Stem Cell Differentiation and Polymers

Abstract

Disclosed herein are compositions and methods related to the differentiation of stem cells into pancreatic cells.In some aspects, the methods provided herein relate to the in vitro generation of pancreatic β cells, α cells, δ cells and EC cells in the presence of polymers, such as water-soluble synthetic polymers.In some aspects, the disclosure provides pharmaceutical compositions comprising the cells produced according to the methods disclosed herein, as well as the methods of treatment using them.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 158,587, filed March 9, 2021, which is incorporated by reference in its entirety. [Background technology]

[0002]

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

[0003] Incorporation by Reference

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

[0004]

[0004] In some aspects, disclosed herein is an in vitro composition comprising a plurality of pancreatic beta cells or precursor cells thereof in a medium comprising a water-soluble synthetic polymer.

[0005] In some examples, the composition comprises pancreatic beta cell precursor cells, and the pancreatic beta cell precursor cells include Sox17-positive cells, FOXA2-positive cells, PDX1-positive cells, NKX6.1-positive cells, ISL1-positive cells, and / or insulin-positive endocrine cells. In some examples, the water-soluble synthetic polymer comprises polyvinyl alcohol, poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), PEG copolymer, poly(N-isopropylacrylamide), or polyacrylamide. In some examples, the water-soluble synthetic polymer comprises polyvinyl alcohol. In some examples, the water-soluble synthetic polymer is present in the medium at a concentration of about 0.005% to about 0.5% (w / v), about 0.01% to about 0.2% (w / v), about 0.02% to about 0.1% (w / v), or about 0.03% to about 0.08% (w / v). In some examples, the water-soluble synthetic polymer is present in the medium at a concentration of about 0.04% to about 0.06% (w / v). In some examples, the water-soluble synthetic polymer is present in the medium at a concentration of about 0.05% (w / v).

[0005]

[0006] In some examples, the composition comprises a plurality of NKX6.1-positive, insulin-positive cells and / or non-native pancreatic beta cells. In some examples, the water-soluble synthetic polymer comprises polyvinyl alcohol that is greater than 85% hydrolyzed. In some examples, the water-soluble synthetic polymer comprises polyvinyl alcohol that is about 87% to 89% hydrolyzed. In some examples, the composition further comprises NKX6.1-positive, ISL1-positive pancreatic endocrine cells. In some examples, the composition further comprises pancreatic alpha cells, pancreatic delta cells, pancreatic F cells, pancreatic epsilon cells, enterochromaffin cells, or any combination thereof. In some examples, the medium further comprises one or more agents selected from the group consisting of a transforming growth factor beta (TGF-β) signaling pathway inhibitor, a thyroid hormone signaling pathway activator, an epigenetic modifying compound, a growth factor from the epidermal growth factor (EGF) family, a retinoic acid (RA) signaling pathway activator, a sonic hedgehog (SHH) pathway inhibitor, a gamma-secretase inhibitor, a protein kinase inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a bone morphogenetic protein (BMP) signaling pathway inhibitor.

[0006]

[0007] In some examples, the composition comprises pancreatic beta cell precursor cells, and the pancreatic beta cell precursor cells comprise a plurality of PDX1-positive, NKX6.1-positive cells. In some examples, the culture medium further comprises one or more agents selected from the group consisting of a protein kinase C activator, a TGF-beta signaling pathway inhibitor, a thyroid hormone signaling pathway activator, an epigenetic modifying compound, a growth factor from the epidermal growth factor (EGF) family, a retinoic acid (RA) signaling pathway activator, a sonic hedgehog (SHH) pathway inhibitor, a gamma-secretase inhibitor, a protein kinase inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a bone morphogenetic protein (BMP) signaling pathway inhibitor.In some examples, the medium comprises: (a) a TGF-β signaling pathway inhibitor selected from the group consisting of Alk5i II, A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB-525334, SD-208, and SB-505124; (b) a thyroid hormone signaling pathway activator, including T3 or GC-1; (c) 3-deazaneplanocin A (DZNep), GSK126, EPZ (d) epigenetic modifying compounds selected from the group consisting of betacellulin or EGF; (e) growth factors from the epidermal growth factor family, including retinoic acid, CD1530, AM580, TTHRB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, and CD2314. (f) a sonic hedgehog pathway inhibitor selected from the group consisting of SANT1, SANT2, SANT4, Cur61414, forskolin, tomatidine, AY9944, triparanol, and cyclopamine; (g) a gamma-secretase inhibitor, including XXI or DAPT; (h) a protein kinase inhibitor, including staurosporine, Ro-31-8220, bicindolylmaleimide (Bis) compounds, 10'-{5"-[(methoxycarbonyl)amino]-2"-methyl}-phenylaminocarbonylstaurosporine, or a staralog; (i) a ROCK inhibitor selected from the group consisting of thiazovivin, Y-27632, Fasudil / HA1077, and 14-1152; (j) a phorbol 12,13-dibutyrate (PDBU), TPB, or phorbol 12-myristate. and / or (k) a protein kinase C activator selected from the group consisting of 13-acetate, and bryostatin 1; and / or (k) a bone morphogenetic protein signaling pathway inhibitor, including LDN193189 or DMH-1. In some examples, the water-soluble synthetic polymer comprises greater than 85% hydrolyzed polyvinyl alcohol.In some examples, the water-soluble synthetic polymer comprises approximately 87% to 89% hydrolyzed polyvinyl alcohol. In some examples, the pancreatic beta cell precursor cells further comprise NKX6.1-positive, ISL1-positive cells.

[0007]

[0008] In some examples, the composition comprises pancreatic beta cell precursor cells, and the pancreatic beta cell precursor cells comprise a plurality of PDX1-positive, NKX6.1-negative cells. In some examples, the medium further comprises one or more agents selected from the group consisting of a protein kinase C activator, a growth factor from the transforming growth factor beta (TGF-β) superfamily, a growth factor from the fibroblast growth factor (FGF) family, a retinoic acid (RA) signaling pathway activator, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a sonic hedgehog (SHH) pathway inhibitor. In some examples, the medium comprises (a) a growth factor from the transforming growth factor beta (TGF-β) superfamily selected from the group consisting of inhibin, activin, Müllerian inhibitory substance (MIS), bone morphogenetic protein (BMP), decapentaplegic (dpp), Vg-1, monoclonal nonspecific inhibitory factor (MNSF), growth differentiation factor 8 (GDF8), and growth differentiation factor 11 (GDF11); (b) a growth factor from the fibroblast growth factor (FGF) family selected from the group consisting of keratinocyte growth factor (KGF), FGF2, FGF10, FGF21, and FGF8B. (c) a retinoic acid (RA) signaling pathway activator selected from the group consisting of retinoic acid, CD1530, AM580, TTHRB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, and CD2314; (d) a ROCK inhibitor selected from the group consisting of thiazovivin, Y-27632, Fasudil / HA1077, and 14-1152; (e) phorbol 12,13-dibutyrate (PDBU), TPB, phorbol 12-myristate and / or (f) a protein kinase C activator selected from the group consisting of SANT1, SANT2, SANT4, Cur61414, forskolin, tomatidine, AY9944, triparanol, and cyclopamine. In some examples, the water-soluble synthetic polymer comprises less than 85% hydrolyzed polyvinyl alcohol.In some examples, the water-soluble synthetic polymer comprises about 80% hydrolyzed polyvinyl alcohol. In some examples, the precursor cells of pancreatic beta cells further comprise PDX1-positive, NKX6.1-positive cells.

[0008]

[0009] In some examples, the composition comprises precursor cells of pancreatic beta cells, and the precursor cells of pancreatic beta cells comprise a plurality of FOXA2-positive gastrula cells. In some examples, the medium further comprises one or more agents selected from the group consisting of a protein kinase C activator, a growth factor from the transforming growth factor beta (TGF-β) superfamily, a bone morphogenetic protein signaling pathway inhibitor, a growth factor from the fibroblast growth factor (FGF) family, a retinoic acid (RA) signaling pathway activator, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a sonic hedgehog (SHH) pathway inhibitor. In some examples, the medium further comprises one or more agents selected from the group consisting of: (a) phorbol 12,13-dibutyrate (PDBU), TPB, phorbol 12-myristate (b) a protein kinase C activator selected from the group consisting of inhibin, activin, Müllerian inhibitory substance (MIS), bone morphogenetic protein (BMP), decapentaplegic (dpp), Vg-1, monoclonal nonspecific inhibitor of growth factor (MNSF), growth differentiation factor 8 (GDF8), and growth differentiation factor 11 (GDF11); (c) a bone morphogenetic protein signaling pathway inhibitor, including LDN193189 or DMH-1; (d) a fibroblast growth factor selected from the group consisting of keratinocyte growth factor (KGF), FGF2, FGF10, FGF21, and FGF8B. (e) a sonic hedgehog pathway inhibitor selected from the group consisting of SANT1, SANT2, SANT4, Cur6l4l4, forskolin, tomatidine, AY9944, triparanol, and cyclopamine; (f) a retinoic acid signaling pathway activator selected from the group consisting of retinoic acid, CD1530, AM580, TTHRB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, and CD2314; and / or (g) a ROCK inhibitor selected from the group consisting of thiazovivin, Y-27632, Fasudil / HA1077, and 14-1152.In some examples, the water-soluble synthetic polymer comprises less than 85% hydrolyzed polyvinyl alcohol. In some examples, the water-soluble synthetic polymer comprises about 80% hydrolyzed polyvinyl alcohol. In some examples, the pancreatic beta cell precursor cells further comprise PDX1-positive, NKX6.1-negative cells.

