efficiently function in the glucose responsiveness in vitro and in vivo. Scientific reports 6:35145 (2016). [0297] Kolesky D B, Homan K A, Skylar-Scott M A and Lewis J A. Three-dimensional bioprinting of thick vascularized tissues. Proc Natl Acad Sci USA; 113:3179-84 (2016). [0298] Kondo Y, Toyoda T, Inagaki N and Osafune K. iPSC technology-based regenerative therapy for diabetes. J Diabetes Investig. (2017). [0299] Lammert E, Cleaver O and Melton D. Induction of pancreatic differentiation by signals from blood vessels. Science; 294:564-7 (2001). [0300] Lang R, et al. Three-dimensional culture of hepatocytes on porcine liver tissue-derived extracellular matrix. Biomaterials 32, 7042-7052 (2011). [0301] Lee V K, Kim D Y, Ngo H, Lee Y, Seo L, Yoo S S, Vincent P A and Dai G. Creating perfused functional vascular channels using 3D bio-printing technology. Biomaterials; 35:8092-102 (2014). [0302] Lee V K, Lanzi A M, Haygan N, Yoo S S, Vincent P A and Dai G. Generation of Multi-Scale Vascular Network System within 3D Hydrogel using 3D Bio-Printing Technology. Cell Mol Bioeng.; 7:460-472 (2014). [0303] Li Y, Xu C, Ma T. In vitro organogenesis from pluripotent stem cells. Organogenesis 10, 159-163 (2014). [0304] Matsuoka T A, Artner I, Henderson E, Means A, Sander M, Stein R. The MafA transcription factor appears to be responsible for tissue-specific expression of insulin. Proc Natl Acad Sci USA 101, 2930-2933 (2004). [0305] Merani S, Toso C, Emamaullee J and Shapiro A M. Optimal implantation site for pancreatic islet transplantation. Br J Surg.; 95:1449-61 (2008). [0306] Miller J S, Stevens K R, Yang M T, Baker B M, Nguyen D H, Cohen D M, Toro E, Chen A A, Galie P A, Yu X, Chaturvedi R, Bhatia S N and Chen C S. Rapid casting of patterned vascular networks for perfusable engineered three-dimensional tissues. Nat Mater.; 11:768-74 (2012). [0307] Millman J R, Xie C, Van Dervort A, Gurtler M, Pagliuca F W and Melton D A. Generation of stem cell-derived beta-cells from patients with type 1 diabetes. Nat Commun.; 7:11463 (2016). [0308] Mirmalek-Sani, Sayed-Hadi, Giuseppe Orlando, John P. McQuilling, Rajesh Pareta, David L. Mack, Marcus Salvatori, Alan C. Farney et al. Porcine pancreas extracellular matrix as a platform for endocrine pancreas bioengineering. Biomaterials 34, no. 22:5488-5495 (2013). [0309] Montazeri L, Hojjati-Emami S, Bonakdar S, Tahamtani Y, Hajizadeh-Saffar E, Noori-Keshtkar M, Najar-Asl M, Ashtiani M K and Baharvand H. Improvement of islet engrafts by enhanced angiogenesis and microparticle-mediated oxygenation. Biomaterials; 89:157-65 (2016). [0310] Munoz-Pinto D J, Jimenez-Vergara A C, Gharat T P and Hahn M S. Characterization of sequential collagen-poly(ethylene glycol) diacrylate interpenetrating networks and initial assessment of their potential for vascular tissue engineering. Biomaterials; 40:32-42 (2015). [0311] Nair G, Hebrok M. Islet formation in mice and men: lessons for the generation of functional insulin-producing beta-cells from human pluripotent stem cells. Curr Opin Genet Dev 32, 171-180 (2015). [0312] Napierala, H., K-H. Hillebrandt, N. Haep, P. Tang, M. Tintemann, J. Gassner, M. Noesser et al. Engineering an endocrine Neo-Pancreas by repopulation of a decellularized rat pancreas with islets of Langerhans. Scientific