Differentiation of Pancreatic Endocrine Cells

Abstract

Disclosed herein are compositions and methods related to the differentiation of stem cells into pancreatic endocrine cells.In some aspects, provided herein are methods related to the production of pancreatic β cells, α cells, δ cells and EC cells in vitro.In some aspects, the present disclosure provides pharmaceutical compositions comprising the cells produced according to the methods disclosed herein, and the methods of treatment that use them.

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 on July 31, 2020, which is hereby incorporated by reference in its entirety.

Background Art

[0002]

[0002] The production of stem - cell - derived β - cells can provide potentially useful steps towards the production of pancreatic islets and pancreatic organs. One rapidly progressive disease that can be treated by tissue derived from stem cells is diabetes. Type 1 diabetes is caused by the autoimmune destruction of β - cells in the pancreatic islets. Type 2 diabetes is caused by insulin resistance in peripheral tissues and β - cell dysfunction. Diabetic patients, especially those suffering from type 1 diabetes, may be cured by the transplantation of new β - cells. Patients who receive transplants of cadaveric human pancreatic islets can become insulin - independent for five years or more by this strategy, but there are limitations to this approach due to the scarcity and quality of donor islets. If an unlimited supply of human β - cells can be produced from stem cells, this therapy could be extended to millions of new patients, which could be an important test case for translating stem - cell biology into the clinic.

[0003] Incorporation by Reference

[0003] All publications, patents, and patent applications described herein are hereby 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, the publications, patents, and patent applications described herein are incorporated by reference in their entirety.

Summary of the Invention

[0004]

[0004] In one aspect, (a) 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 TGF-β superfamily, a growth factor from the FGF family, a RA signaling pathway activator, and a 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 γ-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 a SHH pathway inhibitor for a first period; and (c) 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 γ-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 modification compound for a second period are disclosed herein.

[0005]

[0005] In one aspect, a method is disclosed herein that includes: (a) contacting, during a first period, 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 γ-secretase inhibitor, and an 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 (b) contacting, after the first period, during a second period, 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 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, an ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modification compound.

[0006]

[0006] In some examples, the method further 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.

[0007]

[0007] In one aspect, (a) during a first period, 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, a RA signaling pathway activator, and a SHH pathway inhibitor; (b) after said first period, during a second period, contacting said cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a second composition comprising a PKC activator 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 modification compound; and (c) after said second period, contacting said cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a third composition that differentiates at least a portion of said 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 produced without contacting the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with the PKC activator in the first composition or the second composition, and compared to the corresponding cell population, has (i) an increased percentage of cells expressing glucagon; (ii) a decreased percentage of cells expressing VMAT1; (iii) an increased percentage of cells expressing somatostatin; or (iv) an increased percentage of cells expressing C-peptide. A method is disclosed herein that includes these steps.

[0008]

[0008] In some examples, the third composition comprises a TGF-β signaling pathway inhibitor, a thyroid hormone (TH) signaling pathway activator, and an epigenetic modification 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 modification compound, a growth factor from the EGF family, a RA signaling pathway activator, a SHH pathway inhibitor, a γ-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 modification 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. In some examples, the third composition does not comprise the PKC activator. In some examples, the first composition comprises the ROCK inhibitor, a growth factor from the TGFβ superfamily, a 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 signaling 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 modification compound. In some examples, the cell population comprising NKX6.1-positive, ISL1-positive endocrine cells has (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 the 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 comprising NKX6.1-positive, ISL1-positive endocrine cells, compared to the 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, 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. In some examples, the cell population comprising NKX6.1-positive, ISL1-positive endocrine cells, as measured by flow cytometry, 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. In some examples, the cell population comprising NKX6.1-positive, ISL1-positive endocrine cells, as measured by flow cytometry, compared to the 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, comprises 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. 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 about 500 nM.In some examples, the γ-secretase inhibitor includes XXI. In some examples, the γ-secretase inhibitor is contacted with the cell population comprising PDX1-positive and 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 and NKX6.1-positive pancreatic progenitor cells at a concentration of about 2 μM.

[0009]

[0009] In some examples, the method further comprises obtaining the cell population comprising PDX1-positive and NKX6.1-positive pancreatic progenitor cells by differentiating the cell population comprising PDX1-positive and NKX6.1-negative pancreatic progenitor cells into the PDX1-positive and NKX6.1-positive pancreatic progenitor cells by contacting the cell population comprising PDX1-positive and NKX6.1-negative pancreatic progenitor cells with a composition comprising a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, a RA signaling pathway activator, and a SHH pathway inhibitor. In some examples, the method further comprises differentiating the FOXA2-positive and PDX1-negative foregut cells into the PDX1-positive and NKX6.1-negative pancreatic progenitor cells by contacting the FOXA2-positive and PDX1-negative foregut 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, a SHH pathway inhibitor, and a growth factor from the TGF-β superfamily. In some examples, the method further comprises differentiating the embryonic endoderm cells into the FOXA2-positive and PDX1-negative gut tube cells by contacting the embryonic endoderm cells with a growth factor from the FGF family.

[0010]

[0010] In one aspect, (a) differentiating the pluripotent stem cells into definitive endoderm cells by contacting the pluripotent stem cells in the population 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 foregut cells by contacting the definitive endoderm cells with a growth factor from the FGF family; (c) contacting the FOXA2-positive, PDX1-negative foregut 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, a SHH pathway inhibitor, and a growth factor from the TGF-β superfamily to differentiate the FOXA2-positive, PDX1-negative foregut cells into PDX1-positive, NKX6.1-negative pancreatic progenitor cells; (d) 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 a SHH pathway inhibitor to differentiate 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 for a first period of 1 to 3 days with a first composition comprising a factor selected from the group consisting of a PKC activator, a γ-secretase inhibitor, and a ROCK inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, a RA signaling pathway activator, and a SHH pathway inhibitor; (f) after (e), incubating the PDX1-positive, NKX6.1-positive pancreatic progenitor cells for a second period of 1 to 3 days with a second composition comprising a factor selected from the group consisting of the PKC activator, the γ-secretase inhibitor, and 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 modification compound; (g) after (f), the PDX1-positive, NKX6.A method is disclosed herein comprising the step of differentiating said PDX1-positive, NKX6.1-positive pancreatic progenitor cells into a cell population comprising NKX6.1-positive, ISL1-positive endocrine cells by contacting the positive pancreatic progenitor cells with 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 γ-secretase inhibitor, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modification 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 modification compound comprises DZNep, GSK126, or EPZ6438.

[0011] In one aspect, a method is disclosed herein that includes: (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 a SHH pathway inhibitor, thereby producing a first cell population; (b) contacting the first cell population with a PKC activator and a γ-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 a SHH pathway inhibitor, thereby producing a second cell population; and (c) contacting the second cell population with a PKC activator, a γ-secretase inhibitor, and one or more 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 modification compound, thereby producing a third cell population.

[0012] In one aspect, a method is disclosed herein that includes 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 includes PDX1-positive cells. In some embodiments, the cell population includes PDX1-positive, NKX6.1-negative cells. In some embodiments, the cell population includes PDX1-positive, NKX6.1-positive cells.

[0013]

[0013] In one aspect, a method is disclosed herein that includes: (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 a 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 γ-secretase inhibitor. In some embodiments, the contacting in step (a) is for a period of 4 days or 5 days. In some embodiments, step (b) further includes 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 a SHH pathway inhibitor.

[0014]

[0014] In one aspect, a method is disclosed herein that includes: (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 a SHH pathway inhibitor, thereby producing a first cell population; (b) contacting the first cell population with a PKC activator, which is a benzolactam derivative, 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 a SHH pathway inhibitor, thereby producing a second cell population; and (c) contacting the second cell population with a PKC activator, a γ-secretase inhibitor, and one or more 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 modification compound, thereby producing a third cell population.

[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, 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 modification 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 γ-secretase inhibitor. In some examples, step (d) does not include contacting the third cell population with a 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 comprises (e) contacting the fourth cell population with 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 modification compound, thereby producing a fifth cell population. In some examples, step (e) includes contacting the fourth cell population with a PKC activator.

[0016] In one aspect, (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 a 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, a RA signaling pathway activator, and a 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 γ-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 modification 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 modification 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 modification compound, thereby producing a fifth cell population are disclosed herein.

[0017] In some examples, step (e) includes contacting a fourth cell population with serum albumin protein. In some examples, step (a) is performed over 1, 2, 3, 4, 5, or 6 days. In some examples, step (a) is performed over 3 - 5 days (e.g., 4 days). In some examples, step (b) is performed over 1, 2, 3, or 4 days. In some examples, step (b) is performed over 1 - 3 days (e.g., 2 days). In some examples, step (c) is performed over 1, 2, 3, or 4 days. In some examples, step (c) is performed over 1 - 3 days (e.g., 2 days). In some examples, step (d) is performed over 1, 2, 3, 4, 5, 6, or 7 days. In some examples, step (d) is performed over 4 - 6 days (e.g., 5 days). In some examples, step (e) is performed over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days. In some examples, step (e) is performed over 10 - 12 days. In some examples, the first cell population includes PDX1 - positive, NKX6.1 - negative cells and / or PDX1 - positive, NKX6.1 - positive cells. In some examples, the second cell population includes PDX1 - positive and NKX6.1 - positive cells. In some examples, the third cell population includes 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 includes PDX1 - positive, NKX6.1 - positive, ISL1 - positive cells. In some examples, the fifth cell population includes 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 matostatin 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) includes 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 a SHH pathway inhibitor. In some examples, step (b) includes 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 a SHH pathway inhibitor.In some examples, 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, an 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 modification compound. In some examples, step (d) comprises contacting the third cell population with a serum albumin protein, 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 modification compound. In some examples, the ROCK inhibitor for use in steps (a), (b), (c), (d) and / or (e) is thiazovavin or Y-27632. In some examples, the growth factor from the TGFβ superfamily for use in steps (a) and / or (b) is activin A. In some examples, the growth factor from the FGF family for use in steps (a) and / or (b) is KGF. In some examples, the RA signaling pathway activator for use in steps (a), (b) and / or (c) is retinoic acid. In some examples, the SHH pathway inhibitor for use 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, the γ-secretase inhibitor for use in steps (b) and / or (c) is XXI. In some examples, the TGF-β signaling pathway inhibitor for use in steps (c), (d) and / or (e) is ALK5i. In some examples, the growth factor from the EGF family for use in step (c) is betacellulin.In some examples, the TH signaling pathway activator for use in step (c), (d) and / or (e) is T3, GC-1 or a thyroid hormone derivative. In some examples, the protein kinase inhibitor for use in step (c), (d) and / or (e) is staurosporine. In some examples, the BMP signaling pathway inhibitor for use in step (c), (d) and / or (e) is LDN193189 or DMH-1. In some examples, the epigenetic modification compound for use in step (c), (d) and / or (e) is DZNep.

[0018]

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

[0019]

[0019] In some aspects, an in vitro composition is disclosed herein that includes PDX1-positive, NKX6.1-negative pancreatic progenitor cells; PDX1-positive, NKX6.1-positive pancreatic progenitor cells; a PKC activator; and a γ-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 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the cells in the composition are PDX1-positive, NKX6.1-negative pancreatic progenitor cells.

[0020]

[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 γ-secretase inhibitor is DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester). In some examples, the γ-secretase inhibitor is XXI.

[0021]

[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, provided 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 modification compound, a growth factor from the EGF family, a RA signaling pathway activator, a SHH pathway inhibitor, a protein kinase inhibitor, a ROCK inhibitor, and a BMP signaling pathway inhibitor. In some examples, the composition further comprises a 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 modification compound, a SHH pathway inhibitor, a protein kinase inhibitor, a ROCK inhibitor, and a BMP signaling pathway inhibitor. In some examples, the ROCK inhibitor is thiazovivin. 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 growth factor of the EGF family 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 modification compound is DZNep.

[0024]

[0024] In one aspect, there is provided a composition comprising an in vitro cell population, wherein the cell population comprises (a) at least about 35% of cells that express C-peptide and do not express VMAT1; and (b) at most about 35% of cells that express VMAT1, or at least about 15% of 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, wherein the cell population comprises at least about 35% of cells that express C-peptide and do not express VMAT1; and (i) at most about 35% of cells that express VMAT1, and / or (ii) at least about 15% of cells that express glucagon. In some embodiments, the percentage of cells is measured by flow cytometry.

[0025]

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

[0026]

[0026] In one aspect, a composition comprising a cell population, wherein: 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 the cell population 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) 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 cell population express somatostatin but do not express glucagon is disclosed herein.

[0027]

[0027] In one aspect, a composition comprising a plurality of cells, wherein 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 the cell population 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) 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 cell population express somatostatin but do not express glucagon is disclosed herein.

[0028] In one aspect, a composition comprising 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; 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) 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 cell population express somatostatin but do not express glucagon is disclosed herein.

[0029]

[0029] In one aspect, a composition comprising 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; 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) 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 cell population express somatostatin but do not express glucagon are disclosed herein.

[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]

[0031] In some embodiments of the composition, 40 - 60% of the cells in the cell population express C - peptide and ISL1 but do not express VMAT1; 10 - 25% of the cells in the cell population express glucagon but do not express somatostatin; and 4 - 10% of the cells in the cell population express somatostatin but do not express 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 do not express C - peptide. In some examples, the cell population is produced from stem cells in vitro. In some examples, cells that express C - peptide and do not express VMAT1 exhibit a glucose - stimulated insulin secretion response in vitro. In some examples, in response to a glucose challenge, the secretion of insulin by cells that express C - peptide and do not express VMAT1 is proportional to the glucose concentration of the glucose challenge. In some examples, cells that express C - peptide and do not express VMAT1 secrete insulin in response to one or more glucose challenges. In some examples, 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, in vitro compositions are disclosed herein that include 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 cell composition includes PDX1 - positive, NKX6.1 - negative cells. In some embodiments, the cell composition includes PDX1 - positive, NKX6.1 - positive cells.

[0033]

[0033] In some embodiments, the composition further comprises any one or a combination thereof 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, in vitro compositions are disclosed herein that include PDX1-positive, NKX6.1-negative pancreatic progenitor cells; PDX1-positive, NKX6.1-positive pancreatic progenitor cells; and a γ-secretase inhibitor. In some embodiments, the γ-secretase inhibitor is XXI.

[0035]

[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 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.

[0036]

[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]

[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]

[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 one aspect, there is provided an in vitro differentiated cell population comprising NKX6.1-positive, ISL1-positive cells, and NKX6.1-negative, ISL1-positive cells; 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. In some embodiments, less than 12% of the cells in the population are NKX6.1-negative, ISL1-negative cells.

[0040]

[0040] In one aspect, there is provided an in vitro differentiated cell population 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]

[0041] In one aspect, there is provided 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.

[0042]

[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]

[0043] In some embodiments, the population contains 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]

[0044] In some embodiments, the population contains 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]

[0045] In some embodiments, the population further comprises a culture medium. In some embodiments, the culture medium comprises sugar. In some embodiments, the sugar is sucrose or glucose. In some embodiments, the culture medium comprises 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]

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

[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 an adult subject who is a healthy control. In some embodiments, the population comprises NKX6.1-positive, ISL1-positive cells that express higher levels of MAFB than NKX6.1-positive, ISL1-positive cells from the pancreas of an adult subject who is a healthy control. In some embodiments, 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 from the pancreas of an adult subject who is a healthy control.

[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 within a device for implantation into a subject. In some aspects, the present disclosure provides an encapsulation device for implantation that includes the population. In some embodiments, the device is implanted in a subject having diabetes. In some embodiments, the subject has type I 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 a device in the subject.

[0050]

[0051] In some aspects, the present disclosure provides a pharmaceutical composition comprising a composition disclosed herein, or a cell population generated according to a method disclosed herein, and a pharmaceutically acceptable excipient or carrier.

[0051]

[0052] In some aspects, the present disclosure provides a device comprising a composition disclosed herein, or a cell population generated according to a method disclosed herein, the device being configured to produce and release insulin when implanted in a subject.

[0052]

[0053] In some aspects, the present disclosure provides a method of treating a subject, the method comprising administering to the subject a composition disclosed herein, or a cell population generated according to a method disclosed herein, or a device disclosed herein.

[0053]

[0054] The features of the present disclosure are set forth in detail in the appended claims. A better understanding of the features and advantages of the 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 to the appended drawings.

