Generation of functional human iPSC-derived pancreatic islets in co-culture with isogenic iPSC-derived vascular endothelial cells

Abstract

Diabetes mellitus is a clinical condition affecting millions of people worldwide and is treated by insulin replacement therapy. As an alternative to the limited supply of cadaveric islets or multiple exogenous insulin administrations, new strategies are needed to generate scalable and compatible islets containing insulin-producing β-cells. Improvements are still needed because many immature polyhormonal cells remain and a monohormonal state cannot be achieved. During human development, the pancreas co-develops and shares signals with the endothelium, thereby allowing better maturation of β-cells, which is not included in current differentiation protocols. Organchip-type microfluidic devices allow dynamic co-culture of different cells, thus resembling in vivo physiology. Here, we establish an organchip model in which human iPSC-derived pancreatic precursors are co-cultured with iPSC-derived endothelial cells to obtain more functional and monohormonal iPSC-derived β-cells. TIFF2024506850000009.tif55128

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 USC §119(e) to U.S. Provisional Patent Application No. 63 / 144,155, filed February 1, 2021, the entirety of which is incorporated herein by reference.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Grant No. DK063491 awarded by the National Institutes of Health. The United States Government has certain rights in the invention.

[0003] FIELD OF THEINVENTION The present invention relates to the field of cell culturing, and in particular to the field of culturing pancreatic islet cells together with other cell types. [Background technology]

[0004] background Diabetes mellitus affects millions of people worldwide and is characterized by hyperglycemia, primarily due to the failure of pancreatic islets to produce insufficient or little insulin to meet the body's demand for glucose. Most diabetic patients rely on multiple exogenous insulin injections, which can cause adverse effects, and some patients have undergone cadaveric islet transplants, which are scarce and require long-term immunosuppression. Thus, there is a great need for novel strategies to generate scalable and compatible insulin-producing beta-cell-containing islets. Summary of the Invention

[0005] The following embodiments and aspects thereof are described and illustrated in connection with compositions and methods which are intended to be exemplary and explanatory, and not limiting in scope.

[0006] Various embodiments of the present invention provide methods of generating functional induced pluripotent stem cell (iPSC)-derived pancreatic islets (iIslets), the methods comprising co-culturing a quantity of iPSC-derived endothelial cells (iECs) with a quantity of iPSC-derived islet precursors for about 10-18 days to generate functional iIslets comprising β cells.

[0007] In various embodiments, the co-culturing step may include plating a quantity of iPSC-derived endothelial cells (iECs) onto a MATRIGEL-coated plate and culturing in Phase IV EC medium supplemented with Y27632; plating a quantity of iPSC-derived islets (iIslets) on top of the quantity of iECs and culturing for about 12-16 days in medium comprising about ½ Phase IV iEC medium and about ½ Phase VI islet medium supplemented with Y-27632 or in Phase VI islet medium (islet-only condition) for about 12-16 days to generate functional iIslets comprising β cells.

[0008] In various embodiments, the co-culturing step may include plating a quantity of iPSC-derived vascular endothelial cells (iECs) onto a MATRIGEL-coated plate and culturing in Phase IV EC medium supplemented with Y27632; plating a quantity of iPSC-derived islets (iIslets) on top of the quantity of iECs and culturing either in medium comprising about ½ Phase IV iEC medium and about ½ Phase VI islet medium supplemented with Y-27632 for about 14 days or in Phase VI islet medium (islet-only condition) for about 14 days to generate functional iIslets comprising beta cells.

[0009] In various embodiments, the method may further comprise the step of initially generating iECs by: plating a quantity of induced pluripotent stem cells (iPSCs) onto MATRIGEL; culturing the iPSCs in MATRIGEL for about 2-4 days; culturing in the presence of CHIR99021 for about 1-3 days to generate mesoderm; culturing the mesoderm in the presence of BMP4, FGF2, and VEGF for about 1-3 days to generate vascular precursors; culturing the vascular precursors in the presence of VEGF and Y-27632 for about 3-8 days to generate endothelial cell (EC) precursors.

[0010] In various embodiments, the method may further include the step of initially generating iECs by: plating a quantity of induced pluripotent stem cells (iPSCs) onto MATRIGEL; culturing the iPSCs in MATRIGEL for about 3 days; culturing in the presence of CHIR99021 for about 2 days to generate mesoderm; culturing the mesoderm in the presence of BMP4, FGF2, and VEGF for about 2 days to generate vascular precursors; culturing the vascular precursors in the presence of VEGF and Y-27632 for about 4-7 days to generate endothelial cell (EC) precursors.

[0011] In various embodiments, the method may further comprise initially generating the quantity of islet precursors by: culturing a quantity of induced pluripotent stem cells (iPSCs) in the presence of Activin A, CHIR99021, and Y-27632 for about 1-2 days; culturing in the presence of Activin A and FGF2 for about 1-3 days; culturing in the presence of FGF10, CHIR99021, and Noggin for about 1-3 days to generate posterior foregut cells; culturing the posterior foregut cells in the presence of FGF10, Noggin, RA, and SANT1 for about 3-5 days to generate pancreatic precursors; culturing the pancreatic precursors in the presence of Noggin, EGF, and nicotinamide for about 3-5 days to generate pancreatic endocrine precursors; culturing the pancreatic endocrine precursors in the presence of Noggin, T3, and Alk5i II for about 6-8 days to generate pancreatic islet precursors.

[0012] In various embodiments, the method may further include initially generating the quantity of pancreatic islet precursors by: culturing a quantity of induced pluripotent stem cells (iPSCs) in the presence of activin A, CHIR99021 and Y-27632 for about 1 day; culturing in the presence of activin A and FGF2 for about 2 days; culturing in the presence of FGF10, CHIR99021 and Noggin for about 2 days to generate posterior foregut cells; culturing the posterior foregut cells in the presence of FGF10, Noggin, RA and SANT1 for about 4 days to generate pancreatic precursors; culturing the pancreatic precursors in the presence of Noggin, EGF and nicotinamide for about 4 days to generate pancreatic endocrine precursors; and culturing the pancreatic endocrine precursors in the presence of Noggin, T3 and Alk5i II for about 7 days to generate pancreatic islet precursors.

[0013] In various embodiments, the pancreatic progenitors may express PDX1+ and SOX9+.

[0014] In various embodiments, the pancreatic endocrine precursors can be PDX1+ and NKX6.1+.

[0015] In various embodiments, the iIslet may express C-peptide, glucagon and NKX6.1+.

[0016] In various embodiments, expression of INS, UCN3, NGN3 and CHGA may be upregulated in beta cells produced in islet-only conditions compared to beta cells produced without co-culture with vascular endothelial cells or compared to beta cells produced in cultures without islet-only conditions.

[0017] In various aspects, beta cells are capable of increasing insulin secretion when challenged with high glucose concentrations compared to basal glucose concentrations.

[0018] In various embodiments, the iPSC-derived vascular endothelial cells (iECs) and the iPSC-derived pancreatic islet precursors can be isogenic.

[0019] In various embodiments, the iPSCs used to derive vascular endothelial cells (iECs) and the iPSCs used to derive pancreatic islet precursors may be derived from the same iPSC cell line or the same donor.

[0020] In various embodiments, the iIslet can be a human iIslet.

[0021] Various aspects of the present invention provide induced pluripotent stem cell (iPSC) derived pancreatic islet (iIslet) produced by any one of the methods described herein.In various aspects, induced pluripotent stem cell (iPSC) derived pancreatic islet (iIslet) can express C-peptide, glucagon and NKX6.1+.In various aspects, iIslet can increase insulin secretion when loaded with high glucose concentration compared to basal glucose concentration.

[0022] Various aspects of the present invention provide methods of ameliorating or treating a metabolic disease, disorder, or condition in a subject in need thereof, comprising administering an iIslet of the present invention to the subject to ameliorate or treat the metabolic disease, disorder, or condition. In various aspects, the metabolic disease, disorder, or condition can be diabetes or insulin resistance.

[0023] Various embodiments of the present invention provide a method comprising culturing a quantity of induced pluripotent stem cells (iPSCs) in the presence of Activin A, CHIR99021 and Y-27632 for about 1 day; then culturing in the presence of Activin A and FGF2 for about 2 days; then culturing in the presence of FGF10, CHIR99021 and Noggin for about 2 days to generate posterior foregut cells.

[0024] In various embodiments, the method may further comprise culturing the posterior foregut cells in the presence of FGF10, Noggin, RA and SANT1 for about 4 days to generate pancreatic progenitors.

[0025] In various embodiments, the pancreatic precursor is PDX1 +and SOX9 + may be expressed.

[0026] In various embodiments, the method may further comprise culturing the pancreatic precursors in the presence of noggin, EGF and nicotinamide for about 4 days to generate pancreatic endocrine precursors.

[0027] In various embodiments, the pancreatic endocrine precursor is PDX1 + and NKX6.1 + It could be.

[0028] In various embodiments, the method may further comprise culturing the pancreatic endocrine precursors in the presence of Noggin, T3 and Alk5i II for about 7 days to generate pancreatic islet precursors.

[0029] In various embodiments, the method may further comprise culturing the generated pancreatic islet precursors in the presence of T3, Alk5i II, R428, and N-acetylcysteine ​​(NAC) for about 14 days to generate mature pancreatic islets.

[0030] In various embodiments, the mature pancreatic islets are capable of expressing C-peptide, glucagon and NKX6.1. + may be expressed.

[0031] In various embodiments, the method may further comprise culturing the posterior foregut in the presence of FGF10, Noggin, RA and SANT1 for about 4 days to generate pancreatic precursors; then culturing the pancreatic precursors in the presence of Noggin, EGF and nicotinamide for about 4 days to generate pancreatic endocrine precursors; then culturing the pancreatic endocrine precursors in the presence of Noggin, T3 and Alk5i II for about 7 days to generate pancreatic islet precursors; then culturing the generated pancreatic islet precursors in the presence of T3, Alk5i II, R428 and N-acetylcysteine ​​(NAC) for about 14 days to generate mature pancreatic islets.

[0032] Various embodiments of the present invention provide a quantity of mature pancreatic islets produced by any one of the methods of the invention described herein.

[0033] Various embodiments of the present invention provide a method that includes plating a quantity of induced pluripotent stem cells (iPSCs) onto MATRIGEL; culturing the iPSCs in MATRIGEL for about 3 days; and then culturing in the presence of CHIR99021 to generate mesoderm.

[0034] In various embodiments, the method may further comprise culturing the mesoderm in the presence of BMP4, FGF2, and VEGF for about 2 days to generate vascular precursors.

[0035] In various embodiments, the method may further comprise culturing the vascular progenitors in the presence of VEGF and Y-27632 for about 7 days to generate endothelial cell (EC) progenitors.

[0036] In various embodiments, the method may further comprise culturing the EC precursors with VEGF for about 10 days to generate mature ECs. In various embodiments, the mature ECs are CD31 + , CD144 + , VEGF-A + , VEGFR2 + , and Ac-LDL.

[0037] In various embodiments, the method may further comprise culturing the mesoderm in the presence of BMP4, FGF2, and VEGF for about 2 days to generate vascular precursors; then culturing the vascular precursors in the presence of VEGF and Y-27632 for about 7 days to generate endothelial cell (EC) precursors; then culturing the EC precursors with VEGF for about 10 days to generate mature ECs.

[0038] Various embodiments of the invention provide a quantity of mature ECs produced by any one of the methods of the invention described herein.

[0039] Various embodiments of the present invention provide an assembly comprising a quantity of mature pancreatic islets and a quantity of mature ECs, wherein the mature islets and the mature ECs are isogenic.

[0040] In various embodiments, said quantity of mature pancreatic islets can comprise the mature pancreatic islets of the invention described herein, and said quantity of mature ECs can comprise the mature ECs of the invention described herein.

[0041] In various embodiments, the mature islets, mature ECs, or both can be attached onto the scaffold. In various embodiments, the mature islets, mature ECs, or both can be attached onto the scaffold using a bioink. In various embodiments, the bioink can include fibrin or alginate.

[0042] Also described herein is a method that includes providing a quantity of induced pluripotent stem cells (iPSCs) and culturing them in the presence of Activin A, CHIR99021 and Y-27632 for about 1 day, further culturing them in the presence of Activin A and FGF2 for about 2 days, and additionally culturing them in the presence of FGF10, CHIR99021 and Noggin for about 2 days to generate a posterior foregut.

[0043] In another embodiment, the method includes culturing the posterior foregut in the presence of FGF10, Noggin, RA and SANT1 for about 4 days to generate pancreatic progenitors. + and SOX9 + In another embodiment, the method includes culturing the pancreatic progenitors in the presence of noggin, EGF and nicotinamide for about 4 days to generate pancreatic endocrine progenitors. In another embodiment, the pancreatic endocrine progenitors express PDX1. + and NKX6.1 + In another embodiment, the method comprises culturing the pancreatic endocrine precursors in the presence of noggin, T3 and Alk5i II for about 7 days to generate pancreatic islet precursors. In another embodiment, the method comprises culturing the generated pancreatic islet precursors in the presence of T3, Alk5i II, R428 and N-acetylcysteine ​​(NAC) for about 14 days to generate mature pancreatic islets. In another embodiment, the mature pancreatic islets are expressed in the presence of C-peptide, glucagon and NKX6.1. + is expressed.

[0044] Also described herein is a quantity of mature pancreatic islets produced by the method, which includes providing a quantity of induced pluripotent stem cells (iPSCs) and culturing them in the presence of activin A, CHIR99021 and Y-27632 for about 1 day, further culturing them in the presence of activin A and FGF2 for about 2 days, and additionally culturing them in the presence of FGF10, CHIR99021 and Noggin for about 2 days to produce a posterior foregut. In another embodiment, the method includes culturing the posterior foregut in the presence of FGF10, Noggin, RA and SANT1 for about 4 days to produce a pancreatic progenitor. In another embodiment, the pancreatic progenitor is PDX1. + and SOX9 + In another embodiment, the method includes culturing the pancreatic progenitors in the presence of noggin, EGF and nicotinamide for about 4 days to generate pancreatic endocrine progenitors. In another embodiment, the pancreatic endocrine progenitors express PDX1. + and NKX6.1 + In another embodiment, the method comprises culturing the pancreatic endocrine precursors in the presence of noggin, T3 and Alk5i II for about 7 days to generate pancreatic islet precursors. In another embodiment, the method comprises culturing the generated pancreatic islet precursors in the presence of T3, Alk5i II, R428 and N-acetylcysteine ​​(NAC) for about 14 days to generate mature pancreatic islets. In another embodiment, the mature pancreatic islets are expressed in the presence of C-peptide, glucagon and NKX6.1. + is expressed.

[0045] Also described herein is a method that includes providing a quantity of induced pluripotent stem cells (iPSCs), plating the iPSCs on MATRIGEL, culturing the iPSCs in MATRIGEL for about 3 days, and further culturing in the presence of CHIR99021 to generate mesoderm. In another embodiment, the method includes culturing the mesoderm in the presence of BMP4, FGF2, and VEGF for about 2 days to generate vascular precursors. In another embodiment, the method includes culturing the vascular precursors in the presence of VEGF and Y-27632 for about 7 days to generate endothelial cell (EC) precursors. In another embodiment, the method includes culturing the EC precursors with VEGF for about 10 days to generate mature ECs. In another embodiment, the method includes culturing the vascular precursors in the presence of EGM-MV2 and VEGF for about 4-6 days to generate endothelial progenitor cells, and culturing the endothelial progenitor cells in the presence of EGM-MV2 and VEGF to generate endothelial cells. In other embodiments, the mature ECs are CD31 + , CD144 + , VEGF-A + , VEGFR2 + , and Ac-LDL.