[0009]

[0010] In some examples, the composition comprises precursor cells of pancreatic beta cells, and the precursor cells of pancreatic beta cells comprise a plurality of SOX17-positive definitive endoderm cells. In some examples, the culture medium further comprises a growth factor from the fibroblast growth factor (FGF) family. In some examples, the growth factor from the fibroblast growth factor (FGF) family is selected from the group consisting of keratinocyte growth factor (KGF), FGF2, FGF10, FGF21, and FGF8B. In some examples, the water-soluble synthetic polymer comprises less than 85% hydrolyzed polyvinyl alcohol. In some examples, the water-soluble synthetic polymer comprises about 80% hydrolyzed polyvinyl alcohol. In some examples, the precursor cells of pancreatic beta cells further comprise FOXA2-positive cells.

[0010]

[0011] In some examples, the composition comprises precursor cells of pancreatic beta cells, and the precursor cells of pancreatic beta cells comprise a plurality of pluripotent stem cells. In some examples, the medium further comprises a growth factor from the transforming growth factor beta (TGF-β) superfamily, a WNT signaling pathway activator, or both. In some examples, the medium comprises (a) a growth factor from the transforming growth factor beta (TGF-β) superfamily selected from the group consisting of inhibin, activin, Müllerian inhibitory substance (MIS), bone morphogenetic protein (BMP), decapentaplegic (dpp), Vg-1, monoclonal nonspecific inhibitory factor (MNSF), growth differentiation factor 8 (GDF8), and growth differentiation factor 11 (GDF11); and / or (b) a growth factor from the transforming growth factor beta (TGF-β) superfamily selected from the group consisting of CHIR99021, 3F8, A 1070722, AR-A 014418, BIO, BIO-acetoxime, FRATide, 10Z-hymenialdisine, indirubin-3'oxime, Kenpaullone, L803, L803-mts, lithium carbonate, NSC 693868, SB 216763, SB 415286, TC-G 24, TCS 2002, TCS 21311, and TWS 119. In some examples, the water-soluble synthetic polymer comprises less than 85% hydrolyzed polyvinyl alcohol. In some examples, the water-soluble synthetic polymer comprises about 80% hydrolyzed polyvinyl alcohol. In some examples, the composition further comprises SOX17-positive cells. In some examples, the pluripotent stem cells comprise human stem cells. In some examples, the pluripotent stem cells comprise embryonic stem cells or induced pluripotent stem cells.

[0011]

[0012] In some cases, the medium does not contain albumin protein. In some cases, the medium does not contain human serum albumin (HSA). In some cases, the medium does not contain serum.

[0013] In some embodiments, methods are disclosed herein that include differentiating a plurality of pancreatic beta cell precursor cells in a serum- or serum albumin-free medium. In some examples, the medium includes a water-soluble synthetic polymer. In some embodiments, methods are disclosed herein that include differentiating a plurality of pancreatic beta cell precursor cells in a medium including a water-soluble synthetic polymer. In some examples, the water-soluble synthetic polymer includes poloxamer, polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol (PEG), a PEG copolymer, poly(N-isopropylacrylamide), or polyacrylamide. In some examples, the water-soluble synthetic polymer includes polyvinyl alcohol. In some examples, the water-soluble synthetic polymer is present in the medium at a concentration of about 0.005% to about 0.5% (w / v), about 0.01% to about 0.2% (w / v), about 0.02% to about 0.1% (w / v), or about 0.03% to about 0.08% (w / v). In some examples, the water-soluble synthetic polymer is present in the medium at a concentration of about 0.04% to about 0.06% (w / v). In some examples, the water-soluble synthetic polymer is present in the medium at a concentration of about 0.05% (w / v).

[0012]

[0014] In some examples, the pancreatic beta cell precursor cells include NKX6.1-positive, ISL1-positive cells. In some examples, the method results in the differentiation of NKX6.1-positive, ISL1-positive cells into pancreatic beta cells. In some examples, the water-soluble synthetic polymer includes polyvinyl alcohol that is greater than 85% hydrolyzed. In some examples, the water-soluble synthetic polymer includes polyvinyl alcohol that is about 87% to 89% hydrolyzed. In some examples, the medium further includes one or more agents selected from the group consisting of a transforming growth factor beta (TGF-β) signaling pathway inhibitor, a thyroid hormone signaling pathway activator, an epigenetic modifying compound, a growth factor from the epidermal growth factor (EGF) family, a retinoic acid (RA) signaling pathway activator, a sonic hedgehog (SHH) pathway inhibitor, a gamma-secretase inhibitor, a protein kinase inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a bone morphogenetic protein (BMP) signaling pathway inhibitor. In some examples, the method includes contacting the NKX6.1-positive, ISL1-positive cells with a water-soluble synthetic polymer for about 7 to about 14 days.

[0013]

[0015] In some examples, the precursor cells of pancreatic beta cells include PDX1-positive, NKX6.1-positive cells. In some examples, the method results in the differentiation of PDX1-positive, NKX6.1-positive cells into NKX6.1-positive, ISL1-positive cells. In some examples, the medium further comprises one or more agents selected from the group consisting of a protein kinase C activator, a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, an epigenetic modifying compound, a growth factor from the epidermal growth factor (EGF) family, a retinoic acid (RA) signaling pathway activator, a sonic hedgehog (SHH) pathway inhibitor, a gamma-secretase inhibitor, a protein kinase inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a bone morphogenetic protein (BMP) signaling pathway inhibitor.In some examples, the medium comprises: (a) a TGF-β signaling pathway inhibitor selected from the group consisting of Alk5i II, A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB-525334, SD-208, and SB-505124; (b) a thyroid hormone signaling pathway activator, including T3 or GC-1; (c) 3-deazaneplanocin A (DZNep), GSK126, E (d) an epigenetic modifying compound selected from the group consisting of PZ6438, KD5170, MC1568, and TMP195; (d) a growth factor from the epidermal growth factor family, including betacellulin or EGF; (e) retinoic acid, CD1530, AM580, TTHRB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, and CD23. (f) a sonic hedgehog pathway inhibitor selected from the group consisting of SANT1, SANT2, SANT4, Cur6l4l4, forskolin, tomatidine, AY9944, triparanol, and cyclopamine; (g) a gamma-secretase inhibitor, including XXI or DAPT; (h) a protein kinase inhibitor, including staurosporine, Ro-31-8220, bicindolylmaleimide (Bis) compounds, 10'-{5"-[(methoxycarbonyl)amino]-2"-methyl}-phenylaminocarbonylstaurosporine, or stalalogues; (i) a ROCK inhibitor selected from the group consisting of thiazovivin, Y-27632, Fasudil / HA1077, and 14-1152; (j) a phorbol 12,13-dibutyrate (PDBU), TPB, or phorbol 12-myristate. and / or (k) a protein kinase C activator selected from the group consisting of 13-acetate, and bryostatin 1; and / or (k) a bone morphogenetic protein signaling pathway inhibitor, including LDN193189 or DMH-1. In some examples, the water-soluble synthetic polymer comprises greater than 85% hydrolyzed polyvinyl alcohol. In some examples, the water-soluble synthetic polymer comprises about 87% to 89% hydrolyzed polyvinyl alcohol.In some examples, the method includes contacting the PDX1-positive, NKX6.1-positive cells with the water-soluble synthetic polymer for about 5 to about 10 days or about 6 to about 9 days, hi some examples, the method includes contacting the PDX1-positive, NKX6.1-positive cells with the water-soluble synthetic polymer for about 5, 6, 7, 8, 9, or 10 days.

[0014]

[0016] In some examples, the precursor cells of pancreatic beta cells include PDX1-positive, NKX6.1-negative cells. In some examples, the method results in the differentiation of PDX1-positive, NKX6.1-negative cells into PDX1-positive, NKX6.1-positive cells. In some examples, the medium further comprises one or more agents selected from the group consisting of a protein kinase C activator, a growth factor from the transforming growth factor beta (TGF-β) superfamily, a growth factor from the fibroblast growth factor (FGF) family, a retinoic acid (RA) signaling pathway activator, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a sonic hedgehog (SHH) pathway inhibitor. In some examples, the medium comprises (a) a growth factor from the transforming growth factor beta (TGF-β) superfamily selected from the group consisting of inhibin, activin, Müllerian inhibitory substance (MIS), bone morphogenetic protein (BMP), decapentaplegic (dpp), Vg-1, monoclonal nonspecific inhibitory factor (MNSF), growth differentiation factor 8 (GDF8), and growth differentiation factor 11 (GDF11); (b) a growth factor from the fibroblast growth factor (FGF) family selected from the group consisting of keratinocyte growth factor (KGF), FGF2, FGF10, FGF21, and FGF8B. (c) a retinoic acid (RA) signaling pathway activator selected from the group consisting of retinoic acid, CD1530, AM580, TTHRB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, and CD2314; (d) a ROCK inhibitor selected from the group consisting of thiazovivin, Y-27632, Fasudil / HA1077, and 14-1152; (e) phorbol 12,13-dibutyrate (PDBU), TPB, phorbol 12-myristate and / or (f) a protein kinase C activator selected from the group consisting of SANT1, SANT2, SANT4, Cur61414, forskolin, tomatidine, AY9944, triparanol, and cyclopamine. In some examples, the water-soluble synthetic polymer comprises less than 85% hydrolyzed polyvinyl alcohol.In some examples, the water-soluble synthetic polymer comprises about 80% hydrolyzed polyvinyl alcohol. In some examples, the method includes contacting the PDX1-positive, NKX6.1-negative cells with the water-soluble synthetic polymer for 4 to 8 days or 5 to 7 days. In some examples, the method includes contacting the PDX1-positive, NKX6.1-negative cells with the water-soluble synthetic polymer for about 4, 5, 6, 7, or 8 days.