reports 7:41777 (2017). [0313] Narayanan, Karthikeyan, Vivian Y. Lim, Jiayi Shen, Zhen Wei Tan, Divya Rajendran, Shyh-Chyang Luo, Shujun Gao, Andrew C A Wan, and Jackie Y. Ying. Extracellular matrix-mediated differentiation of human embryonic stem cells: differentiation to insulin-secreting beta cells. Tissue Engineering Part A 20, no. 1-2:424-433 (2013). [0314] Otonkoski, Timo, Gillian M. Beattie, Martin I. Mally, Camillo Ricordi, and Alberto Hayek. Nicotinamide is a potent inducer of endocrine differentiation in cultured human fetal pancreatic cells. Journal of Clinical Investigation 92, no. 3:1459 (1993). [0315] Pagliuca, Felicia W., Jeffrey R. Millman, Mads Grtler, Michael Segel, Alana Van Dervort, Jennifer Hyoje Ryu, Quinn P. Peterson, Dale Greiner, and Douglas A. Melton. Generation of functional human pancreatic cells in vitro. Cell 159, no. 2:428-439 (2014). [0316] Pan F C, Brissova M. Pancreas development in humans. Curr Opin Endocrinol Diabetes Obes 21, 77-82 (2014). [0317] Peloso, Andrea, Luca Urbani, Paolo Cravedi, Ravi Katari, Panagiotis Maghsoudlou, Mario Enrique Alvarez Fallas, Valeria Sordi et al. The human pancreas as a source of protolerogenic extracellular matrix scaffold for a new-generation bioartificial endocrine pancreas. Annals of surgery 264, no. 1:169-179 (2016). [0318] Pepper A R, Gala-Lopez B, Pawlick R, Merani S, Kin T and Shapiro A M. A prevascularized subcutaneous device-less site for islet and cellular transplantation. Nat Biotechnol.; 33:518-23 (2015). [0319] Perry L, Flugelman M Y and Levenberg S. Elderly Patient-Derived Endothelial Cells for Vascularization of Engineered Muscle. Mol Ther.; 25:935-948 (2017). [0320] Poornejad N, et al. Efficient decellularization of whole porcine kidneys improves reseeded cell behavior. Biomed Mater 11, 025003 (2016). [0321] Poornejad N, et al. The impact of decellularization agents on renal tissue extracellular matrix. J Biomater Appl 31, 521-533 (2016). [0322] Raikwar S P, Kim E M, Sivitz W I, Allamargot C, Thedens D R, Zavazava N. Human iPS cell-derived insulin producing cells form vascularized organoids under the kidney capsules of diabetic mice. PLoS One 10, e0116582 (2015). [0323] Rajaei B, Shamsara M, Massumi M, Sanati M H. Pancreatic Endoderm-Derived from Diabetic Patient-Specific Induced Pluripotent Stem Cell Generates Glucose-Responsive Insulin-Secreting Cells. J Cell Physiol, (2016). [0324] Rana, Deepti, Hala Zreiqat, Nadia Benkirane-Jessel, Seeram Ramakrishna, and Murugan Ramalingam. Development of decellularized scaffolds for stem cell-driven tissue engineering. Journal of tissue engineering and regenerative medicine 11, no. 4:942-965 (2017). [0325] Reinert R B, Cai Q, Hong J Y, Plank J L, Aamodt K, Prasad N, Aramandla R, Dai C, Levy S E, Pozzi A, Labosky P A, Wright C V, Brissova M and Powers A C. Vascular endothelial growth factor coordinates islet innervation via vascular scaffolding. Development; 141:1480-91 (2014). [0326] Rezania A, Bruin J E, Arora P, Rubin A, Batushansky I, Asadi A, O'Dwyer S, Quiskamp N, Mojibian M, Albrecht T, Yang Y H, Johnson J D and Kieffer T J. Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells. Nat Biotechnol.; 32:1121-33 (2014). [0327] Rezania A, Bruin J E, Xu J, Narayan K, Fox J K, O'Neil J J and Kieffer T J. Enrichment of human embryonic stem cell-derived NKX6.1-expressing pancreatic progenitor cells accelerates the maturation of insulin-secreting cells in vivo. Stem Cells; 31:2432-42 (2013). [0328] Rorsman P, Braun M. Regulation of insulin secretion in human pancreatic islets. Annu Rev Physiol 75, 155-179 (2013). [0329] Rose S D, Swift G H, Peyton M J, Hammer R E, MacDonald R J. The role of PTF1-P48 in pancreatic acinar gene expression. J Biol Chem 276, 44018-44026 (2001). [0330] Russ H A, Parent A V, Ringler J J, Hennings T G, Nair G G, Shveygert M, Guo T, Puri S, Haataja L, Cirulli V, Blelloch R, Szot G L, Aryan P and Hebrok M. Controlled induction of human pancreatic progenitors produces functional beta-like cells in vitro. EMBO J 34, 1759-1772 (2015). [0331] Sabetkish, Shabnam, Abdol-Mohammad Kajbafzadeh, Nastaran Sabetkish, Reza Khorramirouz, Aram Akbarzadeh, Sanam Ladi Seyedian, Parvin Pasalar et al. Whole-organ tissue engineering: Decellularization and recellularization of three-dimensional matrix liver scaffolds. Journal of Biomedical Materials Research Part A 103, no. 4:1498-1508 (2015). [0332] Sachlos E, Reis N, Ainsley C, Derby B and Czernuszka J T. Novel collagen scaffolds with predefined internal morphology made by solid freeform fabrication. Biomaterials.; 24:1487-97 (2003). [0333] Salvatori, Marcus, Ravi Katari, Timil Patel, Andrea Peloso, Jon Mugweru, Kofi Owusu, and Giuseppe Orlando. Extracellular matrix scaffold technology for bioartificial pancreas engineering: state of the art and future challenges. Journal of diabetes science and technology 8, no. 1:159-169 (2014). [0334] Sato Y, Endo H, Okuyama H, Takeda T, Iwahashi H, Imagawa A, Yamagata K, Shimomura I and Inoue M. Cellular hypoxia of pancreatic beta-cells due to high levels of oxygen consumption for insulin secretion in vitro. J Biol Chem.; 286:12524-32 (2011). [0335] Seymour P A, Sander M. Historical perspective: beginnings of the beta-cell: current perspectives in beta-cell development. Diabetes 60, 364-376 (2011). [0336] Shaer A, Azarpira N, Karimi M H. Differentiation of human induced pluripotent stem cells into insulin-like cell clusters with miR-186 and miR-375 by using chemical transfection. Appl Biochem Biotechnol 174, 242-258 (2014). [0337] Shaer A, Azarpira N, Vandati A, Karimi M H, Shariati M. Differentiation of human-induced pluripotent stem cells into insulin-producing clusters. Exp Clin Transplant 13, 68-75 (2015). [0338] Shi Y, Glaser K J, Venkatesh S K, Ben-Abraham E I and Ehman R L. Feasibility of using 3D M R elastography to determine pancreatic stiffness in healthy volunteers. J Magn Reson Imaging.; 41:369-75 (2015). [0339] Shih H P, Wang A, Sander M. Pancreas organogenesis: from lineage determination to morphogenesis. Annu Rev Cell Dev Biol 29, 81-105 (2013). [0340] Shim J H, Kim J, Han J, An S Y, Jang Y J, Son J, Woo D H, Kim S K and Kim J H. Pancreatic Islet-Like Three-Dimensional Aggregates Derived From Human Embryonic Stem Cells Ameliorate Hyperglycemia in Streptozotocin-Induced Diabetic Mice. Cell Transplant 24, 2155-2168 (2015). [0341] Song W, An D, Kao D I, Lu Y C, Dai G, Chen S and Ma M. Nanofibrous microposts and microwells of controlled shapes and their hybridization with hydrogels for cell encapsulation. ACS Appl Mater Interfaces; 6:7038-44 (2014). [0342] Song