[0046] Also described herein is a quantity of mature ECs produced by the method, which comprises providing a quantity of induced pluripotent stem cells (iPSCs), plating the iPSCs on MATRIGEL, culturing the iPSCs in MATRIGEL for about 3 days, and further culturing in the presence of CHIR99021 to produce mesoderm. In another embodiment, the method comprises culturing the mesoderm in the presence of BMP4, FGF2, and VEGF for about 2 days to produce vascular precursors. In another embodiment, the method comprises culturing the vascular precursors in the presence of VEGF and Y-27632 for about 7 days to produce endothelial cell (EC) precursors. In another embodiment, the method comprises culturing the EC precursors with VEGF for about 10 days to produce mature ECs. In another embodiment, the method includes culturing the vascular precursors in the presence of EGM-MV2 and VEGF for about 4-6 days to generate endothelial progenitor cells, and culturing the endothelial progenitor cells in the presence of EGM-MV2 and VEGF to generate endothelial cells. + , CD144 + , VEGF-A + , VEGFR2 + , and Ac-LDL.

[0047] Also described herein is an assembly comprising a quantity of mature pancreatic islets and a quantity of mature ECs.In another embodiment, the mature pancreatic islets are produced by the method, which includes the steps of preparing a quantity of induced pluripotent stem cells (iPSCs), culturing them in the presence of activin A, CHIR99021 and Y-27632 for about 1 day, further culturing them in the presence of activin A and FGF2 for about 2 days, and further culturing them in the presence of FGF10, CHIR99021 and Noggin for about 2 days to produce a posterior foregut.In another embodiment, the method includes culturing the posterior foregut in the presence of FGF10, Noggin, RA and SANT1 for about 4 days to produce a pancreatic progenitor.In another embodiment, the pancreatic progenitor is selected from the group consisting of PDX1, PDX2, PDX3, PDX4, PDX5, PDX6, PDX7, PDX8, PDX9, PDX10, PDX11, PDX12, PDX13, PDX14, PDX15, PDX16, PDX17, PDX18, PDX19, PDX21, PDX22, PDX23, PDX24, PDX25, PDX26, PDX27, PDX28, PDX29, PDX31, PDX32, PDX32, PDX33, PDX34, PDX35, PDX35, PDX36, PDX37, PDX38, PDX39, PDX39, PDX39, PDX38, PDX39, PDX39, PDX39, PDX39, PDX39, PDX31, PDX31, PDX32, PDX33, PDX34, PDX35, PDX35, PDX36, PDX37, PDX38, PDX39 + and SOX9 +In another embodiment, the method includes culturing the pancreatic progenitors in the presence of noggin, EGF and nicotinamide for about 4 days to generate pancreatic endocrine progenitors. In another embodiment, the pancreatic endocrine progenitors express PDX1. + and NKX6.1 + In another embodiment, the method comprises culturing the pancreatic endocrine precursors in the presence of noggin, T3 and Alk5i II for about 7 days to generate pancreatic islet precursors. In another embodiment, the method comprises culturing the generated pancreatic islet precursors in the presence of T3, Alk5i II, R428 and N-acetylcysteine ​​(NAC) for about 14 days to generate mature pancreatic islets. In another embodiment, the mature pancreatic islets are expressed in the presence of C-peptide, glucagon and NKX6.1. + In another embodiment, the mature EC is produced by a method comprising the steps of providing a quantity of induced pluripotent stem cells (iPSCs), plating the iPSCs on MATRIGEL, culturing the iPSCs in MATRIGEL for about 3 days, and further culturing in the presence of CHIR99021 to produce mesoderm. In another embodiment, the method comprises culturing the mesoderm in the presence of BMP4, FGF2 and VEGF for about 2 days to produce vascular precursors. In another embodiment, the method comprises culturing the vascular precursors in the presence of VEGF and Y-27632 for about 7 days to produce endothelial cell (EC) precursors. In another embodiment, the method comprises culturing the EC precursors with VEGF for about 10 days to produce mature ECs. In another embodiment, the method includes culturing the vascular precursors in the presence of EGM-MV2 and VEGF for about 4-6 days to generate endothelial progenitor cells, and culturing the endothelial progenitor cells in the presence of EGM-MV2 and VEGF to generate endothelial cells. + , CD144 + , VEGF-A + , VEGFR2 + , and Ac-LDL. In other embodiments, the mature islets and mature ECs are isogenic.

[0048] Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of aspects of the invention. [Brief description of the drawings]

[0049] [Figure 1] FIG. 1 shows protein expression of endocrine pancreatic progenitor markers. [Diagram 2] FIG. 2 shows the mRNA expression of pluripotency marker (Oct4) and endocrine pancreatic markers (Pdx1, Nkx6.1, C-peptide) throughout development. [Diagram 3] FIG. 3 shows the protein expression of endothelial cell markers. [Figure 4] FIG. 4 shows the mRNA expression of endothelial cell markers. [Diagram 5] 5A-5B show the results from the co-culture system. (5A) Representation of the co-culture system; (5B) Results of GSIS using direct co-culture of iECs and iIslets. [Figure 6] FIG. 6 shows the mRNA expression of β-cell markers. [Figure 7] Figures 7A-7D show the perfusion system adapted for the GSIS dynamic system. (A) The perfusion system for perfusing the cells with medium. (B) Plate-cultured iECs transferred to a 3D-printed vascular construct. (C), (D) iECs that remained attached to the cast of the vascular construct for at least 2 weeks. [Figure 8A]Figures 8A-8C show the successful generation of iPSC-derived pancreatic endocrine precursors. 8A. Schematic protocol of differentiation of iPSCs to pancreatic endocrine precursor (PEP) phases by detailed small molecules. 8B. Immunofluorescence images showing the expression of markers at each stage: OCT4 / SSEA4 for iPSCs; FOXA2 / SOX17 for DE; PDX1 / SOX9 for PP; and PDX1 / NKX6.1 for PEP. In this experiment, three different cell lines were used as indicated. The cropped image (inset) corresponds to 1 / 7 of the original image. 8C. mRNA expression of different markers at each stage as indicated. This experiment was performed with 07iCTR-n07 cell line. Data are shown as mean ± SEM (n=3 / experiment). *P<0.05 vs iPSC. DE: definitive endoderm. PFG: posterior foregut. PP: pancreatic precursor. PEP: pancreatic endocrine precursor. Nicotin.: Nicotinamide. [Figure 8B] See legend to Figure 8A. [Figure 8C] See legend to Figure 8A. [Figure 9A]Figures 9A-9D show the generation of iPSC-derived islet precursors. 9A. Schematic of the main signaling pathways tested to optimize the generation of islet precursors (IPs) from pancreatic endocrine precursors (PEPs). 9B. Combinations of small molecules tested to modulate the main pathways were: i) Alk5i and T3; ii) Alk5i and T3 and Noggin; iii) Alk5i and T3 and XXI; and iv) Alk5i and T3 and Noggin and XXI. Immunofluorescence images showing marker expression in islets: C-peptide (C-PEP) and NKX6.1 in β cells; glucagon (GCG) in α cells; somatostatin (SST) in δ cells. The cropped images (insert images) correspond to 1 / 7 of the original images. 9C. Number of cells per well in each condition in B. 9D. Percent expression of each marker and their combinations in B. These experiments were performed with the 07iCTR-n07 cell line. Data are presented as mean ± SEM (n=3 / experiment). * P<0.05 vs Alk5i, T3; ∧ P<0.05 vs Alk5i, T3, Noggin; # P<0.05 vs Alk5i, T3, XXI. PEP: pancreatic endocrine precursors. IP: islet precursors. Nog: Noggin. [Figure 9B] See legend to Figure 9A. [Figure 9C] See legend to Figure 9A. [Figure 9D] See legend to Figure 9A. [Figure 10A]Figures 10A-10D show the optimization of the maturation process of iPSC-derived mature islets (MI). 10A. Schematic of the main signaling pathways tested to optimize maturation from islet precursors (IPs) to mature islets (MIs). 10B. Combinations of small molecules tested to modulate the main pathways were: i) T3; ii) T3 and Alk5i; iii) T3, Alk5i and NAC; and iv) T3, Alk5i, NAC and R428. Immunofluorescence images showing marker expression of islets: C-peptide (C-PEP) and Nkx6.1 in β cells; glucagon (GCG) in α cells; somatostatin (SST) in δ cells. The cropped images (insert images) correspond to 1 / 7 of the original images. 10C. Number of cells per well in each condition in B. Percentage of expression of each marker in B. These experiments were performed with the 07iCTR-n07 cell line. Data are presented as mean ± SEM (n = 3 / experiment). * P < 0.05 vs T3; ∧ P < 0.05 vs T3, Alk5i. PEP: pancreatic endocrine precursor. NAC: N-acetylcysteine. R248: AXL inhibitor R428. [Figure 10B] See legend to Figure 10A. [Figure 10C] See legend to Figure 10A. [Figure 10D] See legend to Figure 10A. [Figure 11A] Figures 11A-11D show the successful generation of iPSC-derived vascular endothelial cells (iECs). 11A. Schematic protocol of differentiation of iPSCs to vascular endothelial cells (iECs). 11B. Immunofluorescence images showing the expression of endothelial markers at day 21: VEGFR2 / CD31 and CD144 / CD31, and 11C. % of expression of DAPI cells. The functionality of the cells was also assayed using Dil-Acil-LDL uptake assay (11B-11C). 11D. mRNA expression of different endothelial markers at different days of differentiation. This experiment was performed with 07iCTR-n07 cell line. Data are shown as mean ± SEM (n=3 / experiment). *P<0.05 vs iPSCs. [Figure 11B] See legend to Figure 11A. [Figure 11C]See legend to Figure 11A. [Figure 11D] See legend to Figure 11A. [Figure 12A] Figures 12A-12D show that direct co-culture of i-mature islets (i-MI) with iECs leads to enhanced functionality of i-MI. 12A. Schematic of direct co-culture: i-MIs were co-cultured on iECs for 2 weeks fed with phase VI i-MI medium. 12B. Immunofluorescence images showing expression of pancreatic endocrine markers C-peptide and NKX.6.1 in i-MIs cultured alone or co-cultured with iECs. The fold change in C-peptide / NKX6.1 expression was two-fold when i-MIs were co-cultured. 12C. Cell functionality was assayed through glucose-stimulated insulin secretion (GSIS). The graph shows the fold change in insulin secretion comparing i-MIs alone and i-MIs co-cultured with iECs, loaded with 2.8 mM glucose and 20 mM glucose. 12D. mRNA expression of different pancreatic endocrine markers after 2 weeks of co-culture. This experiment was performed with the 07iCTR-n07 cell line. Data are shown as mean ± SEM (n=3 / experiment). In Fig. 5C, * p<0.05 vs 2.8 mM glucose i-MI alone; # p<0.05 vs 20 mM i-MI alone. In Fig. 12D, * p<0.05 vs iPSC; ∧ p<0.05 vs i-MI alone. [Figure 12B] See legend to Figure 12A. [Figure 12C] See legend to Figure 12A. [Figure 12D] See legend to Figure 12A. [Figure 13] Figures 13A-13B show cell morphology throughout iEC differentiation. 13A. Brightfield image of iPSC colonies 3 days after passaging and plating onto MATRIGEL-coated plates. 13B. Brightfield image of vascular precursors (VPs) at the end of phase II of iEC differentiation. Peripheral VPs easily float after dissociation and can be replated planarly onto MATRIGEL-coated plates to induce endothelial cell precursors (ECs) during phase III of iEC differentiation. [Figure 14]Figures 14A-14C show quantification of protein expression of markers of pancreatic endocrine precursors (PEP) in different cell lines. Quantification of protein expression of pancreatic endocrine markers PDX1 and NKX6.1 (% expression of DAPI cells) in three cell lines: 07iCTR-n07, EDi028-A and EDi029-A. Data are shown as mean ± SEM (n=3 / experiment). * p<0.05, ** p<0.01, **** p<0.0001. [Figure 15] Figure 15 shows gene expression of pluripotency marker OCT4 throughout differentiation of iPSCs to pancreatic endocrine precursors (PEPs). Real-time qPCR (RT-qPCR) was performed using 07iCTR-n05, NCI-N87 and MKN-45 human gastric cancer cell lines. Data are presented as mean ± SEM (n=3 / experiment). *P<0.05 vs iPSCs. [Figure 16] Figure 16 shows static glucose stimulated insulin secretion (GSIS) in islet precursors (IP). Islet precursors (IP) were loaded into a static GSIS assay; the assay consisted of stimulating the cells with 2.8 mM glucose solution (1 h), then 20 mM glucose solution (1 h), and finally 30 mM KCl solution stimulation (1 h). The experiment was performed using 07iCTR-n07 cell line. Data are presented as mean ± SEM (n=3 / experiment). * P<0.05 vs Alk5i, T3 condition; & P<0.05 vs 2.8 mM glucose. [Figure 17] Figure 17 shows static glucose stimulated insulin secretion (GSIS) in mature islets (MI). Mature islets (MI) were loaded into a GSIS assay; the assay consisted of stimulating the cells with 2.8 mM glucose solution (1 h), then 20 mM glucose solution (1 h), and finally 30 mM KCl solution stimulation (1 h). The experiment was performed with 07iCTR-n07 cell line. Data are presented as mean ± SEM (n=3 / experiment). * P<0.05 vs T3 condition. ∧ P<0.05 vs T3, Alk5i condition. # P<0.05 vs T3, Alk5i, NAC condition; & P<0.05 vs 2.8 mM glucose. [Figure 18] Figures 18A-18B show the trial of different media conditions in phase VI to enhance the functionality of iIslets. 18A. Schematic showing the main factors modulated in the media to optimize the generation of functional mature islets. 18B. The following conditions were tried: i) media with 8 mM glucose and ITS-X; ii) media with 8 mM glucose and no ITS-X; iii) media with 20 mM glucose and ITS-X; and iv) media with 20 mM glucose and no ITS-X. Immunofluorescence images showing islet marker expression: C-peptide (C-PEP) and NKX6.1 for β cells; glucagon (GCG) for α cells; somatostatin (SST) for δ cells. The cropped image (inset) corresponds to 1 / 7 of the original image. Data are shown as mean ± SEM (n=3 / experiment). IP: islet precursors; MI: mature islets; Gluc: glucose; ITS-X: insulin-transferrin-selenium-ethanolamine. [Figure 19A]Figures 19A-19C show the timing trials of reaggregation of mature islets (MI) and enhancing the functionality of functional iIslets. 19A. Schematic showing the key factors modulated to optimize the generation of functional mature iIslets. 19B. The following conditions were tried: i) reaggregating cells on the first day of phase VI and then culturing the cells for 7 days; and ii) reaggregating cells on the first day of phase VI and then culturing the cells for 14 days. Immunofluorescence images showing marker expression of islets: C-peptide (C-PEP) and NKX6.1 for β cells; glucagon (GCG) for α cells. The cropped image (inset) corresponds to 1 / 7 of the original image. 19C. Cells were subjected to static glucose-stimulated insulin secretion (GSIS); this consisted of stimulating the cells with 2.8 mM glucose solution (1 h), then 20 mM glucose solution (1 h), and finally 30 mM KCl solution stimulation (1 h). These experiments were performed with the 07iCTR-n07 cell line. Data are presented as mean ± SEM (n=3 / experiment). * P<0.05 vs 2.8 mM glucose; ∧ P<0.05 vs 20 mM glucose; & P<0.05 vs Reag. day 28. IP: islet precursors. MI: mature islets. Reag.: reaggregates. [Figure 19B] See legend to Figure 19A. [Figure 19C] See legend to Figure 19A. [Figure 20] Figure 20 shows that other cell types after reaggregation may affect the lack of functionality of mature islets. Immunofluorescence images showing marker expression of islets 35 days after reaggregation: C-peptide (C-PEP) and KX6.1 for β cells, glucagon (GCG) for α cells, PDX1 / SOX9 for pancreatic precursors (PP), PDX1 / CK19 for pancreatic duct cells, and PDX1 / CDX2 as gastric / intestinal markers. The cropped image (inset) corresponds to 1 / 7 of the original image. These experiments were performed with the 07iCTR-n07 cell line. Data are shown as mean ± SEM (n=3 / experiment). Reag.: reaggregation. [Figure 21] FIG. 21 shows the pluripotency of iECs. [Figure 22]22A-22B show the development of a protocol to differentiate iPSCs into endothelial cells; (22A) schematic of the protocol, (22B) protein expression of key vascular endothelial markers in iECs at day 21 in five different iPSC lines. [Figure 23] Figures 23A-23B show further characterization of key markers of iECs; (22A) protein expression of endothelial markers CD31 and CD144 assessed by flow cytometry at day 11, (22B) gene expression of pluripotency markers OCT4 and SOX2 assessed by RT-qPCR at multiple days of iEC differentiation. [Figure 24] Figures 24A-24B show the addition of Angiopoietin-1 to enhance expression of VEGFA and CD31 iEC markers until day 11; (24A) protein expression of endothelial markers VEGFA and CD31 at day 11, (24B) percentage of cells expressing iEC markers in different conditions. [Diagram 25] 25A-25B show the addition of angiopoietin 1 to enhance expression of the CD144 marker until day 11; (25A) protein expression of the endothelial marker CD144 at day 11, (25B) percentage of cells expressing iEC markers in different conditions. [Figure 26] 26A-26B show the addition of angiopoietin 1 to enhance expression of VEGFA and CD31 iEC markers until day 21. (26A) Protein expression of endothelial markers VEGFA and CD31 at day 21. (26B) Percentage of cells expressing iEC markers in different conditions. [Figure 27] 27A-27B show the addition of angiopoietin 1 to enhance expression of the CD144 marker until day 21; (27A) Protein expression of the endothelial marker CD144 at day 21. (27B) Percentage of cells expressing iEC markers in different conditions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] Detailed Description All references cited herein are incorporated herein by reference in their entirety as if fully set forth.Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.Singleton et al., Dictionary of Microbiology and Molecular Biology 3rd Edition, Revised Edition, J. Wiley & Sons (New York, NY 2006); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4th Edition, Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012) provide those skilled in the art with a general guide to many of the terms used in this application.