[0015]

[0017] In some examples, the precursor cells of pancreatic beta cells include a plurality of FOXA2-positive cells. In some examples, the method results in the differentiation of FOXA2-positive cells into PDX1-positive, NKX6.1-negative cells. In some examples, the medium further comprises one or more agents selected from the group consisting of a protein kinase C activator, a growth factor from the transforming growth factor beta (TGF-β) superfamily, a bone morphogenetic protein signaling pathway inhibitor, a growth factor from the fibroblast growth factor (FGF) family, a retinoic acid (RA) signaling pathway activator, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a sonic hedgehog (SHH) pathway inhibitor. In some examples, the medium further comprises one or more agents selected from the group consisting of: (a) phorbol 12,13-dibutyrate (PDBU), TPB, phorbol 12-myristate (b) a protein kinase C activator selected from the group consisting of inhibin, activin, Müllerian inhibitory substance (MIS), bone morphogenetic protein (BMP), decapentaplegic (dpp), Vg-1, monoclonal nonspecific inhibitor of growth factor (MNSF), growth differentiation factor 8 (GDF8), and growth differentiation factor 11 (GDF11); (c) a bone morphogenetic protein signaling pathway inhibitor, including LDN193189 or DMH-1; (d) a fibroblast growth factor selected from the group consisting of keratinocyte growth factor (KGF), FGF2, FGF10, FGF21, and FGF8B. (e) a sonic hedgehog pathway inhibitor selected from the group consisting of SANT1, SANT2, SANT4, Cur6l4l4, forskolin, tomatidine, AY9944, triparanol, and cyclopamine; (f) a retinoic acid signaling pathway activator selected from the group consisting of retinoic acid, CD1530, AM580, TTHRB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, and CD2314; and / or (g) a ROCK inhibitor selected from the group consisting of thiazovivin, Y-27632, Fasudil / HA1077, and 14-1152.In some examples, the water-soluble synthetic polymer comprises less than 85% hydrolyzed polyvinyl alcohol. In some examples, the water-soluble synthetic polymer comprises about 80% hydrolyzed polyvinyl alcohol. In some examples, the method includes contacting the FOXA2-positive cells with the water-soluble synthetic polymer for 1-3 days. In some examples, the method includes contacting the FOXA2-positive cells with the water-soluble synthetic polymer for about 1, 2, or 3 days.

[0016]

[0018] In some examples, the precursor cells of pancreatic beta cells include SOX17-positive cells. In some examples, the method results in differentiation of SOX17-positive cells into FOXA2-positive cells. In some examples, the medium further includes a growth factor from the fibroblast growth factor (FGF) family. In some examples, the growth factor from the fibroblast growth factor (FGF) family is selected from the group consisting of keratinocyte growth factor (KGF), FGF2, FGF10, FGF21, and FGF8B. In some examples, the water-soluble synthetic polymer includes less than 85% hydrolyzed polyvinyl alcohol. In some examples, the water-soluble synthetic polymer includes about 80% hydrolyzed polyvinyl alcohol. In some examples, the method includes contacting the SOX17-positive cells with the water-soluble synthetic polymer for 1 to 5 days or 2 to 4 days. In some examples, the method includes contacting the SOX17-positive cells with the water-soluble synthetic polymer for about 1, 2, 3, 4, or 5 days.

[0017]

[0019] In some cases, the precursor cells of pancreatic beta cells include stem cells. In some cases, the method results in the differentiation of stem cells into SOX17-positive cells. In some cases, the stem cells include human stem cells. In some cases, the stem cells include embryonic stem cells or induced pluripotent stem cells. In some cases, the medium further includes a growth factor from the transforming growth factor beta (TGF-β) superfamily, a WNT signaling pathway activator, or both. In some examples, the medium comprises (a) a growth factor from the transforming growth factor beta (TGF-β) superfamily selected from the group consisting of inhibin, activin, Müllerian inhibitory substance (MIS), bone morphogenetic protein (BMP), decapentaplegic (dpp), Vg-1, monoclonal nonspecific inhibitory factor (MNSF), growth differentiation factor 8 (GDF8), and growth differentiation factor 11 (GDF11); and / or (b) a growth factor from the transforming growth factor beta (TGF-β) superfamily selected from the group consisting of CHIR99021, 3F8, A 1070722, AR-A 014418, BIO, BIO-acetoxime, FRATide, 10Z-hymenialdisine, indirubin-3'oxime, Kenpaullone, L803, L803-mts, lithium carbonate, NSC 693868, SB 216763, SB 415286, TC-G 24, TCS 2002, TCS 21311, and TWS 119. In some examples, the water-soluble synthetic polymer comprises less than 85% hydrolyzed polyvinyl alcohol. In some examples, the water-soluble synthetic polymer comprises about 80% hydrolyzed polyvinyl alcohol. In some examples, the method comprises contacting the stem cells with the water-soluble synthetic polymer for 1-5 days or 2-4 days. In some examples, the method comprises contacting the stem cells with the water-soluble synthetic polymer for about 1, 2, 3, 4, or 5 days.

[0018]

[0020] In some cases, the medium does not contain albumin protein. In some cases, the medium does not contain human serum albumin (HSA). In some cases, the medium does not contain serum.

[0021] In some aspects, disclosed herein are methods comprising: (i) differentiating a plurality of PDX1-positive, NKX6.1-negative pancreatic progenitor cells or precursor cells thereof in a medium comprising less than 85% hydrolyzed polyvinyl alcohol, thereby generating a plurality of PDX1-positive, NKX6.1-positive pancreatic progenitor cells; and (ii) culturing a plurality of PDX1-positive, NKX6.1-positive pancreatic progenitor cells in a composition comprising more than 85% hydrolyzed polyvinyl alcohol.

[0019]

[0022] In some examples, polyvinyl alcohol that is less than 85% hydrolyzed is about 80% hydrolyzed. In some examples, polyvinyl alcohol that is more than 85% hydrolyzed is about 87% to about 89% hydrolyzed. In some examples, the culturing results in the differentiation of multiple PDX1-positive, NKX6.1-positive pancreatic progenitor cells into NKX6.1-positive, ISL1-positive endocrine cells. In some examples, the culturing results in the differentiation of multiple PDX1-positive, NKX6.1-positive pancreatic progenitor cells into pancreatic beta cells. In some examples, the culturing can result in the differentiation of multiple PDX1-positive, NKX6.1-positive pancreatic progenitor cells into NKX6.1-positive, ISL1-positive endocrine cells, and the method further includes culturing the NKX6.1-positive, ISL1-positive endocrine cells in a medium containing serum or serum albumin. In some examples, the culturing can result in differentiation of a plurality of PDX1-positive, NKX6.1-positive pancreatic progenitor cells into NKX6.1-positive, ISL1-positive endocrine cells, and the method further includes culturing the NKX6.1-positive, ISL1-positive endocrine cells in a medium that does not contain polyvinyl alcohol. In some examples, the method includes culturing the NKX6.1-positive, ISL1-positive endocrine cells in a medium that contains human serum albumin, optionally at a concentration of about 0.01% to about 0.5%, about 0.05% to about 0.2%, or about 0.08% to about 0.12%, and optionally at a concentration of about 0.1%. In some examples, the composition comprising greater than 85% hydrolyzed polyvinyl alcohol further comprises one or more agents selected from the group consisting of a protein kinase C activator, a growth factor from the transforming growth factor beta (TGF-β) superfamily, a bone morphogenetic protein signaling pathway inhibitor, a growth factor from the fibroblast growth factor (FGF) family, a retinoic acid (RA) signaling pathway activator, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a sonic hedgehog (SHH) pathway inhibitor.

[0020]

[0023] In some examples, the composition comprising greater than 85% hydrolyzed polyvinyl alcohol is selected from the group consisting of: (a) a protein kinase C activator selected from the group consisting of phorbol 12,13-dibutyrate (PDBU), TPB, phorbol 12-myristate 13-acetate, and bryostatin 1; (b) a growth factor from the transforming growth factor beta (TGF-β) superfamily selected from the group consisting of inhibin, activin, Müllerian inhibitory substance (MIS), bone morphogenetic protein (BMP), decapentaplegic (dpp), Vg-1, monoclonal nonspecific suppressor of factor (MNSF), growth differentiation factor 8 (GDF8), and growth differentiation factor 11 (GDF11); (c) a bone morphogenetic protein signaling pathway inhibitor, including LDN193189 or DMH-1; (d) a fibroblast growth factor selected from the group consisting of keratinocyte growth factor (KGF), FGF2, FGF10, FGF21, and FGF8B. (e) a sonic hedgehog pathway inhibitor selected from the group consisting of SANT1, SANT2, SANT4, Cur6l4l4, forskolin, tomatidine, AY9944, triparanol, and cyclopamine; (f) a retinoic acid signaling pathway activator selected from the group consisting of retinoic acid, CD1530, AM580, TTHRB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, and CD2314; and / or (g) a ROCK inhibitor selected from the group consisting of thiazovivin, Y-27632, Fasudil / HA1077, and 14-1152.

