Differentiation of pancreatic endocrine cells

Abstract

To provide compositions and methods related to differentiation of stem cells into pancreatic endocrine cells.SOLUTION: In some aspects, the methods provided herein relate to generation of pancreatic β cells, α cells, δ cells, and EC cells in vitro. In some aspects, the disclosure provides pharmaceutical compositions including the cells generated according to the methods disclosed herein, as well as methods of treatment making use thereof.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 059,433, filed July 31, 2020, which is incorporated by reference in its entirety. [Background technology]

[0002]

[0002] The production of stem cell-derived β-cells could provide a potentially useful step toward the production of pancreatic islets and pancreatic organs. Diabetes mellitus is one rapidly progressing disease treatable with stem cell-derived tissues. Type 1 diabetes results from the autoimmune destruction of β-cells in pancreatic islets. Type 2 diabetes results from insulin resistance and abnormal β-cell function in peripheral tissues. Diabetic patients, particularly those with type 1 diabetes, could potentially be cured by transplantation of new β-cells. Patients who receive transplants of cadaveric human islets can remain insulin-dependent for five or more years with this strategy, but the scarcity and quality of donor islets limits this approach. Generating an unlimited supply of human β-cells from stem cells could expand this therapy to millions of new patients, providing an important test case for translating stem cell biology into clinical practice.

[0003] Incorporation by Reference

[0003] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Unless otherwise indicated, all publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entirety. Summary of the Invention

[0004]

[0004] In some aspects, the method includes the steps of: (a) differentiating PDX1-positive, NKX6.1-negative pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells by contacting the PDX1-positive, NKX6.1-negative pancreatic progenitor cells with a ROCK inhibitor, a growth factor from the TGF-β superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor, thereby producing a cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells; and (b) for a first period, differentiating the cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells from a PKC activator, a γ-secretase inhibitor, a ROCK inhibitor, a TGF-β superfamily growth factor, an activator of the RA signaling pathway, and an SHH pathway inhibitor. and (c) after the first period of time, contacting the cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a first composition comprising a growth factor from the EGF family, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor; and (c) after the first period of time, contacting the cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a second composition comprising a PKC activator, a γ-secretase inhibitor, a TGF-β signaling pathway inhibitor, a growth factor from the EGF family, an RA signaling pathway activator, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound for a second period of time.

[0005] In some embodiments, (a) for a first period, a cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells is treated with a PKC activator, a gamma-secretase inhibitor, and a factor selected from the group consisting of a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an activator of the RA signaling pathway, and an inhibitor of the SHH pathway. and (b) after the first period of time, for a second period of time, contacting the cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a second composition comprising a PKC activator, a gamma-secretase inhibitor, and a factor selected from the group consisting of a TGF-β signaling pathway inhibitor, a growth factor from the EGF family, a RA signaling pathway activator, a SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound.

[0006]

[0006] In some examples, the method further includes, after the second period, contacting the cell population containing PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a third composition that differentiates at least a portion of the PDX1-positive, NKX6.1-positive pancreatic progenitor cells into NKX6.1-positive, ISL1-positive endocrine cells, thereby producing a cell population containing NKX6.1-positive, ISL1-positive endocrine cells.

[0007]

[0007] In some aspects, a method for producing a pancreatic progenitor cell population comprising: (a) contacting, for a first period of time, a cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a first composition comprising a factor selected from the group consisting of a PKC activator, a ROCK inhibitor, a growth factor from the TGF-β superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor; (b) after the first period of time, contacting, for a second period of time, the cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a second composition comprising a PKC activator, a factor selected from the group consisting of a TGF-β signaling pathway inhibitor, a growth factor from the EGF family, an RA signaling pathway activator, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound; and (c) contacting the cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a second composition comprising a PKC activator, a factor selected from the group consisting of a TGF-β signaling pathway inhibitor, a growth factor from the EGF family, an RA signaling pathway activator, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound. and after a second period of time, contacting the cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a third composition that differentiates at least a portion of the PDX1-positive, NKX6.1-positive pancreatic progenitor cells into NKX6.1-positive, ISL1-positive endocrine cells, thereby producing a cell population comprising NKX6.1-positive, ISL1-positive endocrine cells, wherein the cell population comprising NKX6.1-positive, ISL1-positive endocrine cells comprises (i) an increased proportion of cells that express glucagon; (ii) a decreased proportion of cells that express VMAT1; (iii) an increased proportion of cells that express somatostatin; or (iv) an increased proportion of cells that express C-peptide, compared to a corresponding cell population produced without contacting the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with the PKC activator in the first composition or the second composition.

[0008] In some examples, the third composition comprises a TGF-β signaling pathway inhibitor, a thyroid hormone (TH) signaling pathway activator, and an epigenetic modifying compound. In some examples, the third composition comprises a differentiation factor selected from the group consisting of a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, an epigenetic modifying compound, a growth factor from the EGF family, a RA signaling pathway activator, an SHH pathway inhibitor, a gamma-secretase inhibitor, a protein kinase inhibitor, a ROCK inhibitor, and a BMP signaling pathway inhibitor. In some examples, the third composition comprises the TGF-β signaling pathway inhibitor, the thyroid hormone signaling pathway activator, the epigenetic modifying compound, the growth factor from the EGF family, the RA signaling pathway activator, the SHH pathway inhibitor, the gamma-secretase inhibitor, the protein kinase inhibitor, the ROCK inhibitor, and the BMP signaling pathway inhibitor. In some examples, the third composition does not comprise the PKC activator. In some examples, the first composition comprises the ROCK inhibitor, the growth factor from the TGFβ superfamily, the growth factor from the FGF family, the RA signaling pathway activator, and the SHH pathway inhibitor. In some examples, the second composition comprises the TGF-β superfamily. The cell population comprises a signal transduction pathway inhibitor, a growth factor from the EGF family, the RA signaling pathway activator, the SHH pathway inhibitor, the TH signaling pathway activator, the protein kinase inhibitor, the ROCK inhibitor, the BMP signaling pathway inhibitor, and the epigenetic modifying compound. In some examples, the cell population comprising NKX6.1-positive, ISL1-positive endocrine cells comprises (i) an increased proportion of cells expressing somatostatin; (ii) an increased proportion of cells expressing glucagon; (iii) a decreased proportion of cells expressing VMAT1; or (iv) an increased proportion of cells expressing C-peptide, compared to a corresponding cell population produced without contacting the PDX1-positive, NKX6.1-positive pancreatic progenitor cells in the first composition or the second composition with the PKC activator. In some examples, the cell population containing NKX6.1-positive, ISL1-positive endocrine cells comprises (i) an increased proportion of cells expressing somatostatin, (ii) an increased proportion of cells expressing glucagon, (iii) a decreased proportion of cells expressing VMAT1, and (iv) an increased proportion of cells expressing C-peptide, compared to a corresponding cell population produced without contacting the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with the PKC activator in the first composition or the second composition. In some examples, the cell population containing NKX6.1-positive, ISL1-positive endocrine cells comprises at least about 4% of cells expressing somatostatin, at least about 15% of cells expressing glucagon, at most about 35% of cells expressing VMAT1, or at least about 40% of cells expressing C-peptide, as measured by flow cytometry.In some examples, the cell population containing NKX6.1-positive, ISL1-positive endocrine cells contains at least about 100% more cells expressing somatostatin, at least about 200% more cells expressing glucagon, at least about 50% fewer cells expressing VMAT1, or at least about 20% more cells expressing C-peptide, as measured by flow cytometry, compared to a corresponding population of cells produced without contacting the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with the PKC activator in the first composition or the second composition. In some examples, the first period is 1 to 3 days. In some examples, the first period is about 2 days. In some examples, the second period is 1 to 3 days. In some examples, the second period is about 2 days. In some examples, the PKC activator is selected from the group consisting of phorbol 12,13-dibutyrate (PDBU), FR236924, prostratin, SC-9, and TPPB. In some examples, the PKC activator comprises PDBU. In some examples, the PKC activator is contacted with the cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells at a concentration of 100 nM to 1000 nM. In some examples, the PKC activator is contacted with the cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells at a concentration of approximately 500 nM. In some examples, the γ-secretase inhibitor comprises XXI. In some examples, the γ-secretase inhibitor is contacted with the cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells at a concentration of 0.5 μM to 10 μM. In some examples, the γ-secretase inhibitor is contacted with the cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells at a concentration of approximately 2 μM.

[0009]

[0009] In some examples, the method further includes a step of obtaining a cell population comprising PDX1-positive, NKX6.1-negative pancreatic progenitor cells by contacting the cell population comprising PDX1-positive, NKX6.1-negative pancreatic progenitor cells with a composition comprising the PDX1-positive, NKX6.1-negative pancreatic progenitor cells, the composition comprising a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor, and differentiating the PDX1-positive, NKX6.1-negative pancreatic progenitor cells into the PDX1-positive, NKX6.1-positive pancreatic progenitor cells. In some examples, the method includes contacting a FOXA2-positive, PDX1-negative gastrula cell with a ROCK inhibitor, a growth factor from the FGF family, a BMP signaling pathway inhibitor, a PKC activator, a retinoic acid signaling pathway activator, an SHH pathway inhibitor, and a growth factor from the TGF-β superfamily, thereby converting the FOXA2-positive, PDX1-negative gastrula cell into the PDX1-positive, NKX1-negative gastrula cell. In some examples, the method further comprises differentiating the definitive endoderm cells into FOXA2-positive, PDX1-negative pancreatic progenitor cells by contacting the definitive endoderm cells with a growth factor from the FGF family, thereby differentiating the definitive endoderm cells into the FOXA2-positive, PDX1-negative intestinal cells.

[0010]

[0010] In some aspects, the method includes the steps of: (a) differentiating pluripotent stem cells in a population into definitive endoderm cells by contacting the pluripotent stem cells with a growth factor from the TGF-β superfamily and a WNT signaling pathway activator; (b) differentiating the definitive endoderm cells into FOXA2-positive, PDX1-negative primitive gut cells by contacting the definitive endoderm cells with a growth factor from the FGF family; and (c) differentiating the FOXA2-positive, PDX1-negative primitive gut cells into definitive endoderm cells by contacting the definitive endoderm cells with a ROCK inhibitor, a growth factor from the FGF family, a BMP signaling pathway activator, and a WNT signaling pathway activator. (d) differentiating the FOXA2-positive, PDX1-negative primitive gut cells into PDX1-positive, NKX6.1-negative pancreatic progenitor cells by contacting the PDX1-positive, NKX6.1-negative pancreatic progenitor cells with a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, a RA signaling pathway activator, and an SHH pathway inhibitor. (e) differentiating the PDX1-positive, NKX6.1-negative pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells; (e) incubating the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a first composition comprising a PKC activator, a gamma-secretase inhibitor, and a factor selected from the group consisting of a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor for a first period of 1 to 3 days; and (f) after (e), differentiating the PDX1-positive, NKX6.1-negative pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells for 1 to 3 days. and (g) incubating the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with the PKC activator, the γ-secretase inhibitor, and a second composition comprising a factor selected from the group consisting of a TGF-β signaling pathway inhibitor, a growth factor from the EGF family, an RA signaling pathway activator, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound for a second period of time; and (g) after (f), incubating the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with the PKC activator, the γ-secretase inhibitor, and a second composition comprising a factor selected from the group consisting of a TGF-β signaling pathway inhibitor, a growth factor from the EGF family, an RA signaling pathway activator, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound.and differentiating the PDX1-positive, NKX6.1-positive pancreatic progenitor cells into a cell population comprising NKX6.1-positive, ISL1-positive endocrine cells by contacting the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a TGF-β signaling pathway inhibitor, a growth factor from the EGF family, an RA signaling pathway activator, an SHH pathway inhibitor, a TH signaling pathway activator, a γ-secretase inhibitor, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound. In some examples, the SHH pathway inhibitor comprises SANT1, the RA signaling pathway activator comprises retinoic acid, the γ-secretase inhibitor comprises XXI, the growth factor from the EGF family comprises betacellulin, the BMP signaling pathway inhibitor comprises LDN or DMH, the TGF-β signaling pathway inhibitor comprises Alk5 inhibitor II, the thyroid hormone signaling pathway activator comprises GC-1, the protein kinase inhibitor comprises staurosporine, the ROCK inhibitor comprises thiazovivin, or the epigenetic modifying compound comprises DZNep, GSK126, or EPZ6438.

[0011] In some aspects, the method includes the steps of: (a) contacting a plurality of PDX1-positive, NKX6.1-negative pancreatic progenitor cells with one or more of a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor, thereby producing a first cell population; and (b) contacting the first cell population with a PKC activator and a γ-secretase inhibitor, as well as a ROCK inhibitor, a TG inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor, thereby producing a first cell population. Disclosed herein are methods comprising: (a) contacting a first cell population with one or more of a growth factor from the Fβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor, thereby producing a second cell population; and (b) contacting the second cell population with one or more of a PKC activator, a γ-secretase inhibitor, and a TGF-β signaling pathway inhibitor, a growth factor from the EGF family, an RA signaling pathway activator, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound, thereby producing a third cell population.

[0012]

[0012] In some aspects, disclosed herein are methods comprising contacting a cell population with a γ-secretase inhibitor and one or both of a growth factor from the TGFβ superfamily and a growth factor from the FGF family. In some embodiments, the cell population comprises PDX1-positive cells. In some embodiments, the cell population comprises PDX1-positive, NKX6.1-negative cells. In some embodiments, the cell population comprises PDX1-positive, NKX6.1-positive cells.

[0013]

[0013] In some aspects, disclosed herein are methods comprising: (a) contacting a plurality of PDX1-positive, NKX6.1-negative pancreatic progenitor cells with one or more of a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, a RA signaling pathway activator, and an SHH pathway inhibitor for a period of 1 to 5 days or less, thereby producing a first cell population; and (b) contacting the first cell population with a gamma-secretase inhibitor. In some embodiments, the contacting in step (a) is for a period of 4 or 5 days. In some embodiments, step (b) further comprises contacting the first cell population with one or more of a PKC activator, a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, a RA signaling pathway activator, and an SHH pathway inhibitor.

[0014] In some aspects, a method for producing a pancreatic progenitor cell line includes the steps of: (a) contacting a plurality of PDX1-positive, NKX6.1-negative pancreatic progenitor cells with one or more of a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor, thereby producing a first cell population; and (b) contacting the first cell population with a PKC activator, and one or more of a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor, thereby producing a first cell population. wherein the PKC activator is a benzolactam derivative; and (c) contacting the second cell population with one or more of a PKC activator, a gamma-secretase inhibitor, and a TGF-β signaling pathway inhibitor, a growth factor from the EGF family, an RA signaling pathway activator, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound, thereby producing a third cell population.

[0015]

[0015] In some examples, the benzolactam derivative is TPPB. In some examples, step (b) further comprises contacting the first cell population with a γ-secretase inhibitor. In some examples, the method further comprises (d) contacting the third cell population with one or more of a TGF-β signaling pathway inhibitor, an RA signaling pathway activator, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound, thereby producing a fourth cell population. In some examples, step (d) does not include contacting the third cell population with a PKC activator. In some examples, step (d) does not include contacting the third cell population with a PKC activator. In some examples, step (d) does not include contacting the third cell population with a gamma-secretase inhibitor. In some examples, step (d) does not include contacting the third cell population with an SHH pathway inhibitor. In some examples, step (d) does not include contacting the third cell population with a growth factor from the EGF family. In some examples, the method further includes (e) contacting the fourth cell population with one or more of serum albumin protein, vitamin C, a TGF-β signaling pathway inhibitor, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound, thereby producing a fifth cell population. In some examples, step (e) includes contacting the fourth cell population with a PKC activator.

[0016]

[0016] In some aspects, the method includes the steps of: (a) contacting a plurality of PDX1-positive, NKX6.1-negative pancreatic progenitor cells with one or more of a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor, thereby producing a first cell population; (b) contacting the first cell population with a PKC activator, and one or more of a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor, thereby producing a second cell population; and (c) contacting the second cell population with a PKC activator, and one or more of a γ-secretase inhibitor, a TGF-β signaling pathway inhibitor, a growth factor from the EGF family, an RA signaling pathway activator, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, and a ROCK inhibitor. , a BMP signaling pathway inhibitor, and an epigenetic modifying compound, thereby producing a third cell population; (d) contacting the third cell population with one or more of a TGF-β signaling pathway inhibitor, an RA signaling pathway activator, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound, thereby producing a fourth cell population; and (e) contacting the fourth cell population with a PKC activator, and one or more of a serum albumin protein, vitamin C, a TGF-β signaling pathway inhibitor, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound, thereby producing a fifth cell population.

[0017] In some examples, step (e) comprises contacting the fourth cell population with serum albumin protein. In some examples, step (a) is performed for 1, 2, 3, 4, 5, or 6 days. In some examples, step (a) is performed for 3 to 5 days (e.g., 4 days). In some examples, step (b) is performed for 1, 2, 3, or 4 days. In some examples, step (b) is performed for 1 to 3 days (e.g., 2 days). In some examples, step (c) is performed for 1, 2, 3, or 4 days. In some examples, step (c) is performed for 1 to 3 days (e.g., 2 days). In some examples, step (d) is performed for 1, 2, 3, 4, 5, 6, or 7 days. In some examples, step (d) is performed for 4 to 6 days (e.g., 5 days). In some examples, step (e) is performed for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days. In some examples, step (e) is performed for 10 to 12 days. In some examples, the first cell population comprises PDX1-positive, NKX6.1-negative cells and / or PDX1-positive, NKX6.1-positive cells. In some examples, the second cell population comprises PDX1-positive and NKX6.1-positive cells. In some examples, the third cell population comprises PDX1-positive, NKX6.1-positive, ISL1-negative cells and / or PDX1-positive, NKX6.1-positive, ISL1-positive cells. In some examples, the fourth cell population comprises PDX1-positive, NKX6.1-positive, ISL1-positive cells. In some examples, the fifth cell population comprises C-peptide and ISIL1-positive cells. In some examples, 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 30-40%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-90%, 50-80%, 50-70%, 50-60%, 60-90%, 60-80%, 60-70%, 70-90%, 70-80%, 70-90%, 70-80%, or 80-90% of the cells in the fourth cell population express C-peptide and ISL1 but do not express VMAT1. In some cases, 40-60% of the cells in the fourth cell population express C-peptide and ISL1 but do not express VMAT1. In some cases, the fourth cell population includes cells that express glucagon but do not express somatostatin. In some examples, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-40%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15-40%, 15-35%, 15-30%, 15-25%, 15-20%, 20-40%, 20-35%, 20-30%, 20-25%, 25-40%, 25-35%, 25-30%, 30-40%, 30-35%, or 35-40% of the cells in the fourth cell population express glucagon but do not express somatostatin. In some examples, 10-25% of the cells in the fourth cell population express somatostatin but do not express glucagon. In some examples, the fourth cell population includes cells that express somatostatin but do not express glucagon. In some examples, 3-20%, 3-15%, 3-12%, 3-10%, 3-8%, 3-5%, 4-20%, 4-15%, 4-12%, 4-10%, 4-8%, 4-5%, 5-20%, 5-15%, 5-12%, 5-10%, 5-8%, 7-20%, 7-15%, 7-12%, 7-10%, 9-20%, 9-15%, 9-12%, 8-10%, 8-12%, 8-15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15%, or 15-20% of the cells in the fourth cell population express somatostatin but do not express glucagon.In some examples, step (a) comprises contacting a plurality of PDX1-positive, NKX6.1-negative pancreatic progenitor cells with a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, a RA signaling pathway activator, and an SHH pathway inhibitor. In some examples, step (b) comprises contacting a first cell population with a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, a RA signaling pathway activator, and an SHH pathway inhibitor. In some examples, step (c) comprises contacting a second cell population with a gamma-secretase inhibitor, a TGF-β signaling pathway inhibitor, a growth factor from the EGF family, a RA signaling pathway activator, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound. In some examples, step (d) comprises contacting the third cell population with serum albumin protein, a TGF-β signaling pathway inhibitor, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound. In some examples, the ROCK inhibitor used in steps (a), (b), (c), (d) and / or (e) is thiazobavin or Y-27632. In some examples, the growth factor from the TGFβ superfamily used in steps (a) and / or (b) is activin A. In some examples, the growth factor from the FGF family used in steps (a) and / or (b) is KGF. In some examples, the RA signaling pathway activator used in steps (a), (b) and / or (c) is retinoic acid. In some examples, the SHH pathway inhibitor used in steps (a), (b) and / or (c) is Sant-1.In some examples, the PKC activator for use in steps (b), (c) and / or (d) is selected from the group consisting of phorbol 12,13-dibutyrate (PDBU), FR236924, prostratin, SC-9, and TPPB. In some examples, the PKC activator is PDBU. In some examples, steps (b) and / or (d) are used. Or the gamma-secretase inhibitor used in step (c) is XXI. In some examples, the TGF-β signaling pathway inhibitor used in step (c), (d) and / or (e) is ALK5i. In some examples, the growth factor from the EGF family used in step (c) is betacellulin. In some examples, the TH signaling pathway activator used in step (c), (d) and / or (e) is T3, GC-1 or a thyroid hormone derivative. In some examples, the protein kinase inhibitor used in step (c), (d) and / or (e) is staurosporine. In some examples, the BMP signaling pathway inhibitor used in step (c), (d) and / or (e) is LDN193189 or DMH-1. In some examples, the epigenetic modifying compound used in step (c), (d) and / or (e) is DZNep.

[0018]

[0018] In some aspects, disclosed herein is an in vitro composition comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells; NKX6.1-positive, ISL1-positive endocrine cells; a PKC activator; and a gamma-secretase inhibitor.

[0019]

[0019] In some aspects, disclosed herein is an in vitro composition comprising PDX1-positive, NKX6.1-negative pancreatic progenitor cells; PDX1-positive, NKX6.1-positive pancreatic progenitor cells; a PKC activator; and a gamma-secretase inhibitor. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the cells in the composition are PDX1-positive, NKX6.1-positive pancreatic progenitor cells. In some embodiments, less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the cells in the composition are PDX1-positive, NKX6.1-negative pancreatic progenitor cells.

[0020] In some embodiments of the composition, the PKC activator is selected from the group consisting of phorbol 12,13-dibutyrate (PDBU), FR236924, prostratin, SC-9, and TPPB. In some examples, the gamma-secretase inhibitor is DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester). In some examples, the gamma-secretase inhibitor is XXI.

[0021] In some embodiments of the composition, the composition further comprises a growth factor from the FGF family. In some embodiments, the growth factor from the FGF family is KGF. In some embodiments, the composition further comprises a growth factor of the TGFβ superfamily. In some embodiments, the growth factor of the TGFβ superfamily is activin A.

[0022]

[0022] In some aspects, disclosed herein is an in vitro composition comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells; NKX6.1-positive, ISL1-positive endocrine cells; and a PKC activator, wherein the PKC activator is a benzolactam derivative.

[0023]

[0023] In some embodiments of the composition, the PKC activator is TPPB. In some examples, the composition further comprises a γ-secretase inhibitor. In some examples, the γ-secretase inhibitor is DAPT. In some examples, the γ-secretase inhibitor is XXI. In some examples, the composition further comprises a differentiation factor selected from the group consisting of a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, an epigenetic modifying compound, a growth factor from the EGF family, an RA signaling pathway activator, an SHH pathway inhibitor, a protein kinase inhibitor, a ROCK inhibitor, and a BMP signaling pathway inhibitor. In some examples, the composition further comprises serum albumin protein. In some examples, the composition further comprises a serum albumin protein, a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, an epigenetic modifying compound, a growth factor from the EGF family, an RA signaling pathway activator, an SHH pathway inhibitor, a protein kinase inhibitor, a ROCK inhibitor, and a BMP signaling pathway inhibitor. The present invention further includes a hormone signaling pathway activator, an epigenetic modifying compound, an SHH pathway inhibitor, a protein kinase inhibitor, a ROCK inhibitor, and a BMP signaling pathway inhibitor. In some examples, the ROCK inhibitor is thiazobabin. In some examples, the ROCK signaling pathway activator is retinoic acid. In some examples, the SHH pathway inhibitor is Sant-1. In some examples, the TGF-β signaling pathway inhibitor is ALK5i. In some examples, the EGF family growth factor is betacellulin. In some examples, the thyroid hormone signaling pathway activator is T3, GC-1, or a thyroid hormone derivative. In some examples, the protein kinase inhibitor is staurosporine. In some examples, the BMP signaling pathway inhibitor is LDN193189 or DMH-1. In some examples, the epigenetic modifying compound is DZNep.

[0024]

[0024] In some aspects, disclosed herein is a composition comprising an in vitro cell population, the cell population comprising: (a) at least about 35% cells that express C-peptide and do not express VMAT1; and (b) at most about 35% cells that express VMAT1 or at least about 15% cells that express glucagon (e.g., as measured by flow cytometry). In some aspects, the disclosure provides a composition comprising an in vitro cell population, the cell population comprising: at least about 35% cells that express C-peptide and do not express VMAT1; and (i) at most about 35% cells that express VMAT1, and / or (ii) at least about 15% cells that express glucagon. In some embodiments, the percentages of cells are measured by flow cytometry.

[0025] In some examples, the cell population comprises at most about 30% cells that express VMAT1 and at least about 20% cells that express glucagon. In some examples, the cell population comprises at most about 30% cells that express VMAT1 and at least about 20% cells that express glucagon, as measured by flow cytometry. In some examples, the cell population comprises at least about 15% cells that express glucagon and do not express somatostatin. In some examples, the cell population comprises at least about 4% cells that express somatostatin and do not express glucagon.

[0026] In some embodiments, there is provided a composition comprising a cell population, the composition comprising: a) 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 30-40%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-90%, 50-80%, 50-70%, 50-60%, 60-90%, 60-80%, 60-70%, 70-90%, 70-80%, 70-90% of the cell population , 70-80%, or 80-90% of cells express C-peptide and ISL1 but do not express VMAT1; b) 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-40%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15-40%, 15-35%, 15-30%, 15-25%, 15-20%, 20- 40%, 20-35%, 20-30%, 20-25%, 25-40%, 25-35%, 25-30%, 30-40%, 30-35%, or 35-40% of the cells express glucagon but not somatostatin; and / or c) 3-20%, 3-15%, 3-12%, 3-10%, 3-8%, 3-5%, 4-20%, 4-15%, 4-12%, 4-10%, 4-8%, 4-5% of the cells in the cell population , 5-20%, 5-15%, 5-12%, 5-10%, 5-8%, 7-20%, 7-15%, 7-12%, 7-10%, 9-20%, 9-15%, 9-12%, 8-10%, 8-12%, 8-15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15%, or 15-20% of the cells express somatostatin but do not express glucagon.

[0027]

[0027] In some embodiments, a composition comprising a plurality of cells, the composition comprising: a) a population of cells in a range of 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 30-40%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-90%, 50-80%, 50-70%, 50-60%, 60-90%, 60-80%, 60-70%, 70-90%, 70-80%, 70-9 ... b) 0%, 70-80%, or 80-90% of cells express C-peptide and ISL1 but do not express VMAT1; b) 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-40%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15-40%, 15-35%, 15-30%, 15-25%, 15-20%, 20-40%, 20-35%, 20-30%, 20-25%, 25-40%, 25-35%, 25-30%, 30-40%, 30-35%, or 35-40% of the cells express glucagon but not somatostatin; and c) 3-20%, 3-15%, 3-12%, 3-10%, 3-8%, 3-5%, 4-20%, 4-15%, 4-12%, 4-10%, 4-8%, 4-5%, Disclosed herein are compositions wherein 5-20%, 5-15%, 5-12%, 5-10%, 5-8%, 7-20%, 7-15%, 7-12%, 7-10%, 9-20%, 9-15%, 9-12%, 8-10%, 8-12%, 8-15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15%, or 15-20% of the cells express somatostatin but do not express glucagon.

[0028] In some embodiments, a composition comprising a cell population, comprising: a) 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15-35%, 15-30%, 15-25%, 15-20%, 20-35%, 20-30%, 20-25%, 25-35%, 25-30%, or 30-35% of the cell population; cells express VMAT1 but do not express C-peptide; b) 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-40%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15-40%, 15-35%, 15-30%, 15-25%, 15-20%, 20-40%, 20-35%, 20-30% of the cell population %, 20-25%, 25-40%, 25-35%, 25-30%, 30-40%, 30-35%, or 35-40% of the cells express glucagon but not somatostatin; and / or c) 3-20%, 3-15%, 3-12%, 3-10%, 3-8%, 3-5%, 4-20%, 4-15%, 4-12%, 4-10%, 4-8%, 4-5%, 5-20%, 5- Disclosed herein are compositions wherein 15%, 5-12%, 5-10%, 5-8%, 7-20%, 7-15%, 7-12%, 7-10%, 9-20%, 9-15%, 9-12%, 8-10%, 8-12%, 8-15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15%, or 15-20% of the cells express somatostatin but do not express glucagon.

[0029] In some embodiments, a composition includes a cell population, wherein: a) 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15-35%, 15-30%, 15-25%, 15-20%, 20-35%, 20-30%, 20-25%, 25-35%, 25-30%, or 30-35% of the cells in the cell population express VMAT1 but do not express C-peptide; and b) 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-40%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15-40%, 15-35%, 15-30%, 15-25%, 15-20%, 20-40%, 20-35%, 20-30%, 20-25%, 25-40%, 25-35%, 25-30%, 30-40%, 30-35%, or 35-40% of the cells express glucagon but not somatostatin; and c) cells Disclosed herein are compositions wherein 3-20%, 3-15%, 3-12%, 3-10%, 3-8%, 3-5%, 4-20%, 4-15%, 4-12%, 4-10%, 4-8%, 4-5%, 5-20%, 5-15%, 5-12%, 5-10%, 5-8%, 7-20%, 7-15%, 7-12%, 7-10%, 9-20%, 9-15%, 9-12%, 8-10%, 8-12%, 8-15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15%, or 15-20% of the cells in a population of cells express somatostatin but do not express glucagon.

[0030]

[0030] In some embodiments of the composition, 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 30-40%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-90%, 50-80%, 50-70%, 50-60%, 60-90%, 60-80%, 60-70%, 70-90%, 70-80%, 70-90%, 70-80%, or 80-90% of the cells in the cell population express C-peptide and ISL1 but do not express VMAT1.

[0031] In some embodiments of the composition, 40-60% of the cells in the cell population express C-peptide and ISL1 but not VMAT1; 10-25% of the cells in the cell population express glucagon but not somatostatin; and 4-10% of the cells in the cell population express somatostatin but not glucagon. In some examples, less than 25%, less than 20%, less than 18%, less than 15%, less than 12%, or less than 10% of the cells in the cell population express VMAT1 but not C-peptide. In some examples, the cell population is produced in vitro from stem cells. In some examples, cells that express C-peptide but not VMAT1 exhibit a glucose-stimulated insulin secretion response in vitro. In some examples, insulin secretion by cells that express C-peptide but not VMAT1 in response to a glucose challenge is proportional to the glucose concentration of the glucose challenge. In some examples, the cells that express C-peptide and do not express VMAT1 secrete insulin in response to one or more glucose challenges.In some examples, the cells that express C-peptide and do not express VMAT1 secrete insulin in response to a first glucose challenge, a second glucose challenge, and a third glucose challenge, and the first glucose challenge, the second glucose challenge, and the third glucose challenge are applied sequentially.

[0032]

[0032] In some aspects, disclosed herein are in vitro compositions comprising PDX1-positive cells, a γ-secretase inhibitor, and one or both of a growth factor from the TGFβ superfamily and a growth factor from the FGF family. In some embodiments, the composition of cells comprises PDX1-positive, NKX6.1-negative cells. In some embodiments, the composition of cells comprises PDX1-positive, NKX6.1-positive cells.

[0033]

[0033] In some embodiments, the composition further comprises any one or combination of a PKC activator, a growth factor from the FGF family, a ROCK inhibitor, a growth factor from the TGFβ superfamily, a sonic hedgehog pathway inhibitor, and a retinoic acid signaling pathway activator.

[0034]

[0034] In some aspects, disclosed herein is an in vitro composition comprising PDX1-positive, NKX6.1-negative pancreatic progenitor cells; PDX1-positive, NKX6.1-positive pancreatic progenitor cells; and a gamma-secretase inhibitor. In some embodiments, the gamma-secretase inhibitor is XXI.

[0035] In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the cells are PDX1-positive, NKX6.1-positive pancreatic progenitor cells. In some embodiments, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the cells in the composition are PDX1-positive, NKX6.1-negative pancreatic progenitor cells.

[0036] In some embodiments, the composition further comprises a growth factor from the FGF family. In some embodiments, the composition further comprises a sonic hedgehog pathway inhibitor. In some embodiments, the composition further comprises a ROCK inhibitor. In some embodiments, the composition further comprises a growth factor from the TGFβ superfamily. In some embodiments, the composition further comprises a retinoic acid signaling pathway activator. In some embodiments, the composition further comprises a PKC activator.

[0037] In some embodiments, the composition further comprises any two of a PKC activator, a growth factor from the FGF family, a ROCK inhibitor, a growth factor from the TGFβ superfamily, a sonic hedgehog pathway inhibitor, and a retinoic acid signaling pathway activator. In some embodiments, the composition further comprises any three of a PKC activator, a growth factor from the FGF family, a ROCK inhibitor, a growth factor from the TGFβ superfamily, a sonic hedgehog pathway inhibitor, and a retinoic acid signaling pathway activator. In some embodiments, the composition further comprises any four of a PKC activator, a growth factor from the FGF family, a ROCK inhibitor, a growth factor from the TGFβ superfamily, a sonic hedgehog pathway inhibitor, and a retinoic acid signaling pathway activator. In some embodiments, the composition further comprises any five of a PKC activator, a growth factor from the FGF family, a ROCK inhibitor, a growth factor from the TGFβ superfamily, a sonic hedgehog pathway inhibitor, and a retinoic acid signaling pathway activator. In some embodiments, the composition further comprises any six of a PKC activator, a growth factor from the FGF family, a ROCK inhibitor, a growth factor from the TGFβ superfamily, a sonic hedgehog pathway inhibitor, and a retinoic acid signaling pathway activator.

[0038] In some embodiments of the composition, the growth factor from the FGF family is KGF. In some embodiments, the sonic hedgehog pathway inhibitor is SANT-1. In some embodiments, the ROCK inhibitor is thiazovivin. In some embodiments, the growth factor from the TGFβ superfamily is activin A. In some embodiments, the retinoic acid signaling pathway activator is retinoic acid. In some embodiments, the PKC activator is PDBU.

[0039]

[0039] In some aspects, disclosed herein is an in vitro differentiated cell population comprising NKX6.1-positive, ISL1-positive cells and NKX6.1-negative, ISL1-positive cells; the population comprises more NKX6.1-negative, ISL1-positive cells than NKX6.1-positive, ISL1-positive cells; and at least 73% of the cells in the population are ISL1-positive cells. In some embodiments, less than 12% of the cells in the population are NKX6.1-negative, ISL1-negative cells.

[0040]

[0040] In some aspects, disclosed herein are in vitro differentiated cell populations comprising NKX6.1-positive, ISL1-positive cells and NKX6.1-negative, ISL1-positive cells; at least 40% of the cells in the population are NKX6.1-negative, ISL1-positive cells. In some embodiments, less than 12% of the cells in the population are NKX6.1-negative, ISL1-negative cells.

[0041] In some embodiments, NKX6.1 positive, ISL1 positive cells and NKX6.1 negative Disclosed herein is an in vitro differentiated cell population comprising NKX6.1-negative, ISL1-positive cells, wherein less than 12% of the cells in the population are NKX6.1-negative, ISL1-negative cells.