[0051] One skilled in the art will recognize that many methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, and indeed the present invention is not limited to the methods and materials described.

[0052] The term "about" as used herein, when used in conjunction with a referenced numerical indication, means the referenced numerical indication plus or minus 5% unless otherwise specified herein. For example, the expression "about 50%" covers a range of 45% to 55%. In various embodiments, the term "about" when used in conjunction with a referenced numerical indication, when otherwise specified in the claims, can mean the referenced numerical indication plus or minus 4%, 3%, 2%, 1%, 0.5%, or 0.25% of the referenced numerical indication.

[0053] As used herein, the term "glucose responsive" refers to the ability of cells to secrete insulin when challenged in a glucose stimulation assay.

[0054] As used herein, the term "reproducible" when used in conjunction with the various differentiation methods described herein refers to a method that has been successful in at least three independent rounds of differentiation.

[0055] As used herein, "MATRIGEL" refers to solubilized basement membrane matrix secreted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, prepared, for example, by Corning Life Sciences.

[0056] As used herein, "subject" refers to a human or an animal. Typically, an animal is a vertebrate, such as a primate, a rodent, a livestock animal, or a game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaque monkeys, such as Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Livestock and game animals include cattle, horses, pigs, deer, bison, buffalo, felines, such as domestic cats, and canines, such as dogs, foxes, and wolves. The terms "patient," "individual," and "subject" are used interchangeably herein. In some embodiments, the subject is a mammal. The mammal can be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. Additionally, the methods described herein can be used to treat livestock and / or pets. In some embodiments, the subject is a human.

[0057] The subject may be one who has been previously diagnosed or confirmed as suffering from or having a disease, disorder or condition that requires treatment, or one or more complications associated with the disease, disorder or condition, and may optionally have already been treated for the disease, disorder or condition, or one or more complications associated with the disease, disorder or condition. Alternatively, the subject may not have been previously diagnosed with the disease, disorder or condition, or one or more complications associated with the disease, disorder or condition. For example, the subject may or may not exhibit one or more risk factors for the disease, disorder or condition, or one or more complications associated with the disease, disorder or condition. A "subject in need" of treatment for a particular disease, disorder or condition may be one who is suspected of having the disease, disorder or condition, has been diagnosed with the disease, disorder or condition, has already been treated or is being treated for the disease, disorder or condition, is not being treated for the disease, disorder or condition, or is at risk of developing the disease, disorder or condition.

[0058] We herein describe, inter alia, an isogenic co-culture method using iPSC-derived islets (iIslets) and endothelial cells (iECs) from the same donor; this method results in better maturation and functionality of iIslets that show high expression of β-cell markers and synchronous insulin secretion with high glucose loading. To our knowledge, this is the first time that iIslets and iECs have been generated from the same iPSC donor and that their co-culture resulted in significantly more defined and functional β-cells.

[0059] Human induced pluripotent stem cells (iPSCs) are derived from adult somatic cells, such as skin or blood cells, that have been genetically reprogrammed into an embryonic stem cell-like state, endowing them with the ability to grow indefinitely (self-renewal) and to give rise to the desired cells of the body from the three germ layers using a specific cocktail of small molecules, transcription factors, and growth factors; thus, iPSCs are a source for generating an unlimited number of differentiated cells. Human embryonic stem cells (ESCs) obtained from discarded embryos have been used in some cases, but this raises several ethical questions. Apart from ethical limitations, β-cell therapy derived from a single allogeneic cell source, such as an ESC line, is likely to be refractory to the recipient due to alloimmunization against human leukocyte antigens (HLA). To avoid possible immunogenicity due to ESC-derived β-cells, a solution would be to utilize iPSC-derived β-cells from the patient's own blood. Alternatively, the use of iPSC haplobanks pooling donors with the most frequent homozygous HLA haplotypes selected for maximum utility to match the intended US recipient population would allow for the scalability of such an approach. Furthermore, most strategies for generating β cells from ESCs have been directed at polyhormonal cells, i.e., insulin-resistant cells. + / Glucagon + / Somatostatin + These cells produce the monohormonal insulin + β-cells cannot maintain their normal glucose state and, moreover, have a low glucose threshold for insulin secretion in vitro that is only amplified several weeks after in vivo transplantation, at which point these cells acquire a more mature profile. Thus, complex signals are likely key to β-cell maturation in vivo, and testing these signals in vitro is urgently needed to develop improved protocols.

[0060] Several signals known to be important for the development and maturation of the endocrine pancreas are provided by the surrounding vascular endothelial cells (ECs), but these signals have not been included in previous protocols for differentiation of β-cells from stem cells. ECs are part of the vasculature and are now considered to be active organs that are important not only for the function of the vasculature but also for the function of organs throughout the body. In particular, in the endocrine pancreas, the islets require a large blood supply from the blood vessels due to their active and active role in maintaining glycemia by sensing external signals such as glucose levels and secreting hormones such as insulin, glucagon, and somatostatin. Although knowledge in this field has progressed, the exact mechanisms and pathways underlying the interaction of ECs with the endocrine pancreas have not been fully elucidated; we believe that they are key to the development and maturation of iPSC-derived β-cells in vitro, and this has been explored in our studies, as further described herein.

[0061] Various embodiments of the present invention provide methods of generating functional induced pluripotent stem cell (iPSC)-derived pancreatic islets (iIslets), the methods comprising co-culturing a quantity of iPSC-derived endothelial cells (iECs) with a quantity of iPSC-derived islet precursors for about 10-18 days to generate functional iIslets comprising β cells.

[0062] In various embodiments, the co-culturing step comprises plating a quantity of iPSC-derived endothelial cells (iECs) on a MATRIGEL-coated plate and culturing in phase IV EC medium supplemented with Y27632; plating a quantity of iPSC-derived islets (iIslets) on the quantity of iECs and culturing for about 12-16 days in medium comprising about 1 / 2 phase IV iEC medium and about 1 / 2 phase VI islet medium supplemented with Y-27632 or in phase VI islet medium (islet-only condition) for about 12-16 days to generate functional iIslets comprising β cells. In various embodiments, the concentration of Y27632 is about 10 μM. In various embodiments, the concentration of Y27632 is about 8-12 μM.

[0063] In various embodiments, the co-culturing step comprises plating a quantity of iPSC-derived vascular endothelial cells (iECs) onto a MATRIGEL-coated plate and culturing in Phase IV EC medium supplemented with Y27632; plating a quantity of iPSC-derived islets (iIslets) on top of the quantity of iECs and culturing either in medium comprising about ½ Phase IV iEC medium and about ½ Phase VI islet medium supplemented with Y-27632 for about 14 days or in Phase VI islet medium (islet-only condition) for about 14 days to generate functional iIslets comprising beta cells.

[0064] In various embodiments, the method includes generating iECs prior to co-culturing, where the iECs are generated by plating a quantity of induced pluripotent stem cells (iPSCs) onto MATRIGEL; culturing the iPSCs in MATRIGEL for about 2-4 days; culturing in the presence of CHIR99021 for about 1-3 days to generate mesoderm; culturing the mesoderm in the presence of BMP4, FGF2, and VEGF for about 1-3 days to generate vascular precursors; and culturing the vascular precursors in the presence of VEGF and Y-27632 for about 3-8 days to generate endothelial cell (EC) precursors.

[0065] In various embodiments, the method includes generating iECs prior to co-culturing, where the iECs are generated by plating a quantity of induced pluripotent stem cells (iPSCs) onto MATRIGEL; culturing the iPSCs in MATRIGEL for about 3 days; culturing in the presence of CHIR99021 for about 2 days to generate mesoderm; culturing the mesoderm in the presence of BMP4, FGF2, and VEGF for about 2 days to generate vascular precursors; and culturing the vascular precursors in the presence of VEGF and Y-27632 for about 4-7 days to generate endothelial cell (EC) precursors.

[0066] In various embodiments for generating iECs, iPSCs are planarly seeded as small cell colonies onto MATRIGEL-coated plates. In various embodiments, the concentration of CHIR99021 is about 4-8 μM. In various embodiments, the concentration of CHIR99021 is about 6 μM. In various embodiments, the concentrations of BMP4, FGF2, and VEGF are about 25 ng / ml (BMP4), about 10 ng / ml (FGF2), and about 50 ng / ml (VEGF). In various embodiments, the concentrations of BMP4, FGF2, and VEGF are about 20-30 ng / ml (BMP4), about 8-12 ng / ml (FGF2), and about 40-60 ng / ml (VEGF). In various embodiments, the medium is changed about every other day. In various embodiments, the medium is changed about every day. In various embodiments, the medium is changed about every other day.

[0067] In various embodiments, the method includes producing the amount of islet precursors prior to co-culturing, wherein the islet precursors are produced by culturing a quantity of induced pluripotent stem cells (iPSCs) in the presence of activin A, CHIR99021, and a ROCK inhibitor (e.g., Y-27632) for about 1-2 days; culturing in the presence of activin A and FGF2 for about 1-3 days; culturing in the presence of FGF10, CHIR99021, and Noggin for about 1-3 days to produce posterior foregut cells; culturing the posterior foregut cells in the presence of FGF10, Noggin, RA, and SANT1 for about 3-5 days to produce pancreatic precursors; culturing the pancreatic precursors in the presence of Noggin, EGF, and nicotinamide for about 3-5 days to produce pancreatic endocrine precursors; and culturing the pancreatic endocrine precursors in the presence of Noggin, T3, and Alk5i II for about 6-8 days to produce islet precursors.

[0068] In various embodiments, the method includes producing the amount of islet precursors prior to co-culturing, wherein the islet precursors are produced by culturing a quantity of induced pluripotent stem cells (iPSCs) in the presence of activin A, CHIR99021 and a ROCK inhibitor (e.g., Y-27632) for about 1 day; culturing in the presence of activin A and FGF2 for about 2 days; culturing in the presence of FGF10, CHIR99021 and Noggin for about 2 days to produce posterior foregut cells; culturing the posterior foregut cells in the presence of FGF10, Noggin, RA and SANT1 for about 4 days to produce pancreatic precursors; culturing the pancreatic precursors in the presence of Noggin, EGF and nicotinamide for about 4 days to produce pancreatic endocrine precursors; culturing the pancreatic endocrine precursors in the presence of Noggin, T3 and Alk5i II for about 7 days to produce pancreatic islet precursors.

[0069] In various embodiments to generate said quantities of islet precursors, iPSCs are dissociated into single cells and replated with a ROCK inhibitor (e.g., Y-27632) at approximately 300,000 cells / cm on MATRIGEL-coated plates. 2 In various embodiments, the density is about 250,000 to 350,000 cells / cm. 2In various embodiments, the concentration of the ROCK inhibitor is about 10 mM. In various embodiments, the concentration of the ROCK inhibitor is about 8-12 mM. In various embodiments, the concentrations of activin A, CHIR99021, and Y-27632 are about 80-120 ng / ml (activin A), about 1-3 μM CHIR99021, and about 8-12 μM (Y-27632). In various embodiments, the concentrations of activin A, CHIR99021, and Y-27632 are about 100 ng / ml (activin A), about 2 μM CHIR99021, and about 10 μM (Y-27632). In various embodiments, the concentrations of activin A and FGF2 are about 80-120 ng / ml (activin A) and about 4-6 ng / ml FGF2 (FGF2). In various embodiments, the concentrations of activin A and FGF2 are about 100 ng / ml (activin A) and about 5 ng / ml FGF2 (FGF2). In various embodiments, the concentrations of FGF10, noggin, RA, and SANT1 are about 40-60 ng / ml FGF10, about 40-60 ng / ml noggin, about 1-3 μM RA, and about 0.2-0.3 μM SANT1. In various embodiments, the concentrations of FGF10, noggin, RA, and SANT1 are about 50 ng / ml FGF10, about 50 ng / ml noggin, about 2 μM RA, and about 0.25 μM SANT1. In various embodiments, the concentrations of noggin, EGF, and nicotinamide are about 40-60 ng / ml noggin, about 80-120 ng / ml EGF, and about 8-12 mM nicotinamide. In various embodiments, the concentrations of Noggin, EGF, and nicotinamide are about 50 ng / ml Noggin, about 100 ng / ml EGF, and about 10 mM nicotinamide. In various embodiments, the concentrations of Noggin, T3, and Alk5i II are about 40-60 ng / ml Noggin, about 0.5-1.5 μM T3, and about 8-10 μM Alk5i II. In various embodiments, the concentrations of Noggin, T3, and Alk5i II are about 50 ng / ml Noggin, about 1 μM T3, and about 10 μM Alk5i II. In various embodiments, the medium is changed about every other day. In various embodiments, the medium is changed about every day. In various embodiments, the medium is changed about every other day.

[0070] In various embodiments, the pancreatic precursors express PDX1+ and SOX9+.