[0021]

[0024] In some aspects, disclosed herein is an in vitro composition comprising a plurality of PDX1-positive, NKX6.1-positive cells, nicotinamide, and a growth factor from the epidermal growth factor (EGF) family.

[0022]

[0025] In some examples, the growth factor from the EGF family includes EGF. In some examples, the composition includes about 1 ng / mL to about 100 ng / mL, about 2 ng / mL to about 50 ng / mL, about 5 ng / mL to about 20 ng / mL, or about 7.5 ng / mL to about 15 ng / mL of EGF. In some examples, the composition includes about 10 ng / mL of EGF. In some examples, the composition does not include betacellulin. In some examples, the composition includes about 1 mM to about 100 mM, about 2 mM to about 50 mM, about 5 mM to about 20 mM, or about 7.5 mM to about 15 mM of nicotinamide. In some examples, the composition includes about 10 mM of nicotinamide. In some examples, the composition further comprises one or more drugs selected from the group consisting of a protein kinase C activator, a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, an epigenetic modifying compound, a retinoic acid (RA) signaling pathway activator, a sonic hedgehog (SHH) pathway inhibitor, a gamma-secretase inhibitor, a protein kinase inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a bone morphogenetic protein (BMP) signaling pathway inhibitor.In some examples, the composition comprises: (a) a TGF-β signaling pathway inhibitor selected from the group consisting of Alk5i II, A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB-525334, SD-208, and SB-505124; (b) a thyroid hormone signaling pathway activator, including T3 or GC-1; (c) 3-deazaneplanocin A (DZNep (d) an epigenetic modifying compound selected from the group consisting of retinoic acid, CD1530, AM580, TTHRB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, and CD2314; (e) sonic hedgehog pathway inhibitors selected from the group consisting of SANT1, SANT2, SANT4, Cur61414, forskolin, tomatidine, AY9944, triparanol, and cyclopamine; (f) gamma-secretase inhibitors, including XXI or DAPT; (g) protein kinase inhibitors, including staurosporine, Ro-31-8220, bicindolylmaleimide (Bis) compounds, 10'-{5"-[(methoxycarbonyl)amino]-2"-methyl}-phenylaminocarbonylstaurosporine, or stalalogues; (h) ROCK inhibitors, selected from the group consisting of thiazovivin, Y-27632, Fasudil / HA1077, and 14-1152; (i) phorbol 12,13-dibutyrate (PDBU), TPB, phorbol 12-myristate 13-acetate, and / or bryostatin 1; and / or (j) a bone morphogenetic protein signaling pathway inhibitor, including LDN193189 or DMH-1.

[0023]

[0026] In some embodiments, methods are disclosed herein that include contacting a plurality of PDX1-positive, NKX6.1-positive cells with a composition comprising nicotinamide and a growth factor from the epidermal growth factor (EGF) family. In some examples, the growth factor from the EGF family includes EGF. In some examples, the composition includes about 1 ng / mL to about 100 ng / mL, about 2 ng / mL to about 50 ng / mL, about 5 ng / mL to about 20 ng / mL, or about 7.5 ng / mL to about 15 ng / mL of EGF. In some examples, the composition includes about 10 ng / mL of EGF. In some examples, the composition does not include betacellulin. In some examples, the composition includes about 1 mM to about 100 mM, about 2 mM to about 50 mM, about 5 mM to about 20 mM, or about 7.5 mM to about 15 mM nicotinamide. In some examples, the composition includes about 10 mM nicotinamide. In some examples, the composition further comprises one or more agents selected from the group consisting of a protein kinase C activator, a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, an epigenetic modifying compound, a retinoic acid (RA) signaling pathway activator, a sonic hedgehog (SHH) pathway inhibitor, a gamma-secretase inhibitor, a protein kinase inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a bone morphogenetic protein (BMP) signaling pathway inhibitor.In some examples, the composition comprises: (a) a TGF-β signaling pathway inhibitor selected from the group consisting of Alk5i II, A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB-525334, SD-208, and SB-505124; (b) a thyroid hormone signaling pathway activator, including T3 or GC-1; (c) 3-deazaneplanocin A (DZNep (d) an epigenetic modifying compound selected from the group consisting of retinoic acid, CD1530, AM580, TTHRB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, and CD2314; (e) sonic hedgehog pathway inhibitors selected from the group consisting of SANT1, SANT2, SANT4, Cur61414, forskolin, tomatidine, AY9944, triparanol, and cyclopamine; (f) gamma-secretase inhibitors, including XXI or DAPT; (g) protein kinase inhibitors, including staurosporine, Ro-31-8220, bicindolylmaleimide (Bis) compounds, 10'-{5"-[(methoxycarbonyl)amino]-2"-methyl}-phenylaminocarbonylstaurosporine, or stalalogues; (h) ROCK inhibitors, selected from the group consisting of thiazovivin, Y-27632, Fasudil / HA1077, and 14-1152; (i) phorbol 12,13-dibutyrate (PDBU), TPB, phorbol 12-myristate and / or (j) a protein kinase C activator selected from the group consisting of 13-acetate, and bryostatin 1; and / or (j) a bone morphogenetic protein signaling pathway inhibitor, including LDN193189 or DMH-1. In some examples, the contacting is performed for about 1 to about 3 days. In some examples, the contacting is performed for about 1, 2, or 3 days.In some examples, the method includes removing nicotinamide and growth factors from the EGF family from the plurality of PDX1-positive, NKX6.1-positive cells for about 1 to about 3 days after contact; and then, after removal, contacting the plurality of PDX1-positive, NKX6.1-positive cells with a composition that does not contain nicotinamide or growth factors from the EGF family. In some examples, the method results in differentiation of the plurality of PDX1-positive, NKX6.1-positive cells into NKX6.1-positive, ISL1-positive cells.

[0024]

[0027] In some aspects, disclosed herein are devices comprising the compositions or cells disclosed herein.

[0028] In some examples, the device is configured to produce and release insulin when implanted in a subject. In some examples, the cells are encapsulated. In some examples, the device further includes a semipermeable membrane configured to retain the cells in the device and allow insulin to pass through.

[0025]

[0029] In some aspects, disclosed herein are methods of treating a subject having a disease characterized by high blood glucose levels over a prolonged period of time, the method comprising administering to the subject a composition or cell disclosed herein, or implanting into the subject a device disclosed herein. In some examples, the disease is diabetes.

[0026]

[0030] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings. [Brief explanation of the drawings]

[0027] [Figure 1A]

[0031] These plots and graphs show the effect of polyvinyl alcohol (PVA) on cell yield (Figure 1A), cell cluster formation (Figure 1B), and cell organization (percentage of PDX1-positive, NKX6.1-positive cells at stage 4) observed in one experiment during differentiation of pancreatic endocrine cells from stem cells. Using a six-stage stepwise protocol as the base protocol for differentiation, different PVA materials, 87-89% PVA, 87-90% PVA, and 99% PVA, were used to replace human serum albumin as a supplement to the medium from stage 1 through stage 4. We found that 87-89% PVA enhanced cell yield (Figure 1A) and increased the percentage of PDX1-positive, NKX6.1-positive cells at stage 4 (Figure 1C). [Figure 1B]

[0031] Plots and graphs showing the effects of polyvinyl alcohol (PVA) observed in one experiment on cell yield (Figure 1A), cell cluster formation (Figure 1B), and cell organization (percentage of PDX1-positive, NKX6.1-positive cells at stage 4) during differentiation of pancreatic endocrine cells from stem cells. [Figure 1C]

[0031] Plots and graphs showing the effects of polyvinyl alcohol (PVA) observed in one experiment on cell yield (Figure 1A), cell cluster formation (Figure 1B), and cell organization (percentage of PDX1-positive, NKX6.1-positive cells at stage 4) during differentiation of pancreatic endocrine cells from stem cells. [Figure 2A]

[0032] These plots and graphs show the effect of polyvinyl alcohol (PVA) on cell yield (Figure 2A) and cell composition (the percentage of PDX1-positive, NKX6.1-positive cells at stage 4, Figure 2B, and the percentage of NKX6.1-positive, ISL1-positive cells at stage 5, Figure 2C) observed in another experiment during differentiation of pancreatic endocrine cells from stem cells. A six-stage stepwise protocol was used as the basic protocol for differentiation. From stage 1 to stage 5, different PVA materials, 80% PVA and 87-89% PVA, were used as supplements to the medium or to supplement HSA ("89 PVA + HSA"). Compared to the HSA control, 80% PVA increased the cell yield and percentage of PDX1-positive, NKX6.1-positive cells up to stage 4 (Figure 2B), but decreased the cell yield and percentage of NKX6.1-positive, ISL1-positive cells at stage 5 (Figure 2C). [Figure 2B] 2A and 2B are plots and graphs showing the effect of polyvinyl alcohol (PVA) observed in another experiment on cell yield (FIG. 2A) and cell composition (percentage of PDX1-positive, NKX6.1-positive cells at stage 4, FIG. 2B; and percentage of NKX6.1-positive, ISL1-positive cells at stage 5, FIG. 2C) during differentiation of pancreatic endocrine cells from stem cells. [Figure 2C] 2A and 2B are plots and graphs showing the effect of polyvinyl alcohol (PVA) observed in another experiment on cell yield (FIG. 2A) and cell composition (percentage of PDX1-positive, NKX6.1-positive cells at stage 4, FIG. 2B; and percentage of NKX6.1-positive, ISL1-positive cells at stage 5, FIG. 2C) during differentiation of pancreatic endocrine cells from stem cells. [Figure 3]