[0042] In some embodiments, less than 10%, less than 8%, less than 6%, or less than 4% of the cells in the population are NKX6.1-negative, ISL1-negative cells. In some embodiments, at least 60%, at least 65%, at least 70%, at least 73%, at least 75%, or at least 80% of the cells in the population are ISL1-positive cells. In some embodiments, 2-12%, 4-12%, 6-12%, 8-12%, 2-8%, 4-8%, 3-6%, or 3-5% of the cells in the population are NKX6.1-negative, ISL1-negative cells. In some embodiments, 50-90%, 50-85%, 50-80%, 50-75%, 50-70%, 50-60%, 60-90%, 60-85%, 60-80%, 60-75%, 60-70%, 65-90%, 65-85%, 65-80%, 65-75%, 65-70%, 70-90%, 70-85%, 70-80%, 70-75%, 75-90%, 75-85%, 75-80%, 80-90%, 80-85%, or 85-90% of the cells in the population are ISL1-positive cells.

[0043] In some embodiments, the population comprises more NKX6.1-negative, ISL1-positive cells than NKX6.1-positive, ISL1-positive cells. In some embodiments, at least 40% of the cells in the population are NKX6.1-negative, ISL1-positive cells. In some embodiments, at least 45%, at least 50%, about 40-50%, about 45-55%, or about 50-55% of the cells in the population are NKX6.1-negative, ISL1-positive cells. In some embodiments, at least 74%, at least 75%, at least 80%, at least 85%, at least 90%, about 85-95%, or about 90-95% of the cells in the population are ISL1-positive cells.

[0044] In some embodiments, the population comprises more stem cell-derived alpha cells than stem cell-derived beta cells. In some embodiments, the cell population is derived from stem cells in vitro.

[0045] In some embodiments, the population further comprises a culture medium. In some embodiments, the culture medium comprises a sugar. In some embodiments, the sugar is sucrose or glucose. In some embodiments, the culture medium comprises a sugar at a concentration of about 0.05% to about 1.5%. In some embodiments, the culture medium is CMRL medium, or the culture medium is HypoThermosol® FRS storage medium.

[0046] In some embodiments, the cell population is in a cell cluster. In some embodiments, the cell population is in one or more cell clusters. In some embodiments, the cell clusters are about 125 to about 225 microns in diameter, about 130 to about 160 microns in diameter, about 170 to about 225 microns in diameter, about 140 to about 200 microns in diameter, about 140 to about 170 microns in diameter, about 160 to about 220 microns in diameter, about 170 to about 215 microns in diameter, or 170 to about 200 microns in diameter.

[0047]

[0047] In some embodiments, the population has a genetic disruption in the beta-2-microglobulin gene.

[0048] In some embodiments, the population comprises NKX6.1-positive, ISL1-positive cells that express lower levels of MAFA than NKX6.1-positive, ISL1-positive cells from the pancreas of a healthy control adult subject. In some embodiments, the population comprises NKX6.1-positive, ISL1-positive cells that express higher levels of MAFB ... The present invention includes NKX6.1-positive, ISL1-positive cells that express higher levels of SIX2, HOPX, IAPP, and / or UCN3 than NKX6.1-positive, ISL1-positive cells derived from the pancreas of an adult subject.

[0048]

[0049] In some embodiments, the population comprises NKX6.1-positive, ISL1-positive cells that do not express MAFA. In some embodiments, the population comprises NKX6.1-positive, ISL1-positive cells that express MAFB.

[0049]

[0050] In some embodiments, the population is contained in a device for implantation into a subject. In some aspects, the present disclosure provides an encapsulated device for implantation comprising the population. In some embodiments, the device is implanted in a subject with diabetes. In some embodiments, the subject has type 1 diabetes. In some aspects, the present disclosure provides a method of treating a subject, the method comprising administering to the subject a composition comprising the population or implanting into the subject a device.

[0050]

[0051] In some aspects, disclosed herein are pharmaceutical compositions comprising a composition disclosed herein, or a cell population produced according to a method disclosed herein, and a pharmaceutically acceptable excipient or carrier.

[0051]

[0052] In some aspects, disclosed herein is a device comprising a composition disclosed herein or a cell population produced according to a method disclosed herein, wherein the device is configured to produce and release insulin when implanted in a subject.

[0052]

[0053] In some aspects, disclosed herein are methods of treating a subject comprising administering to the subject a composition disclosed herein, or a cell population produced according to a method disclosed herein, or a device disclosed herein.

[0053]

[0054] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings. [Brief explanation of the drawings]

[0054] [Figure 1]

[0055] Figure 1 shows the results of single-cell sequencing of in vitro endocrine cell populations produced according to two exemplary differentiation protocols (version A and version B), with or without PDBU applied from S4d5 to S5d2. [Figure 2A]

[0056] Figures 2A-2B summarize the percentage of C-peptide-positive, VMAT1-negative cells in in vitro endocrine cell populations produced following two exemplary differentiation protocols, with or without PDBU applied from S4d5 to S5d2 (Figure 2A), as measured by flow cytometry (Figure 2B). [Figure 2B] Figures 2A-2B summarize the percentage of C-peptide-positive, VMAT1-negative cells in in vitro endocrine cell populations produced following two exemplary differentiation protocols, with or without PDBU applied from S4d5 to S5d2 (Figure 2A), as measured by flow cytometry (Figure 2B). [Figure 3]

[0057] Figure 3 summarizes the percentage of glucagon-positive, somatostatin-negative cells (GCG+ / SST-) in in vitro endocrine cell populations produced according to two exemplary differentiation protocols, with or without PDBU applied from S4d5 to S5d2. [Figure 4]

[0058] Figure 4 summarizes the percentage of somatostatin-positive, glucagon-negative cells (SST+ / GCG-) in in vitro endocrine cell populations produced according to two exemplary differentiation protocols, with or without PDBU applied from S4d5 to S5d2. [Figure 5]

[0059] Figure 5 summarizes the percentage of VMAT1-positive, C-peptide-negative cells (VMAT1+ / c-peptide-) in in vitro endocrine cell populations produced according to two exemplary differentiation protocols, with or without PDBU applied from S4d5 to S5d2. [Figure 6A]

[0060] Figure 6A summarizes the percentage of SOX9-positive cells before reaggregation in in vitro endocrine cell populations produced according to two exemplary differentiation protocols, with or without PDBU applied from S4d5 to S5d2, as measured by flow cytometry. [Figure 6B] Figure 6B summarizes the percentage of SOX9-positive cells after reaggregation, as measured by flow cytometry, in in vitro endocrine cell populations produced according to two exemplary differentiation protocols, with or without PDBU applied from S4d5 to S5d2. [Figure 7]

[0061] Figure 7 summarizes the recovery rates after reaggregation in in vitro endocrine cell populations produced according to two exemplary differentiation protocols, with or without PDBU applied from S4d5 to S5d2. [Figure 8]

[0062] Figure 8 summarizes the glucose-stimulated insulin secretion (GSIS) responses of in vitro endocrine cell populations produced according to two exemplary differentiation protocols, with or without PDBU applied from S4d5 to S5d2. [Figure 9]

[0063] Figure 9 summarizes the insulin content of in vitro endocrine cell populations produced according to two exemplary differentiation protocols, with or without PDBU applied from S4d5 to S5d2. [Figure 10A]

[0064] Figures 10A-10B summarize the percentage of NKX6.1-positive, ISL1-positive cells (Figure 10B) in in vitro cell populations produced following three exemplary differentiation protocols with or without PDBU or PDBU and XXI applied between S4d5 and S5d2, as measured by flow cytometry (Figure 10A). [Figure 10B]

[0064] Figures 10A-10B summarize the percentage of NKX6.1-positive, ISL1-positive cells (Figure 10B) in in vitro cell populations produced according to three exemplary differentiation protocols, with or without PDBU or PDBU and XXI applied between S4d5 and S5d2, as measured by flow cytometry (Figure 10A). [Figure 11A]

[0065] Figures 11A-11C summarize the percentage of NKX6.1-positive / negative cells and ISL1-positive / negative cells (Figure 11B) in in vitro cell populations produced according to three exemplary differentiation protocols: a) Version A without PDBU or TPPB (Version A); b) with PDBU (VA / PDBU); or c) with TPPB (VA / TPPB), as measured by flow cytometry (Figure 11A). Figure 11C shows the cell yields of Version A, VA / PDBU, VA / TPPB, and VA / TPPB+XXI. [Figure 11B] Figures 11A-11C summarize the percentage of NKX6.1-positive / negative cells and ISL1-positive / negative cells (Figure 11B) in in vitro cell populations produced according to three exemplary differentiation protocols: a) Version A without PDBU or TPPB (Version A); b) with PDBU (VA / PDBU); or c) with TPPB (VA / TPPB), as measured by flow cytometry (Figure 11A). Figure 11C shows the cell yields of Version A, VA / PDBU, VA / TPPB, and VA / TPPB+XXI. [Figure 11C]Figures 11A-11C summarize the percentage of NKX6.1-positive / negative cells and ISL1-positive / negative cells (Figure 11B) in in vitro cell populations produced according to three exemplary differentiation protocols: a) Version A without PDBU or TPPB (Version A); b) with PDBU (VA / PDBU); or c) with TPPB (VA / TPPB), as measured by flow cytometry (Figure 11A). Figure 11C shows the cell yields of Version A, VA / PDBU, VA / TPPB, and VA / TPPB+XXI. DETAILED DESCRIPTION OF THE INVENTION

[0055]

[0066] The following description and examples will explain the embodiments of the present disclosure in detail. It should be understood that the present disclosure is not limited to the specific embodiments described herein and may therefore vary. Those skilled in the art will recognize that there are many variations and modifications to the present disclosure, which are encompassed within the scope of the present disclosure.

[0056]

[0067] All terms are intended to be understood as understood by one of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0057]

[0068] The headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0069] Although various features of the present disclosure may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although for clarity the present disclosure may be described herein in the context of separate embodiments, the present disclosure may also be implemented in a single embodiment.

[0058]

[0070] The following definitions supplement the definitions in the art and are directed to this application, and are not attributable to any related or unrelated examples, such as any commonly owned patents or patent applications. Although any methods and materials similar or equivalent to those described herein can be used in the practice of testing the present disclosure, preferred materials and methods are described herein. Therefore, the terms used herein are only for the purpose of describing specific embodiments and are not intended to be limiting.

[0059]

[0071] In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0060]

[0072] The use of "or" in this application means "and / or" unless stated otherwise. As used herein, the terms "and / or" and "any combinations thereof," and their grammatical equivalents, can be used interchangeably. These terms can convey that any combination is specifically intended. For illustrative purposes only, the following phrases, "A, B, and / or C" or "A, B, C, or any combinations thereof," can mean "A individually, B individually, C individually, A and B, B and C, A and C, and A, B, and C." The term "or" can be used conjunctively or disjunctively unless the context specifically implies a disjunctive use.

[0061]

[0073] Furthermore, use of the term "including" and other forms such as "include," "includes," and "included" is not limiting.

[0074] References in the specification to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" include all the features described in connection with those embodiments. This means that a certain feature, structure, or characteristic is present in at least some embodiments of the present disclosure, but not necessarily in all embodiments.

[0062]

[0075] As used in this specification and claims, the word "comprising" and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" ( "includes" and "include" and any other form of "including") or "containing" (and "contains" and "contain" and any other form of "containing") means including. The present disclosure is not intended to be exhaustive or open-ended and does not exclude additional, unrecited elements or method steps. It is intended that any embodiment discussed herein can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, compositions of the disclosure can be used to achieve the methods of the disclosure.

[0063]

[0076] As used herein, the term "about" in connection with a reference numerical value and grammatical equivalents thereof may include the numerical value itself and a range of values ​​plus or minus 10% of that numerical value.

[0077] The terms "about" or "approximately" refer to within an acceptable error range for a particular value, as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within or more than one standard deviation, as practiced in the art. Alternatively, "about" can mean a range of up to 20%, 10%, 5%, or 1% of a given value. In another example, the amount "about 10" includes 10 and any amount from 9 to 11. In yet another example, the term "about" in connection with a reference numerical value can also include ranges of plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of that value. Alternatively, particularly with respect to biological systems or processes, the term "about" can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value. When specific values ​​are described in applications and claims, unless otherwise stated, the term "about" should be assumed to mean within an acceptable error range for the particular value.

[0064]

[0078] As used herein, term " diabetes mellitus " and its grammatical equivalents can refer to the disease characterized by long-term high blood sugar level.For example, as used herein, term " diabetes mellitus " and its grammatical equivalents can refer to all or any type of diabetes, including but not limited to type 1 diabetes, type 2 diabetes, cystic fibrosis-related diabetes, surgical diabetes, gestational diabetes and mitochondrial diabetes.In some cases, diabetes can be a form of hereditary diabetes.

[0065]

[0079] The term "endocrine cells," unless otherwise specified, may refer to hormone-producing cells present in the pancreas of a living organism, such as "pancreatic islets," "pancreatic islet cells," "pancreatic islet equivalents," "pancreatic islet-like cells," "pancreatic islets," and grammatical equivalents thereof. In one embodiment, endocrine cells can be differentiated from pancreatic progenitor cells or precursors. Pancreatic islet cells may include various types of cells, including, but not limited to, pancreatic alpha cells, pancreatic beta cells, pancreatic delta cells, pancreatic F cells, and / or pancreatic epsilon cells. Pancreatic islet cells may also refer to groups of cells, cell clusters, etc.

[0066]

[0080] The terms "progenitor cell" and "precursor" cell are used interchangeably herein to refer to cells that have a more primitive cellular phenotype (e.g., at an earlier step in the developmental pathway or development than a fully differentiated cell) compared to cells that can result from differentiation. Progenitor cells can also often have significant or extremely high proliferative potential. Progenitor cells can give rise to many different differentiated cell types or a single differentiated cell type, depending on the developmental pathway and the environment in which the cells develop and differentiate.

[0067]

[0081] The term "precursor thereof" in relation to an insulin-positive endocrine cell can mean any cell, including, for example, a pluripotent stem cell, a definitive endoderm cell, a primitive gut cell, a pancreatic progenitor cell, or an endocrine progenitor cell, that can differentiate into an insulin-positive endocrine cell when cultured under conditions suitable for differentiating the precursor cell into an insulin-positive endocrine cell.

[0068]

[0082] The terms "stem cell-derived beta cells," "SC-beta cells," "functional beta cells," "functional pancreatic beta cells," "mature SC-beta cells," and their grammatical equivalents can refer to cells (e.g., non-native pancreatic beta cells) that exhibit at least one marker indicative of a pancreatic beta cell (e.g., PDX-1 or NKX6.1), secrete insulin, and exhibit a glucose-stimulated insulin secretion (GSIS) response characteristic of endocrine mature beta cells. In some embodiments, the terms "SC-beta cells" and "non-native beta cells" as used herein are interchangeable. In some embodiments, "SC-beta cells" include mature pancreatic cells. It should be understood that SC-beta cells need not be derived (e.g., directly) from stem cells. This is because the disclosed methods can derive SC-β cells from any insulin-positive endocrine cell or its precursor using any cell as a starting point (the invention is not intended to be limited in this manner, and examples include embryonic stem cells, induced pluripotent stem cells, progenitor cells, partially reprogrammed somatic cells (e.g., somatic cells partially reprogrammed to an intermediate state between an induced pluripotent stem cell and the somatic cell from which it was derived), pluripotent cells, totipotent cells, transdifferentiated versions of any of the above cells, and the like). In some embodiments, the SC-β cells exhibit a response to multiple glucose challenges (e.g., a series of at least one, at least two, or at least three or more glucose challenges). In some embodiments, the response resembles the response of endogenous pancreatic islets (e.g., human pancreatic islets) to multiple glucose challenges. In some embodiments, the morphology of SC-β cells resembles that of endogenous β cells. In some embodiments, the SC-β cells exhibit an in vitro GSIS response similar to that of endogenous β cells. In some embodiments, SC-β cells exhibit an in vivo GSIS response similar to that of endogenous β cells. In some embodiments, SC-β cells exhibit both in vitro and in vivo GSIS responses similar to that of endogenous β cells. The GSIS response of SC-β cells can be observed within 2 weeks after transplantation of SC-β cells into a host (e.g., a human or animal).In some embodiments, SC-β cells package insulin into secretory granules. In some embodiments, SC-β cells exhibit encapsulated crystalline insulin granules. In some embodiments, SC-β cells exhibit a stimulation index greater than 1. In some embodiments, SC-β cells exhibit a stimulation index greater than 1.1. In some embodiments, SC-β cells exhibit a stimulation index greater than 2. In some embodiments, SC-β cells exhibit cytokine-induced apoptosis in response to cytokines. In some embodiments, insulin secretion from SC-β cells is enhanced in response to known antidiabetic agents (e.g., secretagogues). In some embodiments, SC-β cells are monohormonal. In some embodiments, SC-β cells do not abnormally co-express other hormones, such as glucagon, somatostatin, or pancreatic polypeptide. In some embodiments, SC-β cells exhibit a low replication rate. In some embodiments, SC-β cells increase intracellular Ca2+ in response to glucose.

[0069]

[0083] The terms "stem cell-derived α cells," "SC-α cells," "functional α cells," "functional pancreatic α cells," "mature SC-α cells," and their grammatical equivalents can refer to cells (e.g., non-native pancreatic α cells) that exhibit at least one marker indicative of a pancreatic α cell (e.g., expressing glucagon, ISL1, but not NKX6.1), express glucagon, and secrete functional glucagon. In some embodiments, "SC-α cells" do not express somatostatin. In some embodiments, "SC-α cells" do not express insulin. In some embodiments, the terms "SC-α cells" and "non-native α cells" as used herein are interchangeable. In some embodiments, "SC-α cells" include mature pancreatic cells.

[0070]

[0084] The terms "stem cell-derived delta cells," "SC-delta cells," "functional delta cells," "functional pancreatic delta cells," "mature SC-delta cells," and their grammatical equivalents refer to at least one marker indicative of pancreatic delta cells (e.g., somatostatin), and include cells that express and differentiate somatostatin. In some embodiments, "SC-δ cells" may refer to cells that secrete insulin (e.g., non-native pancreatic δ cells). In some embodiments, "SC-δ cells" do not express glucagon. In some embodiments, "SC-δ cells" do not express insulin. In some embodiments, the terms "SC-δ cells" and "non-native δ cells" as used herein are interchangeable. In some embodiments, "SC-δ cells" include mature pancreatic cells.

[0071]

[0085] The terms "stem cell-derived enterochromaffin (EC) cells," "SC-EC cells," and their grammatical equivalents can refer to cells (e.g., non-native pancreatic EC cells) that exhibit at least one marker indicative of pancreatic EC cells (e.g., expressing VMAT1 (vesicular monoamine transporter 1), NKX6.1, but not ISL1). In some embodiments, the terms "SC-EC cells" and "non-native EC cells" as used herein are interchangeable.

[0072]

[0086] It should be understood that, like SC-β cells, SC-α, SC-δ, and SC-EC cells need not be derived (e.g., directly) from stem cells, as the disclosed methods can derive SC-α cells from other progenitor cells produced during in vitro differentiation of SC-β cells as a starting point (e.g., embryonic stem cells, induced pluripotent stem cells, progenitor cells, partially reprogrammed somatic cells (e.g., somatic cells partially reprogrammed to an intermediate state between an induced pluripotent stem cell and the somatic cell from which it was derived), pluripotent cells, totipotent cells, transdifferentiated versions of any of the foregoing cells, etc., can be used, as the invention is not intended to be so limited).

[0073]

[0087] As used herein, the term "insulin-producing cells" and its grammatical equivalents refer to cells that differentiate from pancreatic progenitor cells or their precursors and secrete insulin. Insulin-producing cells, as that term is used herein, can include pancreatic β cells and pancreatic β-like cells (e.g., insulin-positive endocrine cells) that synthesize (e.g., transcribe the insulin gene, translate proinsulin mRNA, and modify proinsulin mRNA to form insulin protein), express (e.g., realize the phenotypic trait conveyed by the insulin gene), or secrete (release insulin into the extracellular space) insulin in a constitutive or inducible manner. For example, a population of insulin-producing cells generated by differentiating insulin-positive endocrine cells or their precursors into SC-β cells according to the methods of the present disclosure can be pancreatic β cells or β-like cells (e.g., cells that have at least one or at least two characteristics of endogenous β cells and exhibit a glucose-stimulated insulin secretion (GSIS) response similar to that of endogenous mature β cells). For example, a population of insulin-producing cells produced by the methods disclosed herein may include mature pancreatic β cells or SC-β cells, and may also include non-insulin-producing cells (e.g., cells that do not produce or secrete insulin but have a cell-like phenotype).

[0074]

[0088] The terms "insulin-positive β-like cells," "insulin-positive endocrine cells," and their grammatical equivalents can refer to cells (e.g., pancreatic endocrine cells) that exhibit at least one marker indicative of pancreatic β cells and also express insulin, but lack the glucose-stimulated insulin secretion (GSIS) response characteristic of endogenous β cells. Exemplary markers of "insulin-positive endocrine cells" include, but are not limited to, NKX6.1 (NK6 homeobox 1), ISL1 (Islet1), and insulin. In some instances, the terms "insulin-positive endocrine cells" and "NKX6.1-positive, ISL1-positive cells" are used interchangeably.

[0075]

[0089] The term "beta cell marker" refers, without limitation, to proteins, peptides, nucleic acids, protein and nucleic acid polymorphisms, splice variants, protein or nucleic acid fragments, elements, and other analytes that are specifically expressed or present in pancreatic beta cells. Markers include, but are not limited to, pancreatic and duodenal homeobox 1 (PDX1) polypeptide, insulin, c-peptide, amylin, E-cadherin, Hnf3β, PCI / 3, B2, Nkx2.2, GLUT2, PC2, ZnT-8, ISL1, Pax6, Pax4, NeuroD, 1 Inf1b, Hnf-6, Hnf-3beta, and MafA, and those described by Zhang et al., Diabetes. 50(10):2231-6 (2001). In some embodiments, the beta cell marker is a nuclear beta-cell marker. In some embodiments, the beta cell marker is PDX1 or PH3.

[0076]

[0090] The term "pancreatic endocrine marker" can refer, without limitation, to proteins, peptides, nucleic acids, protein and nucleic acid polymorphisms, splice variants, protein or nucleic acid fragments, elements, and other analytes that are specifically expressed or present in pancreatic endocrine cells. Exemplary pancreatic endocrine cell markers include, but are not limited to, Ngn-3, NeuroD, and Islet-1.

[0077]

[0091] The terms "pancreatic progenitor cells," "pancreatic endocrine precursor cells," "pancreatic precursors," "pancreatic endocrine precursors," and their grammatical equivalents are used interchangeably herein and can refer to stem cells that can become pancreatic hormone-expressing cells that can form pancreatic endocrine cells, pancreatic exocrine cells, or pancreatic duct cells. These cells are committed to differentiation toward at least one type of pancreatic cell, such as insulin-producing beta cells, glucagon-producing alpha cells, somatostatin-producing delta cells (or D cells), and / or pancreatic polypeptide-producing F cells. Such cells can express at least one of the following markers: NGN3, NKX2.2, NeuroD, ISL-1, Pax4, Pax6, or ARX.

[0078]

[0092] As used herein, the term "PDX1-positive pancreatic progenitor cells" may refer to cells that are pancreatic endoderm (PE) cells capable of differentiating into SC-β cells, such as pancreatic β cells. PDX1-positive pancreatic progenitor cells express the marker PDX1. Other markers include, but are not limited to, Cdcp1, Ptf1a, HNF6, or NRx2.2. PDX1 expression can be assessed by any method known to those skilled in the art, such as immunochemistry using an anti-PDX1 antibody or quantitative RT-PCR. In some cases, PDX1-positive pancreatic progenitor cells lack NKX6.1 expression. In some cases, PDX1-positive pancreatic progenitor cells can also be referred to as PDX1-positive, NKX6.1-negative pancreatic progenitor cells because they lack NKX6.1 expression. In some cases, PDX1-positive pancreatic progenitor cells can also be referred to as "pancreatic foregut endoderm cells."

[0079]

[0093] The terms "PDX1-positive, NKX6.1-positive pancreatic progenitor cells" and "NKX6.1-positive pancreatic precursors" are used interchangeably herein and can refer to cells that are pancreatic endoderm (PE) cells and have the ability to differentiate into insulin-producing cells, such as pancreatic β cells. PDX1-positive, NKX6-1-positive pancreatic progenitor cells express the markers PDX1 and NKX6-1. Other markers include, but are not limited to, Cdcp1, Ptf1a, HNF6, or NRx2.2. NKX6-1 expression can be assessed by any method known to those skilled in the art, such as immunochemistry or quantitative RT-PCR using anti-NKX6-1 antibodies. As used herein, the terms "NKX6.1" and "NKX6-1" are equivalent and interchangeable. In some instances, PDX1-positive, NKX6-1-positive pancreatic progenitor cells can also be referred to as "pancreatic foregut precursor cells."

[0080]

[0094] The terms "NeuroD" and "NeuroD1" are used interchangeably to identify proteins and their encoding genes expressed in pancreatic endocrine precursor cells.

[0095] The term "epigenetics" refers to heritable changes in gene function that do not involve changes in DNA sequence. Epigenetics refers to the genetic alterations of chromosomes that affect gene activity and expression. While the term most often refers to changes in the phenotype of a cell, it can also be used to describe any heritable phenotypic change that does not result from modifications of the genome. Such effects on cellular and physiological phenotypic traits can result from external or environmental factors or be part of the normal developmental program. Epigenetics can also refer to functionally relevant changes in the genome that do not involve changes in the nucleotide sequence. Examples of mechanisms that generate such changes include DNA methylation and histone modifications, each of which alters the way genes are expressed without altering the underlying DNA sequence. Gene expression can be controlled by the action of repressor proteins that bind to silencer regions of DNA. These epigenetic changes can persist through cell divisions during the cell's lifespan and can persist for multiple generations, even though they do not involve changes in the organism's underlying DNA sequence. One example of epigenetics in eukaryotic cell biology is the process of cell differentiation. During morphogenesis, totipotent stem cells can develop into various pluripotent cells, which can then become fully differentiated cells.

[0081]

[0096] The term "epigenetic modifying compound" refers to a compound that induces epigenetic changes in genes, i.e., alters gene expression without altering the DNA sequence. Epigenetics helps determine whether genes are turned on or off and can affect protein production in certain cells, such as beta cells. Epigenetic modifications, such as DNA methylation and histone modifications, alter DNA accessibility and chromatin structure, thereby regulating patterns of gene expression. These processes are important for the normal development and differentiation of unique cell lineages in adult organisms. They can be modified by exogenous influences and therefore may contribute to or be the result of environmental modifications of phenotypes or pathological phenotypes. Importantly, epigenetic modifications play a key role in regulating pluripotency genes, which are inactivated during differentiation. Non-limiting examples of epigenetic modifying compounds include DNA methylation inhibitors, histone acetyltransferase inhibitors, histone deacetylase inhibitors, histone methyltransferase inhibitors, bromodomain inhibitors, or any combination thereof.

[0082]

[0097] The term "differentiated cell" or its grammatical equivalents refers to any primary cell that is not pluripotent in its natural form as that term is defined herein. Alternatively, the term "differentiated cell" can refer to a cell of a more specialized cell type derived from a cell of a less specialized cell type (e.g., a stem cell, such as an induced pluripotent stem cell) during the cell differentiation process. Without wishing to be limited by theory, pluripotent stem cells during normal ontogeny can initially differentiate into endodermal cells, which can form pancreatic cells and other endodermal cell types. Further differentiation of endodermal cells leads to the pancreatic pathway, with approximately 98% of cells becoming exocrine, ductal, or matrix cells, and approximately 2% becoming endocrine cells. Early endocrine cells are pancreatic islet precursor cells, which can then further differentiate into insulin-producing cells (e.g., functional endocrine cells) that secrete insulin, glucagon, somatostatin, or pancreatic polypeptide. Endodermal cells can also differentiate into other cells of endodermal origin, such as lung, liver, intestine, thymus, etc.

[0083]

[0098] As used herein, the term "somatic cell" can refer to any cell that forms an organism, as opposed to a germline cell. In mammals, germline cells (also known as "gametes") are the sperm and eggs that fuse during fertilization to produce a cell called a zygote, from which the entire mammalian embryo develops. Aside from sperm and eggs (gametocytes) and undifferentiated stem cells, from which somatic cells are made, all other cell types in the mammalian body are somatic cells. Internal organs, skin, bone, blood, and connective tissue are all made from somatic cells. In some embodiments, the somatic cells are "non-embryonic somatic cells," which are somatic cells that are not present in or obtained from an embryo and do not result from the in vitro propagation of such cells. In some embodiments, the somatic cell is an "adult somatic cell," which means a cell present in or obtained from an organism other than an embryo or fetus, or resulting from the in vitro propagation of such a cell. Unless otherwise indicated, the method for converting at least one insulin-positive endocrine cell or precursor thereof to an insulin-producing, glucose-responsive cell can be performed both in vivo and in vitro (in vivo performed when at least one insulin-positive endocrine cell or precursor thereof is present in the subject, and in vitro performed with at least one insulin-positive endocrine cell or precursor thereof that has been isolated and maintained in culture).

[0084]

[0099] As used herein, the term "adult cell" may refer to cells found throughout the body after embryonic development.

[0100] As used herein, the term "endodermal cell" can refer to a cell that is from one of the three primary germ cell layers in a very early embryo (the other two being mesoderm and ectoderm). The endoderm is the innermost of the three layers. Endodermal cells differentiate to give rise first to the embryonic gut, then to the lining of the respiratory and digestive tracts (e.g., the intestine), the liver, and the pancreas.

[0085]

[0101] As used herein, the term "cells of endodermal origin" refers to any cell that develops or differentiates from endodermal cells. For example, cells of endodermal origin include cells of the liver, lung, pancreas, thymus, intestine, stomach, and thyroid. Without wishing to be bound by theory, liver and pancreatic progenitor cells (also called pancreatic precursor cells) develop from endodermal cells in the embryonic foregut. Shortly after their identification, liver and pancreatic precursor cells rapidly acquire distinct cellular functions and regenerative capabilities. These changes are driven by inductive signals and genetic regulatory factors that are highly conserved among vertebrates. Interest in organ development and regeneration has been fueled by the strong need for hepatocytes and pancreatic β cells in the therapeutic treatment of liver failure and type 1 diabetes. Studies in various model organisms and humans have revealed a network of evolutionarily conserved inductive signals and transcription factors that trigger hepatocyte and pancreatic cell differentiation and provide guidance on how to promote hepatocyte and β cell differentiation from diverse stem and progenitor cell types.

[0086]

[0102] As used herein, the term "definitive endoderm" may refer to cells that can differentiate from endoderm cells and differentiate into SC-β cells (e.g., pancreatic β cells). Definitive endoderm cells express the marker Sox17. Other markers characteristic of definitive endoderm cells include, but are not limited to, MIXL2, GATA4, HNF3B, GSC, FGF17, VWF, CALCR, FOXQ1, CXCR4, Cerberus, OTX2, goosecoid, C-Kit, CD99, CMKOR1, and CRIP1. In particular, definitive endoderm cells herein express Sox17 and in some embodiments, Sox17 and HNF3B, but do not express significant levels of GATA4, SPARC, APF, or DAB. Definitive endoderm cells are not positive for the marker PDX1 (e.g., they are PDX1 negative). Definitive endoderm cells have the potential to differentiate into cells including liver, lung, pancreatic, thymus, intestine, stomach, and thyroid cells. Expression of Sox17 and other definitive endoderm markers can be assessed by any method known to those skilled in the art, such as immunochemistry using anti-Sox17 antibodies or quantitative RT-PCR.

[0087]

[0103] The term "pancreatic endoderm" can refer to cells of endodermal origin that can differentiate into multiple pancreatic lineages, including pancreatic beta cells, but no longer have the capacity to differentiate into non-pancreatic lineages.

[0104] As used herein, the term "primitive gut cells" or "gut cells" may refer to cells that can differentiate from endoderm cells and differentiate into SC-β cells (e.g., pancreatic β cells). Primitive gut cells express at least one of the following markers: HNP1-β, HNF3-β, or HNF4-α. In some examples, primitive gut cells are FOXA2-positive and SOX2-positive. In some cases, primitive gut cells are FOXA2-positive and PDX1-negative, i.e., they express both FOXA2 (also known as HNF3-β) and SOX2. In some cases, primitive gut cells are FOXA2-positive and PDX1-negative, i.e., they express FOXA2 but not PDX1. Primitive gut cells have the potential to differentiate into cells including lung, liver, pancreatic, stomach, and intestinal cells. Expression of HNF1-β and other primitive gut markers can be assessed by any method known to those skilled in the art, such as immunochemistry using anti-HNF1-β antibodies.

[0088]

[0105] As used herein, the term "stem cell" may refer to an undifferentiated cell that can proliferate and give rise to more progenitor cells that have the capacity to produce a large number of mother cells that can then give rise to differentiated or differentiable daughter cells. The daughter cells themselves can be induced to proliferate and generate progeny that subsequently differentiate into one or more mature cell types while retaining one or more cells with the developmental potential of the parent. The term "stem cell" may refer to a subset of progenitor cells that, under certain circumstances, have the ability or potential to differentiate into a more specialized or differentiated phenotype and, under certain circumstances, retain the ability to proliferate substantially without differentiation. In one embodiment, the term stem cell generally refers to a naturally occurring mother cell whose descendants (progeny) specialize through differentiation, often in different directions, e.g., by acquiring entirely distinct characteristics, as occurs in the gradual diversification of cells and tissues in an embryo. Cell differentiation is a complex process that typically occurs through many cell divisions. Differentiated cells can be derived from pluripotent cells, which themselves are derived from pluripotent cells, and so on. Although each of these pluripotent cells is considered a stem cell, the range of cell types each can give rise to can vary considerably. Some differentiated cells also have the ability to give rise to cells with greater developmental potential. Such ability can be natural or artificially induced by treatment with various factors. In many biological examples, stem cells are also "pluripotent" because they can generate progeny with two or more different cell types, but this is not necessary for them to be "stem." "Self-renewal" is another classic part of the definition of stem cells, and it is important as used herein. Theoretically, self-renewal can occur through one of two main mechanisms: stem cells can divide asymmetrically, with one daughter retaining the stem cell state and the other daughter expressing several different, other specific functions and phenotypes. Alternatively, some stem cells in a population can divide symmetrically into two stem cells, thereby maintaining some stem cells in the population as a whole while other cells in the population give rise only to differentiated progeny.Although technically, cells that begin as stem cells progress toward a differentiated phenotype, it is also possible for them to "reverse" and reverse the stem cell phenotype. This is often referred to by those skilled in the art as "dedifferentiation," "reprogramming," or "reverse differentiation." As used herein, the term "pluripotent stem cells" includes embryonic stem cells, induced pluripotent stem cells, placental stem cells, and the like.

[0089]

[0106] As used herein, the term "pluripotent" can refer to cells that have the ability to differentiate under various conditions into two or more differentiated cell types, preferably into cell types characteristic of all three germ cell layers. Pluripotent cells are primarily characterized by their ability to differentiate into two or more cell types, preferably into all three germ layers, using, for example, a nude mouse teratoma formation assay. While pluripotency can also be demonstrated by the expression of embryonic stem (ES) cell markers, the preferred test for pluripotency is to demonstrate the ability to differentiate into cells of each of the three germ layers. Note that simply culturing such cells does not, in and of itself, render them pluripotent. Reprogrammed pluripotent cells (e.g., iPS cells, as that term is defined herein) are also characterized by their ability to be passaged for extended periods without loss of growth potential, compared to primary parent cells, which generally have the ability to divide only a limited number of times in culture.