[0071] In various embodiments, the pancreatic endocrine precursors are PDX1+ and NKX6.1+.

[0072] In various embodiments, the iIslet expresses C-peptide, glucagon, and NKX6.1+.

[0073] In various embodiments, expression of INS, UCN3, NGN3 and CHGA is upregulated in beta cells produced in islet-only conditions compared to beta cells produced without co-culture with vascular endothelial cells or compared to beta cells produced in cultures without islet-only conditions.

[0074] In various embodiments, the beta cells increase insulin secretion when challenged with a high glucose concentration compared to a basal glucose concentration. In various embodiments, the beta cells increase insulin secretion by at least 50% when challenged with a high glucose concentration compared to a basal glucose concentration. In various embodiments, the beta cells increase insulin secretion by at least 100% when challenged with a high glucose concentration compared to a basal glucose concentration. In various embodiments, the beta cells increase insulin secretion by at least 200% when challenged with a high glucose concentration compared to a basal glucose concentration. In various embodiments, the beta cells increase insulin secretion by at least 300% when challenged with a high glucose concentration compared to a basal glucose concentration. In various embodiments, the beta cells increase insulin secretion by at least 400% when challenged with a high glucose concentration compared to a basal glucose concentration. In various embodiments, the beta cells increase insulin secretion by at least 500% when challenged with a high glucose concentration compared to a basal glucose concentration.

[0075] In various embodiments, the iPSC-derived vascular endothelial cells (iECs) and the iPSC-derived pancreatic islet precursors are isogenic.

[0076] In various embodiments, the iPSCs used to derive vascular endothelial cells (iECs) and the iPSCs used to derive pancreatic islet precursors are from the same iPSC cell line or from the same donor.

[0077] In various embodiments, the iPSCs used to induce endothelial cells (iECs) and the iPSCs used to induce islet precursors are generated from the subject who will receive the iIslet. That is, the source cells of the iPSCs and the generated iIslets are personalized to the same person.

[0078] In various embodiments, the iIslet is a human iIslet.

[0079] Various aspects of the present invention provide iIslets made by any one of the methods of the invention described herein.

[0080] Various aspects of the present invention provide iIslet.In various aspects, iIslet expresses C-peptide, glucagon and NKX6.1+.In various aspects, iIslet increases insulin secretion when loaded with high glucose concentration compared to basal glucose concentration.

[0081] In various embodiments, iIslet is from the composition comprising iIslet produced by the inventive method described herein.For example, iIslet is produced by the inventive method of the present invention, and then the iIslet is frozen for storage.In another example, iIslet is produced by the inventive method of the present invention, and the iIslet is passaged multiple times.Therefore, these passaged iIslet are included in the composition comprising iIslet produced by the inventive method described herein, even if they are not produced directly by the inventive method described herein.

[0082] Various aspects of the invention provide methods of ameliorating or treating a metabolic disease, disorder, or condition in a subject in need thereof, comprising administering an iIslet of the invention to the subject to ameliorate or treat the condition. In various aspects, the metabolic disease, disorder, or condition is diabetes or insulin resistance.

[0083] Various aspects of the present invention provide methods of administering an iIslet of the present invention to a subject in need thereof, the methods comprising administering an iIslet of the present invention to a subject in need thereof, the subject being in need of amelioration or treatment of a metabolic disease, disorder or condition. In various aspects, the metabolic disease, disorder or condition is diabetes or insulin resistance.

[0084] Various embodiments described herein provide a method of cell differentiation, comprising providing a quantity of induced pluripotent stem cells (iPSCs), culturing them in the presence of activin A, CHIR99021 and Y-27632 for about 1 day, further culturing them in the presence of activin A and FGF2 for about 2 days, and further culturing them in the presence of FGF10, CHIR99021 and Noggin for about 2 days to produce a posterior foregut. In other embodiments, the method comprises culturing the posterior foregut in the presence of FGF10, Noggin, RA and SANT1 for about 4 days to produce a pancreatic progenitor. In other embodiments, the pancreatic progenitor is a PDX1 + and SOX9 + In other embodiments, the pancreatic precursor expresses one or more of PDX1. + and SOX9 + In another embodiment, the method includes culturing the pancreatic progenitors in the presence of noggin, EGF and nicotinamide for about 4 days to generate pancreatic endocrine progenitors. In another embodiment, the pancreatic endocrine progenitors express PDX1. + and NKX6.1 + In other embodiments, the pancreatic endocrine precursor expresses one or more of PDX1. + and NKX6.1 +In other embodiments, the method comprises culturing the pancreatic endocrine precursors in the presence of noggin, T3 and Alk5i II for about 7 days to generate pancreatic islet precursors. In some embodiments, the medium is changed every other day. In other embodiments, the method comprises culturing the generated pancreatic islet precursors in the presence of T3, Alk5i II, R428 and N-acetylcysteine ​​(NAC) for about 14 days to generate mature pancreatic islets. In some embodiments, the medium is changed every other day. In other embodiments, the mature pancreatic islets are expressed in C-peptide, glucagon and NKX6.1. + In other embodiments, the mature pancreatic islets express one or more of C-peptide, glucagon and NKX6.1. + In other embodiments, the mature pancreatic islets express one or more of insulin (INS), urocortin 3 (UCN3), neurogenin 3 (NGN3) and chromogranin A (CHGA). In other embodiments, the mature pancreatic islets express insulin (INS), urocortin 3 (UCN3), neurogenin 3 (NGN3) and chromogranin A (CHGA). In various embodiments, the pancreatic islet precursors are multihormonal. In various embodiments, the mature pancreatic islets are monohormonal. For example, the multihormonal cells express insulin, + / Glucagon + / Somatostatin + On the other hand, monohormonal cells express C-peptide + / Glucagon - In various embodiments, the mature islets secrete insulin and / or are glucose responsive. Each of the above growth factors is added at a concentration of 0.25ng / ml to 250ng / ml, and the small molecules are added at 0.1μM to 2.5mM. In various embodiments, each of the above growth factors is added at a concentration of about 0.25ng / ml to 1ng / ml, or 1ng / ml to 10ng / ml, or 10ng / ml to 100ng / ml, or 100ng / ml to 200ng / ml, or 200ng / ml to 300ng / ml. In various embodiments, each of the above small molecules is added at a concentration of about 0.1μM to 1μM, or 1μM to 10μM, or 10μM to 50μM, or 50μM to 100μM, or 100μM to 1mM, or 1mM to 5mM.

[0085] For example, iPSCs (OCT4 expression >90%) maintained on MATRIGEL-coated plates were dissociated into single cells with Accutase, re-seeded with the ROCK inhibitor Y-27632 (10 mM, R&D Systems), and incubated with mTeSR + 300,000 cells / cm using culture medium 2 After 24 hours, iPSCs were directed toward definitive endoderm (DE - Phase I) using a combination of Activin A (100ng / ml, R&D Systems), CHIR99021 (2μM, XcessBio), and Y-27632 (10μM) for 1 day, followed by Activin A (100ng / ml) and FGF2 (5ng / ml, PeproTech) for 2 days. Next, for posterior foregut (PFG - Phase II) patterning, a combination of FGF10 (50ng / ml, PeproTech), CHIR99021 (0.25μM), and Noggin (50ng / ml, PeproTech) was used for 2 days. The cells were then cultured as PDX1 + / SOX9 + To direct pancreatic progenitors (PP - phase III), a combination of FGF10 (50ng / ml), Noggin (50ng / ml), RA (2μM, Cayman) and SANT1 (0.25μM, Sigma) was used for 4 days, followed by treatment with Noggin (50ng / ml), EGF (100ng / ml, PeproTech) and nicotinamide (10mM, Sigma) for 4 days to induce PDX1 + / NKX6.1 +Pancreatic endocrine precursors (PEP - phase IV) were induced. To generate islet precursors (IP - phase V), a combination of Noggin (50 ng / ml), T3 (1 μM, Sigma), and Alk5i II (10 μM, Axxora) was used for 7 days, during which the medium was changed every other day. For islet maturation (MI - phase VI), a combination of T3 (1 μM), Alk5i II (10 μM), AXL inhibitor R428 (2 mM, Selleckchem), and antioxidant N-acetylcysteine ​​NAC (1 mM, Sigma) was used for 14 days, during which the medium was changed every other day.

[0086] In various embodiments, iPSCs are dissociated into single cells and re-seeded with a ROCK inhibitor (e.g., Y-27632) at approximately 300,000 cells / cm. 2 The cells were planarly seeded onto MATRIGEL-coated plates at a density of about 250,000-350,000 cells / cm. In various embodiments, the density is about 250,000-350,000 cells / cm. 2In various embodiments, the concentration of the ROCK inhibitor is about 10 mM. In various embodiments, the concentration of the ROCK inhibitor is about 8-12 mM. In various embodiments, the concentrations of activin A, CHIR99021, and Y-27632 are about 80-120 ng / ml (activin A), about 1-3 μM CHIR99021, and about 8-12 μM (Y-27632). In various embodiments, the concentrations of activin A, CHIR99021, and Y-27632 are about 100 ng / ml (activin A), about 2 μM CHIR99021, and about 10 μM (Y-27632). In various embodiments, the concentrations of activin A and FGF2 are about 80-120 ng / ml (activin A) and about 4-6 ng / ml FGF2 (FGF2). In various embodiments, the concentrations of activin A and FGF2 are about 100 ng / ml (activin A) and about 5 ng / ml FGF2 (FGF2). In various embodiments, the concentrations of FGF10, noggin, RA, and SANT1 are about 40-60 ng / ml FGF10, about 40-60 ng / ml noggin, about 1-3 μM RA, and about 0.2-0.3 μM SANT1. In various embodiments, the concentrations of FGF10, noggin, RA, and SANT1 are about 50 ng / ml FGF10, about 50 ng / ml noggin, about 2 μM RA, and about 0.25 μM SANT1. In various embodiments, the concentrations of noggin, EGF, and nicotinamide are about 40-60 ng / ml noggin, about 80-120 ng / ml EGF, and about 8-12 mM nicotinamide. In various embodiments, the concentrations of Noggin, EGF, and nicotinamide are about 50 ng / ml Noggin, about 100 ng / ml EGF, and about 10 mM nicotinamide. In various embodiments, the concentrations of Noggin, T3, and Alk5i II are about 40-60 ng / ml Noggin, about 0.5-1.5 μM T3, and about 8-10 μM Alk5i II. In various embodiments, the concentrations of Noggin, T3, and Alk5i II are about 50 ng / ml Noggin, about 1 μM T3, and about 10 μM Alk5i II. In various embodiments, the medium is changed about every other day. In various embodiments, the medium is changed about every day. In various embodiments, the medium is changed about every other day.

[0087] Various embodiments described herein provide a quantity of mature pancreatic islets derived from induced pluripotent stem cells (iPSCs). In other embodiments, the mature pancreatic islets express C-peptide, glucagon and NKX6.1. + In other embodiments, the mature pancreatic islets express one or more of C-peptide, glucagon and NKX6.1. + In other embodiments, the mature pancreatic islets express one or more of insulin (INS), urocortin 3 (UCN3), neurogenin 3 (NGN3) and chromogranin A (CHGA). In other embodiments, the mature pancreatic islets express insulin (INS), urocortin 3 (UCN3), neurogenin 3 (NGN3) and chromogranin A (CHGA). In various embodiments, the pancreatic islet precursors are multihormonal. In various embodiments, the mature pancreatic islets are monohormonal. For example, the multihormonal cells express insulin, + / Glucagon + / Somatostatin + On the other hand, monohormonal cells express C-peptide + / Glucagon - In various embodiments, the mature islet secretes insulin and / or is glucose responsive. In various embodiments, the mature islet cell is glucose responsive in glucose stimulated insulin secretion (GSIS) assay. In various embodiments, the GSIS assay is static. In various embodiments, the GSIS assay is dynamic. In various embodiments, GSIS includes mature islet cells forming an assembly with mature endothelial cells organized as a vascularized channel, where the vascularized channel can provide glucose loading to the mature islet cell.

[0088] Various embodiments described herein provide a quantity of mature pancreatic islets produced by the method, which comprises providing a quantity of induced pluripotent stem cells (iPSCs) and culturing them in the presence of activin A, CHIR99021 and Y-27632 for about 1 day, further culturing them in the presence of activin A and FGF2 for about 2 days, and further culturing them in the presence of FGF10, CHIR99021 and Noggin for about 2 days to produce a posterior foregut. In other embodiments, the method comprises culturing the posterior foregut in the presence of FGF10, Noggin, RA and SANT1 for about 4 days to produce pancreatic progenitors. In other embodiments, the pancreatic progenitors are PDX1. + and SOX9 + In other embodiments, the pancreatic precursor expresses one or more of PDX1. + and SOX9 + In another embodiment, the method includes culturing the pancreatic progenitors in the presence of noggin, EGF and nicotinamide for about 4 days to generate pancreatic endocrine progenitors. In another embodiment, the pancreatic endocrine progenitors express PDX1. + and NKX6.1 + In other embodiments, the pancreatic endocrine precursor expresses one or more of PDX1. + and NKX6.1 + In other embodiments, the method comprises culturing the pancreatic endocrine precursors in the presence of noggin, T3 and Alk5i II for about 7 days to generate pancreatic islet precursors. In some embodiments, the medium is changed every other day. In other embodiments, the method comprises culturing the generated pancreatic islet precursors in the presence of T3, Alk5i II, R428 and N-acetylcysteine ​​(NAC) for about 14 days to generate mature pancreatic islets. In some embodiments, the medium is changed every other day. In other embodiments, the mature pancreatic islets are expressed in C-peptide, glucagon and NKX6.1. + In other embodiments, the mature pancreatic islets express one or more of C-peptide, glucagon and NKX6.1. +In other embodiments, the mature pancreatic islets express one or more of insulin (INS), urocortin 3 (UCN3), neurogenin 3 (NGN3) and chromogranin A (CHGA). In other embodiments, the mature pancreatic islets express two or more of insulin (INS), urocortin 3 (UCN3), neurogenin 3 (NGN3) and chromogranin A (CHGA). In other embodiments, the mature pancreatic islets express three or more of insulin (INS), urocortin 3 (UCN3), neurogenin 3 (NGN3) and chromogranin A (CHGA). In other embodiments, the mature pancreatic islets express insulin (INS), urocortin 3 (UCN3), neurogenin 3 (NGN3) and chromogranin A (CHGA). In various embodiments, the pancreatic islet precursors are polyhormonal. In various embodiments, the mature pancreatic islets are monohormonal. For example, multihormonal cells can express insulin + / Glucagon + / Somatostatin + On the other hand, monohormonal cells may be + / Glucagon - In various embodiments, mature pancreatic islets secrete insulin and / or are glucose responsive. In various embodiments, mature pancreatic islet cells are glucose responsive in glucose stimulated insulin secretion (GSIS) assay. In various embodiments, GSIS assay is static. In various embodiments, GSIS assay is dynamic. In various embodiments, GSIS comprises mature pancreatic islet cells forming an assembly with mature endothelial cells organized as angiogenic channels, where the angiogenic channels can provide glucose loading to mature pancreatic islet cells.

[0089] Various embodiments described herein provide a method of cell differentiation, the method comprising providing a quantity of induced pluripotent stem cells (iPSCs), plating the iPSCs on MATRIGEL, culturing the iPSCs in MATRIGEL for about 3 days, and further culturing in the presence of CHIR99021 to generate mesoderm. In other embodiments, the method comprises culturing the mesoderm in the presence of BMP4, FGF2, and VEGF for about 2 days to generate vascular precursors.

