SERUM-FREE IN VITRO DIRECTED DIFFERENTIATION PROTOCOL FOR GENERATING STEM CELL-DERIVED BETA CELLS AND USES THEREOF
Disclosed herein are methods for generating SC-β cells using chemically defined, completely serum free media, and isolated populations of SC-β cells for use in various applications, such as cell therapy.
1 - 31 . (canceled)
32 . An in vitro composition comprising a population of cells suspended in a culture medium, wherein said population of cells comprises PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells in said culture medium, and wherein said culture medium is serum free and is supplemented with a transforming growth factor β (TGF-β) signaling pathway inhibitor and a thyroid hormone signaling pathway activator.
33 . The composition of claim 32 , wherein said PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells express one or more of NGN3, NeuroD1, ISL1, PDX1, NKX6.1, PAX4, ARX, NKX2.2, or PAX6.
34 . The composition of claim 32 , wherein said PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells are genetically modified.
35 . The composition of claim 32 , wherein said PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells do not exhibit in vitro glucose-stimulated insulin secretion (GSIS) response.
36 . The composition of claim 32 , wherein said culture medium comprises an effective amount of said TGF-β signaling pathway inhibitor and said thyroid hormone signaling pathway activator to facilitate differentiation of PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells that do not exhibit GSIS in vitro in said population of cells into mature β cells exhibiting GSIS response in vitro.
37 . The composition of claim 32 , wherein said culture medium is further supplemented with a differentiation factor selected from the group consisting of: a sonic hedgehog (SHH) signaling pathway inhibitor, an epidermal growth factor (EGF) family growth factor, a retinoic signaling pathway activator, a bone morphogenic protein (BMP) signaling pathway inhibitor, a γ-secretase inhibitor, a protein kinase inhibitor, and a Rho-associated protein kinase (ROCK) inhibitor.
38 . The composition of claim 32 , wherein said culture medium is further supplemented with bovine serum albumin.
39 . The composition of claim 32 , wherein said TGF-β signaling pathway inhibitor comprises ALK5 inhibitor II, or an analog or derivative of ALK5 inhibitor II.
40 . The composition of claim 32 , wherein said thyroid hormone signaling pathway activator comprises triiodothyronine, or an analog or derivative of triiodothyronine.
41 . The composition of claim 32 , wherein said population of cells further comprises PDX1-positive, NKX6.1-positive pancreatic progenitor cells.
42 . An in vitro method of generating at least one mature β cell that exhibits a glucose-stimulated insulin secretion (GSIS) response in vitro, the method comprising:
culturing a population of cells comprising PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells in a serum free medium that is supplemented with a transforming growth factor β (TGF-β) signaling pathway inhibitor and a thyroid hormone signaling pathway activator, thereby generating the at least one mature β cell exhibiting a GSIS response in vitro.
43 . The method of claim 42 , further comprising washing the population of cells comprising PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells with a culture medium before treatment with the serum free medium supplemented with a TGF-β signaling pathway inhibitor and thyroid hormone signaling pathway inhibitor.
44 . The method of claim 42 , wherein the serum free medium supplemented with a TGF-β signaling pathway inhibitor and thyroid hormone signaling pathway inhibitor comprises the TGF-β signaling pathway inhibitor at a concentration of 0.1 μM to 110 μM.
45 . The method of claim 42 , wherein the serum free medium supplemented with a TGF-β signaling pathway inhibitor and thyroid hormone signaling pathway inhibitor comprises the thyroid hormone signaling pathway activator at a concentration of 0.1 μM to 110 μM.
46 . The method of claim 42 , wherein the TGF-β signaling pathway inhibitor comprises ALK5 inhibitor II, or an analog or derivative of ALK5 inhibitor II.
47 . The method of claim 42 , wherein the thyroid hormone signaling pathway activator comprises triiodothyronine, or an analog or derivative of triiodothyronine.
48 . The method of claim 42 , further comprising obtaining the cell population comprising the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells by differentiating PDX1-positive, NKX6.1-positive pancreatic progenitor cells in a culture medium that is serum free.
49 . A method of generating PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells in vitro, comprising:
(a) differentiating pluripotent stem cells into SOX17-positive definitive endoderm cells in a medium supplemented with a growth factor from transforming growth factor β (TGF-β) superfamily and a WNT signaling pathway activator;
(b) differentiating said SOX17-positive definitive endoderm cells into FOXA2-positive foregut endoderm cells in a medium supplemented with a growth factor from fibroblast growth factor (FGF) family;
(c) differentiating said FOXA2-positive foregut endoderm cells into PDX1-positive pancreatic progenitor cells in a medium supplemented with a growth factor from FGF family, a bone morphogenic protein (BMP) signaling pathway inhibitor, a protein kinase C (PKC) activator, a Sonic Hedgehog (SHH) signaling pathway inhibitor, and a Rho-associated protein kinase (ROCK) inhibitor;
(d) differentiating said PDX1-positive pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells in a medium supplemented with a growth factor from FGF family, a RA signaling pathway activator, a SHH signaling pathway inhibitor, and a ROCK inhibitor; and
(e) differentiating said PDX1-positive, NKX6.1-positive pancreatic progenitor cells into a cell population comprising at least one ISL1-positive pancreatic endocrine cell in a serum-free medium supplemented with a TGF-β signaling pathway inhibitor and a thyroid hormone signaling pathway activator.
50 . The method of claim 49 , wherein said serum-free medium supplemented with a TGF-β signaling pathway inhibitor and thyroid hormone signaling pathway activator is further supplemented with a differentiation factor selected from the group consisting of: a SHH signaling pathway inhibitor, an epidermal growth factor (EGF) family growth factor, a retinoic signaling pathway activator, a BMP signaling pathway inhibitor, a γ-secretase inhibitor, a protein kinase inhibitor, and a ROCK inhibitor.
51 . The method of claim 49 , wherein all the media used in (a), (b), (c), and (d) of the method are serum free.