[0090]

[0107] As used herein, the terms "iPS cells" and "induced pluripotent stem cells" are used interchangeably and refer to cells that have been induced, for example, by inducing the forced expression of one or more genes. By this it can mean pluripotent stem cells that have been artificially derived (eg, induced or by complete reversion) from non-pluripotent cells, typically from adult somatic cells.

[0091]

[0108] The term "phenotype" can refer to all of the one or several biological characteristics that define a cell or organism under a particular set of environmental conditions and factors, regardless of the actual genotype.

[0092]

[0109] The terms "subject," "patient," or "individual" are used interchangeably herein and may refer to an animal, e.g., a human, from which cells are obtained and / or to which treatment, including prophylactic treatment, using the cells described herein is provided. For treatment of an infection, condition, or disease state specific to a particular animal, such as a human subject, the term subject may refer to that particular animal. "Non-human animal" and "non-human mammal," used interchangeably herein, include mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The term "subject" also encompasses any vertebrate, including, but not limited to, mammals, reptiles, amphibians, and fish. Advantageously, however, the subject is a mammal, such as a human, or other mammal, e.g., a dog, cat, horse, other livestock, or cows, sheep, pigs, or other production mammals. "A patient in need thereof" or "subject in need thereof," as used herein, refers to a patient diagnosed with or suspected of having a disease or disorder, for example, but not limited to, diabetes.

[0093]

[0110] As used herein, "administering" can mean providing one or more compositions described herein to a patient or subject. By way of example and not limitation, administration, e.g., injection, of a composition can be performed via intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im) injection. One or more such routes can be employed. Parenteral administration can be, for example, via bolus injection or gradual perfusion over time. Alternatively, or concurrently, administration can be via the oral route. Additionally, administration can involve surgical deposition of a bolus or pellet of cells or positioning of a medical device. In one embodiment, a composition of the present disclosure can comprise engineered or host cells expressing a nucleic acid sequence described herein or a vector comprising at least one nucleic acid sequence described herein in an amount effective to treat or prevent a proliferative disorder. A pharmaceutical composition can include a cell population described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include a buffer such as neutral buffered saline or phosphate buffered saline, a carbohydrate such as glucose, mannose, sucrose, or dextran, mannitol, a protein, polypeptide, or an amino acid such as glycine, an antioxidant, a chelating agent such as EDTA or glutathione, an adjuvant (e.g., aluminum hydroxide), and a preservative.

[0094]

[0111] Some numerical values ​​disclosed throughout are referred to as, for example, "X is at least or at least about 100, or 200 [or any number]." This includes the number itself and the following: i) X is at least 100; ii) X is at least 200; iii) X is at least about 100, and iv) X is at least about 200 All of the above are included.

[0095]

[0112] All of these different combinations are contemplated by the numerical values ​​disclosed throughout. Unless otherwise specifically indicated to the contrary, all numerical values ​​disclosed, whether in terms of therapeutic administration, days, months, years, weights, dosages, etc., are to be interpreted in this manner. This should be explained.

[0096]

[0113] The generally disclosed ranges may be referred to, for example, as "X is administered on or about days 1-2, or on or about days 2-3 [or any range of numbers]." The ranges may include both the numbers themselves (e.g., the endpoints of the ranges) and the following: i) X is administered between days 1 and 2; ii) X is administered between days 2 and 3; iii) X is administered between about days 1 and 2; iv) X is administered between about days 2 and 3; v) X is administered between day 1 and about day 2; vi) X is administered between day 2 and about day 3; vii) X is administered between about day 1 and about day 2, and viii) X is administered between about day 2 and about day 3 All of the above are included.

[0097]

[0114] All of these different combinations are contemplated by the ranges disclosed throughout. Unless otherwise specifically indicated to the contrary, all disclosed ranges, whether in terms of administration of therapeutic agents, days, months, years, weights, dosages, etc., should be interpreted in this manner.

[0098]

[0115] In some embodiments, the present disclosure provides compositions and methods for differentiating pancreatic progenitor cells. The compositions and methods provided herein can, in some embodiments, provide pancreatic beta cells, cell populations, or cell clusters with high purity pancreatic beta cells, high insulin content, excellent glucose-dependent insulin secretion response, and an appropriate percentage of pancreatic alpha and delta cells and enterochromaffin cells, which can be structurally and functionally similar to native pancreatic islets.

[0099]

[0116] In some embodiments, methods for differentiating pancreatic endocrine cells are provided herein. In some examples, the methods result in the production of increased pancreatic beta cells, increased pancreatic alpha cells, increased pancreatic delta cells, decreased enterochromaffin cells (EC cells), or any combination thereof. In some examples, the methods result in the production of an in vitro cell composition comprising approximately 30%-40% pancreatic beta cells, 30%-40% pancreatic alpha cells, 3-10% pancreatic delta cells, and / or less than 20% EC cells. In some examples, the cell compositions produced according to the methods disclosed herein have improved glucose-stimulated insulin secretion (GSIS) responses compared to cell compositions produced according to conventional methods. In some examples, the cell compositions disclosed herein have dynamic GSIS responses that approximate those of native pancreatic islets.

[0100]

[0117] In some embodiments, the methods provided herein utilize PKC activation during or after the induction of NKX6.1 expression in PDX1 positive pancreatic progenitor cells, for example, in the final stage of differentiating PDX1 positive pancreatic progenitor cells into PDX1 positive, NKX6.1 positive pancreatic progenitor cells.Without being bound by any particular theory, the activation of PKC signaling in PDX1 positive, NKX6.1 positive pancreatic progenitor cells can affect the differentiation fate of certain cells, leading to an increase in the percentage of pancreatic alpha cells and a decrease in the percentage of EC cells.

[0101]

[0118] In some aspects, the disclosure provides a method for producing a pancreatic progenitor cell line comprising the steps of: (a) contacting a plurality of PDX1-positive, NKX6.1-negative pancreatic progenitor cells with one or more of a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor, thereby producing a first cell population; and (b) contacting the first cell population with a PKC activator and a γ-secretase inhibitor, as well as a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor, thereby producing a first cell population. (c) contacting the second cell population with one or more of a PKC activator, a gamma-secretase inhibitor, and a TGF-β signaling pathway inhibitor, a growth factor from the EGF family, a RA signaling pathway activator, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound, thereby producing a third cell population.

[0102]

[0119] In some embodiments, the present disclosure provides a method comprising contacting a cell population with a gamma-secretase inhibitor and one or both of a growth factor from the TGFβ superfamily and a growth factor from the FGF family.In some embodiments, the cell population comprises PDX1 positive cells.In some embodiments, the cell population comprises PDX1 positive, NKX6.1 negative cells.In some embodiments, the cell population comprises PDX1 positive, NKX6.1 positive cells.

[0103]

[0120] In some aspects, the present disclosure provides a method comprising: (a) contacting a plurality of PDX1-positive, NKX6.1-negative pancreatic progenitor cells with one or more of a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, a RA signaling pathway activator, and an SHH pathway inhibitor for a period of 1 to 5 days or less, thereby producing a first cell population; and (b) contacting the first cell population with a gamma-secretase inhibitor. In some embodiments, the contacting in step (a) is for a period of 4 or 5 days. In some embodiments, step (b) further comprises contacting the first cell population with one or more of a PKC activator, a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, a RA signaling pathway activator, and an SHH pathway inhibitor.

[0104]

[0121] In some aspects, the disclosure provides a method for producing a pancreatic progenitor cell line, comprising: (a) contacting a plurality of PDX1-positive, NKX6.1-negative pancreatic progenitor cells with one or more of a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor, thereby producing a first cell population; and (b) contacting the first cell population with a PKC activator, and one or more of a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor, thereby producing a first cell population. (c) contacting the second cell population with one or more of a PKC activator, a γ-secretase inhibitor, a TGF-β signaling pathway inhibitor, a growth factor from the EGF family, an RA signaling pathway activator, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound, thereby producing a third cell population. In some examples, the benzolactam derivative is TPPB.

[0105]

[0122] In some aspects, the disclosure provides a method for producing a pancreatic progenitor cell line comprising the steps of: (a) contacting a plurality of PDX1-positive, NKX6.1-negative pancreatic progenitor cells with one or more of a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor, thereby producing a first cell population; (b) contacting the first cell population with a PKC activator, and one or more of a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor, thereby producing a second cell population; and (c) contacting the second cell population with a PKC activator, and one or more of a γ-secretase inhibitor, a TGF-β signaling pathway inhibitor, a growth factor from the EGF family, and an SHH pathway inhibitor, thereby producing a second cell population. (d) contacting the third cell population with one or more of a TGF-β signaling pathway inhibitor, a RA signaling pathway activator, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound, thereby producing a third cell population; (d) contacting the third cell population with one or more of a TGF-β signaling pathway inhibitor, a RA signaling pathway activator, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound, thereby producing a fourth cell population; and (e) contacting the fourth cell population with a PKC activator, and one or more of a serum albumin protein, vitamin C, a TGF-β signaling pathway inhibitor, a SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound, thereby producing a fifth cell population.

[0106]

[0123] In some embodiments, the methods disclosed herein include differentiating PDX1-positive pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells by contacting the PDX1-positive pancreatic progenitor cells with a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, a RA signaling pathway activator, and an SHH pathway inhibitor, thereby differentiating the PDX1-positive pancreatic progenitor cells into PDX1-positive pancreatic progenitor cells, thereby producing a cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells. In some examples, the methods include contacting a cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a first composition comprising a PKC activator, a gamma-secretase inhibitor, a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, a RA signaling pathway activator, and an SHH pathway inhibitor for a first period of time. In some examples, the method includes, after the first period, contacting the cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a second composition comprising a PKC activator, a gamma-secretase inhibitor, a TGF-β signaling pathway inhibitor, a growth factor from the EGF family, a RA signaling pathway activator, a SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound for a second period. In some examples, the method includes, after the second period, contacting the cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a third composition that differentiates at least a portion of the PDX1-positive, NKX6.1-positive pancreatic progenitor cells into NKX6.1-positive, ISL1-positive endocrine cells, thereby producing a cell population comprising NKX6.1-positive, ISL1-positive endocrine cells.

[0107]

[0124] In some examples, the present disclosure provides an in vitro composition comprising a cell population, the cell population comprising (a) at least about 35% cells that express C-peptide and do not express VMAT1; and (b) at least about 35% cells that express VMAT1 or at least about 15% cells that express glucagon (e.g., as measured by flow cytometry). In some aspects, the present disclosure provides a composition comprising an in vitro cell population, the cell population comprising at least about 35% cells that express C-peptide and do not express VMAT1; and (i) at least about 35% cells that express VMAT1, and / or (ii) at least about 15% cells that express glucagon. In some embodiments, the percentage of cells is measured by flow cytometry. In some examples, the cell population comprises at least about 30% cells that express VMAT1 and at least about 20% cells that express glucagon.

[0108]

[0125] In some examples, provided herein are in vitro compositions comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells; NKX6.1-positive, ISL1-positive endocrine cells; and a PKC activator, wherein the PKC activator is a benzolactam derivative.

[0109]

[0126] In some examples, a composition comprising a cell population, comprising: (a) a cell population having 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 30-40%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-90%, 50-80%, 50-70%, 50-60%, 60-90%, 60-80%, 60-70%, 70-90%, 70-80%, 70-90%, 70-90%, 70-70%, 70-9 ... ~80% or 80-90% of cells express C-peptide and ISL1 but not VMAT1; (b) 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-40%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15-40%, 15-35%, 15-30%, 15-25%, 15-20%, 2% of the cell population. (c) 0-40%, 20-35%, 20-30%, 20-25%, 25-40%, 25-35%, 25-30%, 30-40%, 30-35%, or 35-40% of the cells express glucagon but not somatostatin; (d) 3-20%, 3-15%, 3-12%, 3-10%, 3-8%, 3-5%, 4-20%, 4-15%, 4-12%, 4-10%, 4-8%, 4-5%, 5- Provided herein are compositions wherein 20%, 5-15%, 5-12%, 5-10%, 5-8%, 7-20%, 7-15%, 7-12%, 7-10%, 9-20%, 9-15%, 9-12%, 8-10%, 8-12%, 8-15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15%, or 15-20% of the cells express somatostatin but do not express glucagon.

[0110]

[0127] In some examples, a composition comprising a cell population, comprising: (a) a cell population having 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 30-40%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-90%, 50-80%, 50-70%, 50-60%, 60-90%, 60-80%, 60-70%, 70-90%, 70-80%, 70-90%, 70-90%, 70-70%, 70-9 ... ~80%, or 80-90% of cells express C-peptide and ISL1 but not VMAT1; (b) 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-40%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15-40%, 15-35%, 15-30%, 15-25%, 15-20%, 20% of the cell population. ~40%, 20-35%, 20-30%, 20-25%, 25-40%, 25-35%, 25-30%, 30-40%, 30-35%, or 35-40% of the cells express glucagon but not somatostatin; and (c) 3-20%, 3-15%, 3-12%, 3-10%, 3-8%, 3-5%, 4-20%, 4-15%, 4-12%, 4-10%, 4-8%, 4-5%, Provided herein are compositions wherein 5-20%, 5-15%, 5-12%, 5-10%, 5-8%, 7-20%, 7-15%, 7-12%, 7-10%, 9-20%, 9-15%, 9-12%, 8-10%, 8-12%, 8-15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15%, or 15-20% of the cells express somatostatin but do not express glucagon.

[0111]

[0128] In some examples, the methods provided herein include PKC activation when differentiating PDX1-positive, NKX6.1-positive pancreatic progenitor cells into NKX6.1-positive, ISL1-positive endocrine cells. For example, a PKC activator can be introduced during the initial period of contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a differentiation factor that directs the differentiation of PDX1-positive, NKX6.1-positive pancreatic progenitor cells into NKX6.1-positive, ISL1-positive endocrine cells. In some examples, the methods include: (a) contacting a cell population containing PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a first composition containing a PKC activator, a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor for 1 to 2 days, thereby obtaining a first transformed cell population containing PDX1-positive, NKX6.1-positive pancreatic progenitor cells; and and (b) contacting a first transformed cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a PKC activator, a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, and an epigenetic modifying compound for 1 to 2 days, thereby obtaining a second transformed cell population comprising NKX6.1-positive, ISL1-positive endocrine cells.

[0112] Methods for Producing Endocrine Cells

[0129] In aspects, the present disclosure relates to compositions and methods for producing endocrine cells from pancreatic progenitor or precursor cells. Certain exemplary detailed protocols for producing endocrine cells to provide at least one SC-β cell are described in U.S. Patent Application Publication Nos. 2015 / 0240212 and 2015 / 0218522, each of which is incorporated herein by reference in its entirety.

[0113]

[0130] In some examples, the method for producing a population of endocrine cells, when producing pancreatic beta cells, results in an increased percentage of pancreatic alpha and / or delta cells and a decreased percentage of pancreatic EC cells. In some embodiments, the methods disclosed herein can be used to obtain an enriched population of alpha cells. In some embodiments, the methods disclosed herein can be used to obtain an enriched population of delta cells. In some examples, the method for producing an endocrine cell population includes: (a) contacting a cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a PKC activator for a first period of time; and (b) after the first period of time, contacting the cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a composition comprising a TGF-beta signaling pathway inhibitor, a thyroid hormone signaling pathway activator, and an epigenetic modifying compound, thereby producing a cell population comprising pancreatic endocrine cells. In some examples, the cell population produced according to the methods disclosed herein exhibits (i) an increased percentage of cells expressing somatostatin; (ii) an increased percentage of cells expressing glucagon; (iii) a decreased percentage of cells expressing VMAT1; or (iv) an increased percentage of cells expressing C-peptide, compared to a corresponding cell population produced without contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a PKC activator for the first period of time.

[0114]

[0131] In some examples, the method includes contacting a cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a first composition comprising a factor selected from the group consisting of a PKC activator, a gamma-secretase inhibitor, a growth factor from the TGF-β superfamily, a growth factor from the FGF family, a RA signaling pathway activator, and an SHH pathway inhibitor for a first period of time; and after the first period of time, contacting the cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a second composition comprising a factor selected from the group consisting of a PKC activator, a gamma-secretase inhibitor, and a TGF-β signaling pathway inhibitor, a growth factor from the EGF family, a RA signaling pathway activator, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound for a second period of time. In some examples, the first composition comprises a PKC activator, a gamma-secretase inhibitor, a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor. In some examples, the second composition comprises a PKC activator, a gamma-secretase inhibitor, a TGF-β signaling pathway inhibitor, a growth factor from the EGF family, an RA signaling pathway activator, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound.

[0115]

[0132] In some examples, the composition that differentiates at least a portion of PDX1-positive, NKX6.1-positive pancreatic progenitor cells into NKX6.1-positive, ISL1-positive endocrine cells is a composition that inhibits TGF-β signaling. In some examples, the composition comprises a differentiation factor selected from the group consisting of a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, an epigenetic modifying compound, a growth factor from the EGF family, a RA signaling pathway activator, an SHH pathway inhibitor, a gamma-secretase inhibitor, a protein kinase inhibitor, a ROCK inhibitor, and a BMP signaling pathway inhibitor.

[0116]

[0133] In some examples, the method further includes contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a composition comprising a PKC activator. For example, the method includes (a) contacting a cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a first composition comprising a PKC activator, a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor for 1 to 2 days, thereby obtaining a first transformed cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells; and (b) contacting the first transformed cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a PKC activator, a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, and an epigenetic modifying compound for 1 to 2 days, thereby obtaining a second transformed cell population comprising NKX6.1-positive, ISL1-positive endocrine cells. In some examples, the method further includes contacting the second transformed cell population with a composition comprising a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, and an epigenetic modifying compound, thereby producing a cell population comprising pancreatic endocrine cells.

[0117]

[0134] In some examples, a cell population comprising pancreatic endocrine cells produced according to the methods provided herein comprises at least about 4% of cells expressing somatostatin, at least about 15% of cells expressing glucagon, at least about 35% of cells expressing VMAT1, or at least about 40% of cells expressing C-peptide, as measured by flow cytometry. In some examples, a cell population comprising pancreatic endocrine cells comprises at least about 50% more cells expressing somatostatin, at least about 50% more cells expressing glucagon, at least about 20% less cells expressing VMAT1, or at least about 10% more cells expressing C-peptide, compared to a corresponding cell population produced without contact with a PKC activator. In some examples, the cell population comprising pancreatic endocrine cells comprises at least about 100% more cells expressing somatostatin, at least about 200% more cells expressing glucagon, at least about 50% less cells expressing VMAT1, or at least about 20% more cells expressing C-peptide, compared to a corresponding cell population produced without contact with a PKC activator.

[0118]

[0135] In some embodiments, the present disclosure provides a method comprising contacting a plurality of PDX1-positive, NKX6.1-negative pancreatic progenitor cells with one or more of a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, a RA signaling pathway activator, and an SHH pathway inhibitor, thereby producing a first cell population. In some examples, the method further comprises contacting the first cell population with a PKC activator and a gamma-secretase inhibitor, and one or more of a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, a RA signaling pathway activator, and an SHH pathway inhibitor, thereby producing a second cell population. In some examples, the method further comprises contacting the second cell population with a PKC activator, a gamma-secretase inhibitor, a TGF-β signaling pathway inhibitor, a growth factor from the EGF family, a RA signaling pathway activator, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and and an epigenetic modifying compound, thereby producing a third cell population. In some examples, the method further includes the steps of (a) contacting a plurality of PDX1-positive, NKX6.1-negative pancreatic progenitor cells with one or more of a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor, thereby producing a first cell population; and (b) contacting the first cell population with a PKC activator and a γ-secretase inhibitor, as well as a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor, thereby producing a third cell population. and an SHH pathway inhibitor, thereby producing a second cell population; and (c) contacting the second cell population with one or more of a PKC activator, a gamma-secretase inhibitor, and a TGF-β signaling pathway inhibitor, a growth factor from the EGF family, a RA signaling pathway activator, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound, thereby producing a third cell population. In some examples, the method further includes (d) contacting the third cell population with one or more of serum albumin protein, vitamin C, a TGF-β signaling pathway inhibitor, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound, thereby producing a fourth cell population. In some examples, step (d) includes contacting the third cell population with a PKC activator.

[0119]

[0136] In some aspects, the disclosure provides a method for producing a pancreatic progenitor cell line, comprising: (a) contacting a plurality of PDX1-positive, NKX6.1-negative pancreatic progenitor cells with one or more of a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor, thereby producing a first cell population; and (b) contacting the first cell population with a PKC activator, and one or more of a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor, thereby producing a first cell population. (c) contacting the second cell population with one or more of a PKC activator, a γ-secretase inhibitor, a TGF-β signaling pathway inhibitor, a growth factor from the EGF family, an RA signaling pathway activator, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound, thereby producing a third cell population. In some examples, the benzolactam derivative is TPPB. In some examples, step (b) of producing the second cell population comprises contacting the first cell population with a γ-secretase inhibitor. In some examples, the method further includes (d) contacting the third cell population with one or more of a TGF-β signaling pathway inhibitor, an RA signaling pathway activator, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound, thereby producing a fourth cell population. In some examples, the step (d) of producing the fourth cell population does not include contacting the third cell population with a PKC activator. In some examples, the step (d) of producing the fourth cell population does not include contacting the third cell population with a gamma-secretase inhibitor. In some examples, the step (d) of producing the fourth cell population does not include contacting the third cell population with an SHH pathway inhibitor.In some examples, step (d) of producing the fourth cell population does not include contacting the third cell population with a growth factor from the EGF family.

[0120]

[0137] In some examples, the method further comprises (e) administering to the fourth population of cells a fourth cell population containing a serum albumin protein, vitamin C, a TGF-β signaling pathway inhibitor, an SHH pathway inhibitor, a TH signaling pathway inhibitor, or a TH signaling pathway inhibitor. In some examples, step (e) further comprises contacting the fourth cell population with one or more of a PKC pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound, thereby producing a fifth cell population. In some examples, step (e) comprises contacting the fourth cell population with a PKC activator.

[0121]

[0138] In some aspects, the disclosure provides a method for producing a pancreatic progenitor cell line comprising the steps of: (a) contacting a plurality of PDX1-positive, NKX6.1-negative pancreatic progenitor cells with one or more of a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor, thereby producing a first cell population; (b) contacting the first cell population with a PKC activator and one or more of a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor, thereby producing a second cell population; and (c) contacting the second cell population with a PKC activator and one or more of a γ-secretase inhibitor, a TGF-β signaling pathway inhibitor, a growth factor from the EGF family, an RA signaling pathway activator, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor. (d) contacting the third cell population with one or more of a TGF-β signaling pathway inhibitor, a RA signaling pathway activator, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound, thereby producing a fourth cell population; and (e) contacting the fourth cell population with a PKC activator and one or more of serum albumin protein, vitamin C, a TGF-β signaling pathway inhibitor, a SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound, thereby producing a fifth cell population. In some examples, step (d) of the methods provided herein comprises contacting the fourth cell population with serum albumin protein.

[0122]

[0139] In some examples, step (a) of the methods disclosed herein for producing a first cell population is carried out over about 1, 2, 3, 4, 5, or 6 days. In some examples, step (a) of the methods disclosed herein for producing a first cell population is carried out over 3 to 5 days, e.g., 3 to 4 days, 4 to 5 days, about 3 days, about 4 days, or about 5 days. In some examples, step (a) of the methods disclosed herein for producing a first cell population is carried out over 4 days. In some examples, step (b) of the methods disclosed herein for producing a second cell population is carried out over 1, 2, 3, or 4 days. In some examples, step (b) of the methods disclosed herein for producing a second cell population is carried out over 1 to 3 days, e.g., 1 to 2 days, 2 to 3 days, about 1 day, about 2 days, or about 3 days. In some examples, step (b) of the methods disclosed herein for producing a second cell population is carried out over 2 days. In some examples, step (c) of the methods disclosed herein for producing a third cell population is carried out over 1, 2, 3, or 4 days. In some examples, step (c) of the methods disclosed herein for producing a third cell population is carried out over 1 to 3 days, e.g., 1 to 2 days, 2 to 3 days, about 1 day, about 2 days, or about 3 days. In some examples, step (c) of the methods disclosed herein for producing a third cell population is carried out over 2 days. In some examples, step (d) of the methods disclosed herein for producing a fourth cell population is carried out over 1, 2, 3, 4, 5, 6, or 7 days. In some examples, step (d) of the methods disclosed herein for producing a fourth cell population is carried out over 4 to 6 days, e.g., 5 to 6 days, 4 to 5 days, about 4 days, about 5 days, or about 6 days. In some examples, step (d) of the methods disclosed herein for producing a fourth cell population is carried out over 5 days. In some examples, step (e) for producing the fifth cell population in the methods disclosed herein comprises: In some cases, step (d) for producing the fourth cell population is carried out over a period of 10-12 days, e.g., 10-11 days, 11-12 days, about 10 days, about 11 days, about 12 days.

[0123]

[0140] In some cases, the second cell population comprises PDX1-positive and NKX6.1-positive cells.In some cases, the fourth cell population comprises PDX1-positive, NKX6.1-positive, and ISL1-positive cells.In some cases, the fifth cell population comprises cells that express C-peptide and ISL1, but do not express VMAT1. In some examples, 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 30-40%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-90%, 50-80%, 50-70%, 50-60%, 60-90%, 60-80%, 60-70%, 70-90%, 70-80%, 70-90%, 70-80%, or 80-90% of the cells in the fourth cell population express C-peptide and ISL1 but do not express VMAT1. In some examples, 40-60% of the cells in the fourth cell population express C-peptide and ISL1 but do not express VMAT1. In some examples, the fourth cell population includes cells that express glucagon but do not express somatostatin. In some examples, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-40%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15-40%, 15-35%, 15-30%, 15-25%, 15-20%, 20-40%, 20-35%, 20-30%, 20-25%, 25-40%, 25-35%, 25-30%, 30-40%, 30-35%, or 35-40% of the cells in the fourth cell population express glucagon but do not express somatostatin. In some examples, 10-25% of the cells in the fourth cell population express somatostatin but do not express glucagon. In some examples, the fourth cell population includes cells that express somatostatin but do not express glucagon.In some examples, 3-20%, 3-15%, 3-12%, 3-10%, 3-8%, 3-5%, 4-20%, 4-15%, 4-12%, 4-10%, 4-8%, 4-5%, 5-20%, 5-15%, 5-12%, 5-10%, 5-8%, 7-20%, 7-15%, 7-12%, 7-10%, 9-20%, 9-15%, 9-12%, 8-10%, 8-12%, 8-15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15%, or 15-20% of the cells in the fourth cell population express somatostatin but do not express glucagon.

[0124]

[0141] In some examples, the step of producing a first cell population in the method provided herein comprises contacting a plurality of PDX1-positive, NKX6.1-negative pancreatic progenitor cells with a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor.In some examples, the step of producing a second cell population in the method provided herein comprises contacting the first cell population with a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor.In some examples, the step of producing a third cell population in the method provided herein comprises contacting the second cell population with a gamma-secretase inhibitor, a TGF-β signaling pathway inhibitor, a growth factor from the EGF family, an RA signaling pathway activator, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound. In some examples, producing the fourth cell population in the methods provided herein comprises contacting the third cell population with a serum albumin protein, a TGF-β signaling pathway inhibitor, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound. The ROCK inhibitor used in the method provided herein is thiazobabin.In some examples, the growth factor from the TGFβ superfamily used in the step of producing the first cell population and / or the second cell population in the method provided herein is activin A.In some examples, the growth factor from the FGF family used in the step of producing the first cell population and / or the second cell population in the method provided herein is KGF.In some examples, the RA signaling pathway activator used in the step of producing the first cell population, the second cell population, and / or the third cell population in the method provided herein is retinoic acid.In some examples, the SHH pathway inhibitor used in the step of producing the first cell population, the second cell population, and / or the third cell population in the method provided herein is Sant-1. In some examples, the PKC activator used in the step of producing the second cell population, the third cell population, and / or the fourth cell population in the method provided herein is selected from the group consisting of phorbol 12,13-dibutyrate (PDBU), FR236924, prostratin, SC-9, and TPPB. In some examples, the PKC activator is PDBU. In some examples, the gamma-secretase inhibitor used in the step of producing the second cell population and / or the third cell population in the method provided herein is XXI. In some examples, the TGF-β signaling pathway inhibitor used in the step of producing the third cell population and / or the fourth cell population in the method provided herein is ALK5i. In some examples, the growth factor from the EGF family used in the step of producing the third cell population in the method provided herein is betacellulin. In some examples, the TH signaling pathway activator for use in producing the third and / or fourth cell populations in the methods provided herein is T3, GC-1, or a thyroid hormone derivative.In some examples, the protein kinase inhibitor used in the step of producing the third cell population and / or the fourth cell population in the method provided herein is staurosporine.In some examples, the BMP signaling pathway inhibitor used in the step of producing the third cell population and / or the fourth cell population in the method provided herein is LDN193189 or DMH-1.In some examples, the epigenetic modifying compound used in the step of producing the third cell population and / or the fourth cell population in the method provided herein is DZNep.

[0125]

[0142] In some examples, the first period during which pancreatic progenitor cells are treated with a PKC activator is at least 2 days, 3 days, or 4 days. In some examples, the first period is at most 4 days, 3 days, or 2 days. In some examples, the first period is 2 to 4 days. In some examples, the second period during which pancreatic progenitor cells are treated with a PKC activator is at least 2 days. In some examples, the second period is at most 4 days. In some examples, the second period is 2 to 4 days. In some examples, the treatment with a PKC activator discussed herein during the transition between differentiation of PDX1-positive, NKX6.1-positive pancreatic progenitor cells and differentiation of NKX6.1-positive, ISL1-positive endocrine cells is at least 2 days, 3 days, or 4 days. In some examples, the treatment with a PKC activator contemplated herein during the transition between differentiation of PDX1-positive, NKX6.1-positive pancreatic progenitor cells and differentiation of NKX6.1-positive, ISL1-positive endocrine cells is for at most 2, 3, or 4 days. In some examples, the treatment with a PKC activator contemplated herein during the transition between differentiation of PDX1-positive, NKX6.1-positive pancreatic progenitor cells and differentiation of NKX6.1-positive, ISL1-positive endocrine cells is for 2 to 4 days.

[0126]

[0143] In some embodiments, the PKC activator is contacted with the differentiated cell population at two or more different time points during the differentiation process. In some embodiments, the PKC activator is contacted with the cell population, and the cells comprise PDX1-positive, NKX6.1-negative cells. In some embodiments, A PKC activator is contacted with a cell population, and the cells comprise PDX1-positive, NKX6.1-positive cells. In some embodiments, a PKC activator is contacted with a cell population, and the cells comprise insulin-positive cells. In some embodiments, a PKC activator is contacted with a cell population at each of the following differentiation stages: when the cells comprise PDX1-positive, NKX6.1-negative cells; when the cells comprise PDX1-positive, NKX6.1-positive cells; and when the cells comprise insulin-positive cells. In some embodiments, the same type of PKC activator (e.g., a phorbol ester or a benzolactam derivative) is administered to different cell populations at two or more different time points. For example, in some embodiments, a phorbol ester (e.g., PDBU) is administered to a cell population comprising PDX1-positive, NKX6.1-negative cells, and a phorbol ester (e.g., PDBU) is administered to a cell population comprising PDX1-positive, NKX6.1-positive cells during the same differentiation protocol. In some embodiments, one or more different PKC activators (e.g., a phorbol ester and a benzolactam derivative) are administered to different cell populations at two or more different time points. For example, in some embodiments, a phorbol ester (e.g., PDBU) is administered to a cell population comprising PDX1-positive, NKX6.1-negative cells, and a benzolactam derivative (e.g., TPPB) is administered to a cell population comprising PDX1-positive, NKX6.1-positive cells during the same differentiation protocol.

[0127]

[0144] In some examples, non-limiting examples of PKC activators used in the methods described herein include phorbol 12,13-dibutyrate (PDBU), FR236924, prostratin, SC-9, and TPPB. In some examples, the PKC activator includes PDBU. In some examples, the PKC activator includes TPPB. In some examples, the PKC activator is contacted with a cell population containing PDX1-positive, NKX6.1-positive pancreatic progenitor cells at a concentration of 50 nM to 2000 nM, 75 nM to 1500 nM, 100 nM to 1000 nM, 200 nM to 750 nM, or 400 nM to 600 nM. In some examples, the PKC activator is at a concentration of 100 nM to 1000 nM. In some examples, the PKC activator is at a concentration of at least about 100nM, 200nM, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM, 900nM, or 1000nM. In some examples, the PKC activator is at a concentration of at most about 100nM, 200nM, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM, 900nM, or 1000nM. In some examples, the PKC activator is at a concentration of about 100nM, 200nM, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM, 900nM, or 1000nM. In some examples, the PKC activator is at a concentration of about 500nM.

[0128]

[0145] In some examples, non-limiting examples of gamma secretase inhibitors used in the methods described herein include XXI and DAPT. In some examples, the gamma secretase inhibitor comprises XXI. In some examples, the gamma secretase inhibitor is contacted with a cell population containing PDX1-positive, NKX6.1-positive pancreatic progenitor cells at a concentration of 0.2 μM to 20 μM, 0.3 μM to 15 μM, 0.5 μM to 10 μM, 1 μM to 5 μM, or 1.5 μM to 2.5 μM. In some examples, the gamma secretase inhibitor is at a concentration of about 0.5 μM, 0.75 μM, 1 μM, 1.25 μM, 1.5 μM, 1.75 μM, 2 μM, 2.25 μM, 2.5 μM, 3 μM, 4 μM, 5 μM, 7.5 μM, 10 μM, 15 μM, or 20 μM. In some examples, the gamma secretase inhibitor is at a concentration of at least about 0.5 μM, 0.75 μM, 1 μM, 1.25 μM, 1.5 μM, 1.75 μM, 2 μM, 2.25 μM, 2.5 μM, 3 μM, 4 μM, or 5 μM. In some examples, the gamma secretase inhibitor is at a concentration of at most about 1 μM, 1.25 μM, 1.5 μM, 1.75 μM, 2 μM, 2.25 μM, 2.5 μM, 3 μM, 4 μM, 5 μM, 7.5 μM, 10 μM, 15 μM, or 20 μM.

[0129] cell composition

[0146] In some embodiments, SC-β cells, SC-α cells, SC-δ cells, and SC-E Provided herein is a cell composition comprising C cells. In some cases, provided herein is a cell composition having a desired amount (for example, percentage) of SC-β cells, SC-α cells, and SC-δ cells, and a limited amount of SC-EC cells. In some cases, the cell composition of the cell composition is similar to that of natural pancreatic islets.

[0130]

[0147] In some examples, the SC-β cells of the present disclosure share many characteristic features of β cells that are important for normal β cell function. In some embodiments, the SC-β cells exhibit a glucose-stimulated insulin secretion (GSIS) response in vitro. In some embodiments, the SC-β cells exhibit a GSIS response in vivo. In some embodiments, the SC-β cells exhibit a GSIS response in vitro and in vivo. In some embodiments, the GSIS response resembles the GSIS response of endogenous mature pancreatic β cells. In some embodiments, the SC-β cells exhibit a GSIS response to at least one glucose challenge. In some embodiments, the SC-β cells exhibit a GSIS response to at least two consecutive glucose challenges. In some embodiments, the SC-β cells exhibit a GSIS response to at least three consecutive glucose challenges. In some embodiments, the GSIS response resembles the GSIS response of endogenous human pancreatic islets to multiple glucose challenges. In some embodiments, the GSIS response is observed immediately after transplantation of the cells into a human or animal. In some embodiments, the GSIS response is observed within approximately 24 hours after transplantation of the cells into a human or animal. In some embodiments, the GSIS response is observed within approximately one week after transplantation of the cells into a human or animal. In some embodiments, the GSIS response is observed within approximately two weeks after transplantation of the cells into a human or animal. In some embodiments, the stimulation index of the cells, characterized by the ratio of insulin secreted in response to high glucose concentrations compared to low glucose concentrations, is similar to the stimulation index of endogenous mature pancreatic beta cells. In some embodiments, the SC-beta cells exhibit a stimulation index greater than 1. In some embodiments, the SC-beta cells exhibit a stimulation index greater than or equal to 1. In some embodiments, the SC-beta cells exhibit a stimulation index greater than 1.1. In some embodiments, the SC-beta cells exhibit a stimulation index greater than 1.1. In some embodiments, the SC-beta cells exhibit a stimulation index greater than 2. In some embodiments, the SC-beta cells exhibit a stimulation index greater than or equal to 1.In some embodiments, the SC-beta cells exhibit a stimulation index of at least 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 or greater.