[0090] In various embodiments, iPSCs are planarly seeded as small cell colonies on MATRIGEL-coated plates. In various embodiments, the concentration of CHIR99021 is about 4-8 μM. In various embodiments, the concentration of CHIR99021 is about 6 μM. In various embodiments, the concentrations of BMP4, FGF2, and VEGF are about 25 ng / ml (BMP4), about 10 ng / ml (FGF2), and about 50 ng / ml (VEGF). In various embodiments, the concentrations of BMP4, FGF2, and VEGF are about 20-30 ng / ml (BMP4), about 8-12 ng / ml (FGF2), and about 40-60 ng / ml (VEGF). In various embodiments, the medium is changed about every other day. In various embodiments, the medium is changed about every day. In various embodiments, the medium is changed about every other day.

[0091] In some embodiments, the vascular progenitors are dissociated and cultured at approximately 85,000-100,000 cells / cm. 2 In some embodiments, the vascular progenitors are dissociated and replated at a density of about 85,000 cells / cm. 2 , or approximately 85,000 cells / cm 2 , or approximately 90,000 cells / cm 2 , or approximately 95,000 cells / cm 2 , or approximately 100,000 cells / cm 2 , or approximately 105,000 cells / cm 2In other embodiments, the method includes culturing the vascular progenitors in the presence of VEGF and Y-27632 for about 7 days to generate endothelial cell (EC) progenitors. In some embodiments, the medium is changed every other day. In other embodiments, the EC progenitors are cultured for an additional about 3 days. In other embodiments, the EC progenitors are dissociated and replated on MATRIGEL-coated plates. In other embodiments, the method includes culturing the EC progenitors with VEGF for about 10 days to generate mature ECs. In some embodiments, the mature ECs are replated on MATRIGEL-coated plates. In other embodiments, the method includes culturing the mature ECs with VEGF for about 10 days. In other embodiments, the vascular progenitors and endothelial progenitors are cultured in the presence of angiopoietin 1. In various embodiments, the vascular progenitors and endothelial progenitors are cultured in the presence of angiopoietin 1 from day 4 to day 21 of differentiation. In other embodiments, the method includes culturing the vascular precursors in the presence of EGM-MV2 and VEGF for about 4-6 days to generate endothelial progenitor cells, and culturing the endothelial progenitor cells in the presence of EGM-MV2 and VEGF to generate mature endothelial cells. In some embodiments, the medium is changed every other day. In other embodiments, the mature ECs are CD31 + , CD144 + , VEGF-A + , VEGFR2 + In other embodiments, the mature EC expresses one or more of CD31 + , CD144 + , VEGF-A + , VEGFR2 + , and Ac-LDL. Each of the above growth factors is added at a concentration of 0.25 ng / ml to 250 ng / ml, and small molecules are added at 0.1 μM to 2.5 mM.

[0092] For example, to generate pancreatic islets (iIslets) from iPSCs, we developed a robust and reproducible protocol. Briefly, iPSCs (OCT4 expression >90%) maintained on MATRIGEL-coated plates were dissociated into single cells with Accutase, replated with the ROCK inhibitor Y-27632 (10 mM, R&D Systems), and incubated with mTeSR. + 300,000 cells / cm using culture medium 2 After 24 hours, iPSCs were directed toward definitive endoderm (DE - Phase I) using a combination of Activin A (100ng / ml, R&D Systems), CHIR99021 (2μM, XcessBio), and Y-27632 (10μM) for 1 day, followed by Activin A (100ng / ml) and FGF2 (5ng / ml, PeproTech) for 2 days. Next, for posterior foregut (PFG - Phase II) patterning, a combination of FGF10 (50ng / ml, PeproTech), CHIR99021 (0.25μM), and Noggin (50ng / ml, PeproTech) was used for 2 days. The cells were then cultured as PDX1 + / SOX9 + To direct pancreatic progenitors (PP - phase III), a combination of FGF10 (50ng / ml), Noggin (50ng / ml), RA (2μM, Cayman) and SANT1 (0.25μM, Sigma) was used for 4 days, followed by treatment with Noggin (50ng / ml), EGF (100ng / ml, PeproTech) and nicotinamide (10mM, Sigma) for 4 days to induce PDX1 + / NKX6.1 +Pancreatic endocrine precursors (PEP - phase IV) were induced. To generate islet precursors (IP - phase V), a combination of Noggin (50 ng / ml), T3 (1 μM, Sigma), and Alk5i II (10 μM, Axxora) was used for 7 days, during which the medium was changed every other day. For maturation of islets (MI - phase VI), a combination of T3 (1 μM), Alk5i II (10 μM), AXL inhibitor R428 (2 mM, Selleckchem), and antioxidant N-acetylcysteine ​​NAC (1 mM, Sigma) was used for 14 days, during which the medium was changed every other day. The formulation of the basal medium used throughout differentiation is summarized in Table 2.

[0093] Various embodiments described herein provide mature endothelial cells (ECs) derived from a quantity of induced pluripotent stem cells (iPSCs). In other embodiments, the mature ECs are CD31 + , CD144 + , VEGF-A + , VEGFR2 + In other embodiments, the mature EC expresses one or more of CD31 + , CD144 + , VEGF-A + , VEGFR2 + In another embodiment, the mature EC expresses two or more of CD31 + , CD144 + , VEGF-A + , VEGFR2 + In another embodiment, the mature EC expresses three or more of CD31 + , CD144 + , VEGF-A + , VEGFR2 + In another embodiment, the mature EC expresses four or more of CD31 + , CD144 + , VEGF-A + , VEGFR2 + , and Ac-LDL.

[0094] Various embodiments described herein provide a quantity of mature endothelial cells (ECs) produced by the method; the method includes providing a quantity of induced pluripotent stem cells (iPSCs), plating the iPSCs on MATRIGEL, culturing the iPSCs in MATRIGEL for about 3 days, and further culturing in the presence of CHIR99021 to produce mesoderm. In other embodiments, the method includes culturing the mesoderm in the presence of BMP4, FGF2, and VEGF for about 2 days to produce vascular precursors. In other embodiments, the method includes culturing the vascular precursors in the presence of VEGF and Y-27632 for about 7 days to produce endothelial cell (EC) precursors. In some embodiments, the medium is changed every other day. In other embodiments, the vascular precursors are enzymatically dissociated (e.g., with accutase) and plated on MATRIGEL-coated plates at 85,000-100,000 cells / cm. 2 The EC precursors are then replated at a density of 100-1500 nm to generate EC precursors. In other embodiments, the method includes culturing the EC precursors with VEGF for about 10 days to generate mature ECs. In other embodiments, the vascular precursors and endothelial precursors are cultured in the presence of angiopoietin 1. In various embodiments, the vascular precursors and endothelial precursors are cultured in the presence of angiopoietin 1 from day 4 to day 21 of differentiation. In other embodiments, the method includes culturing the vascular precursors in the presence of EGM-MV2 and VEGF for about 4-6 days to generate mature endothelial progenitor cells, and culturing the endothelial progenitor cells in the presence of EGM-MV2 and VEGF to generate mature endothelial cells. In some embodiments, the medium is changed every other day. In other embodiments, the mature ECs are expressed by CD31 + , CD144 + , VEGF-A + , VEGFR2 + In other embodiments, the mature EC expresses one or more of CD31 + , CD144 + , VEGF-A + , VEGFR2 + In another embodiment, the mature EC expresses two or more of CD31 + , CD144 + , VEGF-A + , VEGFR2+ In another embodiment, the mature EC expresses three or more of CD31 + , CD144 + , VEGF-A + , VEGFR2 + In another embodiment, the mature EC expresses four or more of CD31 + , CD144 + , VEGF-A + , VEGFR2 + , and Ac-LDL.

[0095] Various embodiments described herein provide an assembly comprising a quantity of mature islets and a quantity of mature ECs. In other embodiments, the mature islets and mature ECs are isogenic. In some embodiments, the assembly further comprises a construct such as a scaffold. In various embodiments, the mature islets, mature ECs, or both are attached onto the scaffold. In various embodiments, the mature islets, mature ECs, or both are attached onto the scaffold using a bioink. In various embodiments, the bioink includes fibrin or alginate.

[0096] In another embodiment, the mature pancreatic islets are produced by a method comprising the steps of: preparing a quantity of induced pluripotent stem cells (iPSCs), culturing them in the presence of activin A, CHIR99021 and Y-27632 for about 1 day, further culturing them in the presence of activin A and FGF2 for about 2 days, and further culturing them in the presence of FGF10, CHIR99021 and Noggin for about 2 days to produce a posterior foregut. In another embodiment, the method comprises culturing the posterior foregut in the presence of FGF10, Noggin, RA and SANT1 for about 4 days to produce a pancreatic progenitor. In another embodiment, the pancreatic progenitor is PDX1. + and SOX9 + In other embodiments, the pancreatic precursor expresses one or more of PDX1. + and SOX9 +In another embodiment, the method includes culturing the pancreatic progenitors in the presence of noggin, EGF and nicotinamide for about 4 days to generate pancreatic endocrine progenitors. In another embodiment, the pancreatic endocrine progenitors express PDX1. + and NKX6.1 + In other embodiments, the pancreatic endocrine precursor expresses one or more of PDX1. + and NKX6.1 + In another embodiment, the method comprises culturing the pancreatic endocrine precursors in the presence of noggin, T3 and Alk5i II for about 7 days to generate pancreatic islet precursors. In another embodiment, the method comprises culturing the generated pancreatic islet precursors in the presence of T3, Alk5i II, R428 and N-acetylcysteine ​​(NAC) for about 14 days to generate mature pancreatic islets. In another embodiment, the mature pancreatic islets are expressed in the presence of C-peptide, glucagon and NKX6.1. + In other embodiments, the mature pancreatic islets express one or more of C-peptide, glucagon and NKX6.1. + In other embodiments, the mature pancreatic islets express one or more of insulin (INS), urocortin 3 (UCN3), neurogenin 3 (NGN3) and chromogranin A (CHGA). In other embodiments, the mature pancreatic islets express insulin (INS), urocortin 3 (UCN3), neurogenin 3 (NGN3) and chromogranin A (CHGA).

[0097] In other embodiments, mature ECs are produced by a method comprising providing a quantity of induced pluripotent stem cells (iPSCs), plating the iPSCs on MATRIGEL, culturing the iPSCs in MATRIGEL for about 3 days, and further culturing in the presence of CHIR99021 to produce mesoderm. In other embodiments, the method comprises culturing the mesoderm in the presence of BMP4, FGF2, and VEGF for about 2 days to produce vascular precursors. In other embodiments, the method comprises culturing the vascular precursors in the presence of VEGF and Y-27632 for about 7 days to produce endothelial cell (EC) precursors. In other embodiments, the method comprises culturing the EC precursors with VEGF for about 10 days to produce mature ECs. In other embodiments, the vascular precursors and endothelial precursors are cultured in the presence of angiopoietin 1. In various embodiments, the vascular precursors and endothelial precursors are cultured in the presence of angiopoietin 1 from day 4 to day 21 of differentiation. In another embodiment, the method includes culturing the vascular precursors in the presence of EGM-MV2 and VEGF for about 4-6 days to generate mature endothelial progenitor cells, and culturing the endothelial progenitor cells in the presence of EGM-MV2 and VEGF to generate mature endothelial cells. + , CD144 + , VEGF-A + , VEGFR2 + In other embodiments, the mature EC expresses one or more of CD31 + , CD144 + , VEGF-A + , VEGFR2 + In another embodiment, the mature EC expresses two or more of CD31 + , CD144 + , VEGF-A + , VEGFR2 + In another embodiment, the mature EC expresses three or more of CD31 + , CD144 + , VEGF-A + , VEGFR2 + In another embodiment, the mature EC expresses four or more of CD31 + , CD144+ , VEGF-A + , VEGFR2 + , and Ac-LDL.

[0098] Various embodiments described herein provide a method of administering mature pancreatic islets produced by the methods described herein, endothelial cells produced by the methods described herein, or both to a subject in need thereof. In various embodiments, the subject suffers from a metabolic disease, metabolic disorder, and / or metabolic condition. In various embodiments, the metabolic disorder and / or metabolic condition is diabetes and / or insulin resistance. In various embodiments, the cells are induced pluripotent stem cell (iPSC) derived cells. In various embodiments, the pancreatic islet cells, endothelial cells, or both are isogenic to the subject. In various embodiments, the mature pancreatic islets produced by the methods described herein, endothelial cells produced by the methods described herein, or both can regulate metabolic diseases, disorders, and / or conditions. In various embodiments, the mature pancreatic islets produced by the methods described herein, endothelial cells produced by the methods described herein, or both can treat metabolic diseases, disorders, and / or conditions.

[0099] In another embodiment, mature pancreatic islets are produced by the method, which includes providing a quantity of induced pluripotent stem cells (iPSCs), culturing them in the presence of activin A, CHIR99021 and Y-27632 for about 1 day, further culturing them in the presence of activin A and FGF2 for about 2 days, and further culturing them in the presence of FGF10, CHIR99021 and Noggin for about 2 days to produce a posterior foregut. In another embodiment, the method includes culturing the posterior foregut in the presence of FGF10, Noggin, RA and SANT1 for about 4 days to produce pancreatic progenitors. In another embodiment, the pancreatic progenitors are PDX1. + and SOX9 + In other embodiments, the pancreatic precursor expresses one or more of PDX1. + and SOX9 +In another embodiment, the method includes culturing the pancreatic progenitors in the presence of noggin, EGF and nicotinamide for about 4 days to generate pancreatic endocrine progenitors. In another embodiment, the pancreatic endocrine progenitors express PDX1. + and NKX6.1 + In other embodiments, the pancreatic endocrine precursor expresses one or more of PDX1. + and NKX6.1 + In another embodiment, the method comprises culturing the pancreatic endocrine precursors in the presence of noggin, T3 and Alk5i II for about 7 days to generate pancreatic islet precursors. In another embodiment, the method comprises culturing the generated pancreatic islet precursors in the presence of T3, Alk5i II, R428 and N-acetylcysteine ​​(NAC) for about 14 days to generate mature pancreatic islets. In another embodiment, the mature pancreatic islets are expressed in the presence of C-peptide, glucagon and NKX6.1. + In other embodiments, the mature pancreatic islets express one or more of C-peptide, glucagon and NKX6.1. + In other embodiments, the mature pancreatic islets express one or more of insulin (INS), urocortin 3 (UCN3), neurogenin 3 (NGN3) and chromogranin A (CHGA). In other embodiments, the mature pancreatic islets express two or more of insulin (INS), urocortin 3 (UCN3), neurogenin 3 (NGN3) and chromogranin A (CHGA). In other embodiments, the mature pancreatic islets express three or more of insulin (INS), urocortin 3 (UCN3), neurogenin 3 (NGN3) and chromogranin A (CHGA). In other embodiments, the mature pancreatic islets express insulin (INS), urocortin 3 (UCN3), neurogenin 3 (NGN3) and chromogranin A (CHGA).