Brief Description of the Drawings

[0054]

Figure 1

[0055] Figure 1 shows the results of single-cell sequencing of in vitro endocrine cell populations generated according to two exemplary differentiation protocols (Version A and Version B), regardless of the presence or absence of 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 generated according to two exemplary differentiation protocols, regardless of the presence or absence of PDBU applied from S4d5 to S5d2, as measured by flow cytometry (Figure 2B). (Figure 2A)

Figure 2B

Figure 3

[0057] Figure 3 summarizes the percentage of glucagon positive, somatostatin negative cells (GCG+ / SST-) in in vitro endocrine cell populations generated according to two exemplary differentiation protocols, regardless of the presence or absence of 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 generated according to two exemplary differentiation protocols, regardless of the presence or absence of 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 generated according to two exemplary differentiation protocols, regardless of the presence or absence of PDBU applied from S4d5 to S5d2.

Figure 6A

[0060] Figure 6A summarizes the percentage of SOX9-positive cells before re-aggregation in in vitro endocrine cell populations generated according to two exemplary differentiation protocols, regardless of the presence or absence of PDBU applied from S4d5 to S5d2, when measured by flow cytometry.

Figure 6B

Figure 7

[0061] Figure 7 summarizes the recovery rate after re-aggregation in in vitro endocrine cell populations generated according to two exemplary differentiation protocols, regardless of the presence or absence of PDBU applied from S4d5 to S5d2.

Figure 8

[0062] Figure 8 summarizes the glucose-stimulated insulin secretion (GSIS) response of in vitro endocrine cell populations generated according to two exemplary differentiation protocols, regardless of the presence or absence of PDBU applied from S4d5 to S5d2.

Figure 9

[0063] Figure 9 summarizes the insulin content of in vitro endocrine cell populations generated according to two exemplary differentiation protocols, regardless of the presence or absence of 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 generated according to three exemplary differentiation protocols, regardless of the presence or absence of PDBU or PDBU and XXI applied between S4d5 and S5d2, when 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 generated 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 percentages of NKX6.1-positive / negative and ISL1-positive / negative cells (Figure 11B) in in vitro cell populations generated 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

Figure 11C

[0055]

[0066] The embodiments of the present disclosure will be described in detail by the following description and examples. It should be understood that the present disclosure is not limited to the specific embodiments described herein and can therefore vary. Those skilled in the art will recognize that numerous variations and modifications exist in the present disclosure and that these are included within the scope of the present disclosure.

[0056]

[0067] All terms are intended to be understood as understood by those skilled 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 purposes of organization only and should not be construed as limiting the described subject matter.

[0069] The various features of the present disclosure can be described in relation to a single embodiment, but the features can also be provided individually or in any suitable combination. Conversely, the present disclosure can be described herein in relation to individual embodiments for clarity, but the present disclosure can 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 do not ascribe to any example, whether related or unrelated, such as any widely shared patent or patent application. Any methods and materials similar to or equivalent to the methods and materials described herein can be used in the practice for testing this disclosure, but the preferred materials and methods are described herein. Accordingly, the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0059]

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

[0060]

[0072] In this application, the use of "or" means "and / or" unless otherwise stated. The terms "and / or" and "any combination thereof" and their grammatical equivalents used herein 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 combination thereof", can mean "A individually, B individually, C individually, A and B, B and C, A and C, as well as A, B, and C". The term "or" can be used conjunctively or disjunctively unless the context specifically dictates a disjunctive use.

[0061]

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

[0074] References to "some embodiments", "an embodiment", or "another embodiment" in the specification mean that the particular features, structures, or characteristics described in connection with that embodiment are included in at least some embodiments of the disclosure, but not necessarily all embodiments.

[0062]

[0075] As used in this specification and the 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" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. Any embodiment discussed herein can be practiced with respect to any method or composition of the disclosure, and vice versa is intended. Further, the compositions of the disclosure can be used to achieve the methods of the disclosure.

[0063]

[0076] The term "about" associated with a reference numerical value and its grammatical equivalents as used herein can include the value of that numerical value itself and a range of values plus or minus 10% of that numerical value.

[0077] The terms "about" or "substantially" mean within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will in part depend on how the value is measured or determined, e.g., due to the limitations of the measuring system. For example, "about" can mean within one standard deviation or more than one standard deviation, depending on the convention in the art. Alternatively, "about" can mean within 20%, 10%, 5%, or 1% of a given value. In another example, an amount of "about 10" includes 10 and any amount from 9 to 11. In yet another example, the term "about" in relation to a reference numerical value can also include a range of plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of that value. Alternatively, especially with respect to biological systems or processes, the term "about" can mean within one order of magnitude, preferably within five-fold, more preferably within two-fold of a value. When a particular value is recited in the application and claims, unless otherwise stated, it should be assumed that the term "about", which means within an acceptable error range for the particular value, is intended.

[0064]

[0078] As used herein, the term "diabetes" and its grammatical equivalents can mean a disease characterized by high blood glucose levels over a long period of time. For example, the term "diabetes" and its grammatical equivalents as used herein can mean 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 examples, diabetes can be a form of hereditary diabetes.

[0065]

[0079] The term "endocrine cell", unless otherwise specified, can mean a hormone-producing cell present in the pancreas of a living body, such as "islet", "islet cell", "islet equivalent", "islet-like cell", "pancreatic islet", and its grammatical equivalents. In one embodiment, endocrine cells can differentiate from pancreatic progenitor cells or precursors. Islet cells can 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. Islet cells can also mean a group of cells, a cell cluster, or the like.

[0066]

[0080] The terms "progenitor cell" and "precursor" cell are used interchangeably herein and refer to a cell having a more primitive cell phenotype (e.g., at an earlier step in the developmental pathway or development than a fully differentiated cell) compared to a cell that can arise by differentiation. Progenitor cells can also often have significant or very 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 arise and differentiate.

[0067]

[0081] "Its precursor" as a term related to insulin-positive endocrine cells can mean any cell that can differentiate into insulin-positive endocrine cells when cultured under conditions suitable for differentiating progenitor cells into insulin-positive endocrine cells, such as pluripotent stem cells, embryonic endoderm cells, primitive gut cells, pancreatic progenitor cells, or endocrine progenitor cells.

[0068]

[0082] The terms "stem cell-derived β cells", "SC-β cells", "functional β cells", "functional pancreatic β cells", "mature SC-β cells", and their grammatical equivalents can mean cells (e.g., non-native pancreatic β cells) that exhibit at least one marker indicative of pancreatic β cells (e.g., PDX-1 or NKX6.1), secrete insulin, and exhibit a glucose-stimulated insulin secretion (GSIS) response characteristic of endocrine mature β cells. In some embodiments, the terms "SC-β cells" and "non-native β cells" as used herein are interchangeable. In some embodiments, "SC-β cells" include mature pancreatic cells. It should be understood that SC-β cells need not be derived (e.g., directly) from stem cells. This is because the methods of the present disclosure can induce 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 for example, embryonic stem cells, induced pluripotent stem cells, progenitor cells, partially reprogrammed somatic cells (e.g., somatic cells partially reprogrammed to an intermediate state between induced pluripotent stem cells and the somatic cells from which they were derived), totipotent cells, pluripotent cells, differentiated and transformed versions of any of the above cells, etc. can be used). In some embodiments, SC-β cells exhibit a response to multiple glucose challenges (e.g., at least 1, at least 2, or at least 3 or more glucose challenges in a series). In some embodiments, the response is similar to the response of endogenous islets (e.g., human islets) to multiple glucose challenges. In some embodiments, the morphology of SC-β cells is similar to the morphology of endogenous β cells. In some embodiments, SC-β cells exhibit an in vitro GSIS response similar to the GSIS response of endogenous β cells. In some embodiments, SC-β cells exhibit an in vivo GSIS response similar to the GSIS response of endogenous β cells. In some embodiments, SC-β cells exhibit both in vitro and in vivo GSIS responses similar to the GSIS response of endogenous β cells. The GSIS response of SC-β cells can be observed within 2 weeks after transplanting the SC-β cells into a host (e.g., a human or an animal).In some embodiments, SC-β cells pack insulin into secretory granules. In some embodiments, SC-β cells present 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 anti-diabetic drugs (e.g., secretagogues). In some embodiments, SC-β cells are monoclonal. In some embodiments, SC-β cells do not aberrantly 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 cell", "SC-α cell", "functional α cell", "functional pancreatic α cell", "mature SC-α cell" and their grammatical equivalents refer to cells that express at least one marker indicative of pancreatic α cells (e.g., express glucagon and ISL1 but not NKX6.1), express glucagon, and secrete functional glucagon (e.g., non-native pancreatic α cells). 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 cell" and "non-native α cell" as used herein are interchangeable. In some embodiments, "SC-α cells" include mature pancreatic cells.

[0070]

[0084] The terms "stem cell-derived δ cells", "SC-δ cells", "functional δ cells", "functional pancreatic δ cells", "mature SC-δ cells", and their grammatical equivalents denote cells that exhibit at least one marker indicative of pancreatic δ cells (e.g., somatostatin) and that express and secrete somatostatin (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 denote cells (e.g., non-native pancreatic EC cells) that exhibit at least one marker indicative of pancreatic EC cells (e.g., VMAT1 (vesicular monoamine transporter 1), express NKX6.1 but do not express ISL1). In some embodiments, the terms "SC-EC cells" and "non-native EC cells" as used herein are interchangeable.

[0072]

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

[0073]

[0087] As used herein, the term "insulin-producing cell" and its grammatical equivalents mean cells that differentiate from pancreatic progenitor cells or their precursors and secrete insulin. Insulin-producing cells include pancreatic β-cells, as well as cells that synthesize (e.g., transcribe the insulin gene, translate proinsulin mRNA, and modify proinsulin mRNA into insulin protein), express (e.g., realize the phenotypic traits carried by the insulin gene), or secrete (release insulin into the extracellular space) insulin in a structural or induced manner, such as pancreatic β-like cells (e.g., insulin-positive endocrine cells), as the term is described herein. 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 may 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 generated 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 have a cell-like phenotype except that they do not generate or secrete insulin).

[0074]

[0088] The terms "insulin-positive β-like cells", "insulin-positive endocrine cells", and their grammatical equivalents may mean cells (e.g., pancreatic endocrine cells) that exhibit at least one marker indicative of pancreatic β-cells, 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 examples, the terms "insulin-positive endocrine cells" and "NKX6.1-positive, ISL1-positive cells" are used interchangeably.

[0075]

[0089] The term "β-cell marker" means, without limitation, a protein, peptide, nucleic acid, polymorphism of a protein and a nucleic acid, splice variant, fragment of a protein or nucleic acid, element, and other analyte that is specifically expressed or present in pancreatic β-cells. Exemplary β-cell 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-3 beta, and MafA, as well as those described by Zhang et al., Diabetes. 50(10):2231-6 (2001). In some embodiments, the β-cell marker is a nuclear β-cell marker. In some embodiments, the β-cell marker is PDX1 or PH3.

[0076]

[0090] The term "pancreatic endocrine marker" means, without limitation, a protein, peptide, nucleic acid, polymorphism of a protein and a nucleic acid, splice variant, fragment of a protein or nucleic acid, element, and other analyte that may be 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 cell", "pancreatic endocrine progenitor cell", "pancreatic precursor", "pancreatic endocrine precursor", and their grammatical equivalents are used interchangeably herein and can mean a stem cell that can become a pancreatic hormone-expressing cell that can form pancreatic endocrine cells, pancreatic exocrine cells, or pancreatic duct cells. These cells are involved in the differentiation towards at least one type of pancreatic cell, for example, β-cells that produce insulin, α-cells that produce glucagon, δ-cells (or D-cells) that produce somatostatin, and / or F-cells that produce pancreatic polypeptide. 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" can mean cells that are pancreatic endoderm (PE) cells with the ability to differentiate 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, or Ptf1a, or HNF6 or NRx2.2. Expression of PDX1 can be evaluated by any method known to those skilled in the art, such as immunochemistry using anti-PDX1 antibody or quantitative RT-PCR. In some examples, PDX1-positive pancreatic progenitor cells lack expression of NKX6.1. In some examples, since PDX1-positive pancreatic progenitor cells lack expression of NKX6.1, they can also be referred to as PDX1-positive, NKX6.1-negative pancreatic progenitor cells. In some examples, PDX1-positive pancreatic progenitor cells can also be named "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 with 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, or Ptf1a, or HNF6 or NRx2.2. Expression of NKX6-1 can be evaluated by any method known to those skilled in the art, such as immunochemistry using anti-NKX6-1 antibody or quantitative RT-PCR. As used herein, the terms "NKX6.1" and "NKX6-1" are equivalent and interchangeable. In some examples, PDX1-positive, NKX6-1-positive pancreatic progenitor cells can also be named "pancreatic foregut precursor cells".

[0080]

[0094] The terms "NeuroD" and "NeuroD1" are used interchangeably to identify a protein expressed in pancreatic endocrine progenitor cells and the gene encoding it.

[0095] The term "epigenetics" refers to heritable changes in gene function that do not involve changes in the DNA sequence. Epigenetics most often refers to changes in chromosomes that affect gene activity and expression, but can also be used to describe any heritable change in phenotype that does not arise from a modification of the genome. Such effects on cell and physiological phenotype traits may be due to external or environmental factors, or may be part of the normal developmental program. Epigenetics can also refer to functionally related 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 modification, each of which modifies the way in which 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 epigenetics can persist through cell division during the life of the cell and can persist over multiple generations even though they do not involve changes in the DNA sequence underlying the living organism. One example of epigenetics in eukaryotic cell biology is the process of cell differentiation. During morphogenesis, totipotent stem cells can become various pluripotent cells, which can then become fully differentiated cells.

[0081]

[0096] The term "epigenetic modification compound" means a compound that causes epigenetics in a gene, that is, a compound that changes the expression of a gene without changing the DNA sequence. Epigenetics helps to determine whether a gene is turned on or off and can affect protein production in certain cells, such as beta cells. Epigenetic modifications such as DNA methylation and histone modification alter DNA accessibility and chromatin structure, thereby regulating the pattern of gene expression. These processes are important for the normal development and differentiation of unique cell lineages in adult organisms. These can be modified by exogenous influences and can therefore contribute to or be the result of environmental modification of the phenotype or disease phenotype. Importantly, epigenetic modification plays an important role in the regulation of pluripotent genes, which are inactivated during differentiation. Examples of non-limiting epigenetic modification 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 means any primary cell that is not pluripotent in its native form as the term is defined herein. In other words, the term "differentiated cell" can mean a cell of a highly specialized cell type derived from a cell of a less specialized cell type (such as a stem cell such as an induced pluripotent stem cell) in the cell differentiation process. Without wishing to be limited by theory, pluripotent stem cells in the process of normal ontogeny can differentiate into endodermal cells that can first form pancreatic cells and other endodermal cell types. Further differentiation of endodermal cells is directed along the pancreatic pathway, and approximately 98% of the cells become exocrine, ductal, or matrix cells, and approximately 2% become endocrine cells. Early endocrine cells are pancreatic progenitor cells, which can then further differentiate into insulin-producing cells (such as functional endocrine cells) that secrete insulin, glucagon, somatostatin, or pancreatic polypeptide. Endodermal cells can also differentiate into other cells of endodermal origin, such as the lung, liver, intestine, thymus, and the like.

[0083]

[0098] As used herein, the term "somatic cell" can mean any cell that forms a living organism, as opposed to germline cells. In mammals, germline cells (also known as "gametes") are sperm and eggs that fuse during fertilization to produce a cell called a zygote, from which the entire mammalian embryo develops. With the exception of sperm and eggs (germ cells) 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, somatic cells are "non-embryonic somatic cells", which means somatic cells that do not exist in or are not obtained from an embryo and do not result from the in vitro expansion of such cells. In some embodiments, somatic cells are "adult somatic cells", which means cells that exist in or are obtained from a living organism other than an embryo or fetus, or that result from the in vitro expansion of such cells. Unless otherwise indicated, a method for converting at least one insulin-positive endocrine cell or a precursor thereof into an insulin-producing and glucose-responsive cell can be carried out both in vivo and in vitro (in vivo if at least one insulin-positive endocrine cell or a precursor thereof is present in a subject, and in vitro using at least one insulin-positive endocrine cell or a precursor thereof isolated and maintained in culture).