W, Lu Y C, Frankel A S, An D, Schwartz R E and Ma M. Engraftment of human induced pluripotent stem cell-derived hepatocytes in immunocompetent mice via 3D co-aggregation and encapsulation. Sci Rep.; 5:16884 (2015). [0343] Song, Jeremy J., and Harald C. Ott. Organ engineering based on decellularized matrix scaffolds. Trends in molecular medicine 17, no. 8:424-432 (2011). [0344] Stefan Y, Orci L, Malaisse-Lagae F, Perrelet A, Patel Y, Unger R H. Quantitation of endocrine cell content in the pancreas of nondiabetic and diabetic humans. Diabetes 31, 694-700 (1982). [0345] Steiner D J, Kim A, Miller K, Hara M. Pancreatic islet plasticity: interspecies comparison of islet architecture and composition. Islets 2, 135-145 (2010). [0346] Stendahl, John C., Dixon B. Kaufman, and Samuel I. Stupp. Extracellular matrix in pancreatic islets: relevance to scaffold design and transplantation. Cell transplantation 18, no. 1:1-12 (2009). [0347] Swisa A, Glaser B and Dor Y. Metabolic Stress and Compromised Identity of Pancreatic Beta Cells. Front Genet.; 8:21 (2017). [0348] Takeuchi H, Nakatsuji N, Suemori H. Endodermal differentiation of human pluripotent stem cells to insulin-producing cells in 3D culture. Sci Rep 4, 4488 (2014). [0349] Tapias, Luis F., and Harald C. Ott. Decellularized scaffolds as a platform for bioengineered organs. Current opinion in organ transplantation 19, no. 2:145 (2014). [0350] Tateishi K, He J, Taranova O, Liang G, D'Alessio A C, Zhang Y. Generation of insulin-secreting islet-like clusters from human skin fibroblasts. J Biol Chem 283, 31601-31607 (2008). [0351] Tuch et al. Scaffolds for islets and stem cells differentiated into insulin-secreting cells. Front Biosci (Landmark Ed), 19:126-38 (2014). [0352] Vegas A J, Veiseh O, Gurtler M, Millman J R, Pagliuca F W, Bader A R, Doloff J C, Li J, Chen M, Olejnik K, Tam H H, Jhunjhunwala S, Langan E, Aresta-Dasilva S, Gandham S, McGarrigle J J, Bochenek M A, Hollister-Lock J, Oberholzer J, Greiner D L, Weir G C, Melton D A, Langer R and Anderson D G. Long-term glycemic control using polymer-encapsulated human stem cell-derived beta cells in immune-competent mice. Nat Med 22, 306-311 (2016). [0353] Veiseh O, Doloff J C, Ma M, Vegas A J, Tam H H, Bader A R, Li J, Langan E, Wyckoff J, Loo W S, Jhunjhunwala S, Chiu A, Siebert S, Tang K, Hollister-Lock J, Aresta-Dasilva S, Bochenek M, Mendoza-Elias J, Wang Y, Qi M, Lavin D M, Chen M, Dholakia N, Thakrar R, Lacik I, Weir G C, Oberholzer J, Greiner D L, Langer R and Anderson D G. Size- and shape-dependent foreign body immune response to materials implanted in rodents and non-human primates. Nat Mater.; 14:643-51 (2015). [0354] Vorotnikova E, et al. Extracellular matrix-derived products modulate endothelial and progenitor cell migration and proliferation in vitro and stimulate regenerative healing in vivo. Matrix Biol 29, 690-700 (2010). [0355] Wang X, Ye K. Three-dimensional differentiation of embryonic stem cells into islet-like insulin-producing clusters. Tissue Eng Part A 15, 1941-1952 (2009). [0356] Wang Y C, Gallego-Arteche E, lezza G, Yuan X, Matli M R, Choo S P, Zuraek M B, Gogia R, Lynn F C, German M S, Bergsland E K, Donner D B, Warren R S and Nakakura E K. Homeodomain transcription factor NKX2.2 functions in immature cells to control