[0100] In other embodiments, mature ECs are produced by the method, which includes providing a quantity of induced pluripotent stem cells (iPSCs), plating the iPSCs on MATRIGEL, culturing the iPSCs in MATRIGEL for about 3 days, and further culturing in the presence of CHIR99021 to produce mesoderm. In other embodiments, the method includes culturing the mesoderm in the presence of BMP4, FGF2, and VEGF for about 2 days to produce vascular precursors. In other embodiments, the method includes culturing the vascular precursors in the presence of VEGF and Y-27632 for about 7 days to produce endothelial cell (EC) precursors. In other embodiments, the method includes culturing the EC precursors with VEGF for about 10 days to produce mature ECs. In other embodiments, the vascular precursors and endothelial precursors are cultured in the presence of angiopoietin 1. In various embodiments, the vascular precursors and endothelial precursors are cultured in the presence of angiopoietin 1 from day 4 to day 21 of differentiation. In another embodiment, the method includes culturing the vascular precursors in the presence of EGM-MV2 and VEGF for about 4-6 days to generate mature endothelial progenitor cells, and culturing the endothelial progenitor cells in the presence of EGM-MV2 and VEGF to generate mature endothelial cells. + , CD144 + , VEGF-A + , VEGFR2 + In other embodiments, the mature EC expresses one or more of CD31 + , CD144 + , VEGF-A + , VEGFR2 + In another embodiment, the mature EC expresses two or more of CD31 + , CD144 + , VEGF-A + , VEGFR2 + In another embodiment, the mature EC expresses three or more of CD31 + , CD144 + , VEGF-A + , VEGFR2 + In another embodiment, the mature EC expresses four or more of CD31 +, CD144 + , VEGF-A + , VEGFR2 + , and Ac-LDL. Further information can be found in U.S. Provisional Application No. 62 / 647,548 and PCT Application No. PCT / US2019 / 023749, which are incorporated by reference in their entireties. EXAMPLES

[0101] Example 1 Investigating the contribution of specific cell populations of hiPSC-derived endothelial cells (iECs) and pancreatic islets (iIslets) in promoting the generation of in vitro induced functional iIslets The present inventors isolated iIslet precursors (PDX1 + / NKX6.1 + We have successfully developed a method to generate iIslet precursors and vascular iECs. After generating these cells, we tested different ways to co-culture iIslet precursors with iECs and found the optimal approach that allows iECs to improve iIslet insulin secretion when challenged with glucose. However, the exact cellular composition and signals exchanged between iECs and iIslets that contribute to enhanced iIslet function remain to be investigated. Therefore, we explore the essence of the cellular composition and mechanisms differentially expressed in iIslets co-cultured with iECs by deep single cell transcriptomic analyses using single-cell RNA sequencing (sc RNA-seq).

[0102] Without wishing to be bound by any particular theory, the inventors believe that because iIslets co-cultured with iECs have enhanced functionality compared to iIslets generated alone, there are more monohormonal β cells in iIslets co-cultured with iECs, leading to upregulation of pathways related to the insulin secretion mechanism.

[0103] Ex vivo dynamic perfusion system A novel ex vivo dynamic perfusion system is established to test the functionality of human iIslets. Using a static assay (GSIS), we found that iIslets co-cultured with iECs exhibit increased insulin secretion compared to iIslets cultured alone when loaded with high concentrations of glucose. To better characterize the functionality of iIslets in a more physiological manner ex vivo, we plan to evaluate the functionality of iIslets by dynamic GSIS in a novel 3D bioprinted vascularized iIslet platform; this platform is composed of a perfusion system in which iIslets are dynamically loaded with glucose through iEC vascularized channels.

[0104] Without wishing to be bound by any particular theory, it is believed that iIslets generated in co-culture with iECs in this novel ex vivo 3D bioprinted human vascularized iIslet platform exhibit higher insulin secretion in dynamic assays.

[0105] For the first time, iIslets have been co-cultured with isogenic iECs (from the same donor) to obtain better maturity and functionality of iIslets in vitro and in an ex vivo 3D bioprinted human vascularized iIslet system. Importantly, we will be able to show the mechanisms and pathways underlying this interaction using the state-of-the-art tool sc RNA-seq.

[0106] significance The fact that islets and ECs are generated from the same hiPSC donor is a crucial approach because these cells can be transplanted together back into the same diabetic patient, minimizing the immune response of the transplant. Furthermore, this application brings the novelty to better understand the heterogeneity of the cell population using sc RNAseq analysis and how specific cell types contribute to the enhanced functionality of iIslets co-cultured with iECs, along with the possible pathways that contribute to this phenotype. In particular, by combining powerful iPSC and 3D bioprinting technologies, we propose to develop a novel ex vivo 3D bioprinted human vascularized (with iECs) iIslet prototype to better characterize the functionality of iIslets; this would very closely mimic the physiology of β-cells with respect to insulin secretion in vivo.

[0107] Experimental design The overall goal is to understand the essence of the cellular composition and mechanisms differentially expressed in iIslets co-cultured with iECs, and to establish a novel 3D bioprinted vascularized iIslet platform to better characterize iIslet functions, which closely mimics human physiology.

[0108] To investigate the contribution of specific cell populations of hiPSC-derived endothelial cells (iECs) and pancreatic islets (iIslets) in promoting the generation of in vitro induced functional iIslets, we cultured multiple hiPSC lines with a high percentage of PDX1 + / NKX6.1 +We developed a robust and reproducible protocol for differentiation of hiPSC-containing ECs into pancreatic islet (iIslet) precursors in plate cultures. For each cell line, at least three rounds of differentiation were performed, and protein expression of the three different cell lines tested is shown in Figure 1; Figure 1 demonstrates the robustness of the method. Importantly, as shown in Figure 2, these cells increased mRNA expression of endocrine markers throughout their differentiation compared to the iPSC stage, while at the same time decreasing expression of pluripotency markers. iIslet precursors with high expression of endocrine markers and cells containing a marker of beta cell precursors (C-peptide), in addition to high insulin content, were also generated. However, these cells did not reliably secrete insulin even under repeated glucose loading. We therefore speculated that we could attempt to improve the functionality of iIslets by generating hiPSC-derived ECs (iECs) and co-culturing them with iIslet precursors.

[0109] To generate iECs, we developed a protocol that resulted in iECs that highly expressed multiple endothelial markers, such as CD31, VEGF-A, VEGF-A receptor (VEGFR2), and CD144. The reproducibility and efficiency of this iEC protocol in multiple hiPSC lines (four hiPSC lines) is shown from two cell lines at day 20 (Figure 3). Furthermore, our iECs showed increased mRNA expression of vascular EC markers throughout their differentiation compared to the iPSC stage, which was similar to the expression of markers in human umbilical vein cells (HUVECs) used as a positive control (Figure 4). Importantly, these cells were functional, as measured by uptake of acetylated low-density lipoprotein (Ac-LDL) from the medium (Figure 3); Ac-LDL uptake is a functional assay that has been used to test EC functionality over time.

[0110] We next tested various methods to co-culture iIslet precursors with iECs (Figure 5A). Our first attempt was to co-culture them using transwells; in which signals are exchanged between cells through the medium, but there is no direct cell-cell contact. To do so, we introduced iECs into iIslet precursors from the same donor and co-cultured them for 2 weeks; at this point the iIslet precursors had further developed into more mature iIslets. We tested iECs on top of the insert and iIslet precursors on the bottom of the plate, and vice versa. Using this method, we observed higher expression of C-peptide protein in iIslets at the end of the culture when iECs were cultured on the insert and iIslets on the bottom of the plate. However, none of the methods of co-culturing iIslets with iECs on transwells improved the functionality of iIslets; because although iIslets still contained large amounts of insulin, they did not respond to glucose challenge in a physiological manner. Based on previous evidence that cell-cell interactions between ECs and pancreatic cells are crucial during the development of the endocrine pancreas, we decided to directly co-culture iIslet precursors with iECs from the same donor again for 2 weeks. We tried various methods, such as placing iECs on the bottom of the plate and iIslets on top of iECs, or vice versa. We found that when iECs were placed under iIslets for 2 weeks, the iIslets at the end of differentiation responded to glucose challenge in a physiological manner, i.e., they increased insulin secretion when challenged with a medium with a high glucose concentration. This was not observed for iIslets when cultured alone (Figure 5B). Importantly, iIslets directly co-cultured with iECs also showed higher mRNA expression of multiple markers of mature pancreatic β-cells, including insulin (INS), urocortin 3 (UCN3), neurogenin 3 (NGN3), and chromogranin A (CHGA), compared to the iPSC stage and iIslets cultured alone (Figure 6).

[0111] Taken together, these results indicate that co-culturing iIslet precursors with iECs during islet development results in iIslets with enhanced maturity and functionality. However, the exact cellular composition and signals exchanged between iECs and iIslets that contribute to enhanced iIslet function remain to be investigated. Therefore, we propose here to explore the essence of the cellular composition and mechanisms differentially expressed in iIslets co-cultured with iECs by deep single-cell transcriptome analysis using single-cell RNA sequencing (sc RNA-seq). Single-cell RNAseq has the capacity to characterize rare endocrine and endothelial cell populations that are not captured by previous bulk analyses. With the results obtained here, we will further investigate the signaling pathways that contribute to enhanced iIslet function by modulating key pathways in vitro (gain and loss of function). Briefly, at the end of the co-culture, cells are dissociated into single cells, followed by the same steps as bulk RNAseq, i.e., reverse transcription, amplification, library preparation, and sequencing. Individual cells are encapsulated in individual droplets in a microfluidic device, where the reverse transcription reaction takes place. Each droplet carries a DNA barcode that uniquely labels the cDNA derived from a single cell. Once reverse transcription is complete, the cDNA from many cells is mixed together for sequencing; the transcripts from a particular cell are identified by their unique barcode. PCA will be generated similarly to IPA for pathway analysis.

[0112] We will perform single-cell transcriptome analysis using sc-RNAseq from our samples to investigate the cellular composition and mechanisms that are differentially expressed in iIslets co-cultured with iECs. Separately, we will investigate signaling pathways that contribute to enhanced iIslet function by modulating key pathways implicated in bulk mRNA-seq data and data on the developing pancreas together with the endothelium through gain- or loss-of-function studies.

[0113] Establishing a novel ex vivo dynamic perfusion system to test the functionality of human iIslets Using a static assay (GSIS), we found that iIslets co-cultured with iECs exhibited increased insulin secretion when loaded with high concentrations of glucose compared to iIslets cultured alone. To better characterize the functionality of iIslets ex vivo in a more physiological manner, we will evaluate the functionality of iIslets using dynamic GSIS in a novel 3D bioprinted vascularized iIslet platform; this platform consists of a perfusion system in which iIslets are dynamically loaded with glucose through iEC vascularized channels. This dynamic method is also useful for investigating the regulation of insulin release in response to various secretagogues, such as KCl, IBMX, tolbutamide, extenzin 4, and L-arginine.

[0114] We developed a 3D bioprinter with a motorized extruder that can precisely feed and unload extrusions in a compact and rapidly loadable form-factor. Over the past year, we have successfully cultured iECs on these constructs (Figure 7). Briefly, iECs are cultured planarly from hiPSCs until they reach maturity, at which point they are dissociated, mixed with a bioink (fibrin) and bioprinted onto 3D constructs to resemble blood vessels. Cells are continuously supplied through a dynamic system that mimics blood flow in vivo (Figure 7A); we observed that iECs spread and remain attached to the constructs for more than two weeks. Here, we propose a similar approach for iIslets, with or without co-culture with iECs. At the end of the planar co-culture, we dissociate them, mix with the appropriate bioink and bioprint them into 3D vascular constructs. Several studies have shown islet survival in alginate bioinks, which will be the first approach to test. Other bioinks, such as fibrin, will also be tested as alternative methods. Cell viability within the vascular construct will be determined under a confocal microscope by Cell-Dead staining; in this staining, calcein acetoxymethyl ester (calcein AM) stains live cells and ethidium homodimer 2 staining labels nonviable cells. The system will then be used to test the functionality of iIslet by dynamic GSIS after cells have attached and adapted to the system (we plan to test GSIS after different days of seeding on the construct).

[0115] Example 2 Generation of iPSCs The iPSC lines utilized in this study were derived from healthy, lean individuals (BMI < 27 kg / m) by the iPSC Core at Cedars-Sinai Medical Center. 2) from a male control. These control iPSC lines were generated from peripheral blood mononuclear cells (PMBCs) using non-integrating oriP / EBNA1-based episomal plasmid vectors. With this approach, less than 5% of iPSCs had abnormal karyotypes. All undifferentiated iPSCs were cultured in mTeSR on plates coated with BD MATRIGEL™ matrix. + Cell lines used in this study are summarized in Table 1.

[0116] Differentiation of pancreatic islets (iIslets) from iPSCs To generate pancreatic islets (iIslets) from iPSCs, we developed a robust and reproducible protocol. Briefly, iPSCs (OCT4 expression >90%) maintained on MATRIGEL-coated plates were dissociated into single cells with Accutase and then cultured in mTeSR. + 300,000 cells / cm using culture medium 2 The cells were reseeded planarly on MATRIGEL-coated plates at a density of 100 μM with the ROCK inhibitor Y-27632 (10 mM, R&D Systems). After 24 hours, iPSCs were directed towards definitive endoderm (DE - Phase I) with Activin A (100 ng / ml, R&D Systems), CHIR99021 (2 μM, XcessBio), and Y-27632 (10 μM) for 1 day, followed by Activin A (100 ng / ml) and FGF2 (5 ng / ml, PeproTech) for 2 days. Next, for posterior foregut (PFG - Phase II) patterning, FGF10 (50 ng / ml, PeproTech), CHIR99021 (0.25 μM), and Noggin (50 ng / ml, PeproTech) for 2 days. The cells were then cultured as PDX1 + / SOX9 +To direct pancreatic progenitors (PP - phase III), a combination of FGF10 (50ng / ml), Noggin (50ng / ml), RA (2μM, Cayman) and SANT1 (0.25μM, Sigma) was used for 4 days, followed by treatment with Noggin (50ng / ml), EGF (100ng / ml, PeproTech) and nicotinamide (10mM, Sigma) for 4 days to induce PDX1 + / NKX6.1 + Pancreatic endocrine precursors (PEP - phase IV) were induced. To generate islet precursors (IP - phase V), a combination of Noggin (50 ng / ml), T3 (1 μM, Sigma), and Alk5i II (10 μM, Axxora) was used for 7 days, during which the medium was changed every other day. For maturation of islets (MI - phase VI), a combination of T3 (1 μM), Alk5i II (10 μM), AXL inhibitor R428 (2 mM, Selleckchem), and antioxidant N-acetylcysteine ​​NAC (1 mM, Sigma) was used for 14 days, during which the medium was changed every other day. The formulation of the basal medium used throughout the differentiation process is summarized in Table 2.

[0117] Differentiation of endothelial cells (iECs) from iPSCs For generation of vascular endothelial cells (ECs) from iPSCs (iECs), we plated iPSCs (OCT4 expression >90%) as small cell colonies onto MATRIGEL-coated plates, and after 3 days (Figure 13A), induced mesoderm (ME - phase I) using CHIR99021 (6 μM) for 2 days. Then, BMP4 (25 ng / ml, R&D Systems), FGF2 (10 ng / ml), and VEGF were added to induce mesoderm (ME - phase I). 165 (50 ng / ml, PeproTech) combination was used for an additional 2 days to generate vascular precursors (VP ​​- Phase II). After that, when the majority of VPs showed a cobblestone-like morphology at the periphery of the original iPSC colony (Figure 13B), the cells were dissociated with Accutase; the peripheral cells easily lifted off and were then isolated from the VEGF 165(50 ng / ml) and Y-27632 (10 μM) at 85,000–100,000 cells / cm 2 The cells were then planarly replated onto MATRIGEL-coated plates at a density of 100 μg / ml and induced to produce EC precursors (ECP - phase III) for 7 days; medium was changed every other day. For purification and maturation of iECs (phase IV), iECs were dissociated on day 11 and incubated with VEGF 165 (50 ng / ml) and replated at the same cell density onto MATRIGEL-coated plates with medium changes every other day for 10 days. This process was repeated on day 21 and extended for an additional 10 days as needed. The basal medium used for phases I and II was STEMdiff™ APEL™ 2 medium (StemCell Technologies), and the basal medium used for phases III and IV was EC Growth medium MV2 (ECGM-MV2) (PromoCell).