[0084]

[0099] As used herein, the term "adult cell" can mean a cell found throughout the body after embryonic development.

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

[0085]

[0101] As used herein, the term "endoderm-derived cell" can mean any cell that has developed or differentiated from an endoderm cell. For example, endoderm-derived cells include cells of the liver, lung, pancreas, thymus, intestine, stomach, and thyroid. Without wishing to be bound by theory, the progenitor cells of the liver and pancreas (also referred to as pancreatic progenitor cells) arise from endoderm cells within the embryonic foregut. Soon after they are specified, the progenitor cells of the liver and pancreas rapidly acquire quite distinct cell functions and regenerative capacities. These changes are caused by inductive signals and gene regulatory factors that are highly conserved among vertebrates. Interest in organ development and regeneration has been accelerated by the strong need for hepatocytes and pancreatic beta cells in the treatment of liver failure and type I diabetes. Studies in a variety of model organisms and humans have revealed a network of evolutionarily conserved inductive signals and transcription factors that provide guidance on how to induce hepatocyte and pancreatic cell differentiation and promote the differentiation of hepatocytes and beta cells from diverse stem cell and progenitor cell types.

[0086]

[0102] As used herein, the term "definitive endoderm" can mean cells that differentiate from endoderm cells and can 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, the definitive endoderm cells of the present specification express Sox17 and in some embodiments Sox17 and HNF3B, and 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 ability to differentiate into cells including cells of the liver, lung, pancreas, thymus, intestine, stomach, and thyroid. Expression of Sox17 and other definitive endoderm markers can be evaluated 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 mean cells of endodermal origin that can differentiate into multiple pancreatic lineages including pancreatic β cells but no longer have the ability to differentiate into non-pancreatic lineages.

[0104] As used herein, the term "gastrula cell" or "intestinal cell" can mean a cell that differentiates from endoderm cells and can differentiate into SC-β cells (e.g., pancreatic β cells). Gastrula cells express at least one of the following markers: HNP1-β, HNF3-β, or HNF4-α. In some examples, gastrula cells are positive for FOXA2 and SOX2, i.e., express both FOXA2 (also known as HNF3-β) and SOX2. In some examples, gastrula cells are positive for FOXA2 and negative for PDX1, i.e., express FOXA2 but not PDX1. Gastrula cells have the ability to differentiate into cells including cells of the lung, liver, pancreas, stomach, and intestine. The expression of HNF1-β and other gastrula markers can be evaluated by any method known to those skilled in the art, such as immunohistochemistry using an anti-HNF1-β antibody.

[0088]

[0105] As used herein, the term "stem cell" can mean an undifferentiated cell that gives rise to a greater number of progenitor cells having the ability to produce a number of mother cells that can proliferate and then give rise to daughter cells that have differentiated or are capable of differentiating. While the daughter cells themselves can proliferate and are then induced to generate progeny that differentiate into one or more mature cell types, they can retain one or more cells with parental developmental potential. The term "stem cell" can, in certain contexts, mean a subset of progenitor cells that have the ability or potential to differentiate into a more specialized or differentiated phenotype and, in certain contexts, retain the ability to proliferate without substantial differentiation. In one embodiment, the term stem cell generally refers to naturally occurring mother cells whose progeny differentiate, by differentiation, for example by acquiring fully distinct characteristics, often in different directions, as occurs in the progressive diversification of embryonic cells and tissues. Differentiation of cells is typically a complex process that occurs through many cell divisions. Differentiated cells may be derived from totipotent cells, which in turn may be derived from totipotent cells, and so on. Each of these totipotent cells is thought to be a stem cell, but the range of cell types that 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 may be natural or may be artificially induced by treatment with various factors. In many biological examples, stem cells are also "pluripotent" in that they can generate progeny with two or more different cell types, but this is not necessary for "stemness". "Self-renewal" is another classical part of the definition of stem cells and is important as used herein. Theoretically, self-renewal can occur by either of two main mechanisms. Stem cells divide asymmetrically, with one daughter retaining the stem cell state and the other daughter being able to express some different other specific functions and phenotypes. Alternatively, some of the stem cells in a population can divide symmetrically into two stem cells, whereby some of the stem cells in the population are maintained as a whole while the other cells in the population give rise only to differentiated progeny.Formally, cells that began as stem cells progress toward a differentiated phenotype but can also "retrogress" and re-express the stem cell phenotype, which is often referred to by those skilled in the art as "dedifferentiation" or "reprogramming" or "reverse differentiation." As used herein, the term "pluripotent stem cell" includes embryonic stem cells, induced pluripotent stem cells, placental stem cells, and the like.

[0089]

[0106] As used herein, the term "pluripotency" can mean a cell having the ability to differentiate into two or more differentiated cell types under various conditions, 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 all three germ layers, using, for example, the teratoma formation assay in nude mice. Pluripotency is also demonstrated by the expression of embryonic stem (ES) cell markers, but a preferred test for pluripotency is to demonstrate the ability to differentiate into cells of each of the three germ layers. It should be noted that simply culturing such cells by themselves does not, in itself, make these cells pluripotent. Reprogrammed pluripotent cells (e.g., iPS cells as defined by that term herein) are also characterized by having the ability to be passaged for an extended number of times without losing their growth potential, as compared to primary parental cells that generally have the ability to divide only a limited number of times in culture.

[0090]

[0107] As used herein, the terms "iPS cell" and "induced pluripotent stem cell" are used interchangeably and can mean pluripotent stem cells that are artificially derived (e.g., induced or by complete reversal), typically from adult somatic cells, by inducing the forced expression of one or more genes.

[0091]

[0108] The term "phenotype" can mean all of one or several biological characteristics that define a cell or living 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 can mean an animal, such as a human, from which cells are obtained and / or to which a treatment, including a prophylactic treatment using the cells described herein, is provided. For the treatment of an infection, condition, or disease state specific to a particular animal, such as a human subject, the term "subject" can mean that particular animal. As used interchangeably herein, "non-human animal" and "non-human mammal" include mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The term "subject" encompasses any vertebrate animal, including but not limited to mammals, reptiles, amphibians, and fish. However, advantageously, the subject is a mammal such as a human, or another mammal such as a dog, cat, horse, other domestic animal, or cow, sheep, pig, or other production mammal. "A patient in need thereof" or "a subject in need thereof" means, herein, not only a patient diagnosed with or suspected of having a disease or disorder, such as diabetes, but also other diseases or disorders.

[0093]

[0110] As used herein, "administering" can mean providing one or more of the compositions described herein to a patient or subject. By way of non-limiting example, administration of a composition, such as an injection, can be performed by intravenous (i.v.) injection, subcutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, or intramuscular (i.m.) injection. One or more such routes can be employed. Parenteral administration can be, for example, by bolus injection or by gradual perfusion over time. Alternatively, or concurrently, administration can be by the oral route. Additionally, administration can be by surgical deposition of a bolus or pellet of cells or by positioning a medical device. In one embodiment, the compositions of the disclosure can include engineered cells or host cells that express 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 can include buffers such as neutral buffered saline, phosphate buffered saline, carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol, etc., proteins, polypeptides, or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (such as aluminum hydroxide), and preservatives.

[0094]

[0111] Some of the numerical values disclosed throughout, for example, are referred to as "X is at least or at least about 100, or 200 [or any number]." This numerical value 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 are all included.

[0095]

[0112] All disclosed numerical values contemplate all such different combinations. Unless specifically indicated to the contrary, whether for the administration of a therapeutic agent or for days, months, years, weight, dosage, or otherwise, all disclosed numerical values are to be construed in this manner.

[0096]

[0113] Ranges disclosed in general may, for example, be referred to as “X is administered on day 1 to 2 or about day 1 to 2, or on day 2 to 3 or about day 2 to 3 [or any numerical range].” Such a range includes the numbers themselves (e.g., the endpoints of the range) and the following i) X is administered between day 1 and day 2, ii) X is administered between day 2 and day 3, iii) X is administered between about day 1 and day 2, iv) X is administered between about day 2 and day 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 are all included.

[0097]

[0114] All such different combinations are contemplated by the ranges disclosed in general. Unless specifically indicated to the contrary, whether for the administration of a therapeutic agent or for days, months, years, weight, dosage, or otherwise, all disclosed ranges are to be construed in this manner.

[0098]

[0115] In one aspect, the present disclosure provides compositions and methods for differentiating pancreatic progenitor cells. The compositions and methods provided herein can, in some embodiments, provide pancreatic β cells, cell populations, or highly purified pancreatic β cells, high insulin content, excellent glucose-dependent insulin secretion responses, and cell clusters having appropriate percentages of pancreatic α and δ cells and enterochromaffin cells that can be structurally and functionally similar to native 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 β cells, increased pancreatic α cells, increased pancreatic δ cells, decreased enterochromaffin (EC) cells, or any combination thereof. In some examples, the methods result in the production of an in vitro cell composition comprising about 30% - 40% pancreatic β cells, 30% - 40% pancreatic α cells, 3 - 10% pancreatic δ cells, and / or less than 20% EC cells. In some examples, a cell composition produced according to the methods disclosed herein has an improved glucose-stimulated insulin secretion (GSIS) response compared to a cell composition produced according to conventional methods. In some examples, the cell compositions disclosed herein have a dynamic GSIS response similar to that of native islets.

[0100]

[0117] In some embodiments, the methods provided herein utilize PKC activation during or after induction of NKX6.1 expression in PDX1-positive pancreatic progenitor cells, e.g., at a late stage of differentiating PDX1-positive pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells. Without being bound by a particular theory, activation of PKC signaling in PDX1-positive, NKX6.1-positive pancreatic progenitor cells can influence the differentiation fate of certain cells and result in an increase in the percentage of pancreatic α cells and a decrease in the percentage of EC cells.

[0101]

[0118] In one aspect, 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 a SHH pathway inhibitor, thereby producing a first cell population; (b) contacting the first cell population with a PKC activator, a γ-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 a SHH pathway inhibitor, thereby producing a second cell population; and (c) contacting the second cell population with a PKC activator, a γ-secretase inhibitor, and one or more 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 modification compound, thereby producing a third cell population.

[0102]

[0119] In one aspect, the present disclosure provides a method 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.

[0103]

[0120] In some embodiments, 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 a 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 γ-secretase inhibitor. In some embodiments, the contacting in step (a) is for a period of 4 days 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 a SHH pathway inhibitor.

[0104]

[0121] In some embodiments, 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 a SHH pathway inhibitor, thereby producing a first cell population; (b) contacting the first cell population with a PKC activator, which is a benzolactam derivative, 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 a SHH pathway inhibitor, thereby producing a second cell population; and (c) contacting the second cell population with a PKC activator, a γ-secretase inhibitor, and one or more 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 modification compound, thereby producing a third cell population. In some examples, the benzolactam derivative is TPPB.

[0105]

[0122] In one aspect, 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 a 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, a RA signaling pathway activator, and a 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 γ-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 modification 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 modification 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 modification compound, thereby producing a fifth cell population.

[0106]

[0123] In one aspect, the method disclosed herein comprises 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 a SHH pathway inhibitor, thereby producing a cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells. In some examples, the method comprises contacting, for a first period, a cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a first composition comprising a PKC activator, a γ-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 a SHH pathway inhibitor. In some examples, the method comprises, after the first period, for a second period, 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, 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 modification compound. In some examples, the method comprises, 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, provided herein is an in vitro composition comprising a cell population, the cell population comprising (a) at least about 35% of the cells that express C-peptide and do not express VMAT1; and (b) at least about 35% of the cells that express VMAT1, or at least about 15% of the cells that express glucagon (e.g., as measured by flow cytometry). In some embodiments, the present disclosure provides a composition comprising an in vitro cell population, the cell population comprising at least about 35% of the cells that express C-peptide and do not express VMAT1; and (i) at least about 35% of the cells that express VMAT1, and / or (ii) at least about 15% of the 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% of the cells that express VMAT1 and at least about 20% of the cells that express glucagon.

[0108]

[0125] In some examples, provided 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.

[0109]

[0126] In some examples, a composition comprising a cell population, wherein (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 the cell population 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; (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%, 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 cell population express somatostatin but do not express glucagon are provided herein.

[0110]

[0127] In one example, a composition comprising a cell population, wherein (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 the cell population 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) 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 cell population express somatostatin but do not express glucagon is provided herein.

[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 at an early stage of the time when PDX1-positive, NKX6.1-positive pancreatic progenitor cells are contacted with a differentiation factor that directs the differentiation of the PDX1-positive, NKX6.1-positive pancreatic progenitor cells into NKX6.1-positive, ISL1-positive endocrine cells. In some examples, 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, a RA signaling pathway activator, and a 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 modification compound for 1 to 2 days, thereby obtaining a second transformed cell population comprising NKX6.1-positive, ISL1-positive endocrine cells.

[0112] Method for producing endocrine cells

[0129] In one aspect, the disclosure relates to compositions and methods for producing endocrine cells from pancreatic progenitor cells or precursors. One exemplary detailed protocol for producing endocrine cells to provide at least one SC-β cell is described in US Patent Application Publication Nos. 2015 / 0240212 and 2015 / 0218522, which are hereby incorporated by reference in their entireties.

[0113]

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

[0114]

[0131] In some examples, the method comprises contacting, during a first period, 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 γ-secretase inhibitor, a growth factor from the TGFβ superfamily, a growth factor from the FGF family, a RA signaling pathway activator, and a SHH pathway inhibitor; and, after the first period, during a second period, 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 γ-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 modification compound. In some examples, the first composition comprises a PKC activator, a γ-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 a SHH pathway inhibitor. In some examples, the second composition comprises a PKC activator, a γ-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 modification compound.

[0115]

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

[0116]

[0133] In some examples, the method further comprises contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a composition comprising a PKC activator. For example, the method comprises: (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, a RA signaling pathway activator, and a SHH pathway inhibitor for 1 to 2 days to obtain 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 modification compound for 1 to 2 days to obtain a second transformed cell population comprising NKX6.1-positive, ISL1-positive endocrine cells. In some examples, the method further comprises 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 modification compound to produce 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, when measured by flow cytometry, comprises at least about 4% of the cells expressing somatostatin, at least about 15% of the cells expressing glucagon, at least about 35% of the cells expressing VMAT1, or at least about 40% of the cells expressing C-peptide. 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% fewer cells expressing VMAT1, or at least about 10% more cells expressing C-peptide compared to the corresponding cell population produced without contacting with a PKC activator. In some examples, a 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% fewer cells expressing VMAT1, or at least about 20% more cells expressing C-peptide compared to the corresponding cell population produced without contacting with a PKC activator.

[0118]

[0135] In one aspect, 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 a 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 γ-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 a 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 γ-secretase inhibitor, and one or more 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 modification compound, thereby producing a third cell population.In some examples, the method comprises: (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 a SHH pathway inhibitor, thereby producing a first cell population; (b) contacting the first cell population with a PKC activator and a γ-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 a SHH pathway inhibitor, thereby producing a second cell population; (c) contacting the second cell population with a PKC activator, a γ-secretase inhibitor, and one or more 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 modification compound, thereby producing a third cell population. In some examples, the method further comprises: (d) contacting the third cell population with 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 modification compound, thereby producing a fourth cell population. In some examples, step (d) comprises contacting the third cell population with a PKC activator.

[0119]

[0136] In one aspect, 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 a SHH pathway inhibitor, thereby producing a first cell population; (b) contacting the first cell population with a PKC activator, which is a benzolactam derivative, 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 a SHH pathway inhibitor, thereby producing a second cell population; (c) contacting the second cell population with a PKC activator, a γ-secretase inhibitor, and one or more 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 modification 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 includes 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, 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 modification compound, thereby producing a fourth cell population. In some examples, step (d) of producing the fourth cell population does not include contacting the third cell population with a PKC activator. In some examples, step (d) of producing the fourth cell population does not include contacting the third cell population with a γ-secretase inhibitor. In some examples, step (d) of producing the fourth cell population does not include contacting the third cell population with a SHH pathway inhibitor.In some examples, step (d) of producing a 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 includes (e) contacting the fourth cell population with 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 modification compound, thereby producing a fifth cell population. In some examples, step (e) includes contacting the fourth cell population with a PKC activator.