[0131]

[0148] In some embodiments, the present disclosure provides an in vitro composition comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells; a PKC activator; and a gamma-secretase inhibitor. In some embodiments, the present disclosure provides an in vitro composition comprising NKX6.1-positive, ISL1-positive endocrine cells; a PKC activator; and a gamma-secretase inhibitor. In some embodiments, the present disclosure provides an in vitro composition comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells; an NKX6.1-positive, ISL1-positive endocrine cells; a PKC activator; and a gamma-secretase inhibitor. In some embodiments, the PKC activator is selected from the group consisting of phorbol 12,13-dibutyrate (PDBU), FR236924, prostratin, SC-9, and TPPB. In some embodiments, the gamma-secretase inhibitor is DAPT or XXI.

[0132]

[0149] In some aspects, the present disclosure provides an in vitro composition comprising PDX1-positive, NKX6.1-negative pancreatic progenitor cells; PDX1-positive, NKX6.1-positive pancreatic progenitor cells; a PKC activator; and a gamma-secretase inhibitor. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the composition is PDX1-positive, NKX6.1-negative pancreatic progenitor cells; a PKC activator; and a gamma-secretase inhibitor. 0% of the cells are PDX1-positive, NKX6.1-positive pancreatic progenitor cells. In some embodiments, less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the cells in the composition are PDX1-positive, NKX6.1-negative pancreatic progenitor cells. In some embodiments, the PKC activator is selected from the group consisting of phorbol 12,13-dibutyrate (PDBU), FR236924, prostratin, SC-9, and TPPB. In some embodiments, the gamma-secretase inhibitor is DAPT or XXI. In some embodiments, the composition further comprises a growth factor from the FGF family. In some embodiments, the growth factor from the FGF family is KGF. In some embodiments, the composition further comprises a growth factor from the TGFβ superfamily. In some embodiments, the growth factor from the TGFβ superfamily is activin A.

[0133]

[0150] In some aspects, the present disclosure provides an in vitro composition comprising PDX1-positive cells, a gamma-secretase inhibitor, and one or both of a growth factor from the TGFβ superfamily and a growth factor from the FGF family. In some embodiments, the cell composition comprises PDX1-positive, NKX6.1-negative cells. In some embodiments, the cell composition comprises PDX1-positive, NKX6.1-positive cells. In some embodiments, the composition further comprises any one or combination of a PKC activator, a growth factor from the FGF family, a ROCK inhibitor, a growth factor from the TGFβ superfamily, a sonic hedgehog pathway inhibitor, and a retinoic acid signaling pathway activator.

[0134]

[0151] In some aspects, the present disclosure provides a method for the treatment of pancreatic cancer comprising: PDX1-positive, NKX6.1-negative pancreatic progenitor cells; PDX1-positive, NKX6.1-positive pancreatic progenitor cells; and a gamma-secretase inhibitor. In some embodiments, the gamma-secretase inhibitor is XXI. In some embodiments, the gamma-secretase inhibitor is DAPT.

[0135]

[0152] In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the cells in the composition are PDX1-positive, NKX6.1-positive pancreatic progenitor cells. In some embodiments, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the cells in the composition are PDX1-positive, NKX6.1-negative pancreatic progenitor cells.

[0136]

[0153] In some embodiments, the composition further comprises a growth factor from the FGF family. In some embodiments, the composition further comprises a sonic hedgehog pathway inhibitor. In some embodiments, the composition further comprises a ROCK inhibitor. In some embodiments, the composition further comprises a growth factor from the TGFβ superfamily. In some embodiments, the composition further comprises a retinoic acid signaling pathway activator. In some embodiments, the composition further comprises a PKC activator.

[0137]

[0154] In some embodiments, the composition further comprises two or more (e.g., any two, any three, any four, any five, or any six) of a PKC activator, a growth factor from the FGF family, a ROCK inhibitor, a growth factor from the TGFβ superfamily, a sonic hedgehog pathway inhibitor, and a retinoic acid signaling pathway activator. In some embodiments of the composition, the growth factor from the FGF family is KGF. In some embodiments, the sonic hedgehog pathway inhibitor is SANT-1. In some embodiments, the ROCK inhibitor is thiazovivin. In some embodiments, the growth factor from the TGFβ superfamily is activin A. In some embodiments, the retinoic acid signaling pathway activator is retinoic acid. In some embodiments, the PKC activator is PDBU.

[0138]

[0155] In some aspects, the present disclosure provides an in vitro differentiated cell population comprising NKX6.1-positive, ISL1-positive cells and NKX6.1-negative, ISL1-positive cells. In some embodiments, the population comprises more NKX6.1-negative, ISL1-positive cells than NKX6.1-positive, ISL1-positive cells. In some embodiments, at least 73% of the cells in the population are ISL1-positive cells. In some embodiments, at least 40% of the cells in the population are NKX6.1-negative, ISL1-positive cells. In some embodiments, less than 12% of the cells in the population are NKX6.1-negative, ISL1-negative cells.

[0139]

[0156] In some aspects, the disclosure provides an in vitro differentiated cell population comprising NKX6.1-positive, ISL1-positive cells and NKX6.1-negative, ISL1-positive cells, wherein less than 12% (e.g., about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1% or less) of the cells in the population are NKX6.1-negative, ISL1-negative cells. In some embodiments, less than 10%, less than 8%, less than 6%, less than 4%, 1-11%, 2-10%, 2-12%, 4-12%, 6-12%, 8-12%, 2-8%, 4-8%, 3-6%, or 3-5% of the cells in the population are NKX6.1-negative, ISL1-negative cells. In some embodiments, 2-12%, 4-12%, 6-12%, 8-12%, 2-8%, 4-8%, 3-6%, or 3-5% of the cells in the population are NKX6.1-negative, ISL1-negative cells.

[0140]

[0157] In some embodiments, at least 60%, at least 65%, at least 70%, at least 73%, at least 74%, at least 75%, at least 80%, at least 85%, at least 90%, about 85-95%, or about 90-95% of the cells in the population are ISL1 positive cells. In some embodiments, 50-90%, 50-85%, 50-80%, 50-75%, 50-70%, 50-60%, 60-90%, 60-85%, 60-80%, 60-75%, 60-70%, 65-90%, 65-85%, 65-80%, 65-75%, 65-70%, 70-90%, 70-85%, 70-80%, 70-75%, 75-90%, 75-85%, 75-80%, 80-90%, 80-85%, or 85-90% of the cells in the population are ISL1-positive cells. In some embodiments, at least 74%, at least 75%, at least 80%, at least 85%, at least 90%, about 85-95%, or about 90-95% of the cells in the population are ISL1-positive cells, or about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% of the cells in the population are ISL1-positive cells.

[0141]

[0158] In some embodiments, the population comprises more NKX6.1-negative, ISL1-positive cells than NKX6.1-positive, ISL1-positive cells. In some embodiments, at least 40% of the cells in the population are NKX6.1-negative, ISL1-positive cells. In some embodiments, at least 45%, at least 50%, about 40-50%, about 45-55%, or about 50-55% of the cells in the population are NKX6.1-negative, ISL1-positive cells. In some embodiments, about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, or about 55% of the cells in the population are NKX6.1-negative, ISL1-positive cells.

[0142]

[0159] In some aspects, the present disclosure provides an in vitro composition comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells; NKX6.1-positive, ISL1-positive endocrine cells; and a PKC activator, wherein the PKC activator is a benzolactam derivative. In some examples, the benzolactam is TPPB. In some examples, the composition further comprises a gamma-secretase inhibitor. The gamma-secretase inhibitor can be XXI.

[0143]

[0160] In some examples, the compositions provided herein comprise a differentiation factor selected from the group consisting of a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, an epigenetic modifying compound, a growth factor from the EGF family, a RA signaling pathway activator, an SHH pathway inhibitor, a protein kinase inhibitor, a ROCK inhibitor, and a BMP signaling pathway inhibitor. In some examples, the compositions also comprise serum albumin protein.

[0144]

[0161] In some examples, the compositions provided herein comprise a serum albumin protein, a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, an epigenetic modifying compound, an SHH pathway inhibitor, a protein kinase inhibitor, a ROCK inhibitor, and a BMP signaling pathway inhibitor.

[0145]

[0162] In some examples, the ROCK inhibitor is thiazobabin. In some examples, the RA signaling pathway activator is retinoic acid. In some examples, the SHH pathway inhibitor is Sant-1. In some examples, the TGF-β signaling pathway inhibitor is ALK5i. In some examples, the EGF family growth factor is betacellulin. In some examples, the thyroid hormone signaling pathway activator is T3, GC-1, or a thyroid hormone derivative. In some examples, the protein kinase inhibitor is staurosporine. In some examples, the BMP signaling pathway inhibitor is LDN193189 or DMH-1. In some examples, the epigenetic modifying compound is DZNep.

[0146]

[0163] In some cases, the cell composition of the present disclosure has at least about 35% of cells that express C-peptide and do not express VMAT1, as measured by flow cytometry.In some cases, the expression of C-peptide and the absence of VMAT1 in the cells of the cell composition suggest that the cells are SC-β cells.In some cases, the cell composition has at least about 30%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49% or 50% of cells that express C-peptide and do not express VMAT1, as measured by flow cytometry. In some examples, the cell composition has about 30%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60% of cells that express C-peptide and do not express VMAT1, as measured by flow cytometry. In some examples, the cell composition has about 30% to about 60%, about 35% to about 55%, or about 40% to about 50% of cells that express C-peptide and do not express VMAT1, as measured by flow cytometry.

[0147]

[0164] In some examples, the cell composition of the present disclosure has at most about 35% cells that express VMAT1 as measured by flow cytometry. In some examples, the cell composition of the present disclosure has at most about 35% cells that express VMAT1 and do not express C-peptide as measured by flow cytometry. In some examples, the expression of VMAT1 and the absence of C-peptide in cells of the cell composition suggest that the cells are SC-EC cells. In some examples, the cell composition has at most about 35%, 32%, 31%, 30%, 28%, 25%, 24%, 23%, 22%, 21%, or 20% cells that express VMAT1 and do not express C-peptide as measured by flow cytometry. In some examples, the cell composition has at most about 35%, 32%, 31%, 30%, 28%, 25%, 24%, 23%, 22%, 21%, or 20% cells that express VMAT1 and do not express C-peptide as measured by flow cytometry. In some examples, the cell composition has about 15% to about 30%, about 16% to about 25%, about 17% to about 22%, or about 18% to about 20% of cells that express VMAT1 and do not express C-peptide, as measured by flow cytometry.

[0148]

[0165] In some cases, the cell composition comprises at least about 20% of cells that express glucagon, as measured by flow cytometry. In some cases, the cell composition comprises at least about 15% of cells that express glucagon and do not express somatostatin, as measured by flow cytometry. In some cases, the expression of glucagon and the absence of somatostatin in cells of the cell composition suggests that the cells are SC-α cells. In some cases, the cell composition comprises at least about 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, or 22% of cells that express glucagon and do not express somatostatin, as measured by flow cytometry. In some examples, the cell composition comprises about 10% to about 30%, about 12% to about 25%, about 13% to about 22%, about 15% to about 20%, or about 16% to about 18% cells that express glucagon and do not express somatostatin, as measured by flow cytometry. In some examples, the cell composition comprises about 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, or 22% cells that express glucagon and do not express somatostatin, as measured by flow cytometry.

[0149]

[0166] In some cases, the cell composition comprises at least about 4% of cells that express somatostatin and do not express glucagon, as measured by flow cytometry. In some cases, expression of glucagon and non-expression of somatostatin in cells of the cell composition indicates that the cells are SC-δ cells. In some cases, the cell composition comprises at least about 2%, 3%, 4%, 5%, 6%, 7%, or 8% of cells that express somatostatin and do not express glucagon, as measured by flow cytometry. In some cases, the cell composition comprises about 1% to about 9%, about 2% to about 8%, about 3% to about 7%, or about 4% to about 6% of cells that express somatostatin and do not express glucagon, as measured by flow cytometry. In some examples, the cell composition comprises about 2%, 3%, 4%, 5%, 6%, 7%, or 8% cells that express somatostatin and do not express glucagon, as measured by flow cytometry.

[0150]

[0167] In some cases, the cell composition has at least about 35% of cells that express C-peptide and do not express VMAT1, at most about 30% of cells that express VMAT1, and at least about 20% of cells that express glucagon, as measured by flow cytometry.In some cases, the cell composition has at least about 35% of cells that express C-peptide and do not express VMAT1, at most about 30% of cells that express VMAT1, at least about 20% of cells that express glucagon, and at least 4% of cells that express somatostatin and do not express glucagon, as measured by flow cytometry.

[0151]

[0168] In some cases, the cell composition provided herein comprises, as measured by flow cytometry, (a) at least about 35% of cells that express C-peptide and do not express VMAT1; and (b) at least about 10% of cells that express somatostatin. In some cases, as measured by flow cytometry, there are at least about 15% of cells that express somatostatin in the cell composition.

[0152]

[0169] In some examples, a composition comprising a cell population, comprising: (a) a cell population having 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 30-40%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-90%, 50-80%, 50-70%, 50-60%, 60-90%, 60-80%, 60-70%, 60-80%, 60-90%, 60-70%, 60-90%, 60-8 ... (b) 0%, 70-90%, 70-80%, 70-90%, 70-80%, or 80-90% of cells express C-peptide and ISL1 but not VMAT1; (c) 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-40%, 10-35%, 10-40% of cells in the cell population. 0-30%, 10-25%, 10-20%, 10-15%, 15-40%, 15-35%, 15-30%, 15-25%, 15-20%, 20-40%, 20-35%, 20-30%, 20-25%, 25-40%, 25-35%, 25-30%, 30-40%, 30-35%, or 35-40% of cells express glucagon. expresses but does not express somatostatin; and / or (c) 3-20%, 3-15%, 3-12%, 3-10%, 3-8%, 3-5%, 4-20%, 4-15%, 4-12%, 4-10%, 4-8%, 4-5%, 5-20%, 5-15%, 5-12%, 5-10%, 5-8%, 7-20%, 7-15% of the cell population Provided herein are compositions wherein 7-12%, 7-10%, 9-20%, 9-15%, 9-12%, 8-10%, 8-12%, 8-15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15%, or 15-20% of the cells express somatostatin but do not express glucagon.

[0153]

[0170] In some examples, a composition comprising a cell population, comprising: (a) a cell population having 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 30-40%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-90%, 50-80%, 50-70%, 50-60%, 60-90%, 60-80%, 60-70%, 70-90%, 70-80%, 70-90%, 70-90%, 70-70%, 70-9 ... ~80%, or 80-90% of cells express C-peptide and ISL1 but not VMAT1; (b) 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-40%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15-40%, 15-35%, 15-30%, 15-25%, 15-20%, 20% of the cell population. ~40%, 20-35%, 20-30%, 20-25%, 25-40%, 25-35%, 25-30%, 30-40%, 30-35%, or 35-40% of the cells express glucagon but not somatostatin; and (c) 3-20%, 3-15%, 3-12%, 3-10%, 3-8%, 3-5%, 4-20%, 4-15%, 4-12%, 4-10%, 4-8%, 4-5%, Provided herein are compositions wherein 5-20%, 5-15%, 5-12%, 5-10%, 5-8%, 7-20%, 7-15%, 7-12%, 7-10%, 9-20%, 9-15%, 9-12%, 8-10%, 8-12%, 8-15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15%, or 15-20% of the cells express somatostatin but do not express glucagon.

[0154]

[0171] In some examples, in the cell populations provided herein, 40-60% of the cells express C-peptide and ISL1 but do not express VMAT1; 10-25% of the cells express glucagon but do not express somatostatin; and 4-10% of the cells express somatostatin but do not express glucagon. In some examples, in the cell populations provided herein, less than 25%, less than 20%, less than 18%, less than 15%, less than 12%, or less than 10% of the cells express VMAT1 but do not express C-peptide.

[0155]

[0172] Still other embodiments of the present disclosure relate to compositions, such as isolated cell populations or cell cultures, comprising a mixture of SC-β cells and insulin-positive endocrine cells or their precursors from which they differentiate. For example, cell cultures or cell populations can be produced that comprise at least about 5 SC-β cells for about every 95 insulin-positive endocrine cells or their precursors. In other embodiments, cell cultures or cell populations can be produced that comprise at least about 95 SC-β cells for about every 5 insulin-positive endocrine cells or their precursors. Furthermore, other ratios of SC-β cells to insulin-positive endocrine cells or their precursors can be produced. Cell cultures or cell populations comprising cells are contemplated. For example, compositions can be produced that comprise at least about 1 SC-β cell for about every 1,000,000, or at least 100,000, or at least 10,000, or at least 1000 or 500, or at least 250, or at least 100, or at least 100 insulin-positive endocrine cells or their precursors.

[0156]

[0173] In some cases, the cell populations or cell clusters disclosed herein, for example, cell populations or cell clusters isolated without a cell sorting process, are not sorted. In some embodiments, the cell clusters disclosed herein may refer to cell clusters formed by self-aggregation of cells cultured in a given environment, for example, in a 3D suspension culture. In some embodiments, the cell clusters disclosed herein are intermediate cell clusters formed during the differentiation process as described herein. In some examples, intermediate cell clusters, such as cell clusters containing PDX1-positive, NKX6.1-negative pancreatic progenitor cells (e.g., stage 3 cell clusters) or cell clusters containing PDX1-positive, NKX6.1-positive pancreatic progenitor cells (e.g., stage 4 cell clusters), are not subjected to cell sorting. In some examples, cell populations that pass cell sorting may not be able to form the intermediate cell clusters disclosed herein. For example, PDX1-positive pancreatic progenitor cells may not be able to form the cell clusters disclosed herein after undergoing cell sorting.

[0157]

[0174] Cell sorting as described herein may refer to the process of isolating a group of cells from a plurality of cells by relying on differences in cell size, shape (morphology), surface protein expression, expression of endogenous signal proteins, or any combination thereof. In some cases, cell sorting involves subjecting cells to flow cytometry. Flow cytometry can be a biophysical technique based on lasers or impedance. During flow cytometry, cells can be suspended in a fluid stream and passed through an electronic detection device. Fluorescence-activated cell sorting (FACS), a type of flow cytometry, physically separates cells based on one or more parameters of their optical properties (e.g., emission wavelength upon laser excitation), thereby allowing flow cytometry to purify cells of interest. As described herein, unsorted cell clusters can be cell clusters formed by a plurality of cells that have not been subjected to an active cell sorting process, e.g., flow cytometry. In some cases, the flow cytometry discussed herein can be based on one or more signal peptides expressed within the cells. For example, the cell clusters can include cells expressing a signal peptide (e.g., a fluorescent protein, such as green fluorescent protein (GFP) or tdTomato). In some cases, the signal peptide is expressed as an indicator of insulin expression in the cells. For example, the cell clusters can include cells having an exogenous nucleic acid sequence encoding GFP under the control of an insulin promoter. The insulin promoter can be an endogenous or exogenous promoter. In some cases, expression of GFP in these cells can indicate insulin expression in the cells. Thus, the GFP signal can be a marker for pancreatic beta cells. In some cases, the cell sorting described herein can include magnetically activated flow cytometry, in which magnetic antibodies or other ligands are used to label different types of cells and differences in magnetic properties can be used for cell sorting.

[0158]

[0175] The percentage of cells expressing one or more particular markers, such as PDX1, NKX6.1, insulin, NGN3, or CHGA, as described herein, can be a percentage value detected using a technique such as a flow cytometry assay. In some cases, during a flow cytometry assay, the cell populations or cell clusters discussed herein are isolated using trypsin or TrypLE™ Express. The cells are dispersed into a single-cell suspension by incubation in a digestive enzyme such as PBS. The dispersed cells can be washed in a suitable buffer, such as PBS, centrifuged, and then resuspended in a fixation buffer, such as 4% PFA. They can then be incubated with a primary antibody against the cell marker of interest, followed by incubation with a secondary antibody. After antibody incubation, the cells can be washed and subjected to separation by flow cytometry. Techniques other than flow cytometry can also be used to characterize the cells described herein, for example, to determine the percentage of cells. Non-limiting examples of methods for characterizing cells include gene sequencing, microscopy (fluorescence microscopy, atomic force microscopy), karyotyping, isozyme analysis, DNA characterization, and viral susceptibility.

[0159]

[0176] In some aspects, the disclosure relates to compositions comprising a population of glucose-responsive insulin-secreting cells, wherein the cells secrete greater amounts of insulin when induced with KCl (e.g., about 20 to about 50 mM, e.g., about 30 mM) compared to the amount of insulin secreted when induced with glucose. In some embodiments, the population of glucose-responsive insulin-secreting cells secrete at least 1.5-fold, 2-fold, 2.5-fold, or 3-fold greater amounts of insulin when induced with KCl compared to the amount of insulin secreted when induced with glucose.

[0160]

[0177] In some aspects, the present disclosure relates to a composition comprising a population of glucose-responsive insulin-secreting cells, wherein the cells secrete greater amounts of insulin upon induction with KCl and / or glucose in the presence of a signaling factor compared to comparable cells in the absence of the signaling factor. In some embodiments, the cells secrete greater amounts of insulin in the presence of high glucose but not in the presence of low glucose. In some embodiments, the high glucose concentration is about 10-20 mM. In some embodiments, the low glucose concentration is about 2-5 mM.

[0161]

[0178] In some aspects, the present disclosure relates to a composition comprising a population of differentiated pancreatic progenitor cells, wherein the population comprises at least 60% pancreatic beta cells as determined by flow cytometry. In some embodiments, the population comprises at least 65%, 70%, 75%, 80%, 85%, or 90% pancreatic beta cells. In some embodiments, the population comprises a higher percentage of pancreatic beta cells when contacted with a component of a given basal medium compared to a comparable population not contacted with the component of the basal medium.

[0162]

[0179] In vitro matured SC-β cells (e.g., pancreatic β cells) produced according to the methods of the present disclosure described herein demonstrate many advantages, e.g., they perform glucose-stimulated insulin secretion in vitro, resemble human pancreatic islet β cells by gene expression and ultrastructure, secrete human insulin when transplanted into mice, and ameliorate hyperglycemia, providing a new platform for cell therapy (e.g., transplantation into subjects requiring additional and / or functional β cells), drug screening (e.g., insulin production / secretion, survival, dedifferentiation, etc.), research (e.g., determining functional differences between normal and diabetic β cells), and tissue engineering (e.g., using SC-β cells as the first cell type in reconstituting pancreatic islets). Stem Cells and Reprogramming

[0180] Provided herein is the use of stem cells to generate SC-β cells (e.g., mature pancreatic β cells or β-like cells) or their precursors. In one embodiment, embryonic cells may be used in place of or in conjunction with stem cells to provide at least one SC-β cell, using protocols similar to those described in U.S. Patent Application Publication Nos. 2015 / 0240212 and 2015 / 0218522, each of which is incorporated herein by reference in its entirety. Suitable embryonic cells may be harvested, for example, approximately 8-11 weeks after the last menstrual cycle. They can be prepared from primordial embryonic cells present in human fetal material. Illustrative methods for preparing embryonic cells are described, for example, in Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998, and U.S. Patent No. 6,090,622.

[0163]

[0181] Provided herein are compositions and methods for producing SC-β cells (e.g., pancreatic β cells), as well as pancreatic α cells and / or pancreatic δ cells. In some embodiments, the present disclosure provides a method for producing a cell population enriched for pancreatic α cells. In some embodiments, the present disclosure provides a method for producing a cell population enriched for pancreatic δ cells.

[0164]

[0182] Generally, at least one SC-β cell or precursor thereof, e.g., a pancreatic progenitor cell produced by the methods disclosed herein, can comprise a mixture or combination of various cells, such as primitive gut cells, PDX1-positive pancreatic progenitor cells, PDX1-positive, NKX6-1-positive pancreatic progenitor cells, Ngn3-positive endocrine progenitor cells, insulin-positive endocrine cells (e.g., NKX6.1-positive, ISL1-positive cells, or β-like cells), and / or other pluripotent or stem cells.

[0165]

[0183] Any suitable culture protocol can be used to generate at least one pancreatic alpha, beta, and / or delta cell, or a precursor thereof, for differentiating stem cells or pluripotent cells to a desired differentiation stage. In some embodiments, at least one pancreatic alpha, beta, and / or delta cell, or a precursor thereof, is generated by culturing at least one pluripotent cell for a time and under conditions suitable for differentiating the at least one pluripotent cell into at least one pancreatic alpha, beta, and / or delta cell, or a precursor thereof.

[0166]

[0184] In some embodiments, the at least one pancreatic alpha, beta, and / or delta cell or precursor thereof is a substantially pure population of pancreatic alpha, beta, and / or delta cells or precursors thereof. In some embodiments, the population of pancreatic alpha, beta, and / or delta cells or precursors thereof comprises a mixture of pluripotent cells or differentiated cells. In some embodiments, the population of pancreatic alpha, beta, and / or delta cells or precursors thereof is substantially free of or devoid of embryonic stem cells or pluripotent cells or iPS cells.

[0167]

[0185] In some embodiments, somatic cells, e.g., fibroblasts, can be isolated from a subject, e.g., as a tissue biopsy, e.g., a skin biopsy, and reprogrammed into induced pluripotent stem cells for further differentiation to generate at least one pancreatic alpha, beta, and / or delta cell, or precursor thereof, for use in the compositions and methods described herein. In some embodiments, the somatic cells, e.g., fibroblasts, are maintained in culture by methods known to those of skill in the art, and in some embodiments, expanded before being converted into pancreatic alpha, beta, and / or delta cells by the methods disclosed herein.

[0168]

[0186] In some embodiments, at least one pancreatic alpha, beta and / or delta cell or precursor thereof is maintained in culture by methods known to those of skill in the art, and in some embodiments, expanded before being converted into a pancreatic alpha, beta and / or delta cell by methods described herein.

[0169]

[0187] Furthermore, the at least one pancreatic alpha, beta, and / or delta cell or precursor thereof, e.g., pancreatic progenitor, can be from any mammalian species, non-limiting examples of which include murine, bovine, simian, porcine, equine, ovine, or human cells. For clarity and simplicity, the methods described herein refer to at least one mammalian pancreatic alpha, beta, and / or delta cell or precursor thereof, but it should be understood that all of the methods described herein can be readily applied to other cell types of at least one pancreatic alpha, beta, and / or delta cell or precursor thereof. In some embodiments, at least one pancreatic The α, β and / or δ cells or their precursors are derived from a human individual.

[0170] stem cells

[0188] Embodiments of the present disclosure relate to the use of stem cells for the production of pancreatic α, β, and / or δ cells or their precursors. As used herein, the term "stem cell" can refer to a cell (e.g., plant stem cell, vertebrate stem cell) that has the ability to self-renew and generate differentiated cell types (Morrison et al., (1997) Cell 88:287-298). In the context of cytogenetics, the adjectives "differentiated" or "differentiating" are relative terms. A "differentiated cell" can be a cell that has progressed further downstream in the developmental pathway than the cell it is being compared to. Thus, pluripotent stem cells can differentiate into lineage-restricted progenitor cells (e.g., mesodermal stem cells), which can then differentiate into further restricted cells (e.g., neuronal progenitor cells), which can differentiate into end-stage cells (e.g., terminally differentiated cells, e.g., neurons, cardiomyocytes, etc.). End-stage cells play characteristic roles in certain tissue types and may or may not retain the ability to further proliferate. Stem cells can be characterized by the presence or absence of certain markers (e.g., proteins, RNA, etc.). Stem cells can also be identified by functional assays, both in vitro and in vivo, particularly assays related to the ability of stem cells to give rise to multiple differentiated progeny. In one embodiment, the host cells are adult stem cells, somatic stem cells, non-embryonic stem cells, embryonic stem cells, hematopoietic stem cells, including pluripotent stem cells, and trophoblast stem cells.

[0171]

[0189] Stem cells of interest, such as those that can be used in the methods provided herein, can include pluripotent stem cells (PSCs). As used herein, the term "pluripotent stem cells" or "PSCs" refers to stem cells that can generate all cell types in an organism. Thus, PSCs can give rise to cells of all germ layers in an organism (e.g., endoderm, mesoderm, and ectoderm in vertebrates). Pluripotent cells can form teratomas and contribute to ectodermal, mesodermal, or endodermal tissues in an organism. Plant pluripotent stem cells can give rise to all cell types in the plant (e.g., roots, stems, leaves, and other cells).

[0172]

[0190] Embodiments of the present disclosure relate to the use of PSCs for the production of pancreatic α, β, and / or δ cells or their precursors. PSCs in animals can be derived in several different ways. For example, embryonic stem cells (ESCs) can be derived from the inner cell mass of an embryo (Thomson et al., Science. 1998 Nov. 6; 282(5391):1145-7). On the other hand, induced pluripotent stem cells (iPSCs) can be derived from the inner cell mass of an embryo (Thomson et al., Science. 1998 Nov. 6; 282(5391):1145-7). PSCs (e.g., iPSCs) can be derived from somatic cells (Takahashi et al., Cell. 2007 Nov. 30; 131(5):861-72; Takahashi et al., Nat Protoc. 2007; 2(12):3081-9; Yu et al., Science. 2007 Dec. 21; 318(5858):1917-20. Epub 2007 Nov. 20). Because the term PSC can refer to pluripotent stem cells regardless of their origin, the term PSC can encompass the terms ESC and iPSC, as well as fetal embryonic stem cells (EGSCs), another example of PSCs. PSCs can be in the form of established cell lines, which can be obtained directly from primary embryonic tissue or can be derived from somatic cells.

[0173]

[0191] Embodiments of the present disclosure relate to the use of ESCs for the production of pancreatic beta cells or their precursors. "Embryonic stem cells (ESCs)" may refer to PSCs isolated from an embryo, typically from the inner cell mass of a blastocyst. ESC lines include, for example, hESBGN-01, hESBGN-02, hESBGN-03, hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3, HES-4, HES-5, HES-6 (ES Cell International); Miz-hES1 (MizMedi Hospital-Seoul National University); HSF-1, HSF-6 (University of California at San Francisco); and H1, H7, H9, H13, H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute)). titute) are listed in the NIH Human Embryonic Stem Cell Registry. Stem cells of interest also include embryonic stem cells from other primates, such as rhesus monkey stem cells and marmoset stem cells. Stem cells can be obtained from any mammalian species, e.g., human, horse, cow, pig, dog, cat, rodent, e.g., mouse, rat, hamster, primate, etc. (Thomson et al., (1998) Science 282:1145; Thomson (1995) Proc. Natl. Acad. Sci. USA 92:7844; Thomson et al. (1996) Biol. Reprod. 55:254; Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). In culture, ESCs can grow as flat colonies with a large nucleus-to-cytoplasm ratio, clear borders, and prominent nuclei. Furthermore, ESCs can express SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase, but not SSEA-1. Examples of methods for generating and characterizing ESCs can be found, for example, in U.S. Patent Nos. 7,029,913, 5,843,780, and 6,200,806, each of which is incorporated herein in its entirety. Methods for expanding undifferentiated forms of hESCs are described in WO 99 / 20741, WO 01 / 51616, and WO 03 / 020920, each of which is incorporated herein in its entirety.

[0174]

[0192] "Fetal embryonic stem cells (EGSCs) or fetal embryonic cells" or "EG cells" can refer to embryonic cells and / or embryonic cell precursor cells, such as PSCs derived from primordial embryonic cells, e.g., embryonic cells that can become sperm and eggs. Fetal embryonic cells (EG cells) are believed to have similar properties to the above-mentioned fetal stem cells. Examples of methods for producing and characterizing EG cells can be found, for example, in U.S. Patent No. 7,153,684; Matsui, Y. et al., (1992) Cell 70:841; Shamblott, M. et al., (2001) Proc. Natl. Acad. Sci. USA 98: 113; Shamblott, M. et al., (1998) Proc. Natl. Acad. Sci. USA, 95:13726; and Koshimizu, U. et al., (1996) Development, 122:1235, each of which is incorporated herein in its entirety.

[0175]

[0193] Embodiments of the present disclosure relate to the use of iPSCs for the production of pancreatic alpha, beta, and / or delta cells or their precursors. "Induced pluripotent stem cells" or "iPSCs" can refer to PSCs derived from non-PSC cells (e.g., from cells differentiated into PSCs). iPSCs can be derived from a variety of different cell types, including terminally differentiated cells. iPSCs have an embryonic stem cell-like morphology and can grow as flat colonies with a large nuclear-cytoplasmic ratio, distinct borders, and prominent nuclei. Furthermore, iPSCs can express one or more key pluripotency markers known to those skilled in the art, including, but not limited to, alkaline phosphatase, SSEA3, SSEA4, Sox2, Oct3 / 4, Nanog, TRA160, TRA181, TDGF1, Dnmt3b, FoxD3, GDF3, Cyp26a1, TERT, and zfp42. Examples of methods for generating and characterizing iPSCs can be found, for example, in U.S. Patent Publication Nos. 2009 / 0047263, 2009 / 0068742, 2009 / 0191159, 2009 / 0227032, 2009 / 0246875, and 2009 / 0304646, each of which is incorporated herein in its entirety. Generally, to generate iPSCs, somatic cells are provided with reprogramming factors known in the art (e.g., Oct4, SOX2, KLF4, MYC, Nanog, Lin28, etc.) that reprogram somatic cells to pluripotent stem cells.

[0176]

[0194] Embodiments of the present disclosure relate to the use of somatic cells for the production of pancreatic α, β, and / or δ cells or their precursors. "Somatic cells" can refer to any cell in an organism that does not normally give rise to all cell types in the organism in the absence of experimental manipulation. In other words, somatic cells can be cells that are fully differentiated and therefore do not naturally give rise to cells of all three germ layers of an organism, e.g., ectoderm, mesoderm, and endoderm. For example, somatic cells include neurons and neuronal progenitor cells, and neuronal progenitor cells can give rise to all or some of the cells of the central nervous system. Although they can naturally give rise to several cell types, they cannot give rise to cells of mesodermal or endodermal lineages.

[0177]

[0195] In certain instances, stem cells may be undifferentiated (e.g., cells not committed to a particular lineage) before exposure to at least one differentiation factor or composition according to the methods disclosed herein, while in other instances, it may be desirable to differentiate stem cells into one or more intermediate cell types before exposure to at least one differentiation factor or composition described herein. For example, stem cells may exhibit morphological, biological, or physical characteristics of undifferentiated cells, which can be used to distinguish them from differentiated cells of fetal or adult origin. In some instances, undifferentiated cells may appear microscopically in two dimensions in colonies of cells with a high nucleus / cytoplasm ratio and prominent nuclei. Stem cells may be used by themselves (e.g., substantially free of any undifferentiated cells) or in the presence of differentiated cells. In certain instances, stem cells may be cultured in the presence of suitable nutrients and, optionally, other cells, to grow and optionally differentiate. For example, fetal fibroblasts or fibroblast-like cells may be present in the culture to support stem cell growth. Fibroblasts may be present during one stage of stem cell development, but not necessarily at all stages. For example, fibroblasts may be added to a culture of stem cells at an initial culture stage and not be added to the culture of stem cells at one or more subsequent culture stages.