[0118] Co-culture system of iIslet and iEC iIslets were directly co-cultured with iECs. To do so, in parallel to day 20 of iIslet differentiation, day 11 iECs were plated on the bottom of 24-well MATRIGEL-coated plates with phase IV EC medium supplemented with Y-27632 (10 μM) until the next day. Day 21 iIslets were dissociated and plated on top of iECs at a cell density of 400,000 cells / well with half phase IV iEC medium and half phase VI islet medium supplemented with Y-27632 (10 μM) ("iIslet with 1 / 2 iIslet medium" condition) or they were fed with phase VI medium only ("iIslet with iIslet medium" condition). Co-culture of iIslets with iECs was performed for 14 days, at which point cells were fixed for immunofluorescence or subjected to GSIS assays.

[0119] Immunofluorescence First, cells were fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 20 min, then washed twice with PBS. Fixed cells were then permeabilized and blocked for 1 h with "blocking buffer" containing 10% donkey serum (Millipore) and 0.1% Triton-X (Bio-Rad) in PBS. Primary antibodies were diluted in blocking buffer and placed on the cells overnight at 4°C. The following primary antibodies and dilutions were used: Oct4 (1:250, Stemgent), Ssea4 (1:100, Abcam), Foxa2 (1:100, Novus Biologicals), Sox17 (1:250, Novus Biologicals), Pdx1 (1:100, R&D Systems), Sox9 (1:250, Millipore), Nkx6.1 (1:25, DSHB), C-peptide (1:25, DSHB), Vegfa (1:100, Abcam), Cd31 (1:100, Cell Signaling), Cd144 (1:100, Abcam), and Vegfr2 (1:100, Cell Signaling). The next day, after extensive washing with PBS plus 0.1% Tween-20 (ThermoFisher), cells were incubated with the appropriate species-specific Alexa Fluor-conjugated secondary antibodies (ThermoFisher) diluted in blocking buffer (1:1,000) for 1 h at room temperature. After washing with PBS plus 0.1% Tween-20, cells were incubated in DAPI diluted in PBS (1:2,500) for 15 min. Immunofluorescence images were visualized using an ImageXpress Micro XLS (Molecular Devices) with appropriate fluorescence filters and analyzed using ImageJ software.

[0120] Static glucose-stimulated insulin secretion (GSIS) assay GSIS was performed at the end of iIslet differentiation and at the end of co-culture of iIslets and iECs. To do so, iIslets were washed twice with 2.8 mM glucose Krebs solution (Table 4). Next, cells were pre-incubated with the same 2.8 mM glucose solution for 3 hours of fasting. After that, cells were washed once with 2.8 mM glucose solution and incubated with the same solution for another hour. After this incubation, the supernatant was collected as "low glucose" and cells were washed once with 2.8 mM glucose solution, rinsed, and loaded with high glucose solution (20 mM glucose Krebs solution) (Table 4) for 1 hour. When this loading was performed, the supernatant was collected as "high glucose". After this, cells were washed with PBS and fixed for immunofluorescence assays or collected for gene expression by RT-qPCR at a later date.

[0121] Dil-acetylated LDL uptake assay Acetylated low-density lipoprotein labeled with Dil fluorescent dye (Dil-ac-LDL) (10 μg / ml, Cell Applications) was incubated in phase IV EC medium, HUVEC medium or mTeSR medium as described (Harding et al., 2017). + were added to the culture medium and correspondingly to day 11 and day 21 iECs, HUVECs or iPSCs and incubated for 4 h at 37 °C. Cells were then washed with PBS, fixed and stained with DAPI as described above. Images were captured with ImageXpress Micro XLS and analyzed using ImageJ.

[0122] Real-time quantitative PCR (RT-qPCR) analysis Relative gene expression was quantified using RT-qPCR. To do so, cells were washed with PBS and total RNA was extracted and isolated using the Quick-RNA Mini Prep kit (Zymo Research) according to the manufacturer's instructions. RNA concentration and purity were determined by spectrophotometry (NanoDrop, ThermoFisher); all samples had an A of approximately 2.0. 260 / 280The ratio of cDNA to DNA was 1:1 (Desjardins and Conklin, 2010). RNA (1 μg) was then first treated with DNAse (Promega and ThermoFisher) and then reverse transcribed to cDNA with oligo(dT) using the High Capacity cDNA Reverse Transcription Kit (ThermoFisher). Real-time qPCR was performed in triplicate on a CFX384 Real Time system (Bio-Rad) using SYBR Green Mastermix (Applied Biosystems) and primer sequences specific for each gene (Table 5). Human RPL13 was used as the reference gene, and 2 -ΔΔ Relative expression was measured using the CT method.

[0123] statistical analysis Data are expressed as the mean ± standard error of the mean (SEM). Statistical significance between groups was determined by one-way ANOVA followed by Dunnett's post-test when comparing with control or Tukey's post-test for multiple comparison tests. An unpaired two-tailed Student's test was employed when appropriate. A p value <0.05 was considered statistically significant. Statistical analysis and graphs were prepared using GraphPad Prism 7 for Windows software (GraphPad Software, Inc.).

[0124] Successfully generated pancreatic endocrine precursors (PEPs) from iPSCs The differentiation strategy is summarized in Figure 8A, which details the small molecules used at each phase of differentiation. After determining the optimal and most consistent cell density to initiate differentiation, we demonstrated that our protocol was consistent and reproducible in three different iPSC lines, as shown by protein expression at each phase of differentiation, as seen in Figure 8B. Importantly, we found that PDX1 + / NKX6.1 +High protein expression (>40%) of the cells was reached (Figure 8B, Figure 14); at this point, the cells were characterized as pancreatic endocrine precursors (PEPs). Successful commitment of cell fate to PEPs was also demonstrated by the mRNA expression of additional endocrine markers, namely NGN3 and INS, which increased throughout the differentiation process, reaching a peak in expression at the end of phase IV, as seen in Figure 8C. As the cells were committed to PEPs, there was a concomitant decrease in expression of the pluripotency marker OCT4, as shown in Figure 15.

[0125] Combined inhibition of TGF-β and BMP signaling pathways induced the generation of islet precursors (IPs) from iPSC-derived pancreatic endocrine precursors (PEPs) After successfully generating PEPs in three different iPSC lines, our next step was to direct the cells to pancreatic islet precursors (IPs) and more specifically to β-cells; this was based on our landmark observation that the genes NGN3 and INS, key regulators of pancreatic β-cell differentiation, were already highly expressed at the end of phase IV of differentiation (Figure 8C). This suggested to us that by the end of phase IV, our cells had already passed through the pancreatic bipotent trunk progenitor phase, which can give rise to pancreatic endocrine or ductal cells, and reached an endocrine fate. Therefore, based on previous literature, we investigated three signaling pathways to direct PEPs to IPs, as described in Figure 9A: i) TGF-β inhibition, ii) BMP inhibition, and / or iii) Notch inhibition. Inhibition of TGF-β signaling is a strategy to achieve high expression of β cells; here, we tested inhibition of TGF-β signaling using Alk5 II inhibitor (Alk5i). Another pathway we modulated to direct PEP to IP was inhibition of BMP signaling through the use of Noggin. It is well known that Noggin has high affinity for BMP and prevents the binding of BMP to its receptor. This blockade of BMP signaling results in either mitigation of β cell proliferation or enhancement of β cell proliferation and maintenance of β cell specific gene expression. Our final strategy was to inhibit Notch signaling through the use of gamma secretase inhibitor XXI in an attempt to inhibit differentiation of remaining bipotent stem precursors into pancreatic ductal cells. As shown in previous studies, T3 was used in an attempt to proliferate and mature β cells across all conditions.

[0126] In various combinations tested during phase V, Notch inhibition with the use of XXI reduced the number of cells at the end of phase V compared to conditions without XXI (Figures 9B and 9C); this may indicate less cell proliferation in XXI conditions. Cells treated with XXI also had lower expression of NKX6.1, a key transcription factor present in pancreatic endocrine cells, and higher expression of markers of endocrine cell types other than β cells, such as high expression of somatostatin (SST; δ cells) and glucagon (GCG; α cells). Among the conditions without XXI, the combination of T3 and Alk5i with Noggin showed a trend toward higher expression of C-peptide (P=0.05, t-test) and NKX6.1 / C-peptide (P=0.08, t-test) (Figures 9B and 9C).

[0127] To assess the functionality of β-cells, IPs at the end of phase V were loaded into a static glucose-stimulated insulin secretion (GSIS) assay; in this case, the cells receive different glucose concentrations and, if functional, respond by secreting insulin in correlation to the glucose load. At the end of GSIS, the cells receive a solution containing a high concentration of KCl (30 mM KCl); this solution is able to induce cell membrane depolarization in β-cells and stimulate insulin secretion more potently than a high concentration of glucose. The results of GSIS after phase V can be seen in Figure 16, which shows that insulin secretion did not increase in any condition after stimulation with a high concentration of glucose (20 mM glucose). However, after loading with 30 mM KCl, the cells increased insulin secretion in all conditions, indicating that they were functional only after KCl stimulation. Furthermore, the combination of T3 with Alk5i and Noggin showed higher insulin secretion compared to T3 with Alk5i alone (P=0.02, t-test).

[0128] As the GSIS results showed that none of the conditions contained sufficiently functional cells, and although the combination of T3 + Alk5i + Noggin showed slightly higher expression of C-peptide and NKX6.1 / C-peptide and higher insulin secretion with KCl compared to the T3 + Alk5i only condition, the overall expression of C-peptide was not ideal (approximately 20-25% of cells); this indicates that a subsequent phase is required after phase V to allow IPs to proliferate and mature.

[0129] Inhibition of the antioxidant N-acetylcysteine ​​and AXL pathway enhanced islet precursor (IP) maturation and, more specifically, beta-cell proliferation but not functionality The combination of T3, Alk5i and Noggin used during phase V directed PEPs to a 20% C-peptide expressing population and increased insulin secretion after stimulation with KCl, but it was insufficient to generate fully functional β-cells. Therefore, in an attempt to mature IPs into islets containing functional β-cells, we modulated three major signaling pathways during phase VI based on previous literature: i) inhibition of oxidative stress; ii) continued inhibition of TGF-β signaling; and iii) inhibition of the AXL pathway (Figure 10A). The use of R428, a selective small molecule inhibitor of the tyrosine kinase receptor AXL, and the antioxidant N-acetylcysteine ​​(NAC) was based on the strategy used in (Rezania et al., 2014). These compounds have been identified as key regulators of genes important for β-cell maturation, including those involved in glucose-regulated insulin secretion (Rezania et al., 2014). After 7 days of exposure to the small molecule cocktail in phase VI, as observed in Figure 10B-C, C-peptide protein expression increased compared to the end of phase V, showing approximately 30% C-peptide expression, while the expression of other endocrine cell markers, somatostatin (SST) and glucagon (GCG), was slightly decreased. The combination of T3 and Alk5i and R428 and NAC showed higher expression of C-peptide (P=0.059, t-test vs T3 and Alk5i and NAC), while both conditions with R428 and NAC showed higher SST expression (Figure 10B-C). Despite the higher C-peptide expression compared to the previous phase V, none of the conditions showed increased insulin secretion after stimulation with high glucose. However, when exposed to high concentrations of KCl, all conditions increased insulin secretion, with the combination of T3 and Alk5i and R428 and NAC showing the highest level of insulin secretion, as seen in Figure 17. Therefore, further experiments used a combination of T3, Alk5i, R428 and NAC.

[0130] As β-cell functionality continued to be lacking despite higher C-peptide protein expression and higher insulin secretion after KCl stimulation, we next investigated the concentrations of insulin and glucose in the basal medium of phase VI as potential factors that may moderate β-cell maturation and response to glucose in a physiological manner (Figure 18A). To do so, we probed cells in phase VI medium containing T3, Alk5i, R428 and NAC supplemented with insulin ITS-X (as before) or 8 mM glucose (as before) or 20 mM glucose, or without glucose. When cells were treated with 20 mM glucose medium, we observed that C-peptide expression disappeared and NKX6.1 expression increased after 7 days of treatment compared to cells treated with 8 mM glucose medium, as seen in Figure 18B; this may suggest that exposing IPs to medium containing higher glucose concentrations for 7 days reversed their direction of differentiation towards a more progenitor state. With or without ITS-X, there was no difference in the expression of NKX6.1 or C-peptide proteins, as well as in the expression of SST or GCG (Figure 18B).

[0131] Since adjusting the glucose and / or insulin content in the medium in phase VI was not sufficient to enhance the maturation of β cells, our next attempt to generate functional β cells was to reaggregate cells during phase VI and increase the timing of differentiation (14 days instead of 7 days) (Figure 97A). Dissociation and reaggregation of islet clusters is a strategy previously used in other studies in an attempt to purify the β cell population within the islets and remove other cell types (Stock et al., 2020). Importantly, in these studies, reaggregation of β cells improved GSIS (Lecomte et al., 2016). In our study, cells were dissociated on day 1 of phase VI and plated at the same initial cell density (3 × 10 ) on MATRIGEL-coated plates using phase VI medium containing T3, Alk5i, R428 and NAC. 5 cells / cm2 ) at 28 days. Differentiation was performed for 7 or 14 days. Expression of C-peptide and GCG protein was comparable between day 28 (day 7 of phase VI) and day 35 (day 14 of phase VI), while expression of NKX6.1 was statistically higher at day 35 (Figure 19B). Although the cells were not yet fully functional when challenged with high glucose solution, baseline insulin levels after exposing the cells to 2.8 mM glucose were higher at day 35 compared to day 28, as was the insulin response after KCl challenge (Figure 19C); this suggested to us that performing β-cell differentiation for 35 days would be a better strategy to take in further steps. As shown in Figure 20, the development of other cell types, such as pancreatic duct cells and gastric / intestinal cells, may be associated with the lack of functionality at day 35 after reaggregation.

[0132] Successfully generated functional iPSC-derived endothelial cells (iECs) During endocrine pancreas development, surrounding vascular endothelial cells (ECs) provide signals for the development and maturation of pancreatic β cells, which are considered as important niche components. The fact that here differentiated β cells showed high levels of C-peptide and NKX6.1 protein expression, but still did not show optimal functionality, led us to believe that co-culture with those ECs might enhance their maturation and thus their functionality through exchanged signals. Since an aspect of the present invention is to transplant iPSC-derived β cells back into diabetic patients, we used the same iPSC cell line used for β cell differentiation to generate iPSC-derived ECs (iECs) for co-culture. Thus, we made several modifications to generate iECs, which were mainly related to changes in cell density and plate coating (Figure 11A). iPSC-derived vascular endothelial cells (iECs) generated with our optimized protocol showed high expression of endothelial cell markers such as VEGFR2, CD144, and CD31 (>70% by day 21 of differentiation) (Figure 11B, 11C). These cells were also functional, as demonstrated by Dil-Ac-LDL uptake assay (Figure 11B, 11C). They also highly expressed endothelial cell markers at the mRNA level, which continued to increase until day 21 (Figure 11D). iECs showed minimal expression of pluripotency markers from day 4 to day 31, as shown in Figure 21.