[0033] FIG. 1 is a schematic diagram of five different PVA supplementation paradigms for in vitro pancreatic cell differentiation. [Figure 4A]

[0034] Plots demonstrating the effects of five different PVA supplementation paradigms on cell yield (Figure 4A) and cell composition (percentage of NKX6.1-positive, ISL1-positive cells at stage 5, Figure 4B) are shown. All PVA supplementation paradigms were found to exhibit more consistent cell yields compared to the control HSA paradigm (Figure 4A). Additionally, switching from 80% PVA to 87-89% PVA at stage 5 was found to reverse the decrease in the percentage of NKX6.1-positive, ISL1-positive cells at stage 5, which was seen with all 80% PVA paradigms (Figure 4B). [Figure 4B]

[0034] Plots demonstrating the effect of five different PVA supplementation paradigms on cell yield (Fig. 4A) and cell composition (percentage of NKX6.1-positive, ISL1-positive cells at stage 5, Fig. 4B) are shown. [Figure 5A]

[0035] Figure 5A is a plot showing that replacing betacellulin with nicotinamide and EGF at stage 5 resulted in approximately the same total cell yield and β-cell yield compared to the basic differentiation protocol (control) that included betacellulin. [Figure 5B] FIG. 5B shows the flow cytometry results showing that the percentage of NKX6.1-positive, ISL1-positive cells was higher in the nicotinamide and EGF treatments compared to the betacellulin treatment. [Figure 6]

[0036] Figure 6 shows plots summarizing the effect of replacing betacellulin with nicotinamide and EGF at stage 5 on recovery and total SC-β cell yield at stage 6. The two differentiation conditions were found to result in similar recovery and total SC-β cell yield. DETAILED DESCRIPTION OF THE INVENTION

[0028]

[0037] The following description and examples will explain the embodiments of the present disclosure in detail. It should be understood that the present disclosure is not limited to the specific embodiments described herein and may therefore vary. Those skilled in the art will recognize that there are many variations and modifications to the present disclosure, which are encompassed within the scope of the present disclosure.

[0029]

[0038] All terms are intended to be understood as understood by one of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0030]

[0039] The headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0040] Although various features of the present disclosure may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although for clarity the present disclosure may be described herein in the context of separate embodiments, the present disclosure may also be implemented in a single embodiment.

[0031]

[0041] The following definitions supplement the definitions in the art and are directed to this application, and are not attributable to any related or unrelated examples, such as any commonly owned patents or patent applications. Although any methods and materials similar or equivalent to those described herein can be used in the practice of testing the present disclosure, preferred materials and methods are described herein. Therefore, the terms used herein are only for the purpose of describing specific embodiments and are not intended to be limiting.

[0032]

[0042] In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0033]

[0043] The use of "or" in this application means "and / or" unless stated otherwise. As used herein, the terms "and / or" and "any combinations thereof," and their grammatical equivalents, can be used interchangeably. These terms can convey that any combination is specifically intended. For illustrative purposes only, the following phrases, "A, B, and / or C" or "A, B, C, or any combinations thereof," can mean "A individually, B individually, C individually, A and B, B and C, A and C, and A, B, and C." The term "or" can be used conjunctively or disjunctively unless the context specifically implies a disjunctive use.

[0034]

[0044] Furthermore, use of the term "including" and other forms such as "include," "includes," and "included" is not limiting.

[0045] References in the specification to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least some embodiments of the present disclosure, but not necessarily in all embodiments.

[0035]

[0046] As used in the specification and claims, the words "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") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is intended that any embodiment discussed herein can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, the compositions of the disclosure can be used to achieve the methods of the disclosure.

[0036]

[0047] As used herein, the term "about" in connection with a reference numerical value and grammatical equivalents thereof may include the numerical value itself and a range of values ​​plus or minus 10% of that numerical value.

[0048] The terms "about" or "approximately" refer to within an acceptable error range for a particular value, as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within or more than one standard deviation, as practiced in the art. Alternatively, "about" can mean a range of up to 20%, 10%, 5%, or 1% of a given value. In another example, the amount "about 10" includes 10 and any amount from 9 to 11. In yet another example, the term "about" in connection with a reference numerical value can also include ranges of plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of that value. Alternatively, particularly with respect to biological systems or processes, the term "about" can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value. When specific values ​​are described in applications and claims, unless otherwise stated, the term "about" should be assumed to mean within an acceptable error range for the particular value.

[0037]

[0049] As used herein, term " diabetes mellitus " and its grammatical equivalents can refer to the disease characterized by long-term high blood sugar level.For example, as used herein, term " diabetes mellitus " and its grammatical equivalents can refer to all or any type of diabetes, including but not limited to type 1 diabetes, type 2 diabetes, cystic fibrosis-related diabetes, surgical diabetes, gestational diabetes and mitochondrial diabetes.In some cases, diabetes can be a form of hereditary diabetes.

[0038]

[0050] The term "endocrine cells," unless otherwise specified, may refer to hormone-producing cells present in the pancreas of a living organism, such as "pancreatic islets," "pancreatic islet cells," "pancreatic islet equivalents," "pancreatic islet-like cells," "pancreatic islets," and grammatical equivalents thereof. In one embodiment, endocrine cells can be differentiated from pancreatic progenitor cells or precursors. Pancreatic islet cells may include various types of cells, including, but not limited to, pancreatic alpha cells, pancreatic beta cells, pancreatic delta cells, pancreatic F cells, and / or pancreatic epsilon cells. Pancreatic islet cells may also refer to groups of cells, cell clusters, etc.

[0039]

[0051] The terms "progenitor cell" and "precursor" cell are used interchangeably herein to refer to cells that have a more primitive cellular phenotype (e.g., at an earlier step in the developmental pathway or development than a fully differentiated cell) compared to cells that can result from differentiation. Progenitor cells can also often have significant or extremely high proliferative potential. Progenitor cells can give rise to many different differentiated cell types or a single differentiated cell type, depending on the developmental pathway and the environment in which the cells develop and differentiate.

[0040]

[0052] The term "precursor thereof" in relation to an insulin-positive endocrine cell can mean any cell, including, for example, a pluripotent stem cell, a definitive endoderm cell, a primitive gut cell, a pancreatic progenitor cell, or an endocrine progenitor cell, that can differentiate into an insulin-positive endocrine cell when cultured under conditions suitable for differentiating the precursor cell into an insulin-positive endocrine cell.

[0041]

[0053] The terms "stem cell-derived beta cells," "SC-beta cells," "functional beta cells," "functional pancreatic beta cells," "mature SC-beta cells," and their grammatical equivalents can refer to cells (e.g., non-native pancreatic beta cells) that exhibit at least one marker indicative of a pancreatic beta cell (e.g., PDX-1 or NKX6.1), secrete insulin, and exhibit a glucose-stimulated insulin secretion (GSIS) response characteristic of endocrine mature beta cells. In some embodiments, the terms "SC-beta cells" and "non-native beta cells" as used herein are interchangeable. In some embodiments, "SC-beta cells" include mature pancreatic cells. It should be understood that SC-beta cells need not be derived (e.g., directly) from stem cells. This is because the disclosed methods can derive SC-β cells from any insulin-positive endocrine cell or its precursor using any cell as a starting point (the invention is not intended to be limited in this manner, and examples include embryonic stem cells, induced pluripotent stem cells, progenitor cells, partially reprogrammed somatic cells (e.g., somatic cells partially reprogrammed to an intermediate state between an induced pluripotent stem cell and the somatic cell from which it was derived), pluripotent cells, totipotent cells, transdifferentiated versions of any of the above cells, and the like). In some embodiments, the SC-β cells exhibit a response, e.g., in vitro, to multiple glucose challenges (e.g., a series of at least one, at least two, or at least three or more glucose challenges). In some embodiments, the response resembles the response of endogenous pancreatic islets (e.g., human pancreatic islets) to multiple glucose challenges. In some embodiments, the morphology of SC-β cells resembles that of endogenous β cells. In some embodiments, the SC-β cells exhibit an in vitro GSIS response similar to that of endogenous β cells. In some embodiments, SC-β cells exhibit an in vivo GSIS response similar to that of endogenous β cells. In some embodiments, SC-β cells exhibit both in vitro and in vivo GSIS responses similar to that of endogenous β cells. The GSIS response of SC-β cells can be observed within 2 weeks after transplantation of SC-β cells into a host (e.g., a human or animal).In some embodiments, SC-β cells package insulin into secretory granules. In some embodiments, SC-β cells exhibit encapsulated crystalline insulin granules. In some embodiments, SC-β cells exhibit a stimulation index greater than 1. In some embodiments, SC-β cells exhibit a stimulation index greater than 1.1. In some embodiments, SC-β cells exhibit a stimulation index greater than 2. In some embodiments, SC-β cells exhibit cytokine-induced apoptosis in response to cytokines. In some embodiments, insulin secretion from SC-β cells is enhanced in response to known antidiabetic agents (e.g., secretagogues). In some embodiments, SC-β cells are monohormonal. In some embodiments, SC-β cells do not abnormally co-express other hormones, such as glucagon, somatostatin, or pancreatic polypeptide. In some embodiments, SC-β cells exhibit a low replication rate. In some embodiments, SC-β cells increase intracellular Ca2+ in response to glucose.