[0121]

[0138] In one aspect, 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 a 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, a RA signaling pathway activator, and a 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 γ-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 modification 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 modification compound, thereby producing a fourth cell population; (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 modification 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) for producing the first cell population in the method disclosed herein is performed over about 1, 2, 3, 4, 5, or 6 days. In some examples, step (a) for producing the first cell population is performed over 3 to 5 days, such as 3 to 4 days, 4 to 5 days, about 3 days, about 4 days, or about 5 days. In some examples, step (a) for producing the first cell population is performed over 4 days. In some examples, step (b) for producing the second cell population in the method disclosed herein is performed over 1, 2, 3, or 4 days. In some examples, step (b) for producing the second cell population is performed over 1 to 3 days, such as 1 to 2 days, 2 to 3 days, about 1 day, about 2 days, or about 3 days. In some examples, step (b) for producing the second cell population is performed over 2 days. In some examples, step (c) for producing the third cell population in the method disclosed herein is performed over 1, 2, 3, or 4 days. In some examples, step (c) for producing the third cell population is performed over 1 to 3 days, such as 1 to 2 days, 2 to 3 days, about 1 day, about 2 days, or about 3 days. In some examples, step (c) for producing the third cell population is performed over 2 days. In some examples, step (d) for producing the fourth cell population in the method disclosed herein is performed over 1, 2, 3, 4, 5, 6, or 7 days. In some examples, step (d) for producing the fourth cell population is performed over 4 to 6 days, such as 5 to 6 days, 4 to 5 days, about 4 days, about 5 days, or about 6 days. In some examples, step (d) for producing the fourth cell population is performed over 5 days. In some examples, step (e) for producing the fifth cell population in the method disclosed herein is performed over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days. In some examples, step (d) for producing the fourth cell population is performed over 10 to 12 days, such as 10 to 11 days, 11 to 12 days, about 10 days, about 11 days, or about 12 days.

[0123]

[0140] In some examples, the second cell population includes PDX1-positive and NKX6.1-positive cells. In some examples, the fourth cell population includes PDX1-positive, NKX6.1-positive, and ISL1-positive cells. In some examples, the fifth cell population includes 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 the first cell population in the methods 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, a RA signaling pathway activator, and a SHH pathway inhibitor. In some examples, the step of producing the second cell population in the methods 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, a RA signaling pathway activator, and a SHH pathway inhibitor. In some examples, the step of producing the third cell population in the methods 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, 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 modification compound. In some examples, the step of producing the fourth cell population in the methods provided herein comprises contacting the third cell population with serum albumin protein, 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 modification compound. In some examples, the ROCK inhibitor used for the methods provided herein is thiazovivin. In some examples, the growth factor from the TGFβ superfamily used for the step of producing the first cell population and / or the second cell population in the methods provided herein is activin A. In some examples, the growth factor from the FGF family used for the step of producing the first cell population and / or the second cell population in the methods provided herein is KGF. In some examples, the RA signaling pathway activator used for the step of producing the first cell population, the second cell population, and / or the third cell population in the methods provided herein is retinoic acid.In some examples, the SHH pathway inhibitor for use in the step of producing the first cell population, the second cell population, and / or the third cell population in the methods provided herein is Sant-1. In some examples, the PKC activator for use in the step of producing the second cell population, the third cell population, and / or the fourth cell population in the methods 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 γ-secretase inhibitor for use in the step of producing the second cell population and / or the third cell population in the methods provided herein is XXI. In some examples, the TGF-β signaling pathway inhibitor for use in the step of producing the third cell population and / or the fourth cell population in the methods provided herein is ALK5i. In some examples, the growth factor from the EGF family for use in the step of producing the third cell population in the methods provided herein is betacellulin. In some examples, the TH signaling pathway activator for use in the step of producing the third cell population and / or the fourth cell population in the methods provided herein is T3, GC-1 or a thyroid hormone derivative. In some examples, the protein kinase inhibitor for use in the step of producing the third cell population and / or the fourth cell population in the methods provided herein is staurosporine. In some examples, the BMP signaling pathway inhibitor for use in the step of producing the third cell population and / or the fourth cell population in the methods provided herein is LDN193189 or DMH-1. In some examples, the epigenetic modification compound for use in the step of producing the third cell population and / or the fourth cell population in the methods 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 considered herein during the transition between the differentiation of PDX1-positive, NKX6.1-positive pancreatic progenitor cells and the 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 considered herein during the transition between the differentiation of PDX1-positive, NKX6.1-positive pancreatic progenitor cells and the differentiation of NKX6.1-positive, ISL1-positive endocrine cells is at most 2 days, 3 days, or 4 days. In some examples, the treatment with a PKC activator considered herein during the transition between the differentiation of PDX1-positive, NKX6.1-positive pancreatic progenitor cells and the differentiation of NKX6.1-positive, ISL1-positive endocrine cells is 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 include PDX1-positive, NKX6.1-negative cells. In some embodiments, the PKC activator is contacted with the cell population, and the cells include PDX1-positive, NKX6.1-positive cells. In some embodiments, the PKC activator is contacted with the cell population, and the cells include insulin-positive cells. In some embodiments, the PKC activator is contacted with the cell population at each of the following differentiation stages: when the cells include PDX1-positive, NKX6.1-negative cells; when the cells include PDX1-positive, NKX6.1-positive cells; and when the cells include insulin-positive cells. In some embodiments, the same type of PKC activator (e.g., phorbol ester or 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 that includes PDX1-positive, NKX6.1-negative cells, and a phorbol ester (e.g., PDBU) is administered to a cell population that includes PDX-positive, NKX6.1-positive cells during the same differentiation protocol. In some embodiments, one or more different PKC activators (e.g., phorbol ester and 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 that includes PDX1-positive, NKX6.1-negative cells, and a benzolactam derivative (e.g., TPPB) is administered to a cell population that includes PDX-positive, NKX6.1-positive cells during the same differentiation protocol.

[0127]

[0144] In some examples, non-limiting examples of PKC activators for 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 comprising 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 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, or 1000 nM. In some examples, the PKC activator is at a concentration of at most about 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, or 1000 nM. In some examples, the PKC activator is at a concentration of about 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, or 1000 nM. In some examples, the PKC activator is at a concentration of about 500 nM.

[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 includes XXI. In some examples, the gamma secretase inhibitor is contacted with a cell population comprising 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 aspects, cell compositions comprising SC-β cells, SC-α cells, SC-δ cells, and SC-EC cells are provided herein. In some examples, the cell compositions provided herein have a desired amount (e.g., percentage) of SC-β cells, SC-α cells, and SC-δ cells, and a limited amount of SC-EC cells. In some examples, the cell composition of the cells is similar to that of natural 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 both in vitro and in vivo. In some embodiments, the GSIS response is similar to 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 is similar to the GSIS response of endogenous human islets to multiple glucose challenges. In some embodiments, the GSIS response is observed immediately after transplanting the cells into a human or an animal. In some embodiments, the GSIS response is observed within approximately 24 hours after transplanting the cells into a human or an animal. In some embodiments, the GSIS response is observed within approximately one week after transplanting the cells into a human or an animal. In some embodiments, the GSIS response is observed within approximately two weeks after transplanting the cells into a human or an animal. In some embodiments, the stimulation index of the cells, characterized by the ratio of insulin secreted in response to a high glucose concentration compared to a low glucose concentration, is similar to the stimulation index of endogenous mature pancreatic β cells. In some embodiments, the SC-β cells exhibit a stimulation index greater than 1. In some embodiments, the SC-β cells exhibit a stimulation index greater than or equal to 1. In some embodiments, the SC-β cells exhibit a stimulation index greater than 1.1. In some embodiments, the SC-β cells exhibit a stimulation index greater than or equal to 1.1. In some embodiments, the SC-β cells exhibit a stimulation index greater than 2. In some embodiments, the SC-β cells exhibit a stimulation index greater than or equal to 1.In some embodiments, the SC-β 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 γ-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 γ-secretase inhibitor. In some embodiments, the present disclosure provides 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 γ-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 γ-secretase inhibitor is DAPT or XXI.

[0132]

[0149] In one aspect, 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 γ-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 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% 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 γ-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 of the TGFβ superfamily. In some embodiments, the growth factor of the TGFβ superfamily is activin A.

[0133]

[0150] In one aspect, the present disclosure provides an in vitro composition 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 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 a 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 one aspect, 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; and a γ-secretase inhibitor. In some embodiments, the γ-secretase inhibitor is XXI. In some embodiments, the γ-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 one aspect, the present disclosure provides a population of in vitro differentiated cells 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. In some embodiments, 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 embodiments, 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 γ-secretase inhibitor. The γ-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 modification compound, a growth factor from the EGF family, a RA signaling pathway activator, a SHH pathway inhibitor, a protein kinase inhibitor, a ROCK inhibitor, and a BMP signaling pathway inhibitor. In some examples, the composition also comprises serum albumin protein.

[0144]

[0161] In some examples, the compositions provided herein comprise serum albumin protein, a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, an epigenetic modification compound, a 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 thiazobabine. 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 growth factor of the EGF family 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 modification compound is DZNep.

[0146]

[0163] In some examples, 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 examples, 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 examples, 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%, 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 compositions of the present disclosure have at most about 35% of the cells that express VMAT1 as measured by flow cytometry. In some examples, the cell compositions of the present disclosure have at most about 35% of the 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 the 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% of the cells that express VMAT1 and do not express C-peptide as measured by flow cytometry. In some examples, the cell composition has about 35%, 32%, 31%, 30%, 28%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, or 15% of the 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 25%, about 17% to about 22%, about 18% to about 20% of the cells that express VMAT1 and do not express C-peptide as measured by flow cytometry.

[0148]

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

[0149]

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

[0150]

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

[0151]

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

[0152]

[0169] In some examples, a composition comprising a cell population, wherein (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 the cell population 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) 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 cell population express somatostatin but do not express glucagon are provided herein.

[0153]

[0170] In some examples, a composition comprising a cell population, wherein (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 the cell population 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) 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 cell population express somatostatin but do not express glucagon are provided herein.

[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 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 are differentiated. For example, a cell culture or cell population can be produced that comprises at least about 5 SC - β cells for every about 95 insulin - positive endocrine cells or their precursors. In other embodiments, a cell culture or cell population can be produced that comprises at least about 95 SC - β cells for every about 5 insulin - positive endocrine cells or their precursors. Additionally, cell cultures or cell populations comprising SC - β cells relative to insulin - positive endocrine cells or their precursors at other ratios are contemplated. For example, a composition can be generated that comprises at least about 1 SC - β cell for every about 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 10 insulin - positive endocrine cells or their precursors.

[0156]

[0173] In some cases, the cell populations or cell clusters disclosed herein, such as cell populations or cell clusters isolated without passing through 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, such as 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 passing through 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] The cell sorting described herein can refer to a 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 signaling proteins, or any combination thereof. In some cases, cell sorting includes subjecting the cells to flow cytometry. Flow cytometry can be a biophysical technique based on lasers or impedance. During flow cytometry, the cells can be suspended in a fluid stream and passed through an electronic detection device. In fluorescence-activated cell sorting (FACS), which is a type of flow cytometry, physical separation is performed based on one or more parameters of the optical properties of the cells (e.g., emission wavelength upon laser excitation), thereby enabling purification of the target cells using flow cytometry. As described herein, an unsorted cell cluster can be a cell cluster formed by a plurality of cells that have not been subjected to an active cell sorting process, such as flow cytometry. In some cases, the flow cytometry discussed herein can be based on one or more signal peptides expressed within the cell. For example, a cell cluster can include cells that express 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 within the cell. For example, a cell cluster 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, the expression of GFP within these cells can indicate insulin expression within the cell. Thus, the GFP signal can be a marker for pancreatic β cells. In some cases, the cell sorting described herein can include magnetically activated flow cytometry, which uses magnetic antibodies or other ligands to label different types of cells and can utilize differences in magnetic properties for cell sorting.

[0158]

[0175] The percentage of cells expressing one or more specific markers such as PDX1, NKX6.1, insulin, NGN3, or CHGA described herein can be a percentage value detected using techniques such as flow cytometry assays. In some cases, during a flow cytometry assay, the cell population or cell cluster discussed herein is dispersed into a single cell suspension by incubation in a digestive enzyme such as trypsin or TrypLE™ Express. 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. Incubation can then be performed 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 techniques (fluorescence microscopy, atomic force microscopy), karyotyping, isozyme analysis, DNA characterization, and virus susceptibility.

[0159]

[0176] In some aspects, the disclosure relates to a composition comprising a population of glucose-responsive insulin-secreting cells, wherein the cells secrete a greater amount of insulin when induced with KCl (e.g., from 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 secretes at least 1.5-fold, 2-fold, 2.5-fold, 3-fold more insulin when induced with KCl compared to the amount of insulin secreted when induced with glucose.

[0160]

[0177] In some embodiments, the present disclosure relates to a composition comprising a population of glucose-responsive insulin-secreting cells, wherein the cells secrete a greater amount of insulin when induced with KCl and / or glucose in the presence of a signaling factor as compared to equivalent cells in the absence of the signaling factor. In some embodiments, the cells secrete a greater amount of insulin in the presence of high glucose but do not secrete 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 embodiments, the present disclosure relates to a composition comprising a population of differentiated pancreatic progenitor cells, wherein the population comprises at least 60% pancreatic β-cells as determined by flow cytometry. In some embodiments, the population comprises at least 65%, 70%, 75%, 80%, 85%, or 90% pancreatic β-cells. In some embodiments, the population comprises a higher percentage of pancreatic β-cells when contacted with the components of a given basal medium as compared to an equivalent population not contacted with the components 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, such as performing glucose-stimulated insulin secretion in vitro, resembling human pancreatic islet β-cells in gene expression and ultrastructure, secreting human insulin when transplanted into mice, improving hyperglycemia, and providing a new platform for cell therapy (e.g., transplantation into subjects in need of 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 when reconstructing islets). Stem Cells and Reprogramming

[0180] The use of stem cells to generate SC-β cells (e.g., mature pancreatic β cells or β-like cells) or their precursors is provided herein. In one embodiment, a protocol similar to that described in U.S. Patent Application Publication Nos. 2015 / 0240212 and 2015 / 0218522, each of which is incorporated herein by reference in its entirety, may be used to provide at least one SC-β cell, using embryonic cells instead of, or in addition to, stem cells. Suitable embryonic cells can be prepared, for example, from primordial germ cells present in human fetal material collected about 8 to 11 weeks after the last menstrual period. Exemplary 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] Compositions and methods for producing SC-β cells (e.g., pancreatic β cells), as well as pancreatic α cells, and / or pancreatic δ cells are provided herein. In some embodiments, the disclosure provides a method for producing a cell population enriched for pancreatic α cells. In some embodiments, the disclosure provides a method for producing a cell population enriched for pancreatic δ cells.

[0164]

[0182] Generally, at least one SC-β cell or its precursor, e.g., pancreatic progenitor cells generated by the methods disclosed herein, can include a mixture or combination of various cells, such as primitive gut tube 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 mixtures of pluripotent cells or stem cells, etc.

[0165]

[0183] To differentiate stem cells or pluripotent cells into a desired differentiation stage, at least one pancreatic α, β, and / or δ cell or a precursor thereof can be generated according to any suitable culture protocol. In some embodiments, at least one pancreatic α, β, and / or δ cell or a precursor thereof is generated by culturing at least one pluripotent cell under time and conditions suitable for differentiating at least one pluripotent cell into at least one pancreatic α, β, and / or δ cell or a precursor thereof.

[0166]

[0184] In some embodiments, at least one pancreatic α, β, and / or δ cell or a precursor thereof is a substantially pure population of pancreatic α, β, and / or δ cells or a precursor thereof. In some embodiments, the population of pancreatic α, β, and / or δ cells or a precursor thereof comprises a mixture of pluripotent cells or differentiated cells. In some embodiments, the population of pancreatic α, β, and / or δ cells or a precursor thereof is substantially free of or lacks embryonic stem cells or pluripotent cells or iPS cells.