[0133] An optimized model of co-culturing iPSC-derived islets (iIslets) with endothelial cells (iECs) improved β-cell maturation and functionality iPSCs from the same donor were differentiated to islet precursors (IPs) as shown in Figure 2 using T3, Alk5i and Noggin. At the end of phase V, IPs were dissociated using Accutase and re-plated at a cell density ratio of 1:4 on top of day 11 iECs generated from the same iPSC line (07iCTR) as shown in the differentiation schematic in Figure 12A. IPs were further differentiated to mature islets (MIs) as shown in Figure 3 using T3, Alk5i, R428 and NAC either co-cultured with iECs or not co-cultured with them for 14 days. IPs and iEC combinations were fed with phase VI medium every other day as described previously. Alternatively, co-cultures of cells were fed with a combination of phase VI medium and iEC medium (50% each); however, the results (not shown here) were not as promising as when co-cultures of cells were fed with phase VI medium alone. The protein expression of C-peptide / NKX6.1 in the "islet-only condition" was not significantly different from that in "islets co-cultured with iECs" as shown in Figure 12B. However, when islets co-cultured with iECs were challenged with a high glucose concentration solution (20 mM glucose), they responded to increase insulin secretion compared to basal glucose concentration solution (2.8 mM glucose) (Figure 12C); whereas, the "islet-only condition" did not respond to high glucose challenge; this indicates that co-culture of islets with iECs activates mechanisms that have yet to be elucidated, resulting in enhanced functionality of the islets. To confirm the enhanced functionality probed by GSIS shown in Figure 12C, RNA was extracted from either islets co-cultured with iECs or not, and key markers expressed by islets containing mature β cells were examined. Interestingly, when islets were co-cultured with iECs, we observed upregulation of markers such as INS, UCN3, NGN3, and CHGA compared to the "islet-only condition" (Figure 12D). These results support the hypothesis based on earlier studies that co-culture of pancreatic β cells with ECs enhances β cell maturation and functionality, although the mechanism remains to be elucidated.

[0134] Table 1. Characterization of iPSC lines utilized in this study. TIFF2024506850000002.tif44160* PMBC: Peripheral blood mononuclear cells

[0135] Table 2. Basal medium formulation for the culture of iPSC-derived islets TIFF2024506850000003.tif15160TIFF2024506850000004.tif244160* BSA: bovine serum albumin; ITS-X: insulin-transferrin-selenium-ethanolamine supplement

[0136] Table 3. Media formulation for culturing HUVEC cultures TIFF2024506850000005.tif79160* FBS: Fetal bovine serum

[0137] Table 4: Krebs buffer TIFF2024506850000006.tif80160* BSA: Bovine serum albumin

[0138] Table 5. List of oligonucleotides used for qPCR TIFF2024506850000007.tif194160

[0139] Table 6. Media formulation for culturing iPSC-derived endothelial cells TIFF2024506850000008.tif100160

[0140] The various methods and techniques described above provide many ways to implement the present invention. Of course, it should be understood that not all of the stated objectives or advantages are necessarily achieved according to the particular embodiment described herein. Thus, for example, one skilled in the art will recognize that the present method can be implemented in a manner that achieves or optimizes one or a group of advantages taught herein without necessarily achieving other objectives or advantages that may be taught or suggested herein. Various advantageous and disadvantageous alternatives are mentioned herein. It should be understood that some preferred embodiments specifically include one, another, or several advantageous features, other preferred embodiments specifically exclude one, another, or several disadvantageous features, and still other preferred embodiments specifically alleviate the disadvantageous feature at hand by including one, another, or several advantageous features.

[0141] Moreover, one of ordinary skill in the art will recognize the applicability of various features from different embodiments. Similarly, the various elements, features, and steps described above, as well as other known equivalents to each of such elements, features, or steps, may be mixed and matched by one of ordinary skill in the art to implement methods in accordance with the principles described herein. Of the various elements, features, and steps, some will be specifically included in various embodiments, while others will be specifically excluded.

[0142] While the present invention has been disclosed with respect to particular embodiments and examples, it will be appreciated by those skilled in the art that the aspects of the invention extend beyond the specifically disclosed embodiments to other alternative embodiments and / or uses, as well as modifications and equivalents thereof.

[0143] Many variations and alternative elements are disclosed in the embodiments of the present invention. Further variations and alternative elements will be apparent to those skilled in the art. These variations do not include the compositions and methods related to iPSCs, pancreatic islet cells, endothelial cells, the techniques and solutions used therein, and the specific uses of the products produced by the teachings of the present invention. Various embodiments of the present invention can specifically include or exclude any of these variations or elements.

[0144] In some embodiments, numerical values ​​expressing properties such as amounts of ingredients, concentrations, reaction conditions, etc., used to describe and claim certain embodiments of the present invention should be understood as being modified in some cases by the term "about". Thus, in some embodiments, the numerical parameters set forth in this specification and the appended claims are approximations that may vary depending on the desired properties sought to be obtained in a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as practicable. The numerical values ​​set forth in some embodiments of the present invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0145] In some embodiments, the terms "a" and "an" and "the" and similar referents used in the context of describing certain embodiments of the invention (particularly in the context of the claims that follow) can be interpreted to cover both the singular and the plural. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each value falling within the range. Unless otherwise indicated herein, each value is incorporated herein as if it were individually set forth herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein with respect to certain embodiments is intended merely to facilitate understanding of the invention and does not impose limitations on the scope of the invention as otherwise claimed. No language in this specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0146] Groupings of alternative elements or aspects of the invention disclosed herein should not be construed as limitations. Each group member may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in or deleted from a group for reasons of convenience and / or patentability. When such inclusion or deletion occurs, the specification shall be deemed to include the group as modified to satisfy all Markush group descriptions used in the appended claims.

[0147] Preferred embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred embodiments will become apparent to those skilled in the art upon reading the foregoing description. It is contemplated that such variations may be employed by those skilled in the art and may be practiced in ways other than as specifically described herein. Accordingly, many aspects of the invention include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0148] Additionally, throughout this specification, numerous patents and publications are referenced. Each of the above cited references and publications is herein individually incorporated by reference in its entirety.

[0149] Finally, it is to be understood that the embodiments of the invention disclosed herein reflect the principles of the invention. Other modifications that can be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the invention can be utilized in accordance with the teachings herein. Thus, the embodiments of the invention are not limited to that precisely as shown and described.

Claims

1. 1. A method for generating functional induced pluripotent stem cell (iPSC)-derived pancreatic islets (iIslets), comprising: co-culturing a quantity of iPSC-derived endothelial cells (iECs) with a quantity of iPSC-derived pancreatic islet precursors for about 10-18 days to generate functional iIslets that include β cells. The method comprising:

2. The co-culturing step comprises: (i) plating a quantity of iPSC-derived endothelial cells (iECs) onto a MATRIGEL-coated plate and culturing them in Phase IV EC medium supplemented with Y27632; generating functional iIslets containing β cells by plating a quantity of iPSC-derived islets (iIslets) on top of the quantity of iECs and culturing for about 12-16 days in medium containing about ½ Phase IV iEC medium and about ½ Phase VI islet medium supplemented with Y-27632 or in Phase VI islet medium (islet only condition) for about 12-16 days; or (ii) plating a quantity of iPSC-derived endothelial cells (iECs) onto a MATRIGEL-coated plate and culturing them in Phase IV EC medium supplemented with Y27632; generating functional iIslets containing beta cells by plating a quantity of iPSC-derived islets (iIslets) on top of the quantity of iECs and culturing them for about 14 days in a medium containing about 1 / 2 Phase IV iEC medium and about 1 / 2 Phase VI islet medium supplemented with Y-27632 or in Phase VI islet medium (islet only condition) for about 14 days. and / or (i) plating a quantity of induced pluripotent stem cells (iPSCs) onto MATRIGEL; Culture the iPSCs in MATRIGEL for approximately 2-4 days; Culturing in the presence of CHIR99021 for approximately 1-3 days to generate mesoderm; Culturing the mesoderm in the presence of BMP4, FGF2, and VEGF for approximately 1-3 days to generate vascular precursors; The vascular precursors were cultured in the presence of VEGF and Y-27632 for approximately 3-8 days to generate endothelial cell (EC) precursors. or (ii) plating a quantity of induced pluripotent stem cells (iPSCs) onto MATRIGEL; Culture the iPSCs in MATRIGEL for approximately 3 days; Culturing in the presence of CHIR99021 for approximately 2 days to generate mesoderm; Culturing the mesoderm in the presence of BMP4, FGF2, and VEGF for approximately 2 days to generate vascular precursors; The vascular precursors were cultured in the presence of VEGF and Y-27632 for approximately 4-7 days to generate endothelial cell (EC) precursors. and / or generating iECs by (i) culturing a quantity of induced pluripotent stem cells (iPSCs) in the presence of activin A, CHIR99021, and Y-27632 for about 1-2 days; Cultivate in the presence of activin A and FGF2 for approximately 1-3 days; Culturing in the presence of FGF10, CHIR99021, and Noggin for approximately 1-3 days to generate posterior foregut cells; Culturing posterior foregut cells in the presence of FGF10, Noggin, RA, and SANT1 for approximately 3-5 days to generate pancreatic precursors; culturing the pancreatic precursors in the presence of noggin, EGF, and nicotinamide for about 3-5 days to generate pancreatic endocrine precursors; Culturing pancreatic endocrine precursors in the presence of noggin, T3, and Alk5i II for approximately 6-8 days to generate pancreatic islet precursors or (ii) culturing a quantity of induced pluripotent stem cells (iPSCs) in the presence of activin A, CHIR99021, and Y-27632 for about 1 day; Cultivate in the presence of activin A and FGF2 for approximately 2 days; to generate posterior foregut cells by culturing in the presence of FGF10, CHIR99021, and Noggin for approximately 2 days; Culturing posterior foregut cells in the presence of FGF10, Noggin, RA, and SANT1 for approximately 4 days to generate pancreatic precursors; culturing the pancreatic precursors in the presence of noggin, EGF, and nicotinamide for about 4 days to generate pancreatic endocrine precursors; The pancreatic endocrine precursors were cultured in the presence of noggin, T3, and Alk5i II for approximately 7 days to generate pancreatic islet precursors. and further comprising initially producing said amount of islet precursors by The method of claim 1.

3. (i) pancreatic precursors express PDX1+ and SOX9+; (ii) the pancreatic endocrine precursors are PDX1+ and NKX6.1+, or (iii) iIslet expresses C-peptide, glucagon, and NKX6.1+; The method according to any one of claims 1 to 2.

4. The method of any one of claims 2 to 3, wherein expression of INS, UCN3, NGN3, and CHGA is upregulated in beta cells produced in islet-only conditions compared to beta cells produced without co-culture with vascular endothelial cells or compared to beta cells produced in cultures without islet-only conditions.

5. (i) increase insulin secretion when β-cells are challenged with high glucose concentrations compared to basal glucose concentrations; (ii) iPSC-derived endothelial cells (iECs) and iPSC-derived islet precursors are isogenic; (iii) the iPSCs used to derive the iECs and the iPSCs used to derive the islet precursors are from the same iPSC cell line or from the same donor; and / or (iv) the iIslet is a human iIslet; The method according to any one of claims 1 to 4.

6. An induced pluripotent stem cell (iPSC)-derived pancreatic islet (iIslet), optionally expressing C-peptide, glucagon and NKX6.1+, produced by the method of any one of claims 1 to 5.

7. The iIslet of claim 6, which increases insulin secretion when challenged with a high glucose concentration compared to a basal glucose concentration.

8. 8. An iIslet according to claim 6 or 7 for use in therapy.

9. 8. The iIslet of claim 6 or 7 for use in ameliorating or treating a metabolic disease, disorder or condition in a subject in need thereof.

10. The iIslet of claim 9, wherein the metabolic disease, disorder, or condition is diabetes or insulin resistance.

11. Culturing a quantity of induced pluripotent stem cells (iPSCs) in the presence of activin A, CHIR99021, and Y-27632 for about 1 day; then culturing in the presence of activin A and FGF2 for about 2 days; and Then, the cells are cultured in the presence of FGF10, CHIR99021, and Noggin for about 2 days to generate posterior foregut cells. A method comprising:

12. (i) culturing posterior foregut cells in the presence of FGF10, Noggin, RA, and SANT1 for about 4 days to generate pancreatic progenitors, and optionally, the pancreatic progenitors are selected from the group consisting of PDX1, ＋ and SOX9 ＋ Expressing, (ii) culturing the pancreatic progenitors in the presence of noggin, EGF, and nicotinamide for about 4 days to generate pancreatic endocrine progenitors, and optionally, the pancreatic endocrine progenitors are selected from the group consisting of PDX1 and PDX2. ＋ and NKX6.1 ＋ That is, (iii) culturing the pancreatic endocrine precursors in the presence of Noggin, T3, and Alk5i II for about 7 days to generate pancreatic islet precursors; and / or (iv) culturing the generated islet precursors in the presence of T3, Alk5i II, R428, and N-acetylcysteine ​​(NAC) for about 14 days to generate mature islets, and optionally, the mature islets express C-peptide, glucagon, and NKX6.

1. ＋ Expressing, 12. The method of claim 11, further comprising:

13. culturing the posterior foregut in the presence of FGF10, Noggin, RA, and SANT1 for about 4 days to generate pancreatic precursors; then culturing the pancreatic precursors in the presence of noggin, EGF, and nicotinamide for about 4 days to generate pancreatic endocrine precursors; Then, culturing the pancreatic endocrine precursors in the presence of Noggin, T3, and Alk5i II for about 7 days to generate pancreatic islet precursors; and Then, the generated islet precursors are cultured in the presence of T3, Alk5i II, R428, and N-acetylcysteine ​​(NAC) for about 14 days to generate mature islets.

12. The method of claim 11, further comprising:

14. A quantity of mature pancreatic islets produced by the method of any one of claims 12 to 13.

15. plating a quantity of induced pluripotent stem cells (iPSCs) onto MATRIGEL; Culturing the iPSCs in MATRIGEL for about 3 days; and Then, culturing in the presence of CHIR99021 to produce mesoderm. A method comprising:

16. (i) culturing the mesoderm in the presence of BMP4, FGF2, and VEGF for about 2 days to generate vascular precursors; (ii) culturing the vascular precursors in the presence of VEGF and Y-27632 for about 7 days to generate endothelial cell (EC) precursors; (iii) culturing the EC precursors with VEGF for about 10 days to generate mature ECs, and optionally, the mature ECs are ＋ , CD144 ＋ , VEGF-A ＋ , VEGFR2 ＋ and expressing Ac-LDL, and / or (iv) culturing the mesoderm in the presence of BMP4, FGF2, and VEGF for about 2 days to generate vascular precursors; Then, culturing the vascular precursors in the presence of VEGF and Y-27632 for about 7 days to generate endothelial cell (EC) precursors; and Then, the EC precursors are cultured with VEGF for about 10 days to generate mature ECs.

16. The method of claim 15, further comprising:

17. 17. A quantity of mature ECs produced by the method of claim 16.

18. An assembly comprising a quantity of mature pancreatic islets and a quantity of mature ECs, wherein the mature islets and the mature ECs are isogenic.

19. (i) the amount of mature islets comprises a mature islet according to any one of claims 6 to 7 or claim 14, and the amount of mature ECs comprises a mature EC according to claim 17; and / or (ii) mature islets, mature ECs, or both are attached onto a scaffold, optionally, the mature islets, mature ECs, or both are attached onto the scaffold using a bio-ink, or the bio-ink comprises fibrin or alginate; 20. The assembly of claim 18.

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