[0178]

[0196] Stem cells used in all aspects of the invention can be any cells derived from any type of tissue (e.g., embryonic tissue, such as fetal or pre-fetal tissue, or adult tissue), and these stem cells can be characterized by their ability, under appropriate conditions, to generate progeny consisting of various cell types, e.g., derivatives of all or at least one of the three germ layers (endoderm, mesoderm, and ectoderm). These cell types can be provided in the form of established cell lines, or can be obtained directly from primary embryonic tissue and immediately used for differentiation. Cells listed in the NIH Human Embryonic Stem Cell Registry include hESBGN-01, hESBGN-02, hESBGN-03, and hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3, HES-4, HES-5, and HES-6 (ES Cell International); Miz-hES1 (MizMedi Hospital-Seoul National University); HSF-1 and FISF-6 (University of California at San Francisco); and H1, H7, H9, H13, and H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute)). In some embodiments, the source of human stem cells or pluripotent stem cells used for chemically induced differentiation into mature insulin-positive cells did not involve the destruction of a human embryo. In some embodiments, the source of human stem cells or pluripotent stem cells used for chemically induced differentiation into mature insulin-positive cells did not involve the destruction of a human embryo.

[0179]

[0197] In another example, stem cells can be isolated from tissues, including solid tissues. In some embodiments, the tissue is skin, adipose tissue (e.g., adipose tissue), muscle tissue, heart or cardiac tissue. In other embodiments, the tissue is, for example, but not limited to, umbilical cord blood, placenta, bone marrow, or cartilage.

[0180]

[0198] Stem cells that can be used in the methods provided herein include human embryonic stem (hES) cells, as described by Thomson et al. (1998) Science 282:1145, embryonic stem cells from other primates, such as rhesus monkey stem cells (Thomson et al. (1995) Proc. Natl. Acad. Sci. USA 92:7844), marmoset stem cells (Thomson et al. (1996) Biol. Reprod. 55:254), and human fetal embryonic stem cells ( Various types of embryonic cells may also be included, as exemplified by human embryonic gonadotropin (hEG) cells (Shambloft et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). The methods provided herein are also applicable to stem cells committed to lineages such as mesodermal stem cells and other early cardiac developmental cells (Reyes et al., (2001) Blood 98:2615-2625; Eisenberg and Bader, (1996) Circ Res. 78(2):205-16, etc.). (See, e.g., J. Immunol. 2009, 103:131-132, 2009). Stem cells can be obtained from any mammalian species, e.g., human, equine, bovine, porcine, canine, feline, rodent, e.g., mouse, rat, hamster, primate, etc. In some embodiments, human embryos have not been destroyed for the source of pluripotent cells used in the methods and compositions disclosed herein. In some embodiments, human embryos have not been destroyed for the source of pluripotent cells used in the methods and compositions disclosed herein.

[0181]

[0199] A mixture of cells from a suitable source of endothelial, muscle, and / or neuronal stem cells can be harvested from a mammalian donor for the purposes of the present disclosure. A suitable source is the hematopoietic microenvironment. For example, preferably fixed (e.g., mobilized) circulating peripheral blood can be removed from a subject. In one embodiment, the stem cells can be reprogrammed stem cells, such as stem cells derived from somatic cells or differentiated cells. In such embodiments, the dedifferentiated stem cells can be, for example, but not limited to, neoplastic cells, tumor cells, and cancer cells, or induced reprogrammed cells, such as induced pluripotent stem cells or iPS cells.

[0182]

[0200] In some embodiments, the pancreatic alpha, beta and / or delta cells described herein are selected from the group consisting of hair cells, keratinocytes, gonadotrophs, corticotrophs, thyrotrophs, somatotrophs, lactotrophs, chromaffin cells, parafollicular cells, glomus cells, melanocytes, nevus cells, Merkel cells, odontoblasts, cementoblasts, corneal keratinocytes, retinal müller cells, retinal pigment epithelial cells, neurons, glial cells (e.g., oligodendrocytes, astrocytes), ependymal cells, pineal cells, lung cells (e.g., type I pneumocytes and type II pneumocytes), Clara cells, goblet cells, G cells, D cells, ECL cells, gastric chief cells, parietal cells, pituitary cells, K cells, D cells, I cells, goblet cells, Paneth cells, enterocytes, microfold cells, hepatocytes, hepatic stellate cells (e.g., Kupffer cells from mesoderm), gallbladder cells, atrial cells, pancreatic stellate cells, pancreatic α cells, pancreatic β cells, pancreatic δ cells, pancreatic F cells (e.g., PP cells), pancreatic ε cells, thyroid (e.g., follicular cells), parathyroid (e.g., parathyroid chief cells), eosinophilic cells, urothelial cells, osteoblasts, osteocytes, chondroblasts, chondrocytes, fibroblasts, fibrocytes, myoblasts, myocytes, myosatellite cells, tenocytes, cardiac myocytes, lipoblasts, adipocytes, interstitial cells of Cajal, angioblasts, endothelial cells, mesangial cells (e.g., intraglomerular and extraglomerular mesangial cells), juxtaglomerular cells, macula densa cells, stromal cells, interstitial cells, telosoma The cells can be derived from one or more of: uterine simple epithelial cells, podocytes, renal proximal tubule brush border cells, Sertoli cells, Leydig cells, granulosa cells, Pegg cells, germ layer cells, sperm, eggs, lymphocytes, myeloid cells, endothelial progenitor cells, endothelial stem cells, hemangioblasts, mesoangioblasts, pericyte cells, splenocytes (e.g., T lymphocytes, B lymphocytes, dendritic cells, microphages, leukocytes), trophoblast stem cells, or any combination thereof.

[0183] Reprogramming

[0201] As used herein, the term "reprogramming" may refer to the process of altering or reversing the differentiation state of a somatic cell. Cells can be partially or terminally differentiated before reprogramming. Reprogramming may encompass the complete reversal of the differentiation state of a somatic cell to a pluripotent cell. Such complete reversal of differentiation may generate induced pluripotent (iPS) cells. As used herein, reprogramming may also encompass the partial reversal of the differentiation state of a cell, e.g., to a pluripotent state cell or somatic cell, which is a cell that is neither pluripotent nor pluripotent but has lost one or more specific characteristics of the differentiated cell from which it originated, e.g., the direct reprogramming of a differentiated cell into various somatic cell types. Reprogramming involves the reprogramming of a zygote into a variety of somatic cell types. This may include alteration, e.g., reversal, of at least some of the heritable patterns of nucleic acid modification (e.g., methylation), chromatin condensation, epigenetics, genomic imprinting, etc. that occur during cell differentiation during adult development.

[0184]

[0202] As used herein, the term "reprogramming factor" can refer to a molecule associated with the "reprogramming" of a cell, i.e., differentiation, and / or dedifferentiation, and / or transdifferentiation, whereby a cell is converted into a different cell type or phenotype. Reprogramming factors generally affect the expression of genes associated with cell differentiation, dedifferentiation, and / or transdifferentiation. Transcription factors are examples of reprogramming factors.

[0185]

[0203] As used herein, the term "differentiation" and its grammatical equivalents refer to the process by which a less specialized cell (e.g., a more primitive cell with greater cellular potential) becomes a more specialized cell type (e.g., a less primitive cell with less cellular potential); the term "dedifferentiation" refers to the process by which a more specialized cell becomes a less specialized cell type (e.g., a more primitive cell with greater cellular potential); and the term "transdifferentiation" can refer to the process by which a cell of a particular cell type is converted into another cell type without significantly changing its level of "cellular potential" or "primitiveness." Without wishing to be bound by theory, a cell is considered to "transdifferentiate" if it is converted from one lineage-committed or terminally differentiated cell type to another lineage-committed or terminally differentiated cell type without significantly changing its level of "cellular potential" or "primitiveness."

[0186]

[0204] As used herein, the term "cell differentiation potential" should be understood to mean the ability of a cell to differentiate into cells of different lineages. For example, pluripotent cells (e.g., stem cells) have the potential to differentiate into cells of any of the three germ layers, i.e., endoderm (the inner lining of the stomach, the gastrointestinal tract, and the lungs), mesoderm (muscle, bone, blood, and urogenital tract), or ectoderm (epithelial tissue and the nervous system), and therefore have high cell differentiation potential. Pluripotent cells (e.g., stem cells or certain types of induced stem cells) have the ability to give rise to cells from a large but limited number of lineages (e.g., hematopoietic stem cells, cardiac stem cells, or neural stem cells, etc.) and have relatively lower cell differentiation potential than pluripotent cells. Cells committed to a specific lineage or terminally differentiated may have even lower cell differentiation potential. Specific examples of transdifferentiation known in the art include, for example, the conversion of fibroblastic beta cells or exocrine pancreatic cells to beta cells.

[0187]

[0205] Thus, cells can be differentiated into more primitive cells (e.g., terminally differentiated cells can be differentiated to pluripotent or multipotent), or cells can be dedifferentiated into less primitive cells (e.g., pluripotent or multipotent cells can be differentiated into lineage-committed or terminally differentiated cells). However, in one embodiment, cells can be converted or transdifferentiated from one cell type (or phenotype) to another cell type (or phenotype), e.g., at a similar level of cellular potential. Thus, in one embodiment of the present disclosure, the inducing step of the present disclosure can reprogram cells of the present disclosure to differentiate, dedifferentiate, and / or transdifferentiate. In one embodiment of the present disclosure, the inducing step of the present disclosure can reprogram cells to transdifferentiate.

[0188]

[0206] Those skilled in the art are familiar with methods for reprogramming or inducing a particular type of cell to become another type of cell, for example, by differentiation, dedifferentiation, and / or transdifferentiation, using one or more exogenous polynucleotide or polypeptide reprogramming factors. Such methods may rely on the introduction of genetic material encoding one or more transcription factors or other polypeptides involved in cell reprogramming. For example, PDX1, Ngn3, and MafA, or functional fragments thereof, are all known to encode peptides that can induce cell differentiation, dedifferentiation, and / or transdifferentiation of the cells of the present disclosure. Some methods known to those skilled in the art involve the reprogramming of cells. An exogenous polypeptide (e.g., a recombinant polypeptide) encoded by a gene (such as the genes described above) is contacted with a cell to induce, for example, a cell of the present disclosure. Those skilled in the art will recognize that other genes are also involved in cellular reprogramming, and that exogenous molecules (or functional fragments thereof) encoding such genes and the encoded polypeptides are also considered polynucleotide or polypeptide reprogramming factors (e.g., polynucleotides or polypeptides that subsequently affect the expression level of another gene associated with cellular reprogramming). For example, the introduction of exogenous polynucleotide or polypeptide epigenetic gene silencers that reduce p53 inactivation has been shown to increase the efficiency of inducing induced pluripotent stem cells (iPSCs). Thus, exogenous polynucleotides or polypeptides encoding epigenetic silencers, as well as other genes or proteins believed to be directly or indirectly involved in cellular reprogramming or increasing cellular programming efficiency, are considered to constitute exogenous polynucleotide or polypeptide reprogramming factors. Those skilled in the art will recognize that other methods exist for affecting cellular reprogramming, such as the introduction of RNAi molecules (or genetic material encoding RNAi molecules) that can knock down the expression of genes involved in inhibiting cellular reprogramming. Thus, any exogenous polynucleotide or polypeptide molecule that is associated with or promotes cellular reprogramming should be understood to be an exogenous polynucleotide or polypeptide reprogramming factor as described herein.

[0189]

[0207] In some embodiments of the present disclosure, the methods do not include the use of reprogramming factors that are not small molecules. However, it is recognized that the present methods can utilize "routine" tissue culture components such as media, sera, serum replacements, supplements, antibiotics, and the like, such as RPMI, renal epithelial basal medium (REBM), Dulbecco's modified Eagle's medium (DMEM), MCDB131 medium, CMRL1066 medium, F12, fetal calf serum (FCS), fetal bovine serum (FBS), bovine serum albumin (BSA), D-glucose, L-glutamine, GlutaMAX™-1 (dipeptide, L-alanine-L-glutamine), B27, heparin, progesterone, putrescine, laminin, nicotinamide, insulin, transferrin, sodium selenate, selenium, ethanolamine, human epidermal growth factor (hEGF), basic fibroblast growth factor (bFGF), hydrocortisone, epinephrine, normacin, penicillin, streptomycin, gentamicin, and amphotericin. These typical tissue culture components (and other similar tissue culture components routinely used in tissue culture) are not small molecule reprogramming molecules for the purposes of this disclosure: these components are neither small molecules as defined herein nor reprogramming factors as defined herein.

[0190]

[0208] Thus, in one embodiment, the present disclosure does not include culturing cells with one or more exogenous polynucleotide or polypeptide reprogramming factors. Thus, in one embodiment, the methods of the present disclosure do not include the introduction of one or more exogenous polynucleotide or polypeptide reprogramming factors, e.g., by introducing a transposon, a viral transgenic vector (such as a retroviral vector), a plasmid, mRNA, miRNA, a peptide, or a fragment of any of these molecules, that are involved in producing induced α, β, and / or δ cells or otherwise inducing the cells of the present disclosure to differentiate, dedifferentiate, and / or transdifferentiate.

[0191]

[0209] That is, in one embodiment, the method is performed in the absence of one or more exogenous polynucleotide or polypeptide reprogramming factors. Thus, in one embodiment, the disclosed method involves the use of a polypeptide transcription factor, other polypeptide factors specifically associated with inducing differentiation, dedifferentiation, and / or transdifferentiation, polynucleotide sequences encoding polypeptide transcription factors ... It should be understood that small molecules (e.g., HDAC inhibitors) are utilized to reprogram cells without the addition of polynucleotide sequences encoding other polypeptide factors, mRNAs, interfering RNAs, microRNAs, and fragments thereof that are related to the target gene. Methods for producing induced beta cells from stem cells

[0210] Methods for producing SC-β cells (e.g., non-native pancreatic β cells) are provided herein. Detailed protocols for producing endocrine cells from stem cells to provide at least one SC-β cell are described in U.S. Patent Application Publication Nos. 2015 / 0240212 and 2015 / 0218522, each of which is incorporated herein by reference in its entirety.

[0192]

[0211] The endoderm can give rise to the digestive and respiratory tracts, thyroid gland, liver, and pancreas. A representative disease of the endodermal lineage is type 1 diabetes, which results from the destruction of insulin-producing beta cells. The in vitro generation of functional beta cells from human pluripotent stem cells (hPSCs) could provide a practical and renewable cell source for replacement cell therapy of type 1 diabetes. Embryonic stem (ES) cells, generated from the inner cell mass of blast-stage embryos, represent a promising cell source for transplantation of any damaged cells or cell-based therapies. They can be maintained in culture, self-renew, and proliferate indefinitely as undifferentiated ES cells. ES cells can differentiate into all cell types in the body, including ectodermal, mesodermal, and endodermal lineage cells or tissues. The major advantage of ES cells is their potential for stable self-renewal and differentiation in culture.

[0193]

[0212] Definitive endoderm is produced in vivo from the inner cell mass by the embryonic process of gastrulation, during which epiblast cells are directed to form three germ layers. Definitive endoderm can give rise to a variety of cells and tissues that contribute to vital organs, such as pancreatic beta cells, hepatocytes of the liver, alveolar cells of the lung, thyroid gland, thymus, and the epithelial linings of the nutrient and respiratory tracts. This differs from the primitive endoderm, which is an extraembryonic tissue that can give rise to visceral and parietal endoderm. Definitive endoderm derived from ES cells can theoretically become any endodermal derivative, and directing ES cells toward the endodermal lineage is essential for generating therapeutic endodermal derivatives.

[0194]

[0213] The precise patterning of the definitive endoderm along the anterior-posterior axis ultimately leads to the formation of the primitive gut. The primitive gut, derived from the definitive endoderm, gives rise to the pharynx, esophagus, stomach, duodenum, small intestine, and large intestine, as well as related organs such as the pancreas, lungs, thyroid gland, thymus, parathyroid gland, and liver along the anterior-posterior axis. The anterior portion of the foregut of the primitive gut gives rise to the lungs, thyroid gland, esophagus, and stomach. The pancreas, liver, and duodenum originate from the posterior portion of the foregut. The midgut and hindgut of the primitive gut give rise to the small intestine and large intestine. The anterior foregut expresses developmental markers NK2 homeobox (NKX2-1) and SRY (sex-determining region Y)-box 2 (SOX2); the posterior foregut expresses hematopoietically expressed homeobox (HHEX), pancreatic and duodenal homeobox 1 (PDX1), onecut homeobox 1 (ONECUT1, also known as HNF6), and hepatocyte nuclear factor 4 alpha (HNF4A); and the midgut / hindgut expresses caudal homeobox 1 (CDX1), caudal homeobox 2 (CDX2), and motor neuron and pancreatic homeobox 1 (MNX1) (3, 19, 20).

[0195]

[0214] Successful differentiation of pancreatic β cells requires that the differentiated cells synthesize and secrete physiologically relevant amounts of insulin. An exemplary stepwise protocol for directing hPSC cell differentiation has been developed, involving a differentiation process that recapitulates the key stages of normal pancreatic endocrine development (e.g., the Version A protocol in Example 1). Differentiation of hPSC cells into hormone-expressing pancreatic endocrine cells is achieved by passing hPSC cells through key stages consisting of embryonic development, differentiation into mesendoderm and definitive endoderm, establishment of archenteron endoderm, patterning of the posterior foregut, and specification and maturation of pancreatic endoderm and endocrine precursors. Through these stages, hPSC cells differentiate into hormone-expressing pancreatic endocrine cells. PSC cells can acquire a pancreatic endocrine phenotype and the ability to secrete insulin in response to glucose in vitro.

[0196]

[0215] Generally, at least one pancreatic alpha, beta and / or delta cell or precursor thereof, e.g., a pancreatic progenitor cell produced by the methods disclosed herein, can include a mixture or combination of various cells, e.g., PDX1-positive pancreatic progenitor cells, pancreatic progenitor cells co-expressing PDX1 and NKX6-1, Ngn3-positive endocrine progenitor cells, insulin-positive endocrine cells (e.g., NKX6.1-positive, ISL1-positive, or beta-like cells), and / or other pluripotent or stem cells.

[0197]

[0216] The at least one pancreatic alpha, beta, and / or delta cell, or precursor thereof, can be produced according to any suitable culture protocol that differentiates stem cells or pluripotent cells to a desired differentiation stage. In some embodiments, the at least one pancreatic alpha, beta, and / or delta cell, or precursor thereof, is produced by culturing at least one pluripotent cell for a time and under conditions suitable for differentiating the at least one pluripotent cell into at least one pancreatic alpha, beta, and / or delta cell, or precursor thereof.

[0198]

[0217] In some embodiments, the at least one pancreatic alpha, beta, and / or delta cell or precursor thereof is a substantially pure population of pancreatic alpha, beta, and / or delta cells or precursors thereof. In some embodiments, the population of pancreatic alpha, beta, and / or delta cells or precursors thereof comprises a mixture of pluripotent cells or differentiated cells. In some embodiments, the population of pancreatic alpha, beta, and / or delta cells or precursors thereof is substantially free of or devoid of embryonic stem cells or pluripotent cells or iPS cells.

[0199]

[0218] In some embodiments, somatic cells, e.g., fibroblasts, can be isolated from a subject, e.g., as a tissue biopsy, e.g., a skin biopsy, and reprogrammed into induced pluripotent stem cells for further differentiation to generate at least one pancreatic alpha, beta, and / or delta cell, or precursor thereof, for use in the compositions and methods described herein. In some embodiments, the somatic cells, e.g., fibroblasts, are maintained in culture by methods known to those of skill in the art, and in some embodiments, expanded before being converted into pancreatic alpha, beta, and / or delta cells by the methods disclosed herein.

[0200]

[0219] In some embodiments, at least one pancreatic alpha, beta and / or delta cell or precursor thereof is maintained in culture by methods known to those of skill in the art, and in some embodiments expanded before being converted to a pancreatic alpha, beta and / or delta cell by the methods disclosed herein.

[0201]

[0220] Furthermore, the at least one pancreatic α, β, and / or δ cell or precursor thereof, e.g., pancreatic progenitor, may be from any mammalian species, non-limiting examples of which include murine, bovine, simian, porcine, equine, ovine, or human cells. For clarity and simplicity, the methods described herein refer to at least one mammalian pancreatic α, β, and / or δ cell or precursor thereof, but it should be understood that all of the methods described herein can be readily applied to other cell types of at least one pancreatic α, β, and / or δ cell or precursor thereof. In some embodiments, the at least one SC-β cell or precursor thereof is derived from a human individual.

[0202] Definitive endoderm cells

[0221] Embodiments of the present disclosure include definitive endoderm cells. As used herein, definitive endoderm cells can be derived from any source or produced according to any suitable protocol. In some embodiments, pluripotent stem cells, such as iPSCs or hESCs, can be transformed into endoderm cells. In some embodiments, endoderm cells (stage 1) are further differentiated, e.g., into primitive gut cells (stage 2), PDX1-positive pancreatic progenitor cells (stage 3), NKX6.1-positive pancreatic progenitor cells (stage 4), or Ngn3-positive endocrine precursor cells or insulin-positive endocrine cells (stage 5), and then induced or matured into SC-β cells (stage 6).

[0203]

[0222] In some examples, definitive endoderm cells can be obtained by differentiating at least some pluripotent cells in a population into definitive endoderm cells, for example, by contacting a population of pluripotent cells with i) at least one growth factor from the TGF-β superfamily and ii) a WNT signaling pathway activator to induce differentiation of at least some of the pluripotent cells into definitive endoderm cells, wherein the definitive endoderm cells express at least one marker characteristic of definitive endoderm.

[0204]

[0223] Any growth factor from the TGF-β superfamily that can induce pluripotent stem cells (e.g., alone or in combination with a WNT signaling pathway activator) to differentiate into definitive endoderm cells can be used in the methods provided herein. In some examples, the growth factor from the TGF-β superfamily includes activin A. In some examples, the growth factor from the TGF-β superfamily includes growth differentiation factor 8 (GDF8). Any WNT signaling pathway activator that can induce pluripotent stem cells (e.g., alone or in combination with a growth factor from the TGF-β superfamily) to differentiate into definitive endoderm cells can be used in the methods provided herein. In some examples, the WNT signaling pathway activator includes CHIR99Q21. In some examples, the WNT signaling pathway activator includes Wnt3a recombinant protein.

[0205]

[0224] In some examples, differentiating at least some of the pluripotent cells in the population into definitive endoderm cells is accomplished by a process of contacting the population of pluripotent cells with i) activin A, and ii) CHIR99021 for a suitable period of time, for example, about 2 days, about 3 days, about 4 days, or about 5 days, to induce differentiation of at least some of the pluripotent cells in the population into definitive endoderm cells, wherein the definitive endoderm cells express at least one marker characteristic of definitive endoderm.

[0206]

[0225] In some examples, the method includes contacting a population of pluripotent cells with a suitable concentration of a growth factor from the TGF-β superfamily (e.g., activin A), such as about 10 ng / mL, about 20 ng / mL, about 50 ng / mL, about 75 ng / mL, about 80 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 110 ng / mL, about 120 ng / mL, about 130 ng / mL, about 140 ng / mL, about 150 ng / mL, about 175 ng / mL, about 180 ng / mL, about 200 ng / mL, about 250 ng / mL, or about 300 ng / mL, thereby differentiating the pluripotent cells into definitive endoderm cells. In some examples, the method includes using about 100 ng / mL of activin A to differentiate the pluripotent cells into definitive endoderm cells. In some examples, the methods include the use of about 200 ng / mL of activin A for the differentiation of pluripotent cells to definitive endoderm cells.

[0207]

[0226] In some examples, the method includes contacting a population of pluripotent cells with a suitable concentration of a WNT signaling pathway activator (e.g., CHIR99021) at, for example, about 0.01 μM, about 0.05 μM, about 0.1 μM, about 0.2 μM, about 0.5 μM, about 0.8 μM, about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM, about 5 μM, about 8 μM, about 10 μM, about 12 μM, about 15 μM, about 20 μM, about 30 μM, about 50 μM, about 100 μM, or about 200 μM, thereby differentiating the pluripotent cells into definitive endoderm cells. In some examples, the method includes using about 2 μM of CHIR99021 to differentiate the pluripotent cells into definitive endoderm cells. In some examples, the method includes contacting a population of pluripotent cells with, for example, about 0.01 μM, about 0.05 μM, about 0.1 μM, about 0.2 μM, about 0.5 μM, about 0.8 μM, about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM, about 5 μM, about 8 μM, about 10 μM, about 12 μM, about 15 μM, about 20 μM, about 30 μM, about 50 μM, about 100 μM, or about 200 μM of a WNT signaling pathway activator (e.g., CHIR99021) at, for example, about 2 μM of CHIR9902 Including the use of approximately 5 μM CHIR99021 for differentiation into definitive endoderm cells.

[0208]

[0227] In some examples, the definitive endoderm cells generated by the methods disclosed herein express at least one marker selected from the group consisting of Nodal, Tmprss2, Tmem30b, St14, Spink3, Sh3gl2, Ripk4, Rab1S, Npnt, Clic6, Cldn5, Cacna1b, Bnip1, Anxa4, Emb, FoxA1, Sox17, and Rbm35a, and the expression of the at least one marker is upregulated at a statistically significant amount in the definitive endoderm cells relative to the original pluripotent stem cells from which it was derived. In some examples, the definitive endoderm cells generated by the methods disclosed herein do not express at least one marker selected from the group consisting of Gata4, SPARC, AFP, and Dab2 at a statistically significant amount relative to the original pluripotent stem cells from which it was derived. In some examples, definitive endoderm cells generated by the methods disclosed herein do not express at least one marker selected from the group consisting of Zic1, Pax6, Flk1, and CD31 at a statistically significant level relative to the original pluripotent stem cells from which they were derived. In some examples, definitive endoderm cells generated by the methods disclosed herein have a statistically significant higher level of Smad2 phosphorylation relative to the original pluripotent stem cells from which they were derived. In some examples, definitive endoderm cells generated by the methods disclosed herein have the ability to form a gut tube in vivo. In some examples, definitive endoderm cells generated by the methods disclosed herein can be differentiated into cells with morphology characteristic of intestinal cells, and the cells with morphology characteristic of intestinal cells express FoxA2 and / or Claudin6. In some examples, definitive endoderm cells generated by the methods disclosed herein can be further differentiated into cells of endodermal origin.

[0209]

[0228] In some examples, the population of pluripotent stem cells is cultured in the presence of at least one beta cell differentiation factor before any differentiation or during the first stage of differentiation. Any pluripotent stem cells can be used, such as human pluripotent stem cells or human iPS cells, or any pluripotent stem cells discussed herein or other suitable pluripotent stem cells. In some examples, the beta cell differentiation factors described herein can be present in the culture medium of the population of pluripotent stem cells, or can be added once or periodically during the growth (e.g., replication or expansion) of the population of pluripotent stem cells. In certain examples, the population of pluripotent stem cells can be exposed to at least one beta cell differentiation factor before any differentiation. In other examples, the population of pluripotent stem cells can be exposed to at least one beta cell differentiation factor during the first stage of differentiation.

[0210] gastrula cells

[0229] The embodiments of the present disclosure include primitive gut cells.The primitive gut cells used herein can be derived from any source or produced according to any suitable protocol.In some embodiments, definitive endoderm cells are differentiated into primitive gut cells.In some embodiments, primitive gut cells are further differentiated into, for example, PDX1 positive pancreatic progenitor cells, NKX6.1 positive pancreatic progenitor cells, Ngn3 positive endocrine progenitor cells, insulin positive endocrine cells, and then induce or mature into SC-β cells.

[0211]

[0230] In some examples, primitive gut cells can be obtained by differentiating at least some of the definitive endoderm cells in the population into primitive gut cells, for example, by contacting the definitive endoderm cells with at least one growth factor from the fibroblast growth factor (FGF) family to induce differentiation of at least some of the definitive endoderm cells into primitive gut cells, wherein the primitive gut cells express at least one marker characteristic of primitive gut cells.

[0212]

[0231] Any growth factor from the FGF family that can induce definitive endoderm cells to differentiate into primitive gut cells (e.g., alone or in combination with other factors) can be used in the methods provided herein. In some examples, at least one growth factor from the FGF family can be used. In some examples, the at least one growth factor comprises keratinocyte growth factor (KGF). In some examples, the at least one growth factor from the FGF family comprises FGF2. In some examples, the at least one growth factor from the FGF family comprises FGF8B. In some examples, the at least one growth factor from the FGF family comprises FGF10. In some examples, the at least one growth factor from the FGF family comprises FGF21.

[0213]

[0232] In some examples, primitive gut cells can be obtained by differentiating at least some of the definitive endoderm cells in the population into primitive gut cells, for example, by contacting the definitive endoderm cells with KGF for a period of time, for example, about 1 day, about 2 days, about 3 days, or about 4 days, to induce differentiation of at least some of the definitive endoderm cells into primitive gut cells.

[0214]

[0233] In some examples, the method includes differentiating definitive endoderm cells into primitive gut cells by contacting the cells with a growth factor from the FGF family (e.g., KGF) at a suitable concentration, for example, about 10 ng / mL, about 20 ng / mL, about 50 ng / mL, about 75 ng / mL, about 80 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 110 ng / mL, about 120 ng / mL, about 130 ng / mL, about 140 ng / mL, about 150 ng / mL, about 175 ng / mL, about 180 ng / mL, about 200 ng / mL, about 250 ng / mL, or about 300 ng / mL. In some examples, the method includes using about 50 ng / mL of KGF to differentiate the definitive endoderm cells into primitive gut cells. In some examples, the method includes using about 100 ng / mL of KGF to differentiate the definitive endoderm cells into primitive gut cells.

[0215] PDX1-positive pancreatic progenitor cells

[0234] The embodiments of the present disclosure include PDX1 positive pancreatic progenitor cells.The PDX1 positive pancreatic progenitor cells used herein can be derived from any source or produced according to any suitable protocol.In some embodiments, primitive gut cells are differentiated into PDX1 positive pancreatic progenitor cells.In some embodiments, PDX1 positive pancreatic progenitor cells are further differentiated into, for example, NKX6.1 positive pancreatic progenitor cells, Ngn3 positive endocrine progenitor cells, insulin positive endocrine cells, and then induce or mature into SC-β cells.

[0216]

[0235] In some embodiments, PDX1-positive pancreatic progenitor cells can be obtained by differentiating at least a portion of the primitive gut cells in the population into PDX1-positive pancreatic progenitor cells, for example, by contacting the primitive gut cells with: i) at least one BMP signaling pathway inhibitor; ii) a growth factor from the TGF-β superfamily; iii) at least one growth factor from the FGF family; iv) at least one SHH pathway inhibitor; v) at least one retinoic acid (RA) signaling pathway activator; vi) at least one protein kinase C activator; and vii) a ROCK inhibitor, and inducing at least a portion of the primitive gut cells to differentiate into PDX1-positive pancreatic progenitor cells, wherein the PDX1-positive pancreatic progenitor cells express PDX1.

[0217]

[0236] In some embodiments, PDX1-positive pancreatic progenitor cells can be obtained by differentiating at least a portion of the primitive gut cells in the population into PDX1-positive pancreatic progenitor cells, for example, by contacting the primitive gut cells with i) at least one BMP signaling pathway inhibitor, ii) a growth factor from the TGF-β superfamily, iii) at least one growth factor from the FGF family, iv) at least one SHH pathway inhibitor, v) at least one retinoic acid (RA) signaling pathway activator, and vi) at least one protein kinase C activator, and inducing differentiation of at least a portion of the primitive gut cells into PDX1-positive pancreatic progenitor cells, wherein the PDX1-positive pancreatic progenitor cells express PDX1.

[0218]

[0237] In some cases, PDX1-positive pancreatic progenitor cells are expressed in at least some of the primitive gut tissue in the population. The cells can be differentiated into PDX1-positive pancreatic progenitor cells, for example, by contacting primitive gut cells with i) at least one BMP signaling pathway inhibitor, ii) at least one growth factor from the FGF family, iii) at least one SHH pathway inhibitor, iv) at least one retinoic acid (RA) signaling pathway activator, and v) at least one protein kinase C activator, to induce differentiation of at least a portion of the primitive gut cells into PDX1-positive pancreatic progenitor cells, wherein the PDX1-positive pancreatic progenitor cells express PDX1.

[0219]

[0238] In some examples, PDX1-positive pancreatic progenitor cells can be obtained by differentiating at least some of the primitive gut cells in the population into PDX1-positive pancreatic progenitor cells, for example, by contacting the primitive gut cells with: i) at least one SHH pathway inhibitor; ii) at least one retinoic acid (RA) signaling pathway activator; and iii) at least one protein kinase C activator, wherein the PDX1-positive pancreatic progenitor cells express PDX1.

[0220]

[0239] In some examples, PDX1-positive pancreatic progenitor cells can be obtained by differentiating at least a portion of the primitive gut cells in the population into PDX1-positive pancreatic progenitor cells, for example, by contacting the primitive gut cells with i) at least one growth factor from the FGF family and ii) at least one activator of the retinoic acid (RA) signaling pathway to induce differentiation of at least a portion of the primitive gut cells into PDX1-positive pancreatic progenitor cells, wherein the PDX1-positive pancreatic progenitor cells express PDX1.

[0221]

[0240] Any BMP signaling pathway inhibitor that can induce primitive gut cells to differentiate into PDX1-positive pancreatic progenitor cells (e.g., alone or in combination with a growth factor from the TGF-β superfamily, at least one growth factor from the FGF family, at least one SHH pathway inhibitor, at least one retinoic acid signaling pathway activator, at least one protein kinase C activator, and a ROCK inhibitor) can be used in the methods provided herein. In some examples, the BMP signaling pathway inhibitor includes LDN193189 or DMH-1. In some examples, the method includes contacting the primitive gut cells with a BMP signaling pathway inhibitor (e.g., LDN193189) at a concentration of, for example, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 110 nM, about 120 nM, about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about 200 nM, about 210 nM, about 220 nM, about 230 nM, about 240 nM, about 250 nM, about 280 nM, about 300 nM, about 400 nM, about 500 nM, or about 1 μM. In some examples, the method includes contacting the primitive gut cells with a BMP signaling pathway inhibitor (e.g., DMH-1) at a concentration of, for example, about 0.01 μM, about 0.02 μM, about 0.05 μM, about 0.1 μM, about 0.2 μM, about 0.5 μM, about 0.8 μM, about 1 μM, about 1.2 μM, about 1.5 μM, about 1.75 μM, about 2 μM, about 2.2 μM, about 2.5 μM, about 2.75 μM, about 3 μM, about 3.25 μM, about 3.5 μM, about 3.75 μM, about 4 μM, about 4.5 μM, about 5 μM, about 8 μM, about 10 μM, about 15 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, or about 100 μM.

[0222]

[0241] Any growth factor from the TGF-β superfamily that can induce primitive gut cells to differentiate into PDX1-positive pancreatic progenitor cells (e.g., alone or in combination with at least one BMP signaling pathway inhibitor, a growth factor from the FGF family, at least one SHH pathway inhibitor, at least one retinoic acid signaling pathway activator, at least one protein kinase C activator, and a ROCK inhibitor) can be used. In some examples, the growth factor from the TGF-β family includes activin A. In some examples, the growth factor from the TGF-β family includes activin A or GDF In some examples, the method includes contacting the primitive gut cells with a growth factor from the TGF-β superfamily (e.g., activin A) at a concentration of, for example, about 5 ng / mL, about 7.5 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 11 ng / mL, about 12 ng / mL, about 13 ng / mL, about 14 ng / mL, about 15 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, about 19 ng / mL, about 20 ng / mL, about 21 ng / mL, about 22 ng / mL, about 23 ng / mL, about 24 ng / mL, about 25 ng / mL, about 26 ng / mL, about 27 ng / mL, about 28 ng / mL, about 29 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, or about 100 ng / mL.