[0167]

[0185] In some embodiments, somatic cells, such as fibroblasts, can be isolated from a subject, for example, as a tissue biopsy, such as a skin biopsy, and reprogrammed into induced pluripotent stem cells for further differentiation to generate at least one pancreatic α, β, and / or δ cell or a precursor thereof for use in the compositions and methods described herein. In some embodiments, somatic cells, such as fibroblasts, are maintained in culture by methods known to those skilled in the art and, in some embodiments, are expanded prior to being converted into pancreatic α, β, and / or δ cells by the methods disclosed herein.

[0168]

[0186] In some embodiments, at least one pancreatic α, β, and / or δ cell or a precursor thereof is maintained in culture by methods known to those skilled in the art and, in some embodiments, is expanded prior to being converted into pancreatic α, β, and / or δ cells by the methods described herein.

[0169]

[0187] Furthermore, at least one pancreatic α, β, and / or δ cell or its precursor, e.g., a pancreatic precursor, may be from any mammalian species, and non-limiting examples include cells from the Muridae, Bovidae, Cercopithecidae, Suidae, Equidae, Ovine, or human. For clarity and simplicity, the description of the methods herein refers to at least one pancreatic α, β, and / or δ cell or its precursor of a mammal, 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 its precursor. In some embodiments, at least one pancreatic α, β, and / or δ cell or its precursor is 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 mean a cell that has the ability to self-renew and produce differentiated cell types (e.g., plant stem cells, vertebrate stem cells) (Morrison et al., (1997) Cell 88:287-298). In the context of cytogenesis, the adjectives "differentiated" or "differentiating" are relative terms. A "differentiated cell" can be a cell that has progressed further downstream in the developmental pathway compared to the cell with which it is being compared. Thus, pluripotent stem cells can differentiate into lineage-restricted progenitor cells (e.g., mesenchymal stem cells), which can then differentiate into more 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 a characteristic role in certain tissue types and may or may not retain the ability to further proliferate. Stem cells can be characterized by the presence and absence of specific markers (e.g., proteins, RNAs, etc.). Stem cells can also be identified by both in vitro and in vivo functional assays, particularly assays related to the ability of stem cells to give rise to multiple differentiated progeny. In one embodiment, the host cell is an adult stem cell, somatic stem cell, non-embryonic stem cell, embryonic stem cell, hematopoietic stem cell, and includes pluripotent stem cells, and trophoblast stem cells.

[0171]

[0189] The stem cells of interest, e.g., stem cells that can be used in the methods provided herein, can include pluripotent stem cells (PSCs). As used herein, the term "pluripotent stem cell" or "PSC" means a stem cell that can generate all cell types of a living organism. Thus, PSCs can give rise to cells of all germ layers of a living organism (e.g., the endoderm, mesoderm, and ectoderm of a vertebrate). Pluripotent cells can form teratomas and contribute to tissues of the ectoderm, mesoderm, or endoderm of a living organism. Pluripotent stem cells of a plant can give rise to all cell types of 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 of an animal can be induced in several different ways. For example, embryonic stem cells (ESCs) can be induced 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 induced 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). Since the term PSC can mean pluripotent stem cells regardless of their origin, the term PSC can include the terms ESC and iPSC, as well as the term fetal embryonic stem cells (EGSCs), which are 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 also be induced from somatic cells.

[0173]

[0191] Embodiments of the present disclosure relate to the use of ESCs for the production of pancreatic β cells or their precursors. “Embryonic stem cells (ESCs)” can mean PSCs isolated from an embryo, typically from the inner cell mass of a blastocyst. ESC lines such as 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) are listed in the NIH Human Embryonic Stem Cell Registry. The stem cells of interest also include embryonic stem cells from other primates, such as rhesus stem cells and marmoset stem cells. Stem cells can be obtained from any mammalian species, such as humans, horses, cows, pigs, dogs, cats, rodents, such as mice, rats, hamsters, primates, etc. (Thomson et al., (1998) Science 282:1145; Thomson et al., (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-cytoplasm ratio, distinct boundaries, and prominent nuclei. Additionally, ESCs can express SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase, but not SSEA-1. Examples of methods for producing 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 by reference in its entirety.Methods for growing hESCs in an undifferentiated form are described in WO 99 / 20741, WO 01 / 51616, and WO 03 / 020920, each of which is incorporated herein by reference in its entirety.

[0174]

[0192] "Fetal embryonic stem cells (EGSCs) or fetal embryonic cells" or "EG cells" can mean pluripotent stem cells derived from germ cells and / or germ cell progenitor cells, such as primordial germ cells, such as germ cells that can become sperm and eggs. Fetal embryonic cells (EG cells) are thought to have properties similar to those of the above-described 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 by reference in its entirety.

[0175]

[0193] Embodiments of the present disclosure relate to the use of iPSCs for the production of pancreatic α, β, and / or δ cells or their precursors. "Induced pluripotent stem cells" or "iPSCs" can mean PSCs derived from cells that are not PSCs (e.g., cells differentiated with respect to PSCs). iPSCs can be derived from a variety of different cell types, including terminally differentiated cells. iPSCs have an ES cell-like morphology and can grow as flat colonies with a large nucleus-cytoplasm ratio, distinct boundaries, and prominent nuclei. Further, iPSCs can express one or more important pluripotency markers known to those of skill 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 producing and characterizing iPSCs can be found, for example, in U.S. Patent Publications Nos. 2009 / 0047263, 2009 / 0068742, 2009 / 0191159, 2009 / 0227032, 2009 / 0246875, and 2009 / 0304646, each of which is incorporated herein by reference in its entirety. Generally, to produce iPSCs, reprogramming factors known in the art for reprogramming somatic cells to pluripotent stem cells (e.g., Oct4, SOX2, KLF4, MYC, Nanog, Lin28, etc.) are provided to the somatic 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 mean any cell in an organism that does not normally occur in all types of cells in the organism in the absence of experimental manipulation. In other words, somatic cells are fully differentiated and thus can be cells that do not naturally produce cells of all three germ layers of the body, e.g., cells of the ectoderm, mesoderm, and endoderm. For example, somatic cells include neurons and neural progenitor cells, which can naturally give rise to all or some cell types of the central nervous system but cannot give rise to cells of the mesoderm or endoderm lineages.

[0177]

[0195] In certain examples, the stem cells may be undifferentiated (e.g., cells not committed to a particular lineage) prior to exposure to at least one differentiation factor or composition by the methods disclosed herein, while in other examples, it may be desirable to differentiate the stem cells into one or more intermediate cell types prior to 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, and these characteristics can be used to distinguish these cells from differentiated cells of fetal or adult origin. In some examples, undifferentiated cells can appear microscopically in two dimensions in colonies of cells having a high nucleus / cytoplasm ratio and prominent nuclei. The stem cells may be by themselves (e.g., in the substantial absence of any undifferentiated cells), or may be used in the presence of differentiated cells. In certain examples, the stem cells can be cultured in the presence of suitable nutrients and optionally in the presence of other cells in order to grow and optionally differentiate. For example, fetal fibroblasts or fibroblast-like cells may be present during the culture to assist in the growth of the stem cells. Fibroblasts may be present during one stage of the growth of the stem cells, but need not be present during all stages. For example, fibroblasts may be added to the culture of the stem cells during the first culture stage and need not be added to the culture of the stem cells during one or more subsequent culture stages.

[0178]

[0196] The stem cells used in all aspects of the present 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 have the characteristic of being able to generate progeny consisting of all or at least one derivative of various cell types, such as the three germ layers (endoderm, mesoderm, and ectoderm), under appropriate conditions. These cell types can be provided in the form of established cell lines or obtained directly from primary embryonic tissue and used immediately for differentiation. Cells listed in the NIH Human Embryonic Stem Cell Registry, such as 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, FISF-6 (University of California at San Francisco); and H1, H7, H9, H13, H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute) are included. 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 include the step of destroying 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 does not include the step of destroying a human embryo.

[0179]

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

[0180]

[0198] Among the stem cells that can be used in the methods provided herein are human embryonic stem (hES) cells described by Thomson et al., (1998) Science 282:1145, embryonic stem cells from other primates such as rhesus 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 germ (hEG) cells (Shambloft et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). Also included in the methods provided herein are stem cells involved in lineages such as mesenchymal stem cells and other early heart development cells (see Reyes et al., (2001) Blood 98:2615-2625, Eisenberg and Bader, (1996) Circ Res. 78(2):205-16, etc.). Stem cells can be obtained from any mammalian species, such as human, horse, cow, pig, dog, cat, rodents such as mouse, rat, hamster, primates, etc. In some embodiments, the human embryo is not destroyed for use as a source of pluripotent cells in the methods and compositions disclosed herein. In some embodiments, the human embryo is not destroyed for use as a source of pluripotent cells 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 immobilized (e.g., mobilized) circulating peripheral blood can be removed from the subject. In one embodiment, the stem cells can be reprogrammed stem cells, such as stem cells derived from somatic or differentiated cells. In such embodiments, the dedifferentiated stem cells can be, but are not limited to, for example, neoplastic cells, tumor cells, and cancer cells, or induced and reprogrammed cells such as induced pluripotent stem cells or iPS cells.

[0182]

[0200] In some embodiments, the pancreatic α, β, and / or δ cells described herein can be derived from one or more of hair cells, keratinocytes, gonadotropin-producing cells, adrenocorticotropic hormone-producing cells, thyroid-stimulating hormone-producing cells, somatotropin-producing cells, mammotropin-producing cells, 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), epithelial cells, pinealocytes, lung cells (e.g., type I and type II pneumocytes), Clara cells, goblet cells, G cells, D cells, ECL cells, gastric chief cells, parietal cells, foveolar 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), eosinophils, urothelial cells, osteoblasts, osteocytes, chondroblasts, chondrocytes, fibroblasts, fibrocytes, myoblasts, muscle cells, muscle satellite cells, tendon cells, cardiomyocytes, adipoblasts, adipocytes, Cajal interstitial cells, angioblasts, endothelial cells, mesangial cells (e.g., intraglomerular mesangial cells and extraglomerular mesangial cells), juxtaglomerular cells, macula densa cells, stromal cells, interstitial cells, telocytes, simple epithelial cells, podocytes, renal proximal tubule brush border cells, Sertoli cells, Leydig cells, granulosa cells, peg cells, germ cells, sperm, eggs, lymphocytes, myeloid cells, endothelial progenitor cells, endothelial stem cells, angioblasts, mesoangioblasts, pericytes, splenocytes (e.g., T lymphocytes, B lymphocytes, dendritic cells, macrophages, leukocytes), trophoblast stem cells, or any combination thereof.

[0183] Reprogramming

[0201] As used herein, the term "reprogramming" can mean a process of modifying or reversing the differentiated state of a somatic cell. The cell can be partially or terminally differentiated prior to reprogramming. Reprogramming can include a complete reversal of the differentiated state of a somatic cell to a pluripotent cell. Such a complete reversal of differentiation can generate induced pluripotent (iPS) cells. Reprogramming as used herein also can include a partial reversal of the differentiated state of a cell to a totipotent state cell or a somatic cell that is not, for example, pluripotent or totipotent but has lost one or more specific characteristics of the original differentiated cell from which it originated, for example, direct reprogramming of a differentiated cell into various somatic cell types. Reprogramming can include at least some modification, for example reversal, of heritable patterns such as nucleic acid modifications (e.g., methylation), chromatin condensation, epigenetics, genome imprinting, etc. that occur during cell differentiation as the zygote grows into an adult.

[0184]

[0202] As used herein, the term "reprogramming factor" can mean a molecule associated with "reprogramming" of a cell, i.e., differentiation, and / or dedifferentiation, and / or transdifferentiation by which the 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 mean the process by which a less specialized cell (e.g., a more primitive cell with higher cellular potency) becomes a more specialized cell type (e.g., a less primitive cell with lower cellular potency), the term "dedifferentiation" means the process by which a more specialized cell becomes a less specialized cell type (e.g., a more primitive cell with higher cellular potency), and the term "transdifferentiation" may mean the process by which a cell of a particular cell type is converted into another cell type without a significant change in its level of "cellular potency" or "primitiveness". Without wishing to be bound by theory, a cell is considered to "transdifferentiate" when it is converted from one lineage-committed cell type or terminally differentiated cell type to another lineage-committed cell type or terminally differentiated cell type without a significant change in its level of "cellular potency" 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, namely the endoderm (inner lining of the stomach, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, urogenital), or ectoderm (epithelial tissue and nervous system), and thus have high cell differentiation potential. Totipotent cells (e.g., stem cells or certain types of induced stem cells) have the ability to give rise to cells from many but a limited number of lineages (e.g., hematopoietic stem cells, cardiac stem cells, or neural stem cells, etc.) and have a relatively lower cell differentiation potential than pluripotent cells. Lineage-committed or terminally differentiated cells may have an even lower cell differentiation potential. Specific examples of transdifferentiation known in the art include, for example, the conversion of fibroblast beta cells, or pancreatic exocrine cells to beta cells.

[0187]

[0205] Thus, cells can be differentiated into more primitive cells (e.g., terminally differentiated cells can be differentiated into totipotent or pluripotent cells), or cells can be dedifferentiated into less primitive cells (e.g., totipotent or pluripotent cells can be differentiated into lineage-committed cells 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) at, for example, a similar cell potency level. Thus, in one embodiment of the present disclosure, the inducing step of the present disclosure can reprogram, differentiate, dedifferentiate, and / or transdifferentiate the cells of the present disclosure. In one embodiment of the present disclosure, the inducing step of the present disclosure can reprogram the cells to transdifferentiate.

[0188]

[0206] Methods of reprogramming or inducing a particular type of cell to become another type of cell using one or more exogenous polynucleotide or polypeptide reprogramming factors, for example, by differentiation, dedifferentiation, and / or transdifferentiation, are known to those skilled in the art. Such methods may rely on the introduction of genetic material encoding one or more transcription factors or other polypeptides associated with 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. In some methods known to those skilled in the art, an exogenous polypeptide (e.g., a recombinant polypeptide) encoded by a reprogramming gene (such as the above genes) contacts the cell to induce, for example, the cells of the present disclosure. Those skilled in the art will recognize that other genes are also associated with cell reprogramming, and exogenous molecules (or functional fragments thereof) encoding such genes and the encoded polypeptides are also polynucleotide or polypeptide reprogramming factors (e.g., polynucleotides or polypeptides that subsequently affect the expression levels of other genes associated with cell reprogramming). For example, it has been shown that the introduction of an epigenetic gene silencer of an exogenous polynucleotide or polypeptide that reduces the inactivation of p53 increases the efficiency of inducing induced pluripotent stem cells (iPSCs). Therefore, exogenous polynucleotides or polypeptides encoding epigenetic silencers, as well as other genes or proteins that are thought to be directly or indirectly involved in increasing the efficiency of cell reprogramming or cell programming, are considered to constitute exogenous polynucleotide or polypeptide reprogramming factors. Those skilled in the art will recognize that there are other methods of affecting cell reprogramming, such as the introduction of RNAi molecules (or genetic material encoding RNAi molecules) that can knockdown the expression of genes involved in inhibiting cell reprogramming.Thus, any exogenous polynucleotide molecule or polypeptide molecule related to or promoting cellular reprogramming is to be understood as a reprogramming factor of the exogenous polynucleotides or polypeptides described herein.

[0189]

[0207] In some embodiments of the present disclosure, the method does not include the use of reprogramming factors that are not small molecules. However, the method may utilize "routine" tissue culture components such as media, serum, serum replacements, supplements, antibiotics, and others, for example, RPMI, renal epithelial basal medium (REBM), Dulbecco's modified Eagle medium (DMEM), MCDB131 medium, CMRL1066 medium, F12, fetal bovine 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 selenite, selenium, ethanolamine, human epidermal growth factor (hEGF), basic fibroblast growth factor (bFGF), hydrocortisone, epinephrine, normocin, penicillin, streptomycin, gentamicin, and amphotericin. It is recognized that 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 the present disclosure. These components are neither small molecules as defined herein nor reprogramming factors as defined herein.

[0190]

[0208] Accordingly, in one embodiment, the present disclosure does not include culturing cells with one or more exogenous polynucleotides or polypeptides as reprogramming factors. Thus, in one embodiment, the method of the present disclosure involves producing induced α, β, and / or δ cells, or otherwise inducing the cells of the present disclosure to differentiate, dedifferentiate, and / or transdifferentiate, for example, by introducing a transposon, a viral transgenic vector (such as a retroviral vector), a plasmid, an mRNA, an miRNA, a peptide, or a fragment of any of these molecules, without including the introduction of one or more exogenous polynucleotides or polypeptides as reprogramming factors.