[0042]

[0054] The terms "stem cell-derived α cells," "SC-α cells," "functional α cells," "functional pancreatic α cells," "mature SC-α cells," and their grammatical equivalents can refer to cells (e.g., non-native pancreatic α cells) that exhibit at least one marker indicative of a pancreatic α cell (e.g., expressing glucagon, ISL1, but not NKX6.1), express glucagon, and secrete functional glucagon. In some embodiments, "SC-α cells" do not express somatostatin. In some embodiments, "SC-α cells" do not express insulin. In some embodiments, the terms "SC-α cells" and "non-native α cells" as used herein are interchangeable. In some embodiments, "SC-α cells" include mature pancreatic cells.

[0043]

[0055] 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 can refer to cells (e.g., non-native pancreatic δ cells) that exhibit at least one marker indicative of pancreatic δ cells (e.g., somatostatin) and express and secrete somatostatin. 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.

[0044]

[0056] The terms "stem cell-derived enterochromaffin (EC) cells," "SC-EC cells," and their grammatical equivalents can refer to cells (e.g., non-native pancreatic EC cells) that exhibit at least one marker indicative of a pancreatic EC cell (e.g., expressing VMAT1, NKX6.1, but not ISL1). In some embodiments, the terms "SC-EC cells" and "non-native EC cells" used herein are interchangeable.

[0045]

[0057] It should be understood that, like SC-β cells, SC-α, SC-δ, and SC-EC cells need not be derived (e.g., directly) from stem cells, as the disclosed methods can derive SC-α cells from other progenitor cells produced during in vitro differentiation of SC-β cells as a starting point (e.g., embryonic stem cells, induced pluripotent stem cells, progenitor cells, partially reprogrammed somatic cells (e.g., somatic cells partially reprogrammed to an intermediate state between an induced pluripotent stem cell and the somatic cell from which it was derived), pluripotent cells, totipotent cells, transdifferentiated versions of any of the foregoing cells, etc., can be used, as the invention is not intended to be so limited).

[0046]

[0058] As used herein, the term "insulin-producing cells" and its grammatical equivalents refer to cells that differentiate from pancreatic progenitor cells or their precursors and secrete insulin. Insulin-producing cells, as that term is used herein, can include pancreatic β cells and pancreatic β-like cells (e.g., insulin-positive endocrine cells) that synthesize (e.g., transcribe the insulin gene, translate proinsulin mRNA, and modify proinsulin mRNA to form insulin protein), express (e.g., realize the phenotypic trait conveyed by the insulin gene), or secrete (release insulin into the extracellular space) insulin in a constitutive or inducible manner. For example, a population of insulin-producing cells generated by differentiating insulin-positive endocrine cells or their precursors into SC-β cells according to the methods of the present disclosure can be pancreatic β cells or β-like cells (e.g., cells that have at least one or at least two characteristics of endogenous β cells and exhibit a glucose-stimulated insulin secretion (GSIS) response similar to that of endogenous mature β cells). For example, a population of insulin-producing cells produced by the methods disclosed herein may include mature pancreatic β cells or SC-β cells, and may also include non-insulin-producing cells (e.g., cells that do not produce or secrete insulin but have a cell-like phenotype).

[0047]

[0059] The terms "insulin-positive β-like cells," "insulin-positive endocrine cells," and their grammatical equivalents can refer to cells (e.g., pancreatic endocrine cells) that exhibit at least one marker indicative of pancreatic β cells and also express insulin, but lack the glucose-stimulated insulin secretion (GSIS) response characteristic of endogenous β cells. Exemplary markers of "insulin-positive endocrine cells" include, but are not limited to, NKX6.1, ISL1, and insulin. In some instances, the terms "insulin-positive endocrine cells" and "NKX6.1-positive, ISL1-positive cells" are used interchangeably.

[0048]

[0060] The term "β cell marker" refers, without limitation, to proteins, peptides, nucleic acids, protein and nucleic acid polymorphisms, splice variants, protein or nucleic acid fragments, elements, and other analytes that are specifically expressed or present in pancreatic 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.

[0049]

[0061] The term "pancreatic endocrine marker" can refer, without limitation, to proteins, peptides, nucleic acids, protein and nucleic acid polymorphisms, splice variants, protein or nucleic acid fragments, elements, and other analytes that are specifically expressed or present in pancreatic endocrine cells. Exemplary pancreatic endocrine cell markers include, but are not limited to, Ngn-3, NeuroD, and Islet-1.

[0050]

[0062] The terms "pancreatic progenitor cells," "pancreatic endocrine precursor cells," "pancreatic precursors," "pancreatic endocrine precursors," and their grammatical equivalents are used interchangeably herein and can refer to stem cells that can become pancreatic hormone-expressing cells that can form pancreatic endocrine cells, pancreatic exocrine cells, or pancreatic duct cells. These cells are committed to differentiation toward at least one type of pancreatic cell, such as insulin-producing beta cells, glucagon-producing alpha cells, somatostatin-producing delta cells (or D cells), and / or pancreatic polypeptide-producing F cells. Such cells can express at least one of the following markers: NGN3, NKX2.2, NeuroD, ISL1, Pax4, Pax6, or ARX.

[0051]

[0063] As used herein, the term "PDX1-positive pancreatic progenitor cells" may refer to cells that are pancreatic endoderm (PE) cells capable of differentiating into SC-β cells, such as pancreatic β cells. PDX1-positive pancreatic progenitor cells express the marker PDX1. Other markers include, but are not limited to, Cdcp1, Ptf1a, HNF6, or NRx2.2. PDX1 expression can be assessed by any method known to those skilled in the art, such as immunochemistry using an anti-PDX1 antibody or quantitative RT-PCR. In some cases, PDX1-positive pancreatic progenitor cells lack NKX6.1 expression. In some cases, PDX1-positive pancreatic progenitor cells can also be referred to as PDX1-positive, NKX6.1-negative pancreatic progenitor cells because they lack NKX6.1 expression. In some cases, PDX1-positive pancreatic progenitor cells can also be referred to as "pancreatic foregut endoderm cells."

[0052]

[0064] The terms "PDX1-positive, NKX6.1-positive pancreatic progenitor cells" and "NKX6.1-positive pancreatic precursors" are used interchangeably herein and can refer to cells that are pancreatic endoderm (PE) cells and have the ability to differentiate into insulin-producing cells, such as pancreatic β cells. PDX1-positive, NKX6-1-positive pancreatic progenitor cells express the markers PDX1 and NKX6-1. Other markers include, but are not limited to, Cdcp1, Ptf1a, HNF6, or NRx2.2. NKX6-1 expression can be assessed by any method known to those skilled in the art, such as immunochemistry or quantitative RT-PCR using anti-NKX6-1 antibodies. As used herein, the terms "NKX6.1" and "NKX6-1" are equivalent and interchangeable. In some instances, PDX1-positive, NKX6-1-positive pancreatic progenitor cells can also be referred to as "pancreatic foregut precursor cells."

[0053]

[0065] The terms "NeuroD" and "NeuroD1" are used interchangeably to identify proteins and their encoding genes expressed in pancreatic endocrine precursor cells.

[0066] The term "epigenetics" refers to heritable changes in gene function that do not involve changes in DNA sequence. While epigenetics most often refers to chromosomal changes that affect gene activity and expression, it can also be used to describe any heritable phenotypic change that does not result from modifications to the genome. Such effects on cellular and physiological phenotypic traits can result from external or environmental factors or be part of a normal developmental program. Epigenetics can also refer to functionally relevant changes in the genome that do not involve changes in the nucleotide sequence. Examples of mechanisms that generate such changes include DNA methylation and histone modifications, each of which alters the way genes are expressed without altering the underlying DNA sequence. Gene expression can be controlled by the action of repressor proteins that bind to silencer regions of DNA. These epigenetics can persist through cell divisions during the lifespan of a cell and can persist for multiple generations, even though they do not involve changes in the organism's underlying DNA sequence. One example of epigenetics in eukaryotic cell biology is the process of cellular differentiation. During morphogenesis, totipotent stem cells give rise to a variety of pluripotent cells, which can then give rise to fully differentiated cells.

[0054]

[0067] The term "epigenetic modifying compound" refers to a compound that induces epigenetic changes in genes, i.e., alters gene expression without altering the DNA sequence. Epigenetics helps determine whether genes are turned on or off and can affect protein production in certain cells, such as beta cells. Epigenetic modifications, such as DNA methylation and histone modifications, alter DNA accessibility and chromatin structure, thereby regulating patterns of gene expression. These processes are important for the normal development and differentiation of unique cell lineages in adult organisms. They can be modified by exogenous influences and therefore may contribute to or be the result of environmental modifications of phenotypes or pathological phenotypes. Importantly, epigenetic modifications play a key role in regulating pluripotency genes, which are inactivated during differentiation. Non-limiting examples of epigenetic modifying compounds include DNA methylation inhibitors, histone acetyltransferase inhibitors, histone deacetylase inhibitors, histone methyltransferase inhibitors, bromodomain inhibitors, or any combination thereof.