[0223]

[0242] Any growth factor from the FGF family that can induce primitive gut cells to differentiate into PDX1-positive pancreatic progenitor cells (e.g., alone or in combination with at least one BMP signaling pathway inhibitor, a growth factor from the TGF-β superfamily, at least one SHH pathway inhibitor, at least one retinoic acid signaling pathway activator, at least one protein kinase C activator, and a ROCK inhibitor) can be used. In some examples, the at least one growth factor from the FGF family includes keratinocyte growth factor (KGF). In some examples, the at least one growth factor from the FGF family is selected from the group consisting of FGF2, FGF8B, FGF10, and FGF21. In some examples, the method includes contacting the primitive gut cells with a growth factor from the FGF family (e.g., KGF) at a concentration of, for example, about 10 ng / mL, about 20 ng / mL, about 50 ng / mL, about 75 ng / mL, about 80 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 110 ng / mL, about 120 ng / mL, about 130 ng / mL, about 140 ng / mL, about 150 ng / mL, about 175 ng / mL, about 180 ng / mL, about 200 ng / mL, about 250 ng / mL, or about 300 ng / mL.

[0224]

[0243] Any SHH pathway inhibitor that can induce primitive gut cells to differentiate into PDX1-positive pancreatic progenitor cells (e.g., alone or in combination with at least one BMP signaling pathway inhibitor, at least one growth factor from the FGF family, a growth factor from the TGF-β superfamily, at least one retinoic acid signaling pathway activator, at least one protein kinase C activator, and a ROCK inhibitor) can be used. In some examples, the SHH pathway inhibitor comprises Sant1. In some examples, the method comprises administering to the primitive gut cells at a concentration of, for example, about 0.001 μM, about 0.002 μM, about 0.005 μM, about 0.01 μM, about 0.02 μM, about 0.03 μM, about 0.05 μM, about 0.08 μM, about 0.1 μM, about 0.12 μM, about 0.13 μM, about 0.14 μM, about 0.15 μM, about 0.16 μM, about 0.17 μM, about 0.18 μM, about 0.19 μM, about 0.2 μM, about 0.21 μM, about 0.22 μM, about 0.23 μM, about 0.24 μM, about 0.25 μM, about 0.26 μM, about 0.27 μM, about 0.28 μM, about 0.29 μM, about 0.30 μM, about 0.31 μM, about 0.32 μM, about 0.33 μM, about 0.34 μM, about 0.35 μM, about 0.36 μM, about 0.37 μM, about 0.38 μM, about 0.39 μM, about 10 ... The method includes contacting the cells with an SHH pathway inhibitor (e.g., Sant1) at a concentration of about 0.24 μM, about 0.25 μM, about 0.26 μM, about 0.27 μM, about 0.28 μM, about 0.29 μM, about 0.3 μM, about 0.31 μM, about 0.32 μM, about 0.33 μM, about 0.34 μM, about 0.35 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about 0.6 μM, about 0.8 μM, about 1 μM, about 2 μM, or about 5 μM.

[0225]

[0244] Any RA signaling pathway activator that can induce primitive gut cells to differentiate into PDX1-positive pancreatic progenitor cells (e.g., alone or in combination with at least one BMP signaling pathway inhibitor, at least one growth factor from the FGF family, at least one SHH pathway inhibitor, at least one protein kinase C activator, and a ROCK inhibitor) can be used. In some examples, the RA signaling pathway activator comprises retinoic acid. In some examples, the method can involve inducing primitive gut cells to differentiate into PDX1-positive pancreatic progenitor cells at concentrations of, for example, about 0.02 μM, about 0.1 μM, about 0.2 μM, about 0.25 μM, about 0.3 μM, about 0.4 μM, or about 1 μM. 0.45μM, about 0.5μM, about 0.55μM, about 0.6μM, about 0.65μM, about 0.7μM, about 0.75μM, about 0.8μM, about 0.85μM, about 0.9μM, about 1μM, about 1.1μM, about 1.2μM, about 1.3μM, about 1.4μM, Approximately 1.5μM, approximately 1.6μM, approximately 1.7μM, approximately 1.8μM, approximately 1.9μM, approximately 2μM, approximately 2.1μM, approximately 2.2μM, approximately 2.3μM, approximately 2.4μM, approximately 2.5μM, approximately 2.6μM, approximately 2.7μM, approximately 2.8μM, approximately 3μM, approximately 3.2μM , about 3.4 μM, about 3.6 μM, about 3.8 μM, about 4 μM, about 4.2 μM, about 4.4 μM, about 4.6 μM, about 4.8 μM, about 5 μM, about 5.5 μM, about 6 μM, about 6.5 μM, about 7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM, about 10 μM, about 12 μM, about 14 μM, about 15 μM, about 16 μM, about 18 μM, about 20 μM, about 50 μM, or about 100 μM of a RA signaling pathway activator (e.g., retinoic acid).

[0226]

[0245] Any PKC activator that can induce primitive gut cells to differentiate into PDX1-positive pancreatic progenitor cells (e.g., alone or in combination with at least one BMP signaling pathway inhibitor, at least one growth factor from the FGF family, at least one SHH pathway inhibitor, at least one RA signaling pathway activator, and a ROCK inhibitor) can be used. In some examples, the PKC activator comprises PdBU. In some examples, the PKC activator comprises TPPB. In some examples, the method includes administering to the primitive gut cells a 200 nM or 300 nM or 350 nM or 400 nM or 450 nM or 500 nM or 550 nM or 600 nM or 650 nM or 700 nM or 750 nM or 800 nM or 850 nM or 900 nM or 950 nM or 1 μM or 10 μM or 20 μM or 50 μM or 75 μM or 80 μM or 100 μM or 120 μM or 140 μM or 150 μM or 175 μM or 180 μM or 200 μM or 210 μM or 220 μM or 240 μM or 250 μM or 260 μM or 270 μM or 280 μM or 290 μM or 300 μM or 310 μM or 320 μM or 330 μM or 340 μM or 350 μM or 360 μM or 370 μM or 380 μM or 390 μM or 400 μM or 410 μM or 420 μM or 430 μM or 440 μM or 450 μM or 460 μM or 470 μM or 480 μM or 500 μM or 550 nM or 600 nM or 650 nM or 700 nM or 750 nM or 800 nM or 850 nM or 900 nM or 950 nM or 1 μM or 10 μM or 20 μM or 50 μM or 75 μM or 80 μM or 100 μM or 120 μM The method includes contacting the PKC activator (e.g., PdBU or TPPB) at a concentration of about 0 μM, about 280 μM, about 300 μM, about 320 μM, about 340 μM, about 360 μM, about 380 μM, about 400 μM, about 420 μM, about 440 μM, about 460 μM, about 480 μM, about 500 μM, about 520 μM, about 540 μM, about 560 μM, about 580 μM, about 600 μM, about 620 μM, about 640 μM, about 660 μM, about 680 μM, about 700 μM, about 750 μM, about 800 μM, about 850 μM, about 900 μM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, or about 5 mM. In some embodiments, the method includes contacting the primary gut cells with a PKC activator (e.g., PdBU or TPPB) at a concentration of, for example, 10 nM to 1 mM, 10 nM to 500 μM, 10 nM to 1 μM, 10 to 800 nM, 100 to 900 nM, 300 to 800 nM, 300 to 600 nM, 400 to 600 nM, 450 to 550 nM, or about 500 nM. In some embodiments, the primary gut cells are not treated with a PKC activator (e.g., PDBU).

[0227]

[0246] Any ROCK inhibitor that can induce primitive gut cells to differentiate into PDX1-positive pancreatic progenitor cells (for example, alone or in combination with at least one BMP signaling pathway inhibitor, at least one growth factor from the FGF family, at least one SHH pathway inhibitor, a PKC activator, and at least one RA signaling pathway activator) can be used. In some examples, the ROCK inhibitor comprises thiazovivin, Y-27632, Fasudil / HA1077, or H-1152. In some examples, the ROCK inhibitor comprises Y-27632. In some examples, the ROCK inhibitor comprises thiazovivin. In some examples, the method further comprises administering to the primitive gut cells at a concentration of, for example, about 0.2 μM, about 0.5 μM, about 0.75 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 7.5 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 35 μM, The method includes contacting the cells with a ROCK inhibitor (e.g., Y-27632 or Thiazovivin) at a concentration of about 40 μM, about 50 μM, or about 100 μM.

[0228]

[0247] In some examples, PDX1-positive pancreatic progenitor cells can be obtained by differentiating at least some of the primitive gut cells in the population into PDX1-positive pancreatic progenitor cells, for example, by contacting the primitive gut cells with retinoic acid, KGF, Sant1, DMH-1, PdBU, thiazovivin, and activin A for a suitable period of time, for example, about 1 day, about 2 days, about 3 days, or about 4 days. In some examples, PDX1-positive pancreatic progenitor cells can be obtained by differentiating at least some of the primitive gut cells in the population into PDX1-positive pancreatic progenitor cells, for example, by contacting the primitive gut cells with retinoic acid, KGF, Sant1, DMH-1, PdBU, thiazovivin, and activin A for about 2 days.

[0229] NKX6.1-positive pancreatic progenitor cells

[0248] The embodiments of the present disclosure include NKX6.1 positive pancreatic progenitor cells.The NKX6.1 positive pancreatic progenitor cells used herein can be derived from any source or produced according to any suitable protocol.In some embodiments, PDX1 positive pancreatic progenitor cells are differentiated into NKX6.1 positive pancreatic progenitor cells.In some embodiments, NKX6.1 positive pancreatic progenitor cells are further differentiated into, for example, Ngn3 positive endocrine progenitor cells or insulin positive endocrine cells, and then induce or mature into SC-β cells.

[0230]

[0249] In some aspects, a method for generating NKX6.1-positive pancreatic progenitor cells from PDX1-positive pancreatic progenitor cells includes contacting a population of cells comprising PDX1-positive pancreatic progenitor cells with at least two beta cell differentiation factors (e.g., under conditions that promote cell clustering and / or promote cell survival) comprising a) at least one growth factor from the fibroblast growth factor (FGF) family, b) a sonic hedgehog pathway inhibitor, and optionally c) a low concentration of a retinoic acid (RA) signaling pathway activator to induce differentiation of at least one PDX1-positive pancreatic progenitor cell in the population into an NKX6.1-positive pancreatic progenitor cell, wherein the NKX6.1-positive pancreatic progenitor cell expresses NKX6.1.

[0231]

[0250] In some examples, the PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) a low concentration of an RA signaling pathway activator to induce differentiation of at least some of the PDX1-positive pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells, wherein the PDX1-positive, NKX6.1-positive pancreatic progenitor cells express PDX1 and NKX6.1.

[0232]

[0251] In some examples, PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) a low concentration of an RA signaling pathway activator, iv) a ROCK inhibitor, and v) at least one growth factor from the TGF-β superfamily to induce differentiation of at least some of the PDX1-positive pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells. In some embodiments, 3, 4, or 5 days after the contacting, the PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting the PDX1-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) a low concentration of an activator of the RA signaling pathway, iv) a ROCK inhibitor, and v) at least one growth factor from the TGF-β superfamily, and then the cells are contacted with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) a low concentration of an activator of the RA signaling pathway, iv) a ROCK inhibitor, and v) at least one growth factor from the TGF-β superfamily. In some cases, PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive pancreatic progenitor cells with at least one growth factor from the FGF family under conditions that promote cell clustering.In some cases, the growth factor from the FGF family is KGF.

[0233]

[0252] In some examples, the PDX1-positive pancreatic progenitor cells are generated from a population of pluripotent cells. In some examples, the PDX1-positive pancreatic progenitor cells are generated from a population of iPS cells. In some examples, the PDX1-positive pancreatic progenitor cells are generated from a population of ESC cells. In some examples, the PDX1-positive pancreatic progenitor cells are generated from a population of definitive endoderm cells. In some examples, the PDX1-positive pancreatic progenitor cells are generated from a population of primitive gut cells.

[0234]

[0253] Any growth factor from the FGF family that can induce PDX1-positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (for example, alone or in combination with at least one SHH pathway inhibitor, ROCK inhibitor, growth factor from the TGF-β superfamily, and at least one retinoic acid signaling pathway activator) can be used in the methods provided herein. In some examples, the at least one growth factor from the FGF family includes keratinocyte growth factor (KGF). In some examples, the at least one growth factor from the FGF family is selected from the group consisting of FGF2, FGF8B, FGF10, and FGF21. In some examples, the method includes contacting PDX1-positive pancreatic progenitor cells with a growth factor from the FGF family (e.g., KGF) at a concentration of, for example, about 10 ng / mL, about 20 ng / mL, about 50 ng / mL, about 75 ng / mL, about 80 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 110 ng / mL, about 120 ng / mL, about 130 ng / mL, about 140 ng / mL, about 150 ng / mL, about 175 ng / mL, about 180 ng / mL, about 200 ng / mL, about 250 ng / mL, or about 300 ng / mL.

[0235]

[0254] Any SHH pathway inhibitor that can induce PDX1-positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (for example, alone or in combination with at least one growth factor from the FGF family, at least one retinoic acid signaling pathway activator, a ROCK inhibitor, and at least one growth factor from the TGF-β superfamily) can be used in the methods provided herein. In some examples, the SHH pathway inhibitor comprises Sant1. In some examples, the method comprises administering to the patient a dose of PDX1-positive pancreatic progenitor cells at a concentration of, for example, about 0.001 μM, about 0.002 μM, about 0.005 μM, about 0.01 μM, about 0.02 μM, about 0.03 μM, about 0.05 μM, about 0.08 μM, about 0.1 μM, about 0.12 μM, about 0.13 μM, about 0.14 μM, about 0.15 μM, about 0.16 μM, about 0.17 μM, about 0.18 μM, about 0.19 μM, about 0.2 μM, about 0.21 μM, about 0.22 μM, about 0.24 μM, about 0.25 μM, about 0.26 μM, about 0.27 μM, about 0.28 μM, about 0.29 μM, about 0.30 μM, about 0.31 μM, about 0.32 μM, about 0.33 μM, about 0.34 μM, about 0.35 μM, about 0.36 μM, about 0.37 μM, about 0.38 μM, about 0.39 μM, about 10 μM, about 10 μM, about 110 μM, about 111 μM, about 112 μM, about 113 μM, about 114 μM, about 115 μM, about 116 μM, about 117 μM, about 118 μM, about 119 μM, about 120 μM, about 121 μM, about 122 μM, about 123 μM, about 124 μM, about 125 μM, about 126 μM, The method includes contacting the cells with an SHH pathway inhibitor (e.g., Sant1) at a concentration of about 0.23 μM, about 0.24 μM, about 0.25 μM, about 0.26 μM, about 0.27 μM, about 0.28 μM, about 0.29 μM, about 0.3 μM, about 0.31 μM, about 0.32 μM, about 0.33 μM, about 0.34 μM, about 0.35 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about 0.6 μM, about 0.8 μM, about 1 μM, about 2 μM, or about 5 μM.

[0236]

[0255] Any RA signaling pathway activator that can induce PDX1-positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (e.g., alone or in combination with at least one growth factor from the FGF family, at least one SHH pathway inhibitor, a ROCK inhibitor, and at least one growth factor from the TGF-β superfamily) can be used. In some examples, the RA signaling pathway activator comprises retinoic acid. In some examples, the method can induce PDX1-positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells at concentrations of, for example, about 0.02 μM, about 0.1 μM, about 0.2 μM, about 0.25 μM, about 0.3 μM, about 0.4 μM, or about 0. 45μM, about 0.5μM, about 0.55μM, about 0.6μM, about 0.65μM, about 0.7μM, about 0.75μM, about 0.8μM, about 0.85μM, about 0.9μM, about 1μM, about 1.1μM, about 1.2μM, about 1.3μM, about 1.4μM, about 1.5μM, about 1.6μM, about 1.7μM, about 1.8μM, about 1.9μM, about 2μM, about 2.1μM, about 2.2μM, about 2.3μM, about 2.4μM, about 2.5μM, about 2.6μM, about 2.7μM, about 2.8μM, about 3μM, about 3.2μM, The method includes contacting the subject with an RA signaling pathway activator (e.g., retinoic acid) at a concentration of about 3.4 μM, about 3.6 μM, about 3.8 μM, about 4 μM, about 4.2 μM, about 4.4 μM, about 4.6 μM, about 4.8 μM, about 5 μM, about 5.5 μM, about 6 μM, about 6.5 μM, about 7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM, about 10 μM, about 12 μM, about 14 μM, about 15 μM, about 16 μM, about 18 μM, about 20 μM, about 50 μM, or about 100 μM.

[0237]

[0256] Any ROCK inhibitor that can induce PDX1-positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (for example, alone or in combination with at least one growth factor from the FGF family, at least one SHH pathway inhibitor, RA signaling pathway activator, and at least one growth factor from the TGF-β superfamily) can be used.In some examples, the ROCK inhibitor includes Thiazovivin, Y-27632, Fasudil / HA1077, or 14-1152. In some examples, the methods involve administering to the patient PDX1-positive pancreatic progenitor cells at a concentration of, for example, about 0.2 μM, about 0.5 μM, about 0.75 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 7.5 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, or about 20 μM. The method includes contacting the subject with a ROCK inhibitor (e.g., Y-27632 or Thiazovivin) at a concentration of about 1 μM, about 19 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 35 μM, about 40 μM, about 50 μM, or about 100 μM.

[0238]

[0257] Any activator from the TGF-β superfamily that can induce PDX1-positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (for example, alone or in combination with at least one growth factor from the FGF family, at least one SHH pathway inhibitor, RA signaling pathway activator, and a ROCK inhibitor) can be used. In some examples, the activator from the TGF-β superfamily includes activin A or GDF8. In some examples, the method comprises administering to a patient a culture of PDX1-positive pancreatic progenitor cells at a concentration of, for example, about 0.1 ng / mL, about 0.2 ng / mL, about 0.3 ng / mL, about 0.4 ng / mL, about 0.5 ng / mL, about 0.6 ng / mL, about 0.7 ng / mL, about 0.8 ng / mL, about 1 ng / mL, about 1.2 ng / mL, about 1.4 ng / mL, about 1.6 ng / mL, about 1.8 ng / mL, about 2 ng / mL, about 2.2 ng / mL, about 2.4 ng / mL, about 2.6 ng / mL, about 2.8 ng / mL, about 3 ng / mL, about 3.2 ng / mL, about 3.4 ng / mL, about 3.6 ng / mL, about 3.8 ng / mL, about 4 ng / mL, or about 5 ng / mL. The method includes contacting the PDX1-positive pancreatic progenitor cells with a growth factor from the TGF-β superfamily (e.g., activin A) at a concentration of about 4.2ng / mL, about 4.4ng / mL, about 4.6ng / mL, about 4.8ng / mL, about 5ng / mL, about 5.2ng / mL, about 5.4ng / mL, about 5.6ng / mL, about 5.8ng / mL, about 6ng / mL, about 6.2ng / mL, about 6.4ng / mL, about 6.6ng / mL, about 6.8ng / mL, about 7ng / mL, about 8ng / mL, about 9ng / mL, about 10ng / mL, about 20ng / mL, about 30ng / mL, or about 50ng / mL. In some examples, the method includes contacting the PDX1-positive pancreatic progenitor cells with a growth factor from the TGF-β superfamily (e.g., activin A) at a concentration of, for example, about 5ng / mL.

[0239]

[0258] In some cases, PDX1-positive, NKX6.1-positive pancreatic progenitor cells The pancreatic progenitor cells are obtained by contacting them with KGF, Sant1, and RA for 5 or 6 days under conditions that promote cell clustering. In some examples, PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive pancreatic progenitor cells with KGF, Sant1, RA, thiazovivin, and activin A for 5 or 6 days under conditions that promote cell clustering. In some examples, PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive pancreatic progenitor cells with KGF for 5 days under conditions that promote cell clustering. In some embodiments, PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by: a) contacting PDX1-positive pancreatic progenitor cells with KGF, Sant1, RA, thiazovivin, and activin A for 3, 4, or 5 days, followed by b) contacting the cells of a) with PDBU, XXI, KGF, Sant1, RA, thiazovivin, and activin A for 1, 2, or 3 days.

[0240] Insulin-positive endocrine cells

[0259] Embodiments of the present disclosure include insulin-positive endocrine cells (e.g., NKX6.1-positive, ISL1-positive, or β-like cells). As used herein, insulin-positive endocrine cells can be derived from any source or produced according to any suitable protocol. In some embodiments, NKX6.1-positive pancreatic progenitor cells are insulin-positive endocrine cells (e.g., N In some embodiments, the cells are differentiated into KX6.1-positive, ISL1-positive, or β-like cells. Insulin-positive endocrine cells are further differentiated, for example, by induction or maturation into SC-β cells.

[0241]

[0260] In some embodiments, a method for generating insulin-positive endocrine cells from NKX6.1-positive pancreatic progenitor cells includes contacting a population of cells comprising NKX6.1-positive pancreatic progenitor cells with a) a TGF-β signaling pathway inhibitor and b) a thyroid hormone signaling pathway activator (e.g., under conditions that promote cell clustering) to induce differentiation of at least one NKX6.1-positive pancreatic progenitor cell in the population into an insulin-positive endocrine cell, wherein the insulin-positive endocrine cell expresses insulin. In some examples, the insulin-positive endocrine cell expresses PDX1, NKX6.1, ISL1, NKX2.2, Mafb, glis3, Sur1, Kir6.2, Znt8, SLC2A1, SLC2A3, and / or insulin.

[0242]

[0261] Any TGF-β signaling pathway inhibitor that can induce the differentiation of NKX6.1-positive pancreatic progenitor cells into insulin-positive endocrine cells (for example, alone or in combination with other β cell differentiation factors, such as thyroid hormone signaling pathway activators) can be used. In some cases, the TGF-β signaling pathway comprises TGF-β type I receptor kinase signaling. In some cases, the TGF-β signaling pathway inhibitor comprises Alk5 inhibitor II.

[0243]

[0262] Any thyroid hormone signaling pathway activator that can induce differentiation of NKX6.1-positive pancreatic progenitor cells into insulin-positive endocrine cells (e.g., alone or in combination with other β cell differentiation factors, such as TGF-β signaling pathway inhibitors) can be used. In some examples, the thyroid hormone signaling pathway activator comprises triiodothyronine (T3). In some examples, the thyroid hormone signaling pathway activator comprises GC-1.

[0244]

[0263] In some examples, the method includes contacting the cell population (e.g., NKX6.1-positive pancreatic progenitor cells) with at least one additional factor. In some examples, the method includes contacting the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with: i) an SHH pathway inhibitor; ii) an RA signaling pathway activator; iii) a γ-secretase inhibitor; iv) at least one growth factor from the epidermal growth factor (EGF) family; v) a protein kinase inhibitor. , vi) a TGF-β signaling pathway inhibitor, or vii) a thyroid hormone signaling pathway activator. In some embodiments, the method includes contacting the cell population (e.g., NKX6.1-positive pancreatic progenitor cells) with at least one additional factor. In some examples, the method includes contacting the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with at least one of i) an SHH pathway inhibitor, ii) an RA signaling pathway activator, iii) a gamma-secretase inhibitor, iv) at least one growth factor from the epidermal growth factor (EGF) family, v) a protein kinase inhibitor, vi) a TGF-β signaling pathway inhibitor, vii) a thyroid hormone signaling pathway activator, or vii) a PKC activator.

[0245]

[0264] In some examples, the method includes contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells with at least one of: i) an SHH pathway inhibitor, ii) an RA signaling pathway activator, iii) a gamma-secretase inhibitor, iv) at least one growth factor from the epidermal growth factor (EGF) family, v) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, vi) a TGF-β signaling pathway inhibitor, vii) a thyroid hormone signaling pathway activator, vii) a protein kinase inhibitor, or ix) a ROCK inhibitor.

[0246]

[0265] In some examples, the method includes contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells with at least one of: i) an SHH pathway inhibitor, ii) an RA signaling pathway activator, iii) a gamma-secretase inhibitor, iv) at least one growth factor from the epidermal growth factor (EGF) family, v) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, vi) a TGF-β signaling pathway inhibitor, vii) a thyroid hormone signaling pathway activator, vii) an epigenetic modifying compound, ix) a protein kinase inhibitor, or x) a ROCK inhibitor.

[0247]

[0266] In some embodiments, in the method for generating insulin-positive endocrine cells from PDX1-positive, NKX6.1-positive pancreatic progenitor cells, some of the differentiation factors are present only for the first 1, 2, 3, 4, or 5 days during the differentiation step. In some examples, some of the differentiation factors, such as an SHH pathway inhibitor, an RA signaling pathway activator, a PKC activator, and at least one growth factor from the EGF family, are removed from the culture medium after the first 1, 2, or 3 days of incubation.

[0248]

[0267] Any gamma-secretase inhibitor that can induce the differentiation of NKX6.1-positive pancreatic progenitor cells in the population into insulin-positive endocrine cells (for example, alone or in combination with any TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway activator) can be used. In some examples, the gamma-secretase inhibitor comprises XXI. In some examples, the gamma-secretase inhibitor comprises DAPT. In some examples, the method comprises administering to the patient NKX6.1-positive pancreatic progenitor cells at a concentration of, for example, about 0.01 μM, about 0.02 μM, about 0.05 μM, about 0.075 μM, about 0.1 μM, about 0.2 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1 μM, about 1.1 μM, about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM, about 1.8 μM, about 1.9 μM, about 2 μM, about 2.1 μM, about 2.2 μM, about 2.3 μM, about 2.4 μM, about 2.5 μM, about 2 ... The method includes contacting the antibody with a gamma secretase inhibitor (e.g., XXI) at a concentration of about 0.6 μM, about 2.7 μM, about 2.8 μM, about 2.9 μM, about 3 μM, about 3.2 μM, about 3.4 μM, about 3.6 μM, about 3.8 μM, about 4 μM, about 4.2 μM, about 4.4 μM, about 4.6 μM, about 4.8 μM, about 5 μM, about 5.2 μM, about 5.4 μM, about 5.6 μM, about 5.8 μM, about 6 μM, about 6.2 μM, about 6.4 μM, about 6.6 μM, about 6.8 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 20 μM, about 30 μM, or about 50 μM.

[0249]

[0268] Any growth factor from the EGF family that can induce differentiation of NKX6.1-positive pancreatic progenitor cells in the population into insulin-positive endocrine cells (e.g., alone or in combination with any TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway activator) can be used. In some examples, at least one growth factor from the EGF family includes betacellulin. In some examples, at least one growth factor from the EGF family includes EGF. In some examples, the method includes contacting the NKX6.1-positive pancreatic progenitor cells with a growth factor from the EGF family (e.g., betacellulin) at a concentration of, for example, about 1 ng / mL, about 2 ng / mL, about 4 ng / mL, about 6 ng / mL, about 8 ng / mL, about 10 ng / mL, about 12 ng / mL, about 14 ng / mL, about 16 ng / mL, about 18 ng / mL, about 20 ng / mL, about 22 ng / mL, about 24 ng / mL, about 26 ng / mL, about 28 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 75 ng / mL, about 80 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 150 ng / mL, about 200 ng / mL, about 250 ng / mL, or about 300 ng / mL.

[0250]

[0269] Any RA signaling pathway activator that can induce the differentiation of NKX6.1-positive pancreatic progenitor cells into insulin-positive endocrine cells (for example, alone or in combination with any TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway activator) can be used. In some examples, the RA signaling pathway activator comprises RA. In some examples, the method comprises administering to the patient NKX6.1-positive pancreatic progenitor cells at a concentration of, for example, about 0.02 μM, about 0.1 μM, about 0.2 μM, about 0.25 μM, about 0.3 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about 0.55 μM, about 0.6 μM, about 0.65 μM, about 0.7 μM, about 0.75 μM, about 0.8 μM, about 0.85 μM, about 0.9 μM, about 1 μM, about 1.1 μM, about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM, about 1.8 μM, about 1.9 μM, about 2 μM, about 2.1 μM, about 2.2 μM, about 2.3 μM, about 2.4 μM, about 2 ... The method includes contacting the RA signaling pathway activator (e.g., retinoic acid) at a concentration of about 0.5 μM, about 2.6 μM, about 2.7 μM, about 2.8 μM, about 3 μM, about 3.2 μM, about 3.4 μM, about 3.6 μM, about 3.8 μM, about 4 μM, about 4.2 μM, about 4.4 μM, about 4.6 μM, about 4.8 μM, about 5 μM, about 5.5 μM, about 6 μM, about 6.5 μM, about 7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM, about 10 μM, about 12 μM, about 14 μM, about 15 μM, about 16 μM, about 18 μM, about 20 μM, about 50 μM, or about 100 μM.

[0251]

[0270] Any SHH pathway inhibitor that can induce differentiation of NKX6.1-positive pancreatic progenitor cells into insulin-positive endocrine cells (e.g., alone or in combination with any TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway activator) can be used in the methods provided herein. In some examples, the SHH pathway inhibitor includes Sant1. In some examples, the method involves administering to the patient NKX6.1-positive pancreatic progenitor cells at a concentration of, for example, about 0.001 μM, about 0.002 μM, about 0.005 μM, about 0.01 μM, about 0.02 μM, about 0.03 μM, about 0.05 μM, about 0.08 μM, about 0.1 μM, about 0.12 μM, about 0.13 μM, about 0.14 μM, about 0.15 μM, about 0.16 μM, about 0.17 μM, about 0.18 μM, about 0.19 μM, about 0.2 μM, about 0.21 μM, about 0.22 μM, about The method includes contacting the cells with an SHH pathway inhibitor (e.g., Sant1) at a concentration of 0.23 μM, about 0.24 μM, about 0.25 μM, about 0.26 μM, about 0.27 μM, about 0.28 μM, about 0.29 μM, about 0.3 μM, about 0.31 μM, about 0.32 μM, about 0.33 μM, about 0.34 μM, about 0.35 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about 0.6 μM, about 0.8 μM, about 1 μM, about 2 μM, or about 5 μM.

[0252]

[0271] Differentiation of NKX6.1-positive pancreatic progenitor cells (e.g., alone or in the presence of TGF-β signaling) Any BMP signaling pathway inhibitor that can induce differentiation into insulin-positive endocrine cells (in combination with either a BMP signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator) can be used. In some examples, the BMP signaling pathway inhibitor includes LDN193189 or DMH-1. In some examples, the method includes contacting the NKX6.1-positive pancreatic progenitor cells with a BMP signaling pathway inhibitor (e.g., LDN1931189) at a concentration of, for example, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 110 nM, about 120 nM, about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about 200 nM, about 210 nM, about 220 nM, about 230 nM, about 240 nM, about 250 nM, about 280 nM, about 300 nM, about 400 nM, about 500 nM, or about 1 μM.

[0253]

[0272] Any ROCK inhibitor can be used that can induce the differentiation of NKX6.1 positive pancreatic progenitor cells in the population into insulin-positive endocrine cells (for example, alone or in combination with any TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway activator).In some examples, the ROCK inhibitor comprises Thiazovivin, Y-27632, Fasudil / HA1077, or H-1152.In some examples, the ROCK inhibitor comprises Y-27632.In some examples, the ROCK inhibitor comprises Thiazovivin. In some examples, the method comprises administering to the patient a dose of PDX1-positive, NKX6.1-positive pancreatic progenitor cells at a concentration of, for example, about 0.2 μM, about 0.5 μM, about 0.75 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 7.5 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, or about 18 μM. The method includes contacting the subject with a ROCK inhibitor (e.g., Y-27632 or Thiazovivin) at a concentration of about 1 μM, about 18 μM, about 19 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 35 μM, about 40 μM, about 50 μM, or about 100 μM.

[0254]

[0273] Any epigenetic modification compound can be used that can induce the differentiation of NKX6.1 positive pancreatic progenitor cells in the population into insulin positive endocrine cells (for example, alone or in combination with any TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway activator).In some examples, the epigenetic modification compound comprises a histone methyltransferase inhibitor or an HDAC inhibitor.In some examples, the epigenetic modification compound comprises a histone methyltransferase inhibitor, such as DZNep.In some examples, the epigenetic modification compound comprises an HDAC inhibitor, such as KD5170. In some examples, the method includes contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells with an epigenetic modifying compound (e.g., DZNep or KD5170) at a concentration of, for example, about 0.01 μM, about 0.025 μM, about 0.05 μM, about 0.075 μM, about 0.1 μM, about 0.15 μM, about 0.2 μM, about 0.5 μM, about 0.75 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 7.5 μM, about 8 μM, about 9 μM, about 10 μM, about 15 μM, about 20 μM, about 25 μM, about 30 μM, about 35 μM, about 40 μM, about 50 μM, or about 100 μM.

[0255]

[0274] In some examples, the population of cells is optionally contacted with a protein kinase inhibitor. In some examples, the population of cells is not contacted with a protein kinase inhibitor. In some examples, the population of cells is contacted with a protein kinase inhibitor. Any protein kinase inhibitor capable of inducing differentiation of NKX6.1-positive pancreatic progenitor cells in the population into insulin-positive endocrine cells (e.g., alone or in combination with either a TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator) can be used. In some instances, the protein kinase inhibitor comprises staurosporine.

[0256]

[0275] In some examples, the method includes contacting a population of cells (e.g., NKX6.1-positive pancreatic progenitor cells) with XXI, Alk5i, T3 or GC-1, RA, Sant1, and betacellulin for 7 days to induce differentiation of at least one NKX6.1-positive pancreatic progenitor cell in the population into an insulin-positive endocrine cell, wherein the insulin-positive endocrine cell expresses insulin. In some examples, the method includes contacting a population of cells (e.g., NKX6.1-positive pancreatic progenitor cells) with XXI, Alk5i, T3 or GC-1, RA, Sant1, betacellulin, and LDN193189 for 7 days to induce differentiation of at least one NKX6.1-positive pancreatic progenitor cell in the population into an insulin-positive endocrine cell, wherein the insulin-positive endocrine cell expresses insulin. In some embodiments, one or more differentiation factors are added as part of Stage 5, e.g., only on the first 1, 2, 3, 4, 5, or 6 days of Stage 5, or only on the last 1, 2, 3, 4, 5, or 6 days of Stage 5. In one example, cells are contacted with an SHH signaling pathway inhibitor only on the first 2, 3, 4, or 5 days of Stage 5, after which the SHH signaling pathway inhibitor is removed from the medium. In another example, cells are contacted with a BMP signaling pathway inhibitor only on the first 1, 2, or 3 days of Stage 5, after which the BMP signaling pathway inhibitor is removed from the medium.

[0257]

[0276] In some examples, the method includes culturing a population of cells (e.g., NKX6.1-positive pancreatic progenitor cells) in BE5 medium to induce differentiation of at least one NKX6.1-positive pancreatic progenitor cell in the population into an insulin-positive endocrine cell, wherein the insulin-positive endocrine cell expresses insulin.

[0258]

[0277] An embodiment of the present disclosure involves treating a cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a PKC activator, which can result in an increase in the percentage of pancreatic alpha cells, an increase in the percentage of pancreatic delta cells, an increase in the percentage of pancreatic beta cells, a decrease in the percentage of EC cells, or any combination thereof, in a cell population of pancreatic endocrine cells produced according to the methods disclosed herein.