[0191]

[0209] That is, in one embodiment, the method is performed in the absence of one or more exogenous polynucleotides or polypeptides as reprogramming factors. Thus, in one embodiment, in the method of the present disclosure, without adding a polypeptide transcription factor, other polypeptide factors specifically related to the induction of differentiation, dedifferentiation, and / or transdifferentiation, a polynucleotide sequence encoding a polypeptide transcription factor, a polynucleotide sequence encoding other polypeptide factors specifically related to the induction of differentiation, dedifferentiation, and / or transdifferentiation, an mRNA, an interfering RNA, a microRNA, and fragments thereof, it should be understood that small molecules (such as HDAC inhibitors) are utilized to reprogram the cells. Method for producing β cells derived 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, the thyroid, the liver, and the pancreas. A representative disease of the endoderm lineage is type 1 diabetes, which results from the destruction of insulin-producing β cells. The generation of functional β cells from human pluripotent stem cells (hPSCs) in vitro could be a practical and renewable cell source for replacement cell therapy for type 1 diabetes. Embryonic stem (ES) cells, which are produced from the inner cell mass of the embryo at the blastocyst stage, represent a promising cell source for transplantation of any damaged cell or cell-based therapy. These are maintained in culture, self-renew, and can proliferate infinitely as undifferentiated ES cells. ES cells can differentiate into all cell types of the body as ectoderm, mesoderm, and endoderm lineage cells or tissues. The main advantages of ES cells are stable self-renewal and the potential for differentiation in culture.

[0193]

[0212] The definitive endoderm is produced in vivo from the inner cell mass by the process of gastrulation during embryonic development, in which the epiblast cells are instructed to form the three germ layers. The definitive endoderm can give rise to diverse cells and tissues that contribute to vital organs such as pancreatic β cells, hepatocytes of the liver, alveolar cells of the lung, the thyroid, the thymus, and the epithelial lining of the alimentary and respiratory tracts. This is different from the primitive endoderm of extraembryonic tissues that can give rise to the visceral and parietal endoderm. Definitive endoderm derived from ES cells can theoretically become any endoderm derivative, and directing ES cells into the endoderm lineage is an essential requirement for producing therapeutic endoderm derivatives.

[0194]

[0213] Accurate patterning of the anterior-posterior axis of the embryonic endoderm can ultimately lead to the formation of the primitive gut tube. The primitive gut tube induced by the embryonic endoderm induces the pharynx, esophagus, stomach, duodenum, small intestine, and large intestine, as well as associated organs such as the pancreas, lungs, thyroid, thymus, parathyroid, and liver, along the anterior-posterior axis. The anterior part of the foregut of the primitive gut tube becomes the lungs, thyroid, esophagus, and stomach. The pancreas, liver, and duodenum originate from the posterior part of the foregut. The midgut and hindgut of the primitive gut tube give rise to the small intestine and large intestine. The anterior part of the foregut expresses the developmental markers NK2 homeobox (NKX2-1) and SRY (sex-determining region Y)-box 2 (SOX2), the posterior part of the foregut expresses the hematopoietically expressed homeobox (HHEX), pancreas and duodenum homeobox 1 (PDX1), one cut homeobox 1 (ONECUT1, known as HNF6), and hepatocyte nuclear factor 4 alpha (HNF4A), and the midgut / hindgut expresses caudal type homeobox 1 (CDX1), caudal type homeobox 2 (CDX2), and motor neuron and pancreas homeobox 1 (MNX1) (3, 19, 20).

[0195]

[0214] For the successful differentiation of pancreatic beta cells, it is necessary for the differentiated cells to synthesize and secrete a physiologically appropriate amount of insulin. An exemplary stepwise protocol directed to hPSC cell differentiation has been developed, involving a differentiation process that recapitulates the major stages of normal pancreatic endocrine development (e.g., the version A protocol of Example 1). The differentiation of hPSC cells into hormone-expressing pancreatic endocrine cells is carried out by passing the hPSC cells through the major stages consisting of embryonic development, differentiation into mesendoderm and embryonic endoderm, establishment of the primitive gut endoderm, patterning of the posterior foregut, and specification and maturation of pancreatic endoderm and endocrine precursors. Through these stages, hPSC cells can acquire the pancreatic endocrine phenotype and the ability to secrete glucose-responsive insulin in vitro.

[0196]

[0215] Generally, at least one pancreatic α, β, and / or δ cell or its precursor, e.g., a pancreatic progenitor cell generated 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 cells, or β-like cells), and / or other pluripotent cells or stem cells.

[0197]

[0216] At least one pancreatic α, β, and / or δ cell or its precursor can be generated according to any suitable culture protocol for differentiating stem cells or pluripotent cells to the desired differentiation stage. In some embodiments, at least one pancreatic α, β, and / or δ cell or its precursor is generated by culturing at least one pluripotent cell under time and conditions suitable for differentiating the at least one pluripotent cell into at least one pancreatic α, β, and / or δ cell or its precursor.

[0198]

[0217] In some embodiments, at least one pancreatic α, β, and / or δ cell or its precursor is a substantially pure population of pancreatic α, β, and / or δ cells or their precursors. In some embodiments, the population of pancreatic α, β, and / or δ cells or their precursors includes a mixture of pluripotent cells or differentiated cells. In some embodiments, the population of pancreatic α, β, and / or δ cells or their precursors is substantially free of or lacks embryonic stem cells or pluripotent cells or iPS cells.

[0199]

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

[0200]

[0219] In some embodiments, at least one pancreatic α, β, and / or δ cell or a precursor thereof is maintained in culture by methods known to those skilled in the art and, in some embodiments, is expanded before being converted into pancreatic α, β, and / or δ cells by the methods disclosed herein.

[0201]

[0220] Furthermore, at least one pancreatic α, β, and / or δ cell or a precursor thereof, such as a pancreatic precursor, may be from any mammalian species, and non-limiting examples include murine, bovine, simian, porcine, equine, ovine, or human cells. For clarity and simplicity, the methods described herein refer to at least one pancreatic α, β, and / or δ cell or a precursor thereof of a mammal, 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 a precursor thereof. In some embodiments, at least one SC-β cell or a precursor thereof is derived from a human individual.

[0202] Embryonic endoderm cells

[0221] Aspects of the present disclosure include definitive endoderm cells. Definitive endoderm cells as used herein can be derived from any source or produced according to any suitable protocol. In some aspects, pluripotent stem cells, such as iPSCs or hESCs, are differentiated into endoderm cells. In some aspects, endoderm cells (stage 1) are further differentiated, for example, 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 progenitor cells or insulin-positive endocrine cells (stage 5), and subsequently 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 of the 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 the differentiation of at least some of the pluripotent cells into definitive endoderm cells, and 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 to differentiate into definitive endoderm cells (e.g., alone or in combination with a WNT signaling pathway activator) 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 to differentiate into definitive endoderm cells (e.g., alone or in combination with a growth factor from the TGF-β superfamily) 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, the step of differentiating at least some of the pluripotent cells in the population into definitive endoderm cells is achieved by a process of contacting the population of pluripotent cells with i) activin A, and ii) CHIR99021 for a suitable period, such as about 2 days, about 3 days, about 4 days, or about 5 days, to induce the differentiation of at least some of the pluripotent cells in the population into definitive endoderm cells, and the definitive endoderm cells express at least one marker characteristic of definitive endoderm.

[0206]

[0225] In some examples, the method includes the step of differentiating pluripotent cells into definitive endoderm cells by contacting a population of pluripotent cells with a growth factor (such as activin A) from the TGF-β superfamily at a suitable concentration, 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. In some examples, the method includes the use of about 100 ng / mL of activin A for the differentiation of pluripotent cells into definitive endoderm cells. In some examples, the method includes the use of about 200 ng / mL of activin A for the differentiation of pluripotent cells into definitive endoderm cells.

[0207]

[0226] In some examples, the method includes differentiating pluripotent cells into definitive endoderm cells by contacting a population of pluripotent cells with a WNT signaling pathway activator (such as CHIR99021) at a suitable concentration, such as 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. In some examples, the method includes using about 2 μM of CHIR99021 for differentiating pluripotent cells into definitive endoderm cells. In some examples, the method includes using about 5 μM of CHIR99021 for differentiating pluripotent cells 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 in the definitive endoderm cells in a statistically significant amount relative to the pluripotent stem cells from which it was induced. 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 in a statistically significant amount relative to the pluripotent stem cells from which it was induced. 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 Zic1, Pax6, Flk1, and CD31 in a statistically significant amount relative to the pluripotent stem cells from which it was induced. In some examples, the definitive endoderm cells generated by the methods disclosed herein have a statistically significant amount of higher level of phosphorylation of Smad2 relative to the pluripotent stem cells from which it was induced. In some examples, the definitive endoderm cells generated by the methods disclosed herein have the ability to form intestinal tubes in vivo. In some examples, the definitive endoderm cells generated by the methods disclosed herein can differentiate into cells having a morphology characteristic of intestinal cells, and the cells having a morphology characteristic of intestinal cells express FoxA2 and / or Claudin6. In some examples, the definitive endoderm cells generated by the methods disclosed herein can further differentiate into cells of endodermal origin.

[0209]

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

[0210] Gut tube cells

[0229] Aspects of the disclosure include gut tube cells. Gut tube cells as used herein can be derived from any source or produced according to any suitable protocol. In some aspects, definitive endoderm cells differentiate into gut tube cells. In some aspects, gut tube cells further differentiate, for example, into PDX1-positive pancreatic progenitor cells, NKX6.1-positive pancreatic progenitor cells, Ngn3-positive endocrine progenitor cells, insulin-positive endocrine cells, and are subsequently induced or matured into SC-β cells.

[0211]

[0230] In some examples, gut tube cells can be obtained by differentiating at least some of the definitive endoderm cells in the population into gut tube 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 gut tube cells, and the gut tube cells express at least one marker characteristic of gut tube cells.

[0212]

[0231] Any growth factor from the FGF family that can induce embryonic endoderm cells to differentiate into primitive gut tube 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 includes keratinocyte growth factor (KGF). In some examples, at least one growth factor from the FGF family includes FGF2. In some examples, at least one growth factor from the FGF family includes FGF8B. In some examples, at least one growth factor from the FGF family includes FGF10. In some examples, at least one growth factor from the FGF family includes FGF21.

[0213]

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

[0214]

[0233] In some examples, the method includes differentiating embryonic endoderm cells into primitive gut tube cells by contacting the embryonic endoderm cells with a growth factor (e.g., KGF) from the FGF family at a suitable concentration, 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. In some examples, the method includes the use of about 50 ng / mL of KGF for the differentiation of embryonic endoderm cells into primitive gut tube cells. In some examples, the method includes the use of about 100 ng / mL of KGF for the differentiation of embryonic endoderm cells into primitive gut tube cells.

[0215] PDX1-positive pancreatic progenitor cells

[0234] Aspects of the present disclosure include PDX1-positive pancreatic progenitor cells. As used herein, PDX1-positive pancreatic progenitor cells can be derived from any source or produced according to any suitable protocol. In some aspects, foregut cells are differentiated into PDX1-positive pancreatic progenitor cells. In some aspects, 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 subsequently induced or matured into SC-β cells.

[0216]

[0235] In some aspects, PDX1-positive pancreatic progenitor cells can be obtained by differentiating at least some of the foregut cells in a population into PDX1-positive pancreatic progenitor cells, for example, by contacting the foregut 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 some of the foregut cells to differentiate into PDX1-positive pancreatic progenitor cells, and the PDX1-positive pancreatic progenitor cells express PDX1.

[0217]

[0236] In some aspects, PDX1-positive pancreatic progenitor cells can be obtained by differentiating at least some of the foregut cells in a population into PDX1-positive pancreatic progenitor cells, for example, by contacting the foregut 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 some of the foregut cells into PDX1-positive pancreatic progenitor cells, and the PDX1-positive pancreatic progenitor cells express PDX1.

[0218]

[0237] In some examples, PDX1-positive pancreatic progenitor cells can be obtained by differentiating at least some of the foregut cells in the population into PDX1-positive pancreatic progenitor cells. For example, foregut cells are contacted 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 the differentiation of at least some of the foregut cells into PDX1-positive pancreatic progenitor cells. The PDX1-positive pancreatic progenitor cells express PDX1.

[0219]

[0238] In some examples, PDX1-positive pancreatic progenitor cells can be obtained by contacting at least some of the foregut cells in the population 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, thereby differentiating them into PDX1-positive pancreatic progenitor cells. 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 some of the foregut cells in the population into PDX1-positive pancreatic progenitor cells. For example, foregut cells are contacted with i) at least one growth factor from the FGF family and ii) at least one retinoic acid (RA) signaling pathway activator to induce the differentiation of at least some of the foregut cells into PDX1-positive pancreatic progenitor cells. The PDX1-positive pancreatic progenitor cells express PDX1.

[0221]

[0240] Any BMP signaling pathway inhibitor that can induce (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) gut tube cells to differentiate into PDX1-positive pancreatic progenitor cells can be used in the methods provided herein. In some examples, the BMP signaling pathway inhibitor comprises LDN193189 or DMH-1. In some examples, the method comprises contacting gut tube 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 comprises contacting gut tube 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 definitive endoderm 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) to differentiate into PDX1-positive pancreatic progenitor cells can be used. In some examples, the growth factor from the TGF-β family comprises activin A. In some examples, the growth factor from the TGF-β family comprises activin A or GDF8. In some examples, the method comprises contacting definitive endoderm cells with a growth factor (e.g., activin A) from the TGF-β superfamily 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 (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) primitive gut tube cells to differentiate into PDX1-positive pancreatic progenitor cells can be used. In some examples, at least one growth factor from the FGF family comprises keratinocyte growth factor (KGF). In some examples, 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 comprises contacting primitive gut tube cells with a growth factor (e.g., KGF) from the FGF family 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 (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) gut tube cells to differentiate into PDX1-positive pancreatic progenitor cells can be used. In some examples, the SHH pathway inhibitor comprises Sant1. In some examples, the method comprises contacting gut tube cells with an SHH pathway inhibitor (e.g., Sant1) 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 0.3 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 (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) to differentiate into PDX1-positive pancreatic progenitor cells can be used. In some examples, the RA signaling pathway activator comprises retinoic acid. In some examples, the method comprises contacting primitive gut cells with an RA signaling pathway activator (e.g., retinoic acid) 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 2.5 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.

[0226]

[0245] Any PKC activator that can induce (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) primitive gut tube cells to differentiate into PDX1-positive pancreatic progenitor cells can be used. In some examples, the PKC activator includes PdBU. In some examples, the PKC activator includes TPPB. In some examples, the method comprises contacting primitive gut tube cells with a PKC activator (e.g., PdBU or TPPB) at a concentration of, for example, about 10 nM, 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM, 1 μM, 10 μM, about 20 μM, about 50 μM, about 75 μM, about 80 μM, about 100 μM, about 120 μM, about 140 μM, about 150 μM, about 175 μM, about 180 μM, about 200 μM, about 210 μM, about 220 μM, about 240 μM, about 250 μM, about 260 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 comprises contacting primitive gut tube 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 primitive gut tube cells are not treated with a PKC activator (e.g., PDBU).

[0227]

[0246] Any ROCK inhibitor that can induce primitive gut tube 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, a PKC activator, and at least one RA signaling pathway activator) to differentiate into PDX1-positive pancreatic progenitor cells can be used. In some examples, the ROCK inhibitor includes Thiazovivin, Y-27632, Fasudil / HA1077, or H-1152. In some examples, the ROCK inhibitor includes Y-27632. In some examples, the ROCK inhibitor includes Thiazovivin. In some examples, the method includes contacting primitive gut tube cells with a ROCK inhibitor (e.g., Y-27632 or Thiazovivin) 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, 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 tube cells in the population into PDX1-positive pancreatic progenitor cells, for example, by contacting the primitive gut tube cells with retinoic acid, KGF, Sant1, DMH-1, PdBU, thiazovivin, and activin A for a suitable period, such as 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 tube cells in the population into PDX1-positive pancreatic progenitor cells, for example, by contacting the primitive gut tube 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] Aspects of the present disclosure include NKX6.1-positive pancreatic progenitor cells. As used herein, NKX6.1-positive pancreatic progenitor cells can be derived from any source or produced according to any suitable protocol. In some aspects, PDX1-positive pancreatic progenitor cells differentiate into NKX6.1-positive pancreatic progenitor cells. In some aspects, NKX6.1-positive pancreatic progenitor cells further differentiate, for example, into Ngn3-positive endocrine progenitor cells or insulin-positive endocrine cells, and are subsequently induced or matured into SC-β cells.