[0055]

[0068] The term "differentiated cell" or its grammatical equivalents refers to any primary cell that is not pluripotent in its natural form as that term is defined herein. Alternatively, the term "differentiated cell" can refer to a cell of a more specialized cell type derived from a cell of a less specialized cell type (e.g., a stem cell, such as an induced pluripotent stem cell) during the cell differentiation process. Without wishing to be limited by theory, pluripotent stem cells during normal ontogeny can initially differentiate into endodermal cells, which can form pancreatic cells and other endodermal cell types. Further differentiation of endodermal cells leads to the pancreatic pathway, and in some embodiments, approximately 98% of the cells become exocrine, ductal, or matrix cells, and approximately 2% become endocrine cells. Early endocrine cells are pancreatic islet precursor cells, which can then further differentiate into insulin-producing cells (e.g., functional endocrine cells) that secrete insulin, glucagon, somatostatin, or pancreatic polypeptide. Endodermal cells can also differentiate into other cells of endodermal origin, such as lung, liver, intestine, thymus, etc.

[0056]

[0069] As used herein, the term "somatic cell" can refer to any cell that forms an organism, as opposed to a germline cell. In mammals, germline cells (also known as "gametes") are sperm and eggs that fuse during fertilization to produce a cell called a zygote, from which the entire mammalian embryo develops. Aside from sperm and eggs (gametocytes) and undifferentiated stem cells, from which somatic cells are made, all other cell types in the mammalian body are somatic cells. Internal organs, skin, bone, blood, and connective tissue are all made from somatic cells. In some embodiments, the somatic cell is a "non-embryonic somatic cell," which refers to a somatic cell that is not present in or obtained from an embryo and that does not result from in vitro propagation of such a cell. In some embodiments, the somatic cell is an "adult somatic cell," which refers to a cell that is present in or obtained from an organism other than an embryo or fetus, or that results from in vitro propagation of such a cell. Unless otherwise indicated, the method for converting at least one insulin-positive endocrine cell or precursor thereof into an insulin-producing, glucose-responsive cell can be performed both in vivo and in vitro (in vivo performed when at least one insulin-positive endocrine cell or precursor thereof is present in the subject, and in vitro performed using at least one insulin-positive endocrine cell or precursor thereof that has been isolated and maintained in culture).

[0057]

[0070] As used herein, the term "adult cell" may refer to cells found throughout the body after embryonic development.

[0071] As used herein, the term "endodermal cell" can refer to a cell that is from one of the three primary germ cell layers in a very early embryo (the other two being mesoderm and ectoderm). Endoderm is the innermost of the three layers. Endoderm cells can differentiate to give rise first to the embryonic gut, and then to the lining of the respiratory and digestive tract (e.g., intestine), liver, and pancreas.

[0058]

[0072] As used herein, the term "cells of endodermal origin" refers to any cell that develops or differentiates from endodermal cells. For example, cells of endodermal origin include cells of the liver, lung, pancreas, thymus, intestine, stomach, and thyroid. Without wishing to be bound by theory, hepatic and pancreatic progenitor cells (also called pancreatic precursor cells) can develop from endodermal cells in the embryonic foregut. Shortly after their identification, hepatic and pancreatic precursor cells rapidly acquire distinct cellular functions and regenerative capabilities. These changes are driven by inductive signals and genetic regulatory factors that are highly conserved among vertebrates. Interest in organ development and regeneration has been fueled by the strong need for hepatocytes and pancreatic β cells in the therapeutic treatment of liver failure and type 1 diabetes. Studies in various model organisms and humans have revealed a network of evolutionarily conserved inductive signals and transcription factors that trigger hepatic and pancreatic cell differentiation and provide guidance on how to promote hepatic and β cell differentiation from diverse stem and progenitor cell types.

[0059]

[0073] As used herein, the term "definitive endoderm" may refer to cells that can differentiate from endoderm cells and differentiate into SC-β cells (e.g., pancreatic β cells). Definitive endoderm cells express the marker Sox17. Other markers characteristic of definitive endoderm cells include, but are not limited to, MIXL2, GATA4, HNF3B, GSC, FGF17, VWF, CALCR, FOXQ1, CXCR4, Cerberus, OTX2, goosecoid, C-Kit, CD99, CMKOR1, and CRIP1. In particular, definitive endoderm cells herein express Sox17 and in some embodiments, Sox17 and HNF3B, but do not express significant levels of GATA4, SPARC, APF, or DAB. Definitive endoderm cells are not positive for the marker PDX1 (e.g., they are PDX1 negative). Definitive endoderm cells have the potential to differentiate into cells including liver, lung, pancreatic, thymus, intestine, stomach, and thyroid cells. Expression of Sox17 and other definitive endoderm markers can be assessed by any method known to those skilled in the art, such as immunochemistry using anti-Sox17 antibodies or quantitative RT-PCR.

[0060]

[0074] The term "pancreatic endoderm" can refer to cells of endodermal origin that can differentiate into multiple pancreatic lineages, including pancreatic beta cells, but no longer have the capacity to differentiate into non-pancreatic lineages.

[0075] As used herein, the terms "primitive gut cells," "primitive gut," or "gut cells" can refer to cells that differentiate from endoderm cells and can differentiate into SC-β cells (e.g., pancreatic β cells). Primitive gut cells express at least one of the following markers: HNF1-β, HNF3-β, or HNF4-α. In some cases, primitive gut cells are FOXA2-positive and SOX2-positive, i.e., express both FOXA2 (also known as HNF3-β) and SOX2. In some cases, primitive gut cells are FOXA2-positive and PDX1-negative, i.e., express FOXA2 but not PDX1. Primitive gut cells have the potential to differentiate into cells including lung, liver, pancreatic, stomach, and intestinal cells. Expression of HNF1-β and other primitive gut markers can be assessed by any method known to those skilled in the art, such as immunochemistry using an anti-HNF1-β antibody.

[0061]

[0076] As used herein, the term "stem cell" may refer to an undifferentiated cell that can proliferate and give rise to more progenitor cells that have the capacity to produce a large number of mother cells that can then give rise to differentiated or differentiable daughter cells. The daughter cells themselves can be induced to proliferate and generate progeny that subsequently differentiate into one or more mature cell types while retaining one or more cells with the developmental potential of the parent. The term "stem cell" may refer to a subset of progenitor cells that, under certain circumstances, have the ability or potential to differentiate into a more specialized or differentiated phenotype and, under certain circumstances, retain the ability to proliferate substantially without differentiation. In one embodiment, the term stem cell generally refers to a naturally occurring mother cell whose descendants (progeny) specialize through differentiation, often in different directions, e.g., by acquiring entirely distinct characteristics, as occurs in the gradual diversification of cells and tissues in an embryo. Cell differentiation is a complex process that typically occurs through many cell divisions. Differentiated cells can be derived from pluripotent cells, which themselves are derived from pluripotent cells, and so on. Although each of these pluripotent cells is considered a stem cell, the range of cell types each can give rise to can vary considerably. Some differentiated cells also have the ability to give rise to cells with greater developmental potential. Such ability can be natural or artificially induced by treatment with various factors. In many biological examples, stem cells are also "pluripotent" because they can generate progeny with two or more different cell types, but this is not necessary for them to be "stem." "Self-renewal" is another classic part of the definition of stem cells, and it is important as used herein. Theoretically, self-renewal can occur through one of two main mechanisms: stem cells can divide asymmetrically, with one daughter retaining the stem cell state and the other daughter expressing several different, other specific functions and phenotypes. Alternatively, some stem cells in a population can divide symmetrically into two stem cells, thereby maintaining some stem cells in the population as a whole while other cells in the population give rise only to differentiated progeny.Although technically, cells that begin as stem cells progress toward a differentiated phenotype, it is also possible for them to "reverse" and reverse the stem cell phenotype. This is often referred to by those skilled in the art as "dedifferentiation," "reprogramming," or "reverse differentiation." As used herein, the term "pluripotent stem cells" includes embryonic stem cells, induced pluripotent stem cells, placental stem cells, and the like.

[0062]

[0077] As used herein, the term "pluripotent" can refer to cells that have the ability to differentiate under various conditions into two or more differentiated cell types, preferably into cell types characteristic of all three germ cell layers. Pluripotent cells are primarily characterized by their ability to differentiate into two or more cell types, preferably into all three germ layers, using, for example, a nude mouse teratoma formation assay. While pluripotency can also be demonstrated by the expression of embryonic stem (ES) cell markers, the preferred test for pluripotency is to demonstrate the ability to differentiate into cells of each of the three germ layers. Note that simply culturing such cells does not, in and of itself, render them pluripotent. Reprogrammed pluripotent cells (e.g., iPS cells, as that term is defined herein) are also characterized by their ability to be passaged for extended periods without loss of growth potential, compared to primary parent cells, which generally have the ability to divide only a limited number of times in culture.

[0063]

[0078] As used herein, the terms "iPS cells" and "induced pluripotent stem cells" are used interchangeably and may refer to pluripotent stem cells that have been artificially derived (e.g., induced or by complete reversion) from non-pluripotent cells, typically adult somatic cells, for example, by inducing forced expression of one or more genes.