enteroendocrine differentiation and is expressed in gastrointestinal neuroendocrine tumors. Endocr Relat Cancer; 16:267-79 (2009). [0357] Wang, Weiwei, Sha Jin, and Kaiming Ye. Development of Islet Organoids from H9 Human Embryonic Stem Cells in Biomimetic 3D Scaffolds. Stem cells and development 26, no. 6:394-404 (2017). [0358] Wen Y, Jin S. Production of neural stem cells from human pluripotent stem cells. J Biotechnol 188, 122-129 (2014). [0359] Wilding L and Gannon M. The role of pdx1 and HNF6 in proliferation and differentiation of endocrine precursors. Diabetes Metab Res Rev.; 20:114-23 (2004). [0360] Wong D Y, Ranganath T and Kasko A M. Low-Dose, Long-Wave U V Light Does Not Affect Gene Expression of Human Mesenchymal Stem Cells. PLoS One.; 10:e0139307 (2015). [0361] Wu D, et al. 3D Culture of MIN-6 Cells on Decellularized Pancreatic Scaffold: In Vitro and In Vivo Study. BioMed research international 2015, 432645 (2015). [0362] Wu W, DeConinck A and Lewis J A. Omnidirectional printing of 3D microvascular networks. Adv Mater.; 23:H178-83 (2011). [0363] Yamaguchi T, Sato H, Kato-Itoh M, Goto T, Hara H, Sanbo M, Mizuno N, Kobayashi T, Yanagida A, Umino A, Ota Y, Hamanaka S, Masaki H, Rashid S T, Hirabayashi M and Nakauchi H. Interspecies organogenesis generates autologous functional islets. Nature; 542:191-196 (2017). [0364] Ye K, Jin S and Schultz J S. Genetically engineered fluorescent cell marker for labeling CD34+ hematopoietic stem cells. Biotechnol Prog.; 20:561-5 (2004). [0365] Ye M, Sanchez H M, Hultz M, Yang Z, Bogorad M, Wong A D and Searson P C. Brain microvascular endothelial cells resist elongation due to curvature and shear stress. Sci Rep.; 4:4681 (2014). [0366] Yesmin, S., M. B. Paget, H. E. Murray, and R. Downing. Bio-scaffolds in organ-regeneration: Clinical potential and current challenges. Current Research in Translational Medicine (2017). [0367] Yu, Yaling, Ali Alkhawaji, Yuqiang Ding, and Jin Mei. Decellularized scaffolds in regenerative medicine. Oncotarget 7, no. 36:58671 (2016). [0368] Zhang B, Jin S, Jin J, Li F and Montelaro R C. A tumor necrosis factor receptor family protein serves as a cellular receptor for the macrophage-tropic equine lentivirus. Proc Natl Acad Sci USA; 102:9918-23 (2005). [0369] Zhang D, Jiang W, Liu M, Sui X, Yin X, Chen S, Shi Y and Deng H. Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells. Cell Res.; 19:429-38 (2009). [0370] Zhang, C, T. Moriguchi, M. Kajihara, R. Esaki, A. Harada, H. Shimohata, H. Oishi, M. Hamada, N. Morito, K. Hasegawa, T. Kudo, J. D. Engel, M. Yamamoto, S. Takahashi, MafA is a key regulator of glucose-stimulated insulin secretion, Mol Cell Biol 25(12):4969-76 (2005). [0371] Zhao X, Liu L, Wang J, Xu Y, Zhang W, Khang G and Wang X. In vitro vascularization of a combined system based on a 3D printing technique. J Tissue Eng Regen Med.; 10:833-842 (2016). [0372] Zhou, Pengcheng, Yibing Guo, Yan Huang, Mingyan Zhu, Xiangjun Fan, Lei Wang, Yao Wang et al. The dynamic three-dimensional culture of islet-like clusters in decellularized liver scaffolds. Cell and tissue research 365, no. 1:157-171 (2016). [0373] Zhu S, Russ H A, Wang X, Zhang M, Ma T, Xu T, Tang S, Hebrok M and Ding S. Human pancreatic beta-like cells converted from fibroblasts. Nat Commun.; 7:10080 (2016).