[0230]

[0249] In some aspects, a method of generating NKX6.1-positive pancreatic progenitor cells from PDX1-positive pancreatic progenitor cells comprises contacting a population of cells comprising PDX1-positive pancreatic progenitor cells (e.g., under conditions that promote cell clustering and / or promote cell survival) with at least two β-cell differentiation factors 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, 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 a 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 inducing the differentiation of PDX1-positive pancreatic progenitor cells into at least some PDX1-positive, NKX6.1-positive pancreatic progenitor cells 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 RA signaling pathway activator, iv) a ROCK inhibitor, and v) at least one growth factor from the TGF-β superfamily. In some embodiments, 3, 4, or 5 days after the contact, 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 RA signaling pathway activator, 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 RA signaling pathway activator, iv) a ROCK inhibitor, and v) at least one growth factor from the TGF-β superfamily, and vi) a PKC activator, and optionally a gamma secretase inhibitor. In some examples, the PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting the PDX1-positive pancreatic progenitor cells with at least one growth factor from the FGF family under conditions that promote cell clustering. In some examples, the growth factor from the FGF family is KGF.

[0233]

[0252] In some examples, PDX1-positive pancreatic progenitor cells are generated from a population of pluripotent cells. In some examples, PDX1-positive pancreatic progenitor cells are generated from a population of iPS cells. In some examples, PDX1-positive pancreatic progenitor cells are generated from a population of ESC cells. In some examples, PDX1-positive pancreatic progenitor cells are generated from a population of embryonic endoderm cells. In some examples, PDX1-positive pancreatic progenitor cells are generated from a population of primitive gut tube 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 (e.g., 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, at least one growth factor from the FGF family includes keratinocyte growth factor (KGF). In some examples, 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 comprises contacting PDX1-positive pancreatic progenitor cells with a growth factor (e.g., KGF) from the FGF family 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 (e.g., 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) to differentiate into NKX6.1-positive pancreatic progenitor cells can be used in the methods provided herein. In some examples, the SHH pathway inhibitor comprises Sant1. In some examples, the method comprises contacting PDX1-positive pancreatic progenitor cells with an SHH pathway inhibitor (e.g., Sant1) 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 0.3 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 capable of inducing 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 comprises contacting the PDX1-positive pancreatic progenitor cells with an RA signaling pathway activator (e.g., retinoic acid) 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 2.5 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.

[0237]

[0256] Any ROCK inhibitor that can induce PDX1-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, an RA signaling pathway activator, and at least one growth factor from the TGF-β superfamily) to differentiate into NKX6.1-positive pancreatic progenitor cells can be used. In some examples, the ROCK inhibitor includes Thiazovivin, Y-27632, Fasudil / HA1077, or 14-1152. In some examples, the method includes contacting the PDX1-positive pancreatic progenitor cells with a ROCK inhibitor (e.g., Y-27632 or Thiazovivin) 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, 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 (e.g., alone or in combination with at least one growth factor from the FGF family, at least one SHH pathway inhibitor, an RA signaling pathway activator, and a ROCK inhibitor) to differentiate into NKX6.1-positive pancreatic progenitor cells can be used. In some examples, the activator from the TGF-β superfamily includes activin A or GDF8. In some examples, the method includes contacting PDX1-positive pancreatic progenitor cells with a growth factor (e.g., activin A) from the TGF-β superfamily 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, about 4.2 ng / mL, about 4.4 ng / mL, about 4.6 ng / mL, about 4.8 ng / mL, about 5 ng / mL, about 5.2 ng / mL, about 5.4 ng / mL, about 5.6 ng / mL, about 5.8 ng / mL, about 6 ng / mL, about 6.2 ng / mL, about 6.4 ng / mL, about 6.6 ng / mL, about 6.8 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, or about 50 ng / mL. In some examples, the method includes contacting PDX1-positive pancreatic progenitor cells with a growth factor (e.g., activin A) from the TGF-β superfamily at a concentration of about 5 ng / mL.

[0239]

[0258] In some examples, PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive pancreatic progenitor cells 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] Aspects of the disclosure include insulin-positive endocrine cells (e.g., NKX6.1-positive, ISL1-positive cells, or β-like cells). Insulin-positive endocrine cells as used herein can be derived from any source or produced according to any suitable protocol. In some aspects, NKX6.1-positive pancreatic progenitor cells differentiate into insulin-positive endocrine cells (e.g., NKX6.1-positive, ISL1-positive cells, or β-like cells). In some aspects, insulin-positive endocrine cells are further differentiated, for example, by induction or maturation into SC-β cells.

[0241]

[0260] In one aspect, a method of generating insulin - positive endocrine cells from NKX6.1 - positive pancreatic progenitor cells comprises contacting a population of cells comprising NKX6.1 - positive pancreatic progenitor cells (e.g., under conditions that promote cell clustering) with a) a TGF - β signaling pathway inhibitor, and b) a thyroid hormone signaling pathway activator, to induce the differentiation of at least one NKX6.1 - positive pancreatic progenitor cell in the population into an insulin - positive endocrine cell, where the insulin - positive endocrine cell expresses insulin. In some examples, the insulin - positive endocrine cells express 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 (e.g., alone or in combination with other β - cell differentiation factors such as a thyroid hormone signaling pathway activator) into insulin - positive endocrine cells can be used. In some examples, the TGF - β signaling pathway includes TGF - β type I receptor kinase signaling. In some examples, the TGF - β signaling pathway inhibitor includes Alk5 inhibitor II.

[0243]

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

[0244]

[0263] In some examples, the method includes contacting a cell population (e.g., NKX6.1-positive pancreatic progenitor cells) with at least one additional factor. 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) a 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 a cell population (e.g., NKX6.1-positive pancreatic progenitor cells) with at least one additional factor. 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) a 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; 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) a 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; vii) a protein kinase inhibitor; or ix) a ROCK inhibitor.

[0246]

[0265] In some examples, the method comprises contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells with at least one of: i) a SHH pathway inhibitor; ii) a 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; vii) an epigenetic modification compound; ix) a protein kinase inhibitor; or x) a ROCK inhibitor.

[0247]

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

[0248]

[0267] Any γ-secretase inhibitor that can induce the differentiation of NKX6.1-positive pancreatic progenitor cells in a population into insulin-positive endocrine cells (e.g., alone or in combination with any of a TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator) can be used. In some examples, the γ-secretase inhibitor includes XXI. In some examples, the γ-secretase inhibitor includes DAPT. In some examples, the method comprises contacting NKX6.1-positive pancreatic progenitor cells with a γ-secretase inhibitor (e.g., XXI) 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 2.6 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 the 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 comprises betacellulin. In some examples, at least one growth factor from the EGF family comprises EGF. In some examples, the method comprises contacting NKX6.1-positive pancreatic progenitor cells with a growth factor (e.g., betacellulin) from the EGF family 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 (e.g., alone or in combination with any TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway activator) into insulin-positive endocrine cells can be used. In some examples, the RA signaling pathway activator comprises RA. In some examples, the method comprises contacting NKX6.1-positive pancreatic progenitor cells with an RA signaling pathway activator (e.g., retinoic acid) 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 2.5 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 capable of inducing the differentiation of NKX6.1-positive pancreatic progenitor cells (e.g., alone or in combination with any TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway activator) into insulin-positive endocrine cells can be used in the methods provided herein. In some examples, the SHH pathway inhibitor comprises Sant1. In some examples, the method comprises contacting NKX6.1-positive pancreatic progenitor cells with an SHH pathway inhibitor (e.g., Sant1) 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 0.3 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] Any BMP signaling pathway inhibitor that can induce the differentiation of NKX6.1-positive pancreatic progenitor cells (e.g., alone or in combination with any TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway activator) into insulin-positive endocrine cells can be used. In some examples, the BMP signaling pathway inhibitor includes LDN193189 or DMH-1. In some examples, the method includes contacting 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 that can induce the differentiation of NKX6.1-positive pancreatic progenitor cells in a 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, the ROCK inhibitor includes Thiazovivin, Y-27632, Fasudil / HA1077, or H-1152. In some examples, the ROCK inhibitor includes Y-27632. In some examples, the ROCK inhibitor includes Thiazovivin. In some examples, the method comprises contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a ROCK inhibitor (e.g., Y-27632 or Thiazovivin) 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, about 40 μM, about 50 μM, or about 100 μM.

[0254]

[0273] Any epigenetic modification compound that can induce the differentiation of NKX6.1-positive pancreatic progenitor cells in a 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, the epigenetic modification compound includes a histone methyltransferase inhibitor or an HDAC inhibitor. In some examples, the epigenetic modification compound includes a histone methyltransferase inhibitor, such as DZNep. In some examples, the epigenetic modification compound includes an HDAC inhibitor, such as KD5170. In some examples, the method includes contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells with an epigenetic modification 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 cell population is optionally contacted with a protein kinase inhibitor. In some examples, the cell population is not contacted with a protein kinase inhibitor. In some examples, the cell population is contacted with a protein kinase inhibitor. Any protein kinase inhibitor that can induce the differentiation of NKX6.1-positive pancreatic progenitor cells in a 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, the protein kinase inhibitor includes 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 the differentiation of at least one NKX6.1-positive pancreatic progenitor cell in the population into insulin-positive endocrine cells, and the insulin-positive endocrine cells express 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 the differentiation of at least one NKX6.1-positive pancreatic progenitor cell in the population into insulin-positive endocrine cells, and the insulin-positive endocrine cells express insulin. In some embodiments, one or more differentiation factors are part of stage 5 and are added only, for example, on the first 1, 2, 3, 4, 5, or 6 days of the period of stage 5, or on the last 1, 2, 3, 4, 5, or 6 days of the period of the stage. In one example, the cells are contacted with an SHH signaling pathway inhibitor only for the first 2, 3, 4, or 5 days between the cells and stage 5, and then the SHH signaling pathway inhibitor is removed from the medium. In another example, the cells are contacted with a BMP signaling pathway inhibitor only for the first 1, 2, or 3 days between the cells and stage 5, and then 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 the differentiation of at least one NKX6.1-positive pancreatic progenitor cell in the population into insulin-positive endocrine cells, and the insulin-positive endocrine cells express insulin.

[0258]

[0277] Aspects of the present disclosure involve treating a cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a PKC activator, and in a cell population of pancreatic endocrine cells produced according to the methods disclosed herein, 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 can be brought about.

[0259]

[0278] In some examples, the method comprises 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, a RA signaling pathway activator, and a 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 modification 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 comprises: (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 comprising 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, v) at least one growth factor from the TGF-β superfamily, and vi) a PKC activator for 1 to 2 days to thereby produce a first transformed cell population comprising PDX1-positive, NKX6.1-positive pancreatic progenitor cells.

[0261]

[0280] In some examples, the method comprises the step of contacting a first population of transformed cells comprising (3) PDX1-positive, NKX6.1-positive pancreatic progenitor cells with i) a SHH pathway inhibitor, ii) a 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 modification compound, ix) a protein kinase inhibitor, x) a ROCK inhibitor, and xi) a PKC activator for 1 to 2 days, thereby producing a second population of transformed cells, and (4) contacting the second population of transformed cells with i) a SHH pathway inhibitor, ii) a 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 modification 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 β cells

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

[0263]

[0282] In some examples, insulin-positive pancreatic endocrine cells produced using the methods provided herein can form cell clusters alone or together with other cell types, such as their precursors, such as stem cells, embryonic endoderm cells, gut tube 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 within a cell cluster. In some aspects, cell clusters are provided herein that are similar in function and characteristics to endogenous islets. Such cell clusters can mimic the function of endogenous islets in metabolism, for example, the regulation of glucose metabolism in a subject. Thus, cell clusters can be transplanted into a subject to treat insufficient islet function, such as diseases resulting from diabetes. The terms "cluster" and "aggregate" can be used interchangeably and refer to a group of cells having close cell-cell contact, and in some examples, the cells within the cluster can adhere to each other. A cell cluster contains a plurality of cells. In some embodiments, the cell cluster contains 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 cluster contains 10 to 10,000 cells, 50 to 10,000, 100 to 10,000, 100 to 10,000, 1,000 to 10,000, 500 to 10,000, 500 to 5,000, 500 to 2,500, 500 to 2,000, 1,000 to 100,000, 1,000 to 50,000, 1,000 to 40,000, 1,000 to 20,000, 1,000 to 10,000, 1,000 to 5,000, and 1,000 to 3,000 cells. In some embodiments, the cell cluster contains at least 500 cells. In some embodiments, the cell cluster contains at least 1,000 cells. In some embodiments, the cell cluster contains at least 2,000 cells. In some embodiments, the cell cluster contains at least 5,000 cells.In some embodiments, the cell cluster comprises 100,000 or fewer, 90,000 or fewer, 80,000 or fewer, 70,000 or fewer, 60,000 or fewer, 50,000 or fewer, 40,000 or fewer, 30,000 or fewer, 20,000 or fewer, 10,000 or fewer, 7,000 or fewer, 5,000 or fewer, 3,000 or fewer, 2,000 or fewer, or 1,000 or fewer cells.

[0265]

[0284] The cell cluster can be of a size similar to that of an endogenous pancreas islet. For example, the cell cluster can have a diameter similar to that of an endogenous pancreas islet. The diameter of the cell cluster can refer to the maximum linear distance between two points on the surface of the cell cluster. In some examples, the diameter of the 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 the cell cluster can be from about 75 μm to about 250 μm. The diameter of the cell cluster can be at most 100 μm.

[0266]

[0285] In some embodiments, the cell cluster has a diameter of about 100 to about 250 microns (e.g., a diameter of 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). For example, in some embodiments, the cell cluster has a diameter of 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.

[0267]

[0286] In some embodiments, the compositions, cells or cell populations of the present disclosure comprise cells having genomic disruption in at least one gene sequence. In some embodiments, the genomic disruption reduces or eliminates the expression of the protein encoded by said 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 composition or cell population has a genomic disruption in the beta-2-microglobulin gene. Additional examples of genes and their genomic disruptions are described in more detail in International Application Publication No. WO2020 / 033879, the relevant content of which is incorporated herein by reference. In some embodiments, the genomic disruption is induced using gene editing techniques (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 derived from the pancreas of an adult subject who is a healthy control. In some embodiments, the composition or cell population comprises NKX6.1-positive, ISL-positive cells that express higher levels of MAFB than NKX6.1-positive, ISL-positive cells derived from the pancreas of an adult subject who is a healthy control. In some embodiments, the composition or cell population comprises NKX6.1-positive, ISL-positive cells that express higher levels of SIX2, HOPX, IAPP and / or UCN3 than NKX6.1-positive, ISL-positive cells derived from the pancreas of an adult subject who is a healthy control.

[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 composition or cell population comprises NKX6.1-positive, ISL-positive cells that express MAFB.

[0270]

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

[0271]

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

[0272]

[0291] In some examples, the methods provided herein comprise 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 modification 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, 5 days. In some examples, the contact 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 cell population containing insulin-positive endocrine cells in MCDB131 medium, which may be supplemented with 2% BSA. In some examples, the 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, the 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 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 to 12 days, during which ZnSO4 is introduced from the 4th day of S6.