[0259]

[0278] In some examples, the method includes the steps of: contacting a cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a first composition comprising a PKC activator, a ROCK inhibitor, a growth factor from the TGF-β superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor for 1 to 2 days, thereby obtaining a first transformed cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells; and contacting the first transformed cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a second composition comprising a PKC activator, a TGF-β signaling pathway inhibitor, a TH signaling pathway activator, and an epigenetic modifying compound for 1 to 2 days, thereby obtaining a second transformed cell population comprising NKX6.1-positive, ISL1-positive endocrine cells.

[0260]

[0279] In some examples, the method includes the steps of: (1) contacting PDX1-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) a low concentration of an RA signaling pathway activator, iv) a ROCK inhibitor, and v) at least one growth factor from the TGF-β superfamily for about 2 to 6 days to induce differentiation of at least a portion of the PDX1-positive pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells; and (2) after (1), contacting the population containing the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, iii) a low concentration of an RA signaling pathway activator, iv) a ROCK inhibitor, and v) at least one growth factor from the TGF-β superfamily for about 2 to 6 days. and vi) contacting the cells with at least one growth factor, and vi) a PKC activator for 1 to 2 days, thereby producing a first transformed cell population containing PDX1-positive, NKX6.1-positive pancreatic progenitor cells.

[0261]

[0280] In some examples, the method further includes (3) contacting a first transformed cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells with i) an SHH pathway inhibitor, ii) an RA signaling pathway activator, iii) a γ-secretase inhibitor, iv) at least one growth factor from the epidermal growth factor (EGF) family, v) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, vi) a TGF-β signaling pathway inhibitor, vii) a thyroid hormone signaling pathway activator, viii) an epigenetic modifying compound, ix) a protein kinase inhibitor, x) a ROCK inhibitor, and xi) a PKC activator for 1 to 2 days, thereby resulting in a second transformed cell population. and (4) contacting the second transformed cell population with i) an SHH pathway inhibitor, ii) an RA signaling pathway activator, iii) a gamma-secretase inhibitor, iv) at least one growth factor from the epidermal growth factor (EGF) family, v) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, vi) a TGF-β signaling pathway inhibitor, vii) a thyroid hormone signaling pathway activator, viii) an epigenetic modifying compound, ix) a protein kinase inhibitor, and x) a ROCK inhibitor, thereby producing a cell population comprising NKX6.1-positive, ISL1-positive endocrine cells.

[0262] Pancreatic beta cells

[0281] Aspects of the present disclosure include producing pancreatic β cells (e.g., non-native pancreatic β cells), which in some instances are similar in morphology and function to endogenous mature β cells, but are nevertheless distinct from native β cells.

[0263]

[0282] In some examples, insulin-positive pancreatic endocrine cells produced using the methods provided herein can form cell clusters, either alone or together with other types of cells, such as their precursors, for example, stem cells, definitive endoderm cells, primitive gut cells, PDX1-positive pancreatic progenitor cells, or NKX6.1-positive pancreatic progenitor cells.

[0264]

[0283] In some embodiments, any of the cells or cell populations disclosed herein are in cell clusters. In some aspects, cell clusters similar to the functions and characteristics of endogenous pancreatic islets are provided herein. Such cell clusters can mimic the function of endogenous pancreatic islets in regulating metabolism, for example, glucose metabolism, in a subject. Thus, cell clusters can be transplanted into a subject to treat diseases caused by insufficient pancreatic islet function, for example, diabetes. The terms "cluster" and "aggregate" can be used interchangeably and refer to a group of cells with close cell-to-cell contact, and in some cases, the cells within the cluster can adhere to each other. A cell cluster comprises multiple cells. In some embodiments, the cell clusters comprise at least 10, at least 50, at least 200, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 20,000, at least 30,000, or at least 50,000 cells. In some embodiments, the cell clusters comprise between 10 and 10,000 cells, 50 and 10,000 cells, 100 and 10,000 cells, 100 and 10,000 cells, 1,000 and 10,000 cells, 500 and 10,000 cells, 500 and 5,000 cells, 500 and 2,500 cells, 500 and 2,000 cells, 1,000 and 100,000 cells, 1,000 and 50,000 cells, 1,000 and 40,000 cells, 1,000 and 20,000 cells, 1,000 and 10,000 cells, 1,000 and 1,000 cells, In some embodiments, the cell clusters comprise 5,000 cells, and 1,000 to 3,000 cells. In some embodiments, the cell clusters comprise at least 500 cells. In some embodiments, the cell clusters comprise at least 1,000 cells. In some embodiments, the cell clusters comprise at least 2,000 cells. In some embodiments, the cell clusters comprise at least 5,000 cells. In some embodiments, the cell clusters comprise no more than 100,000 cells, no more than 90,000 cells, no more than 80,000 cells, no more than 70,000 cells, no more than 60,000 cells, no more than 50,000 cells, no more than 40,000 cells, no more than 30,000 cells, no more than 20,000 cells, no more than 10,000 cells, no more than 7,000 cells, no more than 5,000 cells, no more than 3,000 cells, no more than 2,000 cells, or no more than 1,000 cells.

[0265]

[0284] The cell clusters may be similar in size to endogenous pancreatic islets. For example, the cell clusters may have a diameter similar to that of endogenous pancreatic islets. The diameter of a cell cluster may refer to the maximum linear distance between two points on the surface of the cell cluster. In some examples, the diameter of a cell cluster is at most 300 μm, 200 μm, 150 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, or 40 μm. The diameter of a cell cluster may be about 75 μm to about 250 μm. The diameter of a cell cluster may be at most 100 μm.

[0266]

[0285] In some embodiments, the cell clusters are about 100 to about 250 microns in diameter (e.g., about 125, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 200, about 210, about 215, about 220, or about 225 microns in diameter). For example, in some embodiments, the cell clusters are about 125 to about 225, about 130 to about 160, about 170 to about 225, about 140 to about 200, about 140 to about 170, about 160 to about 220, about 170 to about 215, or about 170 to about 200 microns in diameter.

[0267]

[0286] In some embodiments, the compositions, cells, or cell populations of the present disclosure comprise cells having a genome disruption in at least one gene sequence. In some embodiments, the genome disruption reduces or eliminates the expression of the protein encoded by the gene sequence. In some embodiments, the at least one gene sequence encodes an MHC-class I gene. In some embodiments, the MHC-class I gene encodes beta-2 microglobulin, HLA-A, HLA-B, or HLA-C. In some embodiments, the at least one gene sequence encodes CIITA. For example, in some embodiments, the compositions or cell populations have a genome disruption in the beta-2 microglobulin gene. Additional examples of genes and their genome disruption are described in more detail in International Application Publication No. WO2020 / 033879, the relevant contents of which are incorporated herein by reference. In some embodiments, the genome disruption is induced using gene editing technology (e.g., CRISPR Cas).

[0268]

[0287] In some embodiments, the compositions or cell populations of the present disclosure comprise NKX6.1-positive, ISL-positive cells that express lower levels of MAFA than NKX6.1-positive, ISL-positive cells from the pancreas of a healthy adult subject. In some embodiments, the compositions or cell populations comprise NKX6.1-positive, ISL-positive cells that express higher levels of MAFB than NKX6.1-positive, ISL-positive cells from the pancreas of a healthy adult subject. In some embodiments, the compositions or cell populations comprise NKX6.1-positive, ISL-positive cells that express higher levels of SIX2, HOPX, IAPP, and / or UCN3 than NKX6.1-positive, ISL-positive cells from the pancreas of a healthy adult subject.

[0269]

[0288] In some embodiments, the compositions or cell populations of the present disclosure comprise NKX6.1-positive, ISL-positive cells that do not express MAFA. In some embodiments, the compositions or cell populations comprise NKX6.1-positive, ISL-positive cells that express MAFB.

[0270]

[0289] In some examples, a cell population comprising insulin-positive endocrine cells can be directly induced to mature into SC-β cells without the addition of any exogenous differentiation factors (e.g., an inhibitor of the TGF-β signaling pathway, a thyroid hormone signaling pathway activator, a PKC activator, a growth factor from the TGF-β superfamily, the FGF family, or the EGF family, an SHH signaling pathway inhibitor, a γ-secretase inhibitor, a ROCK inhibitor, or a BMP signaling pathway inhibitor). In some embodiments, the methods provided herein comprise contacting a cell population comprising NKX6.1-positive, ISL1-positive endocrine cells with serum albumin protein, a TGF-β signaling pathway inhibitor, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and / or an epigenetic modifying compound. In some embodiments, the methods provided herein comprise contacting a cell population comprising NKX6.1-positive, ISL1-positive endocrine cells with human serum albumin protein. In some embodiments, the methods provided herein comprise contacting a cell population comprising NKX6.1-positive, ISL1-positive endocrine cells with a PKC activator.

[0271]

[0290] In some examples, a cell population containing insulin-positive endocrine cells can be induced to mature into SC-β cells by contacting insulin-positive endocrine cells with a differentiation factor. The differentiation factor can include at least one inhibitor of the TGF-β signaling pathway and an activator of the thyroid hormone signaling pathway described herein. In some examples, SC-β cells can be obtained by contacting a cell population containing insulin-positive endocrine cells with Alk5i and T3 or GC-1.

[0272]

[0291] In some examples, the methods provided herein include contacting a cell population comprising NKX6.1-positive, ISL1-positive endocrine cells with (i) a growth factor from the FGF family, (ii) a TGF-β signaling pathway inhibitor, (iii) a thyroid hormone signaling pathway activator, (iv) an epigenetic modifying compound, (v) a protein kinase inhibitor, (vi) a ROCK inhibitor, (vii) a BMP signaling pathway inhibitor, and (viii) a lipase inhibitor for about 1, 2, or 5 days. In some examples, the contacting is for about 3 days.

[0273]

[0292] In some examples, insulin-positive endocrine cells can be matured in NS-GFs medium, MCDB131 medium, DMEM medium, or CMRL medium. In some examples, insulin-positive endocrine cells can be matured in CMRL medium supplemented with 10% FBS. In some examples, insulin-positive endocrine cells can be matured in DMEM / F12 medium supplemented with 1% HSA. In other examples, SC-β cells can be obtained by culturing a population of cells containing insulin-positive endocrine cells in MCDB131 medium, which may be supplemented with 2% BSA. In some examples, MCDB131 medium supplemented with 2% BSA for maturing insulin-positive endocrine cells into SC-β cells may not contain the small molecule factors described herein. In some examples, MCDB131 medium supplemented with 2% BSA for maturing insulin-positive endocrine cells into SC-β cells may not contain serum (e.g., FBS). In other examples, SC-β cells can be obtained by culturing a cell population containing insulin-positive endocrine cells in MCDB131 medium, which can be supplemented with 0.05% HSA and vitamin C. In some examples, SC-β cells can be obtained by culturing a cell population containing insulin-positive endocrine cells in MCDB131 medium, which can be supplemented with 0.05% HSA, ITS-X, vitamin C, and glutamine (Gln, e.g., 4 mM). In some examples, the type of medium can be changed during S6. For example, S6 cells are cultured in MCDB131 medium, which can be supplemented with 0.05% HSA and vitamin C, for the first 2 to 4 days, and then cultured in DMEM / F12 medium supplemented with 1% HSA. In some examples, additional factors are introduced into the medium. For example, S6 cells can be cultured in MCDB131 medium, which can be supplemented with 0.05% HSA, ITS-X, vitamin C, and glutamine (Gln, e.g., 4 mM), for 10–12 days, during which ZnSO4 is introduced from day 4 of S6.

[0274]

[0293] In some aspects, the disclosure provides a method of producing SC-β cells from pluripotent cells, the method comprising: a) differentiating pluripotent stem cells in a population into definitive endoderm cells by contacting the pluripotent stem cells with at least one factor from the TGFβ superfamily and a WNT signaling pathway activator for three days; b) differentiating at least a portion of the definitive endoderm cells into primitive gut cells by a process of contacting the definitive endoderm cells with at least one factor from the FGF family for three days; c) differentiating at least a portion of the primitive gut cells into PDX1-positive pancreatic progenitor cells by a process of contacting the PDX1-positive pancreatic progenitor cells with i) a retinoic acid signaling pathway activator, ii) at least one factor from the FGF family, iii) an SHH pathway inhibitor, iv) a BMP signaling pathway inhibitor (e.g., DMH-1 or LDN193189), v) a PKC activator, and vi) a ROCK inhibitor, under conditions that promote cell clustering. and e) differentiating at least a portion of the PDX1-positive pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells by a process of contacting the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with i) a TGF-β signaling pathway inhibitor, ii) at least one SHH pathway inhibitor, and optionally iii) an RA signaling pathway activator, and optionally iv) a ROCK inhibitor, and v) at least one factor from the TGF-β superfamily for 5 days. and f) differentiating at least a portion of the PDX1-positive, NKX6.1-positive pancreatic progenitor cells into PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells by a process of contacting the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a PDX1-positive, NKX6.1-positive, insulin-positive endocrine cell line, the PDX1-positive, NKX6.1-positive, pancreatic progenitor cells with a PDX1-positive, NKX6.1-positive, insulin-positive endocrine cell line, the NKX6.1-positive, insulin-positive, ...PDX1-positive, insulin-positive endocrine cells were cultured in medium without exogenous differentiation factors (e.g., NS-GFs medium, MCDB medium supplemented with BSA, MCDB131 medium, or DMEM / F12 medium) for 7–14 days to differentiate at least a portion of the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells. and differentiating at least a portion of the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells by a process of inducing in vitro maturation, wherein the SC-β cells exhibit a GSIS response in vitro and / or in vivo. In some examples, the GSIS response resembles the GSIS response of endogenous mature β cells.

[0275]

[0294] In some aspects, the disclosure provides a method for producing SC-β cells from pluripotent cells, the method comprising: a) differentiating pluripotent stem cells in a population into definitive endoderm cells by contacting the pluripotent stem cells with at least one factor from the TGFβ superfamily and a WNT signaling pathway activator for three days; b) differentiating at least a portion of the definitive endoderm cells into primitive gut cells by a process of contacting the definitive endoderm cells with at least one factor from the FGF family for three days; and c) differentiating the primitive gut cells into definitive endoderm cells by contacting the definitive endoderm cells with i) a retinoic acid signaling pathway activator, ii) a WNT signaling pathway activator, and iii) a WNT signaling pathway activator. d) differentiating at least a portion of the primitive gut cells into PDX1-positive pancreatic progenitor cells by a process of contacting the PDX1-positive pancreatic progenitor cells with i) at least one factor from the FGF family, ii) at least one SHH pathway inhibitor, iv) a BMP signaling pathway inhibitor, v) a PKC activator, vi) a ROCK inhibitor, and vii) a growth factor from the TGFβ superfamily for two days, under conditions that promote cell clustering; e) differentiating at least a portion of the PDX1-positive pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells by a process of contacting the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with i) a TGF-β signaling pathway inhibitor, ii) a TH signaling pathway activator, iii) at least one SHH pathway inhibitor, iv) an RA signaling pathway activator, v) a γ-secretase inhibitor, and optionally vi) at least one factor from the epidermal growth factor (EGF) family for 5 days; and optionally vii) a BMP signaling pathway inhibitor, for 5 to 7 days; and f) differentiating at least a portion of the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells into PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells by a process of culturing the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells in a medium that does not contain exogenous differentiation factors (e.g., NS-GFs medium, MCDB medium supplemented with BSA, MCDB131 medium, or DMEM / F12 medium) for 7 to 14 days to induce in vitro maturation of at least a portion of the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells, wherein the SC-β cells exhibit a GSIS response in vitro and / or in vivo. In some instances, the GSIS response resembles that of endogenous mature beta cells.

[0276]

[0295] In some aspects, the disclosure provides a method for producing SC-β cells from pluripotent cells, the method comprising: a) differentiating pluripotent stem cells in a population into definitive endoderm cells by contacting the pluripotent stem cells with at least one factor from the TGFβ superfamily and a WNT signaling pathway activator for three days; b) differentiating at least a portion of the definitive endoderm cells into primitive gut cells by a process of contacting the definitive endoderm cells with at least one factor from the FGF family for three days; and c) differentiating the primitive gut cells into definitive endoderm cells by: i) treating the definitive endoderm cells with retinoic acid; ii) a signal transduction pathway activator, ii) at least one factor from the FGF family, iii) an SHH pathway inhibitor, iv) a PKC activator, and v) a ROCK inhibitor, and d) differentiating at least a portion of the primitive gut cells into PDX1-positive pancreatic progenitor cells by a process of contacting the PDX1-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) a RA signal transduction pathway activator, and optionally i) a ROCK inhibitor, under conditions that promote cell clustering. and e) differentiating at least a portion of the PDX1-positive pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells by a process of contacting the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with: i) a TGF-β signaling pathway inhibitor; ii) a TH signaling pathway activator; iii) at least one SHH pathway inhibitor; iv) an RA signaling pathway activator; v) a γ-secretase inhibitor; and optionally vi) epithelial cells. f) differentiating at least a portion of the PDX1-positive, NKX6.1-positive pancreatic progenitor cells into PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells by contacting them with at least one growth factor from the epidermal growth factor (EGF) family for 5 to 7 days; and f) culturing the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells in a medium that does not contain exogenous differentiation factors (e.g., NS-GFs medium, MCDB medium supplemented with BSA, MCDB131 medium, or DMEM / F12 medium) for 7 to 14 days to differentiate them into PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells.and differentiating at least a portion of the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells by a process of inducing in vitro maturation of at least a portion of the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells, wherein the SC-β cells are differentiated in vitro and / or in vivo. Methods are provided for producing a GSIS response in vivo, in some instances, where the GSIS response resembles the GSIS response of endogenous mature beta cells.

[0277]

[0296] In some aspects, the present disclosure provides a method for producing SC-β cells from pluripotent cells, the method comprising: a) differentiating pluripotent stem cells in a population into definitive endoderm cells by contacting the pluripotent stem cells with at least one factor from the TGFβ superfamily and a WNT signaling pathway activator for three days; b) differentiating at least a portion of the definitive endoderm cells into primitive gut cells by a process of contacting the definitive endoderm cells with at least one factor from the FGF family for three days; and c) differentiating the primitive gut cells into: i) retinoblastoma cells; and (d) differentiating at least a portion of the primitive gut cells into PDX1-positive pancreatic progenitor cells by a process of contacting the PDX1-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) an activator of the insulin signaling pathway, iii) an inhibitor of the SHH pathway, iv) an inhibitor of the BMP signaling pathway (e.g., DMH-1 or LDN193189), v) a PKC activator, and vi) a ROCK inhibitor under conditions that promote cell clustering. e) differentiating at least a portion of the PDX1-positive pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells by a process of contacting the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with i) an SHH pathway inhibitor, ii) an RA signaling pathway activator, iii) a γ-secretase inhibitor, iv) an epithelial cell line, and optionally iii) an RA signaling pathway activator, and optionally iv) a ROCK inhibitor, and v) at least one factor from the TGFβ superfamily for 5 or 6 days; At least a portion of the PDX1-positive, NKX6.1-positive pancreatic progenitor cells are induced to be PDX1-positive and NKX6-positive by a process of contacting the cells with at least one growth factor from the epidermal growth factor (EGF) family, v) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, vi) a TGF-β signaling pathway inhibitor, vii) a thyroid hormone signaling pathway activator, viii) an epigenetic modifying compound (e.g., DZNep or KD5170), ix) a protein kinase inhibitor, and x) a ROCK inhibitor for 5 to 7 days.and f) differentiating at least a portion of the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells by a process of culturing the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells in a medium that does not contain exogenous differentiation factors (e.g., NS-GFs medium, MCDB medium supplemented with BSA, MCDB131 medium, or DMEM / F12 medium) for 7 to 14 days to induce in vitro maturation of at least a portion of the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells, where the SC-β cells exhibit a GSIS response in vitro and / or in vivo. In some examples, the GSIS response resembles the GSIS response of endogenous mature β cells.

[0278]

[0297] In some aspects, the disclosure provides a method for producing SC-β cells from pluripotent cells, the method comprising: a) differentiating pluripotent stem cells in a population into definitive endoderm cells by contacting the pluripotent stem cells with at least one factor from the TGFβ superfamily and a WNT signaling pathway activator for three days; b) differentiating at least a portion of the definitive endoderm cells into primitive endoderm cells by a process of contacting the definitive endoderm cells with at least one factor from the FGF family for three days; and c) differentiating the primitive endoderm cells with: i) a retinoic acid signaling pathway activator, ii) at least one factor from the FGF family, iii) an SHH pathway inhibitor, iv) a B and (d) differentiating at least a portion of the primitive gut cells into PDX1-positive pancreatic progenitor cells by a process of contacting the PDX1-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) an RA signaling pathway activator, and optionally iv) a ROCK inhibitor, and v) at least one factor from the TGFβ superfamily, under conditions that promote cell clustering, for 5 or 6 days. e) differentiating at least a portion of the PDX1-positive, NKX6.1-positive pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells; and f) contacting the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with i) a γ-secretase inhibitor, ii) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, iii) a TGF-β signaling pathway inhibitor, iv) a thyroid hormone signaling pathway activator, v) an epigenetic modifying compound (e.g., DZNep or KD5170), vi) a protein kinase inhibitor, and vii) a ROCK inhibitor for 5 to 7 days, and within the first 3 days of the 5 to 7 days, differentiating the PDX1-positive, NKX6.1-positive pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells later. f) differentiating at least a portion of the PDX1-positive, NKX6.1-positive pancreatic progenitor cells into PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells by a process of contacting the progenitor cells with an SHH pathway inhibitor, an RA signaling pathway, and at least one growth factor from the EGF family; and f) differentiating at least a portion of the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells by a process of culturing the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells in a medium that does not contain exogenous differentiation factors (e.g., NS-GFs medium, MCDB medium supplemented with BSA, MCDB131 medium, or DMEM / F12 medium) for 7 to 14 days to induce in vitro maturation of at least a portion of the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells, wherein the SC-β cells exhibit a GSIS response in vitro and / or in vivo. In some instances, the GSIS response resembles that of endogenous mature beta cells.

[0279]

[0298] In some aspects, the present disclosure provides a method for producing SC-β cells from pluripotent cells, the method comprising: a) differentiating pluripotent stem cells in a population into definitive endoderm cells by contacting the pluripotent stem cells with at least one factor from the TGFβ superfamily and a WNT signaling pathway activator for three days; b) differentiating at least a portion of the definitive endoderm cells into primitive gut cells by a process of contacting the definitive endoderm cells with at least one factor from the FGF family for three days; c) differentiating at least a portion of the primitive gut cells into PDX1-positive pancreatic progenitor cells by a process of contacting the primitive gut cells with i) a retinoic acid signaling pathway activator, ii) at least one factor from the FGF family, iii) an SHH pathway inhibitor, iv) a BMP signaling pathway inhibitor (e.g., DMH-1 or LDN193189), v) a PKC activator, and vi) a ROCK inhibitor; and d) differentiating at least a portion of the primitive gut cells into PDX1-positive pancreatic progenitor cells by a process of contacting the PDX1-positive pancreatic progenitor cells with at least one factor from the FGF family for three days. and e) differentiating at least a portion of the PDX1-positive pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells by a process of contacting the PDX1-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) an RA signaling pathway activator, and optionally iv) a ROCK inhibitor, and v) at least one factor from the TGFβ superfamily for 3 or 4 days under conditions that promote cell clustering, followed by contacting the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) an RA signaling pathway activator, and optionally iv) a ROCK inhibitor, v) at least one factor from the TGFβ superfamily, and vi) a PKC activator, and optionally vii) a gamma secretase inhibitor for 1 to 2 days.IgE-positive pancreatic progenitor cells were contacted with i) an SHH pathway inhibitor, ii) an RA signaling pathway activator, iii) a γ-secretase inhibitor, iv) at least one growth factor from the epidermal growth factor (EGF) family, v) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, vi) a TGF-β signaling pathway inhibitor, vii) a thyroid hormone signaling pathway activator, viiii) an epigenetic modifying compound (e.g., DZNep or KD5170), ix) a protein kinase inhibitor, x) a ROCK inhibitor, and xi) a PKC activator for 1-2 days, followed by i) an SHH pathway inhibitor, ii) an RA signaling pathway activator, iii) a γ-secretase inhibitor, iv) an epidermal growth factor (EGF) family growth factor, v) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, vi) a TGF-β signaling pathway inhibitor, vii) a thyroid hormone signaling pathway activator, viii .... f) differentiating at least a portion of the PDX1-positive, NKX6.1-positive pancreatic progenitor cells into PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells by a process of contacting the cells with at least one growth factor from the F) family, v) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, vi) a TGF-β signaling pathway inhibitor, vii) a thyroid hormone signaling pathway activator, viii) an epigenetic modifying compound (e.g., DZNep or KD5170), ix) a protein kinase inhibitor, and x) a ROCK inhibitor for 3 to 6 days; and f) differentiating at least a portion of the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells.

[0280]

[0299] The medium used to culture the cells separated from the first cell cluster can be xeno-free. Xeno-free medium for culturing cells and / or cell clusters of animal origin cannot contain any other animal-derived products. In some cases, xeno-free medium for culturing human cells and / or cell clusters cannot contain any non-human animal-derived products. For example, xeno-free medium for culturing human cells and / or cell clusters can contain human platelet lysate (PLT) instead of fetal bovine serum (FBS). For example, the medium can contain about 1% to about 20%, about 5% to about 15%, about 8% to about 12%, or about 9 to about 11% serum. In some cases, the medium can contain about 10% serum. In some cases, the medium cannot contain small molecules and / or FBS. For example, the medium can contain MCDB131 basal medium supplemented with 2% BSA. In some cases, the medium is serum-free. In some examples, the medium may contain exogenous small molecules or signaling pathway agonists or antagonists, such as growth factors from the fibroblast growth factor family (FGF, e.g., FGF2, FGF8B, FGF10, or FGF21), sonic hedgehog antagonists (e.g., Sant1, Sant2, Sant4, Sant4, Cur61414, forskolin, tomatidine, AY9944, triparanol, cyclopamine, or derivatives thereof), retinoic acid signaling inhibitors, or the like. Signaling agonists (e.g., retinoic acid, CD1530, AM580, TTHPB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, or CD2314), inhibitors of Rho-associated coiled-coil-containing protein kinase (ROCK) (e.g., thiazovivin, Y-27632, fasudil / HA1077, or 14-1152), activators of protein kinase C (PKC) (e.g., phorbol 12,13-dibutyrate (PDBU), TPB, phorbol 12-myristate 13-acetate, bryostatin 1, or their derivatives), antagonists of the TGFβ superfamily (e.g., Alk5 inhibitor II (CAS 446859-33-2), A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB-525334, SD-208, SB-505124, or derivatives thereof), inhibitors of bone morphogenetic protein (BMP) type 1 receptor (e.g., LDN193189 or derivatives thereof), thyroid hormone signaling pathway activators (e.g., T3, GC-1, or derivatives thereof), gamma secretase inhibitors (e.g., XXI, DAPT, or derivatives thereof), activators of the TGF-β signaling pathway (e.g., WNT3a or activin A) The reaggregation medium may not contain growth factors from the epidermal growth factor (EGF) family (e.g., betacellulin or EGF), a wide range of kinases (e.g., staurosporine or derivatives thereof), non-essential amino acids, vitamins or antioxidants (e.g., cyclopamine, vitamin D, vitamin C, vitamin A, or derivatives thereof), or other additives such as N-acetylcysteine, zinc sulfate, or heparin. In some cases, the reaggregation medium may not contain exogenous extracellular matrix molecules. In some cases, the reaggregation medium does not contain Matrigel™. In some cases, the reaggregation medium may contain other extracellular matrix molecules or materials, such as collagen, gelatin, poly-L-lysine, poly-D-lysine, vitrone, The present invention does not include, for example, cutin, laminin, fibronectin, PLO laminin, fibrin, thrombin, and RetroNectin, and mixtures thereof, or lysed cell membrane preparations.

[0281]

[0300] Those skilled in the art will recognize that the concentration of serum albumin supplemented in the medium can vary. For example, the medium (e.g., MCDB131) can contain about 0.01%, 0.05%, 0.1%, 1%, about 2%, about 3%, about 4%, about 5%, about 10%, or about 15% BSA. In other cases, the medium can contain about 0.01%, 0.05%, 0.1%, 1%, about 2%, about 3%, about 4%, about 5%, about 10%, or about 15% HSA. The medium used (e.g., MCDB131 medium) can contain components not found in traditional basal media, such as trace elements, putrescine, adenine, thymidine, and high levels of some amino acids and vitamins. These additives can allow the medium to be supplemented with very low levels of serum or defined components. The medium may be protein and / or growth factor-free and may be supplemented with EGF, hydrocortisone, and / or glutamine. The medium may contain one or more extracellular matrix molecules (e.g., extracellular proteins). Non-limiting exemplary extracellular matrix molecules used in the medium may include collagen, placental matrix, fibronectin, laminin, merosin, tenascin, heparin, heparin sulfate, chondroitin sulfate, dermatan sulfate, aggrecan, biglycan, thrombospondin, vitronectin, and decorin. In some cases, the medium includes laminin, such as LN-332. In some cases, the medium includes heparin.

[0282]

[0301] For example, medium can be periodically changed during culturing, so as to provide the optimal environment for cells in the medium.When culturing the cells separated from the first cell cluster for ...

Claims

1. (a) differentiating PDX1-positive, NKX6.1-negative pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells by contacting the PDX1-positive, NKX6.1-negative pancreatic progenitor cells with a ROCK inhibitor, a growth factor from the TGF-β superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor, thereby producing a cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells; (b) contacting the cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a first composition comprising a PKC activator, a gamma-secretase inhibitor, a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor for a first period of time; (c) after the first period of time, contacting the cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a second composition comprising a PKC activator, a gamma-secretase inhibitor, a TGF-β signaling pathway inhibitor, a growth factor from the EGF family, an RA signaling pathway activator, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound for a second period of time; A method comprising:

2. (a) contacting a cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a first composition comprising a PKC activator, a gamma-secretase inhibitor, and a factor selected from the group consisting of a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor for a first period of time; (b) after the first period of time, for a second period of time, contacting the cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a second composition comprising a factor selected from the group consisting of a PKC activator, a gamma-secretase inhibitor, and a TGF-β signaling pathway inhibitor, a growth factor from the EGF family, an RA signaling pathway activator, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound; A method comprising:

3. The method of claim 1 or 2, further comprising, after the second period of time, contacting the cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a third composition that differentiates at least a portion of the PDX1-positive, NKX6.1-positive pancreatic progenitor cells into NKX6.1-positive, ISL1-positive endocrine cells, thereby producing a cell population comprising NKX6.1-positive, ISL1-positive endocrine cells.

4. (a) contacting a cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a first composition comprising a PKC activator and a factor selected from the group consisting of a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor for a first period of time; (b) after the first period of time, for a second period of time, contacting the cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a second composition comprising a factor selected from the group consisting of a PKC activator and a TGF-β signaling pathway inhibitor, a growth factor from the EGF family, an RA signaling pathway activator, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound; (c) after said second period, said cells containing PDX1-positive, NKX6.1-positive pancreatic progenitor cells; and contacting a cell population comprising NKX6.1-positive, ISL1-positive pancreatic progenitor cells with a third composition that differentiates at least a portion of the PDX1-positive, NKX6.1-positive pancreatic progenitor cells into NKX6.1-positive, ISL1-positive endocrine cells, thereby producing a cell population comprising NKX6.1-positive, ISL1-positive endocrine cells, wherein the cell population comprising NKX6.1-positive, ISL1-positive endocrine cells is more potent than a corresponding cell population produced without contacting the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with the PKC activator in the first composition or the second composition. (i) an increased proportion of cells expressing glucagon; (ii) a reduced proportion of cells expressing VMAT1; (iii) an increased proportion of cells expressing somatostatin; or (iv) A method comprising an increased proportion of cells expressing C-peptide.

5. The method of claim 3 or 4, wherein the third composition comprises a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, and an epigenetic modifying compound.

6. The method of claim 3 or 4, wherein the third composition comprises a differentiation factor selected from the group consisting of a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, an epigenetic modifying compound, a growth factor from the EGF family, an RA signaling pathway activator, an SHH pathway inhibitor, a gamma-secretase inhibitor, a protein kinase inhibitor, a ROCK inhibitor, and a BMP signaling pathway inhibitor.

7. The method of claim 6, wherein the third composition comprises the TGF-β signaling pathway inhibitor, the thyroid hormone signaling pathway activator, the epigenetic modifying compound, the growth factor from the EGF family, the RA signaling pathway activator, the SHH pathway inhibitor, the γ-secretase inhibitor, the protein kinase inhibitor, the ROCK inhibitor, and the BMP signaling pathway inhibitor.

8. The method of claim 1 , wherein the third composition does not comprise the PKC activator.

9. 9. The method of any one of claims 2 to 8, wherein the first composition comprises the ROCK inhibitor, the growth factor from the TGFβ superfamily, the growth factor from the FGF family, the RA signaling pathway activator, and the SHH pathway inhibitor.

10. The method of any one of claims 2 to 9, wherein the second composition comprises the TGF-β signaling pathway inhibitor, the growth factor from the EGF family, the RA signaling pathway activator, the SHH pathway inhibitor, the TH signaling pathway activator, the protein kinase inhibitor, the ROCK inhibitor, the BMP signaling pathway inhibitor, and the epigenetic modifying compound.

11. the cell population comprising NKX6.1-positive, ISL1-positive endocrine cells is compared to a corresponding cell population produced without contacting the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with the PKC activator in the first composition or the second composition; (i) an increased proportion of cells expressing somatostatin; (ii) an increased proportion of cells expressing glucagon; (iii) a decreased proportion of cells expressing VMAT1; or (iv) an increased proportion of cells expressing C-peptide 11. The method of claim 3 or 5 to 10, comprising:

12. the cell population comprising NKX6.1-positive, ISL1-positive endocrine cells is compared to a corresponding cell population produced without contacting the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with the PKC activator in the first composition or the second composition; (i) an increased proportion of cells expressing somatostatin; (ii) an increased proportion of cells expressing glucagon; (iii) a reduced proportion of cells expressing VMAT1; and (iv) an increased proportion of cells expressing C-peptide 12. The method of any one of claims 3 to 11, comprising:

13. The cell population containing NKX6.1-positive, ISL1-positive endocrine cells is measured by flow cytometry, at least about 4% of cells expressing somatostatin; at least about 15% of the cells express glucagon; At most about 35% of cells express VMAT1, or At least about 40% of cells express C-peptide 13. The method of any one of claims 3 to 12, comprising:

14. the cell population comprising NKX6.1-positive, ISL1-positive endocrine cells is compared to a corresponding cell population produced without contacting the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with the PKC activator in the first composition or the second composition, as measured by flow cytometry; at least about 100% of the cells express somatostatin; at least about 200% more cells expressing glucagon; At least about 50% fewer cells express VMAT1; or At least about 20% of cells express C-peptide 14. The method of any one of claims 3 to 13, comprising:

15. 15. The method of any one of claims 1 to 14, wherein the first period of time is 1 to 3 days.

16. 15. The method of any one of claims 1 to 14, wherein the first period of time is about 2 days.

17. 17. The method of any one of claims 1 to 16, wherein the second period of time is 1 to 3 days.

18. 17. The method of any one of claims 1 to 16, wherein the second period of time is about 2 days.

19. 19. The method of any one of claims 1 to 18, wherein the PKC activator is selected from the group consisting of phorbol 12,13-dibutyrate (PDBU), FR236924, prostratin, SC-9, and TPPB.

20. 19. The method of any one of claims 1 to 18, wherein the PKC activator comprises PDBU.