[0274]

[0293] In one aspect, the present disclosure is 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 3 days; b) differentiating at least a portion of the definitive endoderm cells into primitive gut tube cells by a process of contacting the definitive endoderm cells with at least one factor from the FGF family for 3 days; c) differentiating at least a portion of the primitive gut tube cells into PDX1-positive pancreatic progenitor cells by a process of contacting the primitive gut tube cells with i) a retinoic acid signaling pathway activator, ii) at least one factor from the FGF family, iii) a SHH pathway inhibitor, iv) a BMP signaling pathway inhibitor (e.g., DMH-1 or LDN193189), v) a PKC activator, and vi) a ROCK inhibitor; d) 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) a 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 days; e) 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 i) a TGF-β signaling pathway inhibitor, ii) a TH signaling pathway activator, iii) at least one SHH pathway inhibitor, iv) a RA signaling pathway activator, v) a γ-secretase inhibitor, optionally vi) at least one growth factor from the epidermal growth factor (EGF) family, and optionally vii) a BMP signaling pathway inhibitor for 5 to 7 days; f) PDX1-positive, NKX6.Step of differentiating at least a part of PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells by a process of culturing PDX1-positive, insulin-positive endocrine cells in a medium containing no exogenous differentiation factor (for example, NS-GFs medium, MCDB medium supplemented with BSA, MCDB131 medium, or DMEM / F12 medium) for 7 to 14 days and inducing in vitro maturation of at least a part 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 examples, the GSIS response is similar to the GSIS response of endogenous mature β cells.

[0275]

[0294] In one aspect, the present disclosure is 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 3 days; b) differentiating at least a portion of the definitive endoderm cells into primitive gut tube cells by a process of contacting the definitive endoderm cells with at least one factor from the FGF family for 3 days; c) differentiating at least a portion of the primitive gut tube cells into PDX1-positive pancreatic progenitor cells by a process of contacting the primitive gut tube 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, v) a PKC activator, vi) a ROCK inhibitor, and vii) a growth factor from the TGFβ superfamily for 2 days; d) 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) a 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 days; e) 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 i) a TGF-β signaling pathway inhibitor, ii) a TH signaling pathway activator, iii) at least one SHH pathway inhibitor, iv) a RA signaling pathway activator, v) a γ-secretase inhibitor, and optionally vi) at least one growth factor from the epidermal growth factor (EGF) family, and optionally vii) a BMP signaling pathway inhibitor for 5 to 7 days; f) PDX1-positive, NKX6.1.A process of culturing PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells in a medium without 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 part of the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells, and differentiating at least a part 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 examples, the GSIS response is similar to the GSIS response of endogenous mature β cells.

[0276]

[0295] In one aspect, the present disclosure is 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 3 days; b) differentiating at least a portion of the definitive endoderm cells into primitive gut tube cells by a process of contacting the definitive endoderm cells with at least one factor from the FGF family for 3 days; c) differentiating at least a portion of the primitive gut tube cells into PDX1-positive pancreatic progenitor cells by a process of contacting the primitive gut tube cells with i) a retinoic acid signaling pathway activator, ii) at least one factor from the FGF family, iii) a SHH pathway inhibitor, iv) a PKC activator, and v) a ROCK inhibitor; d) 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) a 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 days; e) 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 i) a TGF-β signaling pathway inhibitor, ii) a TH signaling pathway activator, iii) at least one SHH pathway inhibitor, iv) a RA signaling pathway activator, v) a γ-secretase inhibitor, and optionally vi) at least one growth factor from the epithelial growth factor (EGF) family for 5 to 7 days; f) culturing the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells in a medium without exogenous differentiation factors (e.g., NS-GFs medium, MCDB medium supplemented with BSA, MCDB131 medium, or DMEM / F12 medium) for 7 to 14 days, PDX1-positive, NKX6.Differentiating at least a part of PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells by a process that induces in vitro maturation of at least a part of 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 examples, the GSIS response is similar to the GSIS response of endogenous mature β cells.

[0277]

[0296] In one aspect, the present disclosure is 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 3 days; b) differentiating at least a portion of the definitive endoderm cells into primitive gut tube cells by a process of contacting the definitive endoderm cells with at least one factor from the FGF family for 3 days; c) differentiating at least a portion of the primitive gut tube cells into PDX1-positive pancreatic progenitor cells by a process of contacting the primitive gut tube cells with i) a retinoic acid signaling pathway activator, ii) at least one factor from the FGF family, iii) a SHH pathway inhibitor, iv) a BMP signaling pathway inhibitor (e.g., DMH-1 or LDN193189), v) a PKC activator, and vi) a ROCK inhibitor; d) 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) a 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 by a process of contacting the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with i) a SHH pathway inhibitor, ii) a 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 modification compound (e.g., DZNep or KD5170), ix) a protein kinase inhibitor, and x) a ROCK inhibitor for 5-7 days.a step of differentiating into PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells; and f) culturing the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells in a medium without exogenous differentiation factors (for example, NS-GFs medium, MCDB medium supplemented with BSA, MCDB131 medium, or DMEM / F12 medium) for 7 to 14 days, and inducing in vitro maturation of at least a part of the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells, thereby differentiating at least a part 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 examples, the GSIS response is similar to the GSIS response of endogenous mature β cells.

[0278]

[0297] In one aspect, the present disclosure is 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 3 days; b) differentiating at least a portion of the definitive endoderm cells into primitive gut tube cells by a process of contacting the definitive endoderm cells with at least one factor from the FGF family for 3 days; c) differentiating at least a portion of the primitive gut tube cells into PDX1-positive pancreatic progenitor cells by a process of contacting the primitive gut tube cells with i) a retinoic acid signaling pathway activator, ii) at least one factor from the FGF family, iii) a SHH pathway inhibitor, iv) a BMP signaling pathway inhibitor (e.g., DMH-1 or LDN193189), v) a PKC activator, and vi) a ROCK inhibitor; d) 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) a 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 under conditions that promote cell clustering; e) 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 modification 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, contacting the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a SHH pathway inhibitor, a RA signaling pathway, and at least one growth factor from the EGF family that will later be removed from the PDX1-positive, NKX6.1-positive pancreatic progenitor cells by a process of...1 differentiating at least a portion of the positive pancreatic progenitor cells into PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells; and f) culturing the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells in a medium without exogenous differentiation factors (e.g., NS-GFs medium, MCDB medium supplemented with BSA, MCDB131 medium, or DMEM / F12 medium) for 7 to 14 days, and differentiating at least a portion of the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells by a process that induces 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 examples, the GSIS response is similar to the GSIS response of endogenous mature β cells.

[0279]

[0298] In one aspect, the present disclosure provides a method for producing SC-β cells from pluripotent cells. The method includes: 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 3 days; b) differentiating at least a portion of the definitive endoderm cells into primitive gut tube cells by contacting the definitive endoderm cells with at least one factor from the FGF family for 3 days; c) differentiating at least a portion of the primitive gut tube cells into PDX1-positive pancreatic progenitor cells by contacting the primitive gut tube 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; d) differentiating at least a portion of the PDX1-positive pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells 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 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, followed by contacting with: i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) a RA signaling pathway activator, and optionally iv) a ROCK inhibitor, v) at least one factor from the TGFβ superfamily, vi) a PKC activator, and optionally vii) a gamma secretase inhibitor for 1 to 2 days; e) PDX1-positive, NKX6.Contact positive pancreatic progenitor cells with i) an SHH pathway inhibitor, ii) a 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 modification compound (e.g., DZNep or KD5170), ix) a protein kinase inhibitor, x) a ROCK inhibitor, and xi) a PKC activator for 1 to 2 days, and then contact them with i) an SHH pathway inhibitor, ii) a 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, vii) an epigenetic modification compound (e.g., DZNep or KD5170), ix) a protein kinase inhibitor, and x) a ROCK inhibitor for 3 to 6 days, to differentiate at least a part of the PDX1-positive, NKX6.1-positive pancreatic progenitor cells into PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells; and f) differentiating at least a part of the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells. A method is provided that includes these steps.

[0280]

[0299] The medium used to culture cells isolated from the first cell cluster can be xenofree. A xenofree medium for culturing cells and / or cell clusters of animal origin cannot have products derived from other animals. In some cases, a xenofree medium for culturing human cells and / or cell clusters cannot have any products derived from nonhuman animals. For example, a xenofree 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%, 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 also does not contain exogenous small molecules or signal transduction 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 signal transduction 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 derivatives thereof), 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 receptors (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), growth factors from the epidermal growth factor (EGF) family (e.g., betacellulin or EGF), a broad 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 cannot contain exogenous extracellular matrix molecules. In some cases, the reaggregation medium does not contain Matrigel™. In some cases, the reaggregation medium does not contain other extracellular matrix molecules or materials, such as collagen, gelatin, poly-L-lysine, poly-D-lysine, vitronectin, laminin, fibronectin, PLO laminin, fibrin, thrombin, and RetroNectin and mixtures thereof, or, for example, lysed cell membrane preparations.,

[0281]

[0300] One skilled in the art would 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, as well as high levels of some amino acids and vitamins. These additives can enable a medium supplemented with very low levels of serum or defined components. The medium may not contain proteins and / or growth factors, 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 can include collagen, placental matrix, fibronectin, laminin, merosin, tenascin, heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, aggrecan, biglycan, thrombospondin, vitronectin, and decorin. In some cases, the medium contains laminin, e.g., LN-332. In some cases, the medium contains heparin.

[0282]

[0301] The medium can be periodically exchanged during culturing, for example, to provide an optimal environment for the cells in the medium. When culturing cells separated from a first cell cluster for re-aggregation, the medium can be exchanged at least or about every 4 hours, 12 hours, 24 hours, 48 hours, 3 days, or 4 days. For example, the medium can be exchanged about every 48 hours.

[0283]

[0302] In some cases, cells can be cultured under dynamic conditions (e.g., conditions where the cells are subjected to a certain movement or agitation while in suspension culture). To culture cells dynamically, the cells can be cultured in a container (e.g., a non-adhesive container such as a spinner flask (e.g., 200 ml to 3000 ml, e.g., 250 ml; 100 ml; or in a 125 ml Erlenmeyer) etc.) that is connected to a control unit and can thus provide a controlled culture system. In some cases, cells can be cultured under non-dynamic conditions (e.g., static culture) while maintaining their proliferative capacity. To culture cells non-dynamically, the cells can be cultured in an adherent culture vessel. The adherent culture vessel may be coated with any of the substrates for cell adhesion such as an extracellular matrix (ECM) to improve the adhesion of cells to the vessel surface. The substrate for cell adhesion can be any material intended to attach stem cells or feeder cells (if used). Examples of substrates for cell adhesion include collagen, gelatin, poly-L-lysine, poly-D-lysine, vitronectin, laminin, fibronectin, PLO laminin, fibrin, thrombin, and RetroNectin and mixtures thereof, such as Matrigel (trademark), and lysed cell membrane preparations.

[0284]

[0303] The medium in a dynamic cell culture vessel (e.g., a spinner flask) can be agitated (e.g., by a stirrer). The spin rate can correlate with the size of the second cell cluster that re-aggregates. The spin rate can be controlled such that the size of the second cell cluster can be similar to that of endogenous islets. In some cases, the spin rate is controlled such that the size of the second cell cluster can be from about 75 μm to about 250 μm. The spin rate of a dynamic cell culture vessel (e.g., a spinner flask) can be from about 20 revolutions per minute (rpm) to about 100 rpm, e.g., from about 30 rpm to about 90 rpm, from about 40 rpm to about 60 rpm, from about 45 rpm to about 50 rpm. In some cases, the spin rate can be about 50 rpm.

[0285]

[0304] The cells of stage 6 provided herein may or may not be subjected to the separation and re-aggregation process as described herein. In some cases, cell clusters containing insulin-positive endocrine cells can be re-aggregated. By re-aggregating the cell clusters, insulin-positive endocrine cells can be enriched. In some cases, insulin-positive endocrine cells in the cell clusters can be further matured into pancreatic β cells. For example, after re-aggregation, the second cell cluster may exhibit GSIS in vitro, similar to native islets. For example, after re-aggregation, the second cell cluster may contain non-native pancreatic β cells that exhibit GSIS in vitro. In some embodiments, the re-aggregation process may be performed according to the disclosure of PCT application PCT / US2018 / 043179, which is hereby incorporated by reference in its entirety.

[0286]

[0305] Cells at stage 6 obtained according to the methods provided herein can have a high recovery rate after cryopreservation and reaggregation procedures. In some cases, cells at stage 6 obtained in a differentiation process involving treatment with a BMP signaling pathway inhibitor (e.g., DMH-1 or LDN) and a growth factor from the TGF-β superfamily (e.g., activin A) at stage 3 and treatment with an epigenetic modification compound (e.g., a histone methyltransferase inhibitor, e.g., an EZH2 inhibitor, e.g., DZNep) at stage 5 can have a higher recovery rate after cryopreservation following stage 5 compared to the corresponding cell population without such treatment. In some cases, cells at stage 6 obtained in a differentiation process involving treatment with a BMP signaling pathway inhibitor (e.g., DMH-1 or LDN) and a growth factor from the TGF-β superfamily (e.g., activin A) at stage 3 and treatment with an epigenetic modification compound (e.g., a histone methyltransferase inhibitor, e.g., an EZH2 inhibitor, e.g., DZNep) at stage 5 can have a higher recovery rate after cryopreservation following stage 5 compared to the corresponding cell population without treatment with a BMP signaling pathway inhibitor (e.g., DMH-1 or LDN) and a growth factor from the TGF-β superfamily (e.g., activin A) at stage 3. In some cases, cells at stage 6 obtained in a differentiation process involving treatment with a BMP signaling pathway inhibitor (e.g., DMH-1 or LDN) and a growth factor from the TGF-β superfamily (e.g., activin A) at stage 3 and treatment with an epigenetic modification compound (e.g., a histone methyltransferase inhibitor, e.g., an EZH2 inhibitor, e.g., DZNep) at stage 5 can have a recovery rate that is at least about 35%, 37.5%, 40%, 42.5%, 45%, 47.5%, 48%, 49%, or 50% after cryopreservation following stage 5. The recovery rate can be calculated as the percentage of cells that survive, reaggregate, and form reaggregated cell clusters after cryopreservation, thawing and recovery, and reaggregation procedures compared to the cells before cryopreservation.

[0287]

[0306] In some embodiments, the present disclosure relates to cryopreservation of non-natural pancreatic β-cells or precursors thereof obtained using the methods provided herein. In some embodiments, a cell population comprising non-natural pancreatic β-cells can be preserved by cryopreservation. For example, a cell population comprising non-natural β-cells, such as stage 6 cells, may in some cases be isolated in a cell suspension, such as a single cell suspension, and the cell suspension can be cryopreserved, such as by freezing in a cryopreservation solution. Isolation of the cells can be performed by any of the techniques provided herein, such as by enzymatic treatment. The cells can be frozen at a temperature of up to -20°C, up to -30°C, up to -40°C, up to -50°C, up to -60°C, up to -70°C, up to -80°C, up to -90°C, up to -100°C, up to -110°C, up to -120°C, up to -130°C, up to -140°C, up to -150°C, up to -160°C, up to -170°C, up to -180°C, up to -190°C, or the cells can be frozen at a temperature of up to -200°C. In some cases, the cells are frozen at a temperature of about -80°C. In some cases, the cells are frozen at a temperature of about -195°C. To provide the low temperature required for cryopreservation, any cooling method can be used, including but not limited to, electric freezers, solid carbon dioxide, and liquid nitrogen. In some cases, any cryopreservation solution available to those skilled in the art, including both custom-made and commercially available solutions, can be used to incubate the cells for storage at low temperature. For example, a solution containing a cr...

Claims

**Claim 1** A composition comprising a cell population for use in a method of administering a composition to a subject with type I diabetes, the composition comprising NKX6.1-positive and ISL1-positive cells, wherein (i) at least 80% of the cells of the cell population express ISL1; and (ii) less than 6% of the cells of the cell population are NKX6.1-negative and ISL1-negative cells. **Claim 2** The composition according to claim 1, which is administered to a subject with type 1 diabetes by intraportal transplantation. **Claim 3** The composition according to claim 1 or 2, which is administered into a device configured to produce and release insulin when implanted in a subject. **Claim 4** The composition according to any one of claims 1 to 3, wherein the cell population comprises cells having a disruption in the beta-2-microglobulin gene, the disruption reducing or eliminating the expression of the beta-2-microglobulin gene. **Claim 5** The composition according to any one of claims 1 to 4, by means of which a subject comes to have a glucose level of 80 to 120 milligrams per deciliter (mg / dl) before meals. **Claim 6** The composition according to any one of claims 1 to 5, wherein 80% to 90% of the cells of the cell population express ISL1. **Claim 7** The composition according to any one of claims 1 to 6, wherein 85% to 95% of the cells of the cell population express ISL1.

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