21. 21. The method of any one of claims 1 to 20, wherein the PKC activator is contacted with the cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells at a concentration of 100 nM to 1000 nM.

22. 21. The method of any one of claims 1 to 20, wherein the PKC activator is contacted with the cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells at a concentration of about 500 nM.

23. 23. The method of any one of claims 1, 2, or 6-22, wherein the gamma-secretase inhibitor comprises XXI.

24. 24. The method of any one of claims 1 to 3 or 5 to 23, wherein the gamma-secretase inhibitor is contacted with the cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells at a concentration of 0.5 μM to 10 μM.

25. 24. The method of any one of claims 1 to 3 or 5 to 23, wherein the gamma-secretase inhibitor is contacted with the cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells at a concentration of about 2 μM.

26. obtaining a cell population comprising PDX1-positive, NKX6.1-negative pancreatic progenitor cells by contacting the cell population comprising PDX1-positive, NKX6.1-negative pancreatic progenitor cells with a composition comprising the PDX1-positive, NKX6.1-negative pancreatic progenitor cells, the composition comprising a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor, and differentiating the PDX1-positive, NKX6.1-negative pancreatic progenitor cells into the PDX1-positive, NKX6.1-positive pancreatic progenitor cells.

26. The method of any one of claims 2 to 25, further comprising:

27. differentiating the FOXA2-positive, PDX1-negative primitive gut cells into the PDX1-positive, NKX6.1-negative pancreatic progenitor cells by contacting the FOXA2-positive, PDX1-negative primitive gut cells with a ROCK inhibitor, a growth factor from the FGF family, a BMP signaling pathway inhibitor, a PKC activator, a retinoic acid signaling pathway activator, an SHH pathway inhibitor, and a growth factor from the TGF-β superfamily.

27. The method of claim 1 or 26, further comprising:

28. differentiating the definitive endoderm cells into the FOXA2-positive, PDX1-negative intestinal cells by contacting the definitive endoderm cells with a growth factor from the FGF family.

28. The method of claim 27, further comprising:

29. (a) differentiating pluripotent stem cells in a population into definitive endoderm cells by contacting said pluripotent stem cells with a growth factor from the TGF-β superfamily and an activator of the WNT signaling pathway; (b) differentiating the definitive endoderm cells into FOXA2-positive, PDX1-negative primitive gut cells by contacting the definitive endoderm cells with a growth factor from the FGF family; (c) differentiating the FOXA2-positive, PDX1-negative primitive gut cells into PDX1-positive, NKX6.1-negative pancreatic progenitor cells by contacting the FOXA2-positive, PDX1-negative primitive gut cells with a ROCK inhibitor, a growth factor from the FGF family, a BMP signaling pathway inhibitor, a PKC activator, a retinoic acid signaling pathway activator, an SHH pathway inhibitor, and a growth factor from the TGF-β superfamily; (d) differentiating the PDX1-positive, NKX6.1-negative pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells by contacting the PDX1-positive, NKX6.1-negative pancreatic progenitor cells with a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor; (e) incubating the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a first composition comprising a PKC activator, a gamma-secretase inhibitor, and a factor selected from the group consisting of a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor for a first period of 1 to 3 days. Incubating; (f) after (e), incubating the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with the PKC activator, the γ-secretase inhibitor, and a second composition containing a factor selected from the group consisting of a TGF-β signaling pathway inhibitor, a growth factor from the EGF family, an RA signaling pathway activator, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound for a second period of 1 to 3 days; and (g) f) followed by differentiating the PDX1-positive, NKX6.1-positive pancreatic progenitor cells into a cell population comprising NKX6.1-positive, ISL1-positive endocrine cells by contacting the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a TGF-β signaling pathway inhibitor, a growth factor from the EGF family, an RA signaling pathway activator, an SHH pathway inhibitor, a TH signaling pathway activator, a γ-secretase inhibitor, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound. A method comprising:

30. (A) the SHH pathway inhibitor comprises SANT1; (B) the RA signaling pathway activator comprises retinoic acid; (C) the γ-secretase inhibitor comprises XXI; (D) the growth factor from the EGF family includes betacellulin; (E) the BMP signaling pathway inhibitor comprises LDN or DMH; (F) the TGF-β signaling pathway inhibitor comprises Alk5 inhibitor II; (G) the thyroid hormone signaling pathway activator comprises GC-1; (H) the protein kinase inhibitor comprises staurosporine; (I) the ROCK inhibitor comprises thiazovivin; or (J) The method of any one of claims 1 to 29, wherein the epigenetic modification compound comprises DZNep, GSK126, or EPZ6438.

31. (a) contacting a plurality of PDX1-positive, NKX6.1-negative pancreatic progenitor cells with one or more of a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor, thereby producing a first cell population; (b) contacting the first cell population with a PKC activator and a gamma-secretase inhibitor, and one or more of a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor, thereby producing a second cell population; (c) contacting the second cell population with one or more of a PKC activator, a gamma-secretase inhibitor, and a TGF-β signaling pathway inhibitor, a growth factor from the EGF family, an RA signaling pathway activator, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound, thereby producing a third cell population.

32. A method comprising contacting a cell population with a gamma-secretase inhibitor and one or both of a growth factor from the TGFβ superfamily and a growth factor from the FGF family, wherein the cell population comprises PDX1-positive cells.

33. 33. The method of claim 32, wherein the cell population comprises PDX1-positive, NKX6.1-negative cells.

34. 34. The method of claim 33, wherein the cell population comprises PDX1-positive, NKX6.1-positive cells.

35. (a) contacting a plurality of PDX1-positive, NKX6.1-negative pancreatic progenitor cells with one or more of a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor for a period of no more than 1-5 days, thereby producing a first cell population; (b) contacting the first cell population with a gamma secretase inhibitor; A method comprising:

36. 36. The method of claim 35, wherein the contacting in step (a) is for a period of 4 or 5 days.

37. 37. The method of claim 35 or 36, wherein step (b) further comprises contacting the first cell population with one or more of a PKC activator, a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor.

38. (a) contacting a plurality of PDX1-positive, NKX6.1-negative pancreatic progenitor cells with one or more of a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor, thereby producing a first cell population; (b) contacting the first cell population with a PKC activator and one or more of a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor, thereby producing a second cell population, wherein the PKC activator is a benzolactam derivative; (c) contacting the second cell population with one or more of a PKC activator, a gamma-secretase inhibitor, and a TGF-β signaling pathway inhibitor, a growth factor from the EGF family, an RA signaling pathway activator, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound, thereby producing a third cell population.

39. 39. The method of claim 38, wherein the benzolactam derivative is TPPB.

40. 40. The method of claim 38 or 39, wherein step (b) further comprises contacting the first cell population with a gamma secretase inhibitor.

41. The method is (d) contacting the third cell population with one or more of a TGF-β signaling pathway inhibitor, a RA signaling pathway activator, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound, thereby producing a fourth cell population.

41. The method of any one of claims 31 to 40, further comprising:

42. 42. The method of claim 41, wherein step (d) does not include contacting the third cell population with a PKC activator.

43. 43. The method of claim 41 or 42, wherein step (d) does not include contacting the third cell population with a gamma secretase inhibitor.

44. 44. The method of any one of claims 41 to 43, wherein step (d) does not include contacting the third cell population with an SHH pathway inhibitor.

45. 45. The method of any one of claims 41 to 44, wherein step (d) does not include contacting the third cell population with a growth factor from the EGF family.

46. The method is (e) contacting the fourth cell population with one or more of a serum albumin protein, vitamin C, a TGF-β signaling pathway inhibitor, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound, thereby producing a fifth cell population.

46. ​​The method of any one of claims 31 to 45, further comprising:

47. 47. The method of claim 46, wherein step (e) comprises contacting the fourth cell population with a PKC activator.

48. (a) contacting a plurality of PDX1-positive, NKX6.1-negative pancreatic progenitor cells with one or more of a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor, thereby producing a first cell population; (b) contacting the first cell population with a PKC activator and one or more of a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor, thereby producing a second cell population; (c) contacting the second cell population with a PKC activator and one or more of a gamma secretase inhibitor, a TGF-β signaling pathway inhibitor, a growth factor from the EGF family, a RA signaling pathway activator, a SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound, thereby producing a third cell population; (d) contacting the third cell population with one or more of a TGF-β signaling pathway inhibitor, a RA signaling pathway activator, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound, thereby producing a fourth cell population; (e) contacting the fourth cell population with a PKC activator and one or more of a serum albumin protein, vitamin C, a TGF-β signaling pathway inhibitor, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound, thereby producing a fifth cell population; A method comprising:

49. 49. The method of any one of claims 46 to 48, wherein step (e) comprises contacting the fourth cell population with serum albumin protein.

50. 50. The method of any one of claims 31 to 49, wherein step (a) is carried out for 1, 2, 3, 4, 5, or 6 days.

51. 51. The method of claim 50, wherein step (a) is carried out over a period of 3 to 5 days (e.g., 4 days).

52. 52. The method of any one of claims 31 to 51, wherein step (b) is carried out for 1, 2, 3 or 4 days.

53. 53. The method of claim 52, wherein step (b) is carried out for 1 to 3 days (e.g., 2 days).

54. 54. The method of any one of claims 31 to 53, wherein step (c) is carried out for 1, 2, 3, or 4 days.

55. 55. The method of claim 54, wherein step (c) is carried out over a period of 1 to 3 days (e.g., 2 days).

56. 56. The method of any one of claims 31 to 55, wherein step (d) is carried out for 1, 2, 3, 4, 5, 6, or 7 days.

57. 57. The method of claim 56, wherein step (d) is carried out for 4 to 6 days (e.g., 5 days).

58. 58. The method of any one of claims 46-57, wherein step (e) is carried out for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days.

59. 59. The method of claim 58, wherein step (e) is carried out for 10 to 12 days.

60. 60. The method of any one of claims 31 to 59, wherein the first cell population comprises PDX1-positive, NKX6.1-negative cells and / or PDX1-positive, NKX6.1-positive cells.

61. 61. The method of any one of claims 31 to 60, wherein the second cell population comprises PDX1-positive and NKX6.1-positive cells.

62. 61. The method of any one of claims 31 to 60, wherein the third cell population comprises PDX1-positive, NKX6.1-positive, ISL1-negative cells and / or PDX1-positive, NKX6.1-positive, ISL1-positive cells.

63. 63. The method of any one of claims 41 to 62, wherein the fourth cell population comprises PDXl-positive, NKX6.1-positive, ISLl-positive cells.

64. 64. The method of any one of claims 46 to 63, wherein the fifth cell population comprises cells that express C-peptide and ISL1, but do not express VMAT1.

65. 65. The method of claim 64, wherein 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 30-40%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-90%, 50-80%, 50-70%, 50-60%, 60-90%, 60-80%, 60-70%, 70-90%, 70-80%, 70-90%, 70-80%, or 80-90% of the cells in the fourth cell population express C-peptide and ISL1 but do not express VMAT1.

66. 66. The method of claim 65, wherein 40-60% of the cells in the fourth cell population express C-peptide and ISL1 but do not express VMAT1.

67. 67. The method of any one of claims 41 to 66, wherein the fourth cell population comprises cells that express glucagon but do not express somatostatin.

68. 68. The method of claim 67, wherein 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-40%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15-40%, 15-35%, 15-30%, 15-25%, 15-20%, 20-40%, 20-35%, 20-30%, 20-25%, 25-40%, 25-35%, 25-30%, 30-40%, 30-35%, or 35-40% of the cells in the fourth cell population express glucagon but do not express somatostatin.

69. 69. The method of claim 68, wherein 10-25% of the cells in the fourth cell population express somatostatin but do not express glucagon.

70. 70. The method of any one of claims 41 to 69, wherein the fourth cell population comprises cells that express somatostatin but do not express glucagon.

71. 71. The method of claim 70, wherein 3-20%, 3-15%, 3-12%, 3-10%, 3-8%, 3-5%, 4-20%, 4-15%, 4-12%, 4-10%, 4-8%, 4-5%, 5-20%, 5-15%, 5-12%, 5-10%, 5-8%, 7-20%, 7-15%, 7-12%, 7-10%, 9-20%, 9-15%, 9-12%, 8-10%, 8-12%, 8-15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15%, or 15-20% of the cells in the fourth population of cells express somatostatin but do not express glucagon.

72. 72. The method of any one of claims 31 to 71, wherein step (a) comprises contacting a plurality of PDX1-positive, NKX6.1-negative pancreatic progenitor cells with a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor.

73. 73. The method of any one of claims 31 to 72, wherein step (b) comprises contacting the first cell population with a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, a RA signaling pathway activator, and an SHH pathway inhibitor.

74. 74. The method of any one of claims 31 to 73, wherein step (c) comprises contacting the second cell population with a gamma-secretase inhibitor, a TGF-β signaling pathway inhibitor, a growth factor from the EGF family, a RA signaling pathway activator, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound.

75. 75. The method of any one of claims 41 to 74, wherein step (d) comprises contacting the third cell population with a serum albumin protein, a TGF-β signaling pathway inhibitor, an SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modifying compound.

76. 76. The method of claim 31, wherein the ROCK inhibitor for use in steps (a), (b), (c), (d) and / or (e) is thiazobabin or Y-27632.

10. The method according to any one of claims 1 to 9.

77. 77. A method according to any one of claims 31 to 76, wherein the growth factor from the TGFβ superfamily for use in steps (a) and / or (b) is activin A.

78. 78. The method of any one of claims 31 to 77, wherein the growth factor from the FGF family for use in steps (a) and / or (b) is KGF.

79. 79. The method of any one of claims 31 to 78, wherein the RA signaling pathway activator for use in steps (a), (b) and / or (c) is retinoic acid.

80. 80. The method of any one of claims 31 to 79, wherein the SHH pathway inhibitor for use in steps (a), (b) and / or (c) is Sant-1.

81. 81. The method of any one of claims 31 or 41-80, wherein the PKC activator for use in steps (b), (c) and / or (d) is selected from the group consisting of phorbol 12,13-dibutyrate (PDBU), FR236924, prostratin, SC-9, and TPPB.

82. 82. The method of claim 81, wherein the PKC activator is PDBU.

83. 83. The method of any one of claims 31 to 82, wherein the gamma-secretase inhibitor for use in step (b) and / or (c) is XXI.

84. 84. The method of any one of claims 31 to 83, wherein the TGF-β signaling pathway inhibitor for use in steps (c), (d) and / or (e) is an ALK5i.

85. 85. A method according to any one of claims 31 to 84, wherein the growth factor from the EGF family for use in step (c) is betacellulin.

86. 86. The method of any one of claims 31 to 85, wherein the TH signaling pathway activator for use in steps (c), (d) and / or (e) is T3, GC-1 or a thyroid hormone derivative.

87. 87. The method of any one of claims 31 to 86, wherein the protein kinase inhibitor for use in steps (c), (d) and / or (e) is staurosporine.

88. 88. The method of any one of claims 31 to 87, wherein the BMP signaling pathway inhibitor for use in steps (c), (d) and / or (e) is LDN193189 or DMH-1.

89. 89. The method of any one of claims 31 to 88, wherein the epigenetic modifying compound for use in steps (c), (d) and / or (e) is DZNep.

90. In vitro composition comprising PDX1-positive, NKX6.1-negative pancreatic progenitor cells; PDX1-positive, NKX6.1-positive pancreatic progenitor cells; a PKC activator; and a gamma-secretase inhibitor thing.

91. The composition of claim 90, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the cells in the composition are PDX1-positive, NKX6.1-positive pancreatic progenitor cells.

92. 92. The composition of claim 90 or 91, wherein less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% of the cells in the composition are PDX1-positive, NKX6.1-negative pancreatic progenitor cells.

93. 93. The composition of any one of claims 90 to 92, wherein the PKC activator is selected from the group consisting of phorbol 12,13-dibutyrate (PDBU), FR236924, prostratin, SC-9, and TPPB.

94. 94. The composition of any one of claims 90 to 93, wherein the gamma-secretase inhibitor is DAPT.

95. 94. The composition of any one of claims 90 to 93, wherein the gamma-secretase inhibitor is XXI.

96. 96. The composition of any one of claims 90 to 95, wherein the composition further comprises a growth factor from the FGF family.

97. 97. The composition of claim 96, wherein the growth factor from the FGF family is KGF.

98. 98. The composition of any one of claims 90 to 97, wherein the composition further comprises a growth factor of the TGFβ superfamily.

99. 99. The composition of claim 98, wherein the growth factor of the TGFβ superfamily is activin A.

100. An in vitro composition comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells; NKX6.1-positive, ISL1-positive endocrine cells; and a PKC activator, wherein the PKC activator is a benzolactam derivative.

101. 101. The composition of any one of claims 90 to 100, wherein the PKC activator is TPPB.

102. The composition of claim 100 or 101, wherein the composition further comprises a gamma-secretase inhibitor.

103. The composition of claim 102, wherein the gamma-secretase inhibitor is XXI.

104. The composition of claim 102, wherein the gamma-secretase inhibitor is DAPT.

105. 105. The composition of any one of claims 90 to 104, further comprising a differentiation factor selected from the group consisting of a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, an epigenetic modifying compound, a growth factor from the EGF family, an RA signaling pathway activator, an SHH pathway inhibitor, a protein kinase inhibitor, a ROCK inhibitor, and a BMP signaling pathway inhibitor.

106. 106. The composition of any one of claims 90 to 105, further comprising a serum albumin protein.

107. 107. The composition of any one of claims 90 to 106, further comprising a serum albumin protein, a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, an epigenetic modifying compound, an SHH pathway inhibitor, a protein kinase inhibitor, a ROCK inhibitor, and a BMP signaling pathway inhibitor.

108. 108. The composition of any one of claims 105 to 107, wherein the ROCK inhibitor is thiazobabine.

109. 109. The composition of any one of claims 105 to 108, wherein the RA signaling pathway activator is retinoic acid.

110. 110. The composition of any one of claims 105 to 109, wherein the SHH pathway inhibitor is Sant-1.

111. 111. The composition of any one of claims 105 to 110, wherein the TGF-β signaling pathway inhibitor is an ALK5i.

112. 112. The composition of any one of claims 105 to 111, wherein the growth factor from the EGF family is betacellulin.

113. 113. The composition of any one of claims 105 to 112, wherein the thyroid hormone signaling pathway activator is T3, GC-1 or a thyroid hormone derivative.

114. 114. The composition of any one of claims 105 to 113, wherein the protein kinase inhibitor is staurosporine.

115. The composition of any one of claims 105 to 114, wherein the BMP signaling pathway inhibitor is LDN193189 or DMH-1.

116. 116. The composition of any one of claims 105 to 115, wherein the epigenetic modifying compound is DZNep.

117. A composition comprising an in vitro cell population, said cell population comprising: At least about 35% of cells that express C-peptide and do not express VMAT1; and (i) at most about 35% of cells express VMAT1, and / or (ii) at least about 15% of the cells express glucagon A composition comprising:

118. The composition of claim 117, wherein the cell population comprises at most about 30% of cells that express VMAT1 and at least about 20% of cells that express glucagon.

119. 119. The composition of claim 117 or 118, wherein the percentage of cells is measured by flow cytometry.

120. 120. The composition of any one of claims 117 to 119, wherein the cell population comprises at least about 15% of cells that express glucagon and do not express somatostatin.

121. 121. The composition of any one of claims 117 to 120, wherein the cell population comprises at least about 4% of cells that express somatostatin and do not express glucagon.

122. 1. A composition comprising a cell population, a) 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 30-40%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-90%, 50-80%, 50-70%, 50-60%, 60-90%, 60-80%, 60-70%, 70-90%, 70-80%, 70-90%, 70-80%, or 80-90% of the cells in a population of cells express C-peptide and ISL1 but do not express VMAT1; b) 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-40%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15-40%, 15-35%, 15-30%, 15-25%, 15-20%, 20-40%, 20-35%, 20-30%, 20-25%, 25-40%, 25-35%, 25-30%, 30-40%, 30-35%, or 35-40% of the cells in the cell population express glucagon but do not express somatostatin; and / or c) a cell population 3-20%, 3-15%, 3-12%, 3-10%, 3-8%, 3-5%, 4-20%, 4-15%, 4-12%, 4-10%, 4-8%, 4-5%, 5-20%, 5-15%, 5-12%, 5-10%, 5-8%, 7-20%, 7-15%, 7-12%, 7-10%, 9-20%, 9-15%, 9-12%, 8-10%, 8-12%, 8-15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15%, or 15-20% of the cells in the composition express somatostatin but do not express glucagon.

123. 1. A composition comprising a cell population, a) 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 30-40%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-90%, 50-80%, 50-70%, 50-60%, 60-90%, 60-80%, 60-70%, 70-90%, 70-80%, 70-90%, 70-80%, or 80-90% of the cells in a population of cells express C-peptide and ISL1 but do not express VMAT1; b) 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-40%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15-40%, 15-35%, 15-30%, 15-25%, 15-20%, 20-40%, 20-35%, 20-30%, 20-25%, 25-40%, 25-35%, 25-30%, 30-40%, 30-35%, or 35-40% of the cells in the cell population express glucagon but do not express somatostatin; and c) A composition wherein 3-20%, 3-15%, 3-12%, 3-10%, 3-8%, 3-5%, 4-20%, 4-15%, 4-12%, 4-10%, 4-8%, 4-5%, 5-20%, 5-15%, 5-12%, 5-10%, 5-8%, 7-20%, 7-15%, 7-12%, 7-10%, 9-20%, 9-15%, 9-12%, 8-10%, 8-12%, 8-15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15%, or 15-20% of the cells in a population of cells express somatostatin but do not express glucagon.

124. 1. A composition comprising a cell population, a) 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15-35%, 15-30%, 15-25%, 15-20%, 20-35%, 20-30%, 20- 25%, 25-35%, 25-30%, or 30-35% of the cells express VMAT1 but do not express C-peptide; b) 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-40%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15-40%, 15-35%, 15-30%, 15-25%, 15-20%, 20-40%, 20-35%, 20-30%, 20-25%, 25-40%, 25-35%, 25-30%, 30-40%, 30-35%, or 35-40% of the cells in the cell population express glucagon but do not express somatostatin; and / or c) A composition wherein 3-20%, 3-15%, 3-12%, 3-10%, 3-8%, 3-5%, 4-20%, 4-15%, 4-12%, 4-10%, 4-8%, 4-5%, 5-20%, 5-15%, 5-12%, 5-10%, 5-8%, 7-20%, 7-15%, 7-12%, 7-10%, 9-20%, 9-15%, 9-12%, 8-10%, 8-12%, 8-15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15%, or 15-20% of the cells in a population of cells express somatostatin but do not express glucagon.

125. 1. A composition comprising a cell population, a) 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15-35%, 15-30%, 15-25%, 15-20%, 20-35%, 20-30%, 20-25%, 25-35%, 25-30%, or 30-35% of the cells in a population of cells express VMAT1 but do not express C-peptide; b) 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-40%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15-40%, 15-35%, 15-30%, 15-25%, 15-20%, 20-40%, 20-35%, 20-30%, 20-25%, 25-40%, 25-35%, 25-30%, 30-40%, 30-35%, or 35-40% of the cells in the cell population express glucagon but do not express somatostatin; and c) A composition wherein 3-20%, 3-15%, 3-12%, 3-10%, 3-8%, 3-5%, 4-20%, 4-15%, 4-12%, 4-10%, 4-8%, 4-5%, 5-20%, 5-15%, 5-12%, 5-10%, 5-8%, 7-20%, 7-15%, 7-12%, 7-10%, 9-20%, 9-15%, 9-12%, 8-10%, 8-12%, 8-15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15%, or 15-20% of the cells in a population of cells express somatostatin but do not express glucagon.

126. 126. The composition of claim 124 or 125, wherein 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 30-40%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-90%, 50-80%, 50-70%, 50-60%, 60-90%, 60-80%, 60-70%, 70-90%, 70-80%, 70-90%, 70-80%, or 80-90% of the cells in the cell population express C-peptide and ISL1 but do not express VMAT1.

127. a) 40-60% of the cells in the cell population express C-peptide and ISL1, but not VMAT1; b) 10-25% of the cells in the cell population express glucagon but not somatostatin; and c) A composition according to any one of claims 122 to 126, wherein 4-10% of the cells in the cell population express somatostatin but do not express glucagon.

128. The composition of claim 127, wherein less than 25%, less than 20%, less than 18%, less than 15%, less than 12%, or less than 10% of the cells in the cell population express VMAT1 but do not express C-peptide.

129. 129. The composition of any one of claims 117 to 128, wherein the cell population is produced in vitro from stem cells.

130. 130. The composition of any one of claims 117 to 129, wherein cells that express C-peptide and do not express VMAT1 exhibit a glucose-stimulated insulin secretion response in vitro.

131. 131. The composition of claim 130, wherein the secretion of insulin by cells that express C-peptide and do not express VMAT1 in response to a glucose challenge is proportional to the glucose concentration of the glucose challenge.

132. 132. The composition of claim 130 or 131, wherein the cells that express C-peptide and do not express VMAT1 secrete insulin in response to one or more glucose challenges.

133. The composition of claims 130 to 132, wherein cells that express C-peptide and do not express VMAT1 secrete insulin in response to a first glucose challenge, a second glucose challenge, and a third glucose challenge, and wherein the first glucose challenge, the second glucose challenge, and the third glucose challenge are applied sequentially.

134. 134. A pharmaceutical composition comprising a composition according to any one of claims 90 to 133, or a cell population produced according to the method of any one of claims 1 to 89, and a pharmaceutically acceptable excipient or carrier.

135. A device comprising a composition described in any one of claims 90 to 134 or a cell population produced according to the method of any one of claims 1 to 89, wherein the device is configured to produce and release insulin when implanted in a subject.

136. A method of treating a subject, comprising administering to the subject a composition described in any one of claims 90 to 134, or a cell population produced according to a method of any one of claims 1 to 89, or a device described in claim 135.

137. PDX1-positive cells, a gamma-secretase inhibitor, and a growth factor from the TGFβ superfamily or a growth factor from the FGF family or both. vitro composition.

138. The composition of claim 137, wherein the composition of cells comprises PDX1-positive, NKX6.1-negative cells.

139. The composition of claim 138, wherein the composition of cells comprises PDX1-positive, NKX6.1-positive cells.

140. The composition of any one of claims 137 to 139, wherein the composition further comprises any one or combination of a PKC activator, a growth factor from the FGF family, a ROCK inhibitor, a growth factor from the TGFβ superfamily, a sonic hedgehog pathway inhibitor, and a retinoic acid signaling pathway activator.

141. An in vitro composition comprising: PDX1-positive, NKX6.1-negative pancreatic progenitor cells; PDX1-positive, NKX6.1-positive pancreatic progenitor cells; and a gamma-secretase inhibitor.

142. The composition of claim 141, wherein the gamma-secretase inhibitor is XXI.

143. The composition of claim 141 or 142, wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the cells in the composition are PDX1-positive, NKX6.1-positive pancreatic progenitor cells.

144. The composition of any one of claims 141 to 143, wherein less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the cells in the composition are PDX1-positive, NKX6.1-negative pancreatic progenitor cells.

145. 145. The composition of any one of claims 141 to 144, further comprising a growth factor from the FGF family.

146. The composition of claim 145, wherein the growth factor from the FGF family is KGF.

147. 147. The composition of any one of claims 141 to 146, further comprising a sonic hedgehog pathway inhibitor.

148. The composition of claim 147, wherein the sonic hedgehog pathway inhibitor is SANT-1.

149. 149. The composition of any one of claims 141 to 148, further comprising a ROCK inhibitor.

150. The composition of claim 149, wherein the ROCK inhibitor is thiazovivin.

151. 151. The composition of any one of claims 141 to 150, further comprising a growth factor from the TGFβ superfamily.

152. The composition of claim 151, wherein the growth factor from the TGFβ superfamily is activin A.

153. 153. The composition of any one of claims 141 to 152, further comprising a retinoic acid signaling pathway activator.

154. The composition of claim 153, wherein the retinoic acid signaling pathway activator is retinoic acid.

155. 155. The composition of any one of claims 141 to 154, further comprising a PKC activator.

156. 156. The composition of claim 155, wherein the PKC activator is PDBU.

157. The composition comprises a PKC activator, a growth factor from the FGF family, a ROCK inhibitor, a growth factor from the TGFβ superfamily, a sonic hedgehog pathway inhibitor, and a retinal 145. The composition of any one of claims 141 to 144, further comprising any two of the following activators of the noic acid signaling pathway:

158. The composition of any one of claims 141 to 144, wherein the composition further comprises any three of a PKC activator, a growth factor from the FGF family, a ROCK inhibitor, a growth factor from the TGFβ superfamily, a sonic hedgehog pathway inhibitor, and a retinoic acid signaling pathway activator.

159. The composition of any one of claims 141 to 144, wherein the composition further comprises any four of a PKC activator, a growth factor from the FGF family, a ROCK inhibitor, a growth factor from the TGFβ superfamily, a sonic hedgehog pathway inhibitor, and a retinoic acid signaling pathway activator.

160. The composition of any one of claims 141 to 144, wherein the composition further comprises any five of a PKC activator, a growth factor from the FGF family, a ROCK inhibitor, a growth factor from the TGFβ superfamily, a sonic hedgehog pathway inhibitor, and a retinoic acid signaling pathway activator.

161. The composition of any one of claims 141 to 144, wherein the composition further comprises any six of a PKC activator, a growth factor from the FGF family, a ROCK inhibitor, a growth factor from the TGFβ superfamily, a sonic hedgehog pathway inhibitor, and a retinoic acid signaling pathway activator.

162. 162. A device comprising the composition of any one of claims 141 to 161, the device configured to produce and release insulin when implanted in a subject.

163. 162. A method of treating a subject comprising administering to the subject a composition described in any one of claims 141 to 161 or implanting in the subject a device described in claim 162.

164. An in vitro differentiated cell population comprising NKX6.1-positive, ISL1-positive cells and NKX6.1-negative, ISL1-positive cells, wherein the population contains more NKX6.1-negative, ISL1-positive cells than NKX6.1-positive, ISL1-positive cells, and at least 73% of the cells in the population are ISL1-positive cells.

165. An in vitro differentiated cell population comprising NKX6.1-positive, ISL1-positive cells and NKX6.1-negative, ISL1-positive cells, wherein at least 40% of the cells in the population are NKX6.1-negative, ISL1-positive cells.

166. 166. The population of claim 164 or 165, wherein less than 12% of the cells in the population are NKX6.1-negative, ISL1-negative cells.

167. An in vitro differentiated cell population comprising NKX6.1-positive, ISL1-positive cells and NKX6.1-negative, ISL1-positive cells, wherein less than 12% of the cells in the population are NKX6.1-negative, ISL1-negative cells.

168. 168. The population of any one of claims 165 to 167, wherein less than 10%, less than 8%, less than 6%, or less than 4% of the cells in the population are NKX6.1-negative, ISL1-negative cells.

169. 169. The population of any one of claims 165 to 168, wherein at least 60%, at least 65%, at least 70%, at least 73%, at least 75%, or at least 80% of the cells in the population are ISL1 positive cells.

170. 170. The population of any one of claims 165 to 169, wherein 2-12%, 4-12%, 6-12%, 8-12%, 2-8%, 4-8%, 3-6% or 3-5% of the cells in the population are NKX6.1-negative, ISL1-negative cells.

171. 171. The population of any one of claims 165 to 170, wherein 50-90%, 50-85%, 50-80%, 50-75%, 50-70%, 50-60%, 60-90%, 60-85%, 60-80%, 60-75%, 60-70%, 65-90%, 65-85%, 65-80%, 65-75%, 65-70%, 70-90%, 70-85%, 70-80%, 70-75%, 75-90%, 75-85%, 75-80%, 80-90%, 80-85%, or 85-90% of the cells in the population are ISL1 positive cells.

172. 172. The population of any one of claims 165 to 171, wherein the population comprises more NKX6.1-negative, ISL1-positive cells than NKX6.1-positive, ISL1-positive cells.

173. 173. The population of any one of claims 165 to 172, wherein at least 4%, at least 5%, at least 6%, at least 8%, at least 10%, about 4-11%, or about 5-10% of the cells in the population are NKX6.1-negative, ISL1-negative cells.

174. 174. The population of any one of claims 165 to 173, wherein at least 40% of the cells in the population are NKX6.1 negative, ISL1 positive cells.

175. 175. The population of any one of claims 165 to 174, wherein at least 45%, at least 50%, about 40-50%, about 45-55%, or about 50-55% of the cells in the population are NKX6.1 negative, ISL1 positive cells.

176. 176. The population of any one of claims 165 to 175, wherein at least 74%, at least 75%, at least 80%, at least 85%, at least 90%, about 85-95%, or about 90-95% of the cells in the population are ISL1 positive cells.

177. 177. The population of any one of claims 165 to 176, wherein the population comprises more stem cell-derived alpha cells than stem cell-derived beta cells.

178. 178. The population of cells of any one of claims 165 to 177, wherein the population of cells is derived from stem cells in vitro.

179. 179. The population of any one of claims 165 to 178, further comprising a culture medium.

180. 180. The population of claim 179, wherein the medium comprises sugar.

181. 181. The population of claim 180, wherein the sugar is sucrose or glucose.

182. 182. The population of claim 180 or 181, wherein the culture medium comprises sugar at a concentration of about 0.05% to about 1.5%.

183. The medium is CMRL medium or the medium is HypoThermosol® 183. The population of any one of claims 179 to 182, which is FRS Preservation Media.

184. 184. The population of any one of claims 165 to 183, wherein the cell population is in a cell cluster.

185. 184. The population of any one of claims 165 to 183, wherein the cell population is within one or more cell clusters.

186. 186. The population of claim 185, wherein the cell clusters are about 125 to about 225 microns in diameter, about 130 to about 160 microns in diameter, about 170 to about 225 microns in diameter, about 140 to about 200 microns in diameter, about 140 to about 170 microns in diameter, about 160 to about 220 microns in diameter, about 170 to about 215 microns in diameter, or 170 to about 200 microns in diameter.

187. 187. The population of any one of claims 165 to 186, wherein the population has a genetic disruption in the beta-2-microglobulin gene.

188. A population described in any one of claims 165 to 187, wherein the population comprises NKX6.1-positive, ISL1-positive cells that express lower levels of MAFA than NKX6.1-positive, ISL1-positive cells derived from the pancreas of a healthy control adult subject.

189. The population described in any one of claims 165 to 188, wherein the population comprises NKX6.1-positive, ISL1-positive cells that express higher levels of MAFB than NKX6.1-positive, ISL1-positive cells derived from the pancreas of a healthy control adult subject.

190. A population described in any one of claims 165 to 189, wherein the population comprises NKX6.1-positive, ISL1-positive cells that express higher levels of SIX2, HOPX, IAPP and / or UCN3 than NKX6.1-positive, ISL1-positive cells derived from the pancreas of a healthy control adult subject.

191. 191. The population of any one of claims 165 to 190, wherein the population comprises NKX6.1-positive, ISL1-positive cells that do not express MAFA.

192. 192. The population of any one of claims 165 to 191, wherein the population comprises NKX6.1-positive, ISL1-positive cells that express MAFB.

193. 193. The population of any one of claims 165 to 192, contained in a device for implantation into a subject.

194. 193. An implantable encapsulation device comprising the population of any one of claims 165-192.

195. 195. The implantable encapsulation device of claim 194, wherein the device is implanted in a subject with diabetes.

196. 196. The implantable encapsulation device of claim 195, wherein the subject has type 1 diabetes.

197. Administering to a subject a composition comprising the population of any one of claims 165-192 or implanting in a subject a device of any one of claims 193-196. A method of treating a subject, comprising the step of:

Images