[0064]

[0079] The term "phenotype" can refer to all of the one or several biological characteristics that define a cell or organism under a particular set of environmental conditions and factors, regardless of the actual genotype.

[0065]

[0080] The terms "subject," "patient," or "individual" are used interchangeably herein and may refer to an animal, e.g., a human, from which cells are obtained and / or to which treatment, including prophylactic treatment, using the cells described herein is provided. For treatment of an infection, condition, or disease state specific to a particular animal, such as a human subject, the term subject may refer to that particular animal. "Non-human animal" and "non-human mammal," used interchangeably herein, include mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The term "subject" also encompasses any vertebrate, including, but not limited to, mammals, reptiles, amphibians, and fish. Advantageously, however, the subject is a mammal, such as a human, or other mammal, e.g., a dog, cat, horse, other livestock, or cows, sheep, pigs, or other production mammals. "A patient in need thereof" or "subject in need thereof," as used herein, refers to a patient diagnosed with or suspected of having a disease or disorder, for example, but not limited to, diabetes.

[0066]

[0081] As used herein, "administering" can mean providing one or more compositions described herein to a patient or subject. By way of example and not limitation, administration, e.g., injection, of a composition can be performed via intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im) injection. One or more such routes can be employed. Parenteral administration can be, for example, via bolus injection or gradual perfusion over time. Alternatively, or concurrently, administration can be via the oral route. Additionally, administration can involve surgical deposition of a bolus or pellet of cells or positioning of a medical device. In one embodiment, a composition of the present disclosure can comprise engineered or host cells expressing a nucleic acid sequence described herein or a vector comprising at least one nucleic acid sequence described herein in an amount effective to treat or prevent a proliferative disorder. A pharmaceutical composition can include a cell population described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include a buffer such as neutral buffered saline or phosphate buffered saline, a carbohydrate such as glucose, mannose, sucrose, or dextran, mannitol, a protein, polypeptide, or an amino acid such as glycine, an antioxidant, a chelating agent such as EDTA or glutathione, an adjuvant (e.g., aluminum hydroxide), and a preservative.

[0067]

[0082] Some numerical values ​​disclosed throughout are referred to as, for example, "X is at least or at least about 100, or 200 [or any number]." This includes the number itself and the following: i) X is at least 100; ii) X is at least 200; iii) X is at least about 100, and iv) X is at least about 200 All of the above are included.

[0068]

[0083] All of these different combinations are contemplated by the numerical values ​​disclosed throughout, and unless otherwise specifically indicated to the contrary, all numerical values ​​disclosed, whether in terms of therapeutic administration, days, months, years, weights, dosages, etc., should be construed in this manner.

[0069]

[0084] The generally disclosed ranges may be referred to, for example, as "X is administered on or about days 1-2, or on or about days 2-3 [or any range of numbers]." The ranges may include both the numbers themselves (e.g., the endpoints of the ranges) and the following: i) X is administered between days 1 and 2; ii) X is administered between days 2 and 3; iii) X is administered between about days 1 and 2; iv) X is administered between about days 2 and 3; v) X is administered between day 1 and about day 2; vi) X is administered between day 2 and about day 3; vii) X is administered between about day 1 and about day 2, and viii) X is administered between about day 2 and about day 3 All of the above are included.

[0070]

[0085] All of these different combinations are contemplated by the ranges disclosed throughout. Unless otherwise specifically indicated to the contrary, all disclosed ranges, whether in terms of administration of therapeutic agents, days, months, years, weights, dosages, etc., should be interpreted in this manner.

[0071]

[0086] In some aspects, the present disclosure relates to the use of water-soluble synthetic polymers for the in vitro generation and culture of pancreatic endocrine cells, such as pancreatic beta cells, pancreatic alpha cells, pancreatic delta cells, or enterophrenia cells, or pancreatic F cells, or epsilon cells, or their precursor cells.In some examples, compositions and methods are provided herein for the in vitro generation of pancreatic endocrine cells, such as pancreatic beta cells, pancreatic alpha cells, pancreatic delta cells, or enterophrenia cells, or pancreatic F cells, or pancreatic epsilon cells, or their precursor cells.The methods provided herein can lead to the efficient generation of pancreatic endocrine cells or their precursor cells through in vitro differentiation.

[0072]

[0087] In some instances, methods for generating pancreatic beta cells or their precursor cells are provided herein. Historically, serum has been an important component of cell culture methodologies as a provider of complex biological molecules, such as hormones, growth factors, adhesion factors, and numerous low-molecular-weight nutrients. In certain instances, for example, during in vitro cell differentiation, replacement of serum with serum albumin is common and can provide sufficient support for the successful growth of various cell types, the development of permanent cell lines, and, in some instances, the differentiation of cells into certain desired cell types. Albumin is the major protein in serum, typically present at approximately 50 mg / ml, accounting for approximately 60% of total protein. Approximately 60% of total body albumin is present in the extravascular space, such as within the interstitial space of tissues, suggesting its important role in the physiological stability of cells. Without wishing to be bound by any particular theory, the primary functions of albumin include (1) maintaining blood oncotic pressure and pH, (2) binding and transport of physiologically important ligands, including lipids, metal ions, amino acids, and other factors, and (3) antioxidant function, but are summarized primarily in terms of its role in the circulation. These fundamental functions of the albumin molecule also apply to interactions between albumin and cells in animal tissues, or, importantly for this review, between cells grown in culture, whether in the laboratory or on a large-scale commercial scale. Both bovine serum albumin (BSA) and human serum albumin (HSA) are widely used in tissue engineering, e.g., for the in vitro differentiation of pancreatic cells. However, the cost and lot-to-lot variability of serum albumins, e.g., BSA and HSA, remain significant challenges for industrial-scale production of cell products. The methods and compositions provided herein can solve this problem by using water-soluble polymers in place of serum or serum albumin during the in vitro generation of pancreatic beta cells or their precursor cells.

[0073]

[0088] In some examples, the method includes differentiating a plurality of precursor cells of pancreatic beta cells in a serum- or serum albumin-free medium. In some examples, the method includes differentiating a plurality of precursor cells of pancreatic beta cells in a medium containing a water-soluble polymer, for example, a water-soluble synthetic polymer. The method can be applied to any stage of differentiation from stem cells to pancreatic beta cells. The differentiation process can result in the production of precursor cells of pancreatic beta cells, such as Sox17-positive cells, FOXA2-positive cells, PDX1-positive cells, NKX6.1-positive cells, ISL1-positive cells, or insulin-positive endocrine cells, or ultimately mature functional pancreatic beta cells.

[0074]

[0089] In some examples, the present disclosure provides an in vitro composition comprising a plurality of pancreatic beta cells or their precursor cells in a medium containing a water-soluble polymer, e.g., a water-soluble synthetic polymer. The composition can comprise a plurality of pancreatic beta cells or precursor cells of pancreatic beta cells, such as, but not limited to, Sox17-positive cells, FOXA2-positive cells, PDX1-positive cells, NKX6.1-positive cells, ISL1-positive cells, or insulin-positive endocrine cells. In some examples, the compositions provided herein are intermediate-stage compositions during the differentiation of pancreatic beta cells or their precursor cells. In some examples, the compositions are final-stage compositions during the differentiation process. In some examples, the compositions are adapted from the composition during differentiation, for example, cells can be isolated from the medium used for differentiation and reconstituted in a medium containing a water-soluble polymer, e.g., a water-soluble synthetic polymer.

[0075] Water-soluble synthetic polymers

[0090] The water-soluble polymers described herein can refer to any polymer that has hydrophilic properties and is soluble in aqueous solution at room temperature. The water-soluble polymers can be either naturally occurring or synthetic. In some embodiments, the water-soluble polymer is an albumin protein (e.g., human serum albumin or bovine serum albumin). In some embodiments, the water-soluble polymer is a water-soluble synthetic polymer. The water-soluble synthetic polymers described herein can refer to any synthetic polymer that has hydrophilic properties and is soluble in aqueous solution at room temperature. Water-soluble synthetic polymers applicable to the present methods and compositions include, but are not limited to, poloxamer, polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol (PEG), PEG copolymers, poly(N-isopropylacrylamide), and polyacrylamide. The water-soluble synthetic polymer can refer to a polymeric compound or a mixture of polymeric compounds that can have an idealized chemical formula, but can also refer to various derivatives and / or precursors of the idealized formula, depending on the applicable manufacturing method. In some cases, the water-soluble synthetic polymer is used to at least partially replace the serum or serum albumin, such as BSA or HSA, typically used in cell differentiation, for example, differentiation of pancreatic β cells or their precursor cells.In some cases, the water-soluble synthetic polymer replaces 100% of the serum albumin, such as BSA or HSA, typically used in cell differentiation, for example, differentiation of pancreatic β cells or their precursor cells.In some cases, the water-soluble synthetic polymer reduces the amount of serum albumin, such as BSA or HSA, by at least 20%, 30%, 40%, 50%, 60%, 80%, 90%, 95%, or 99% of that typically used in cell differentiation, for example, differentiation of pancreatic β cells or their precursor cells.In some embodiments, the present disclosure provides a composition comprising a population of any of the cells disclosed herein (e.g., pluripotent stem cells; endoderm cells; gastrula cells; PDX1-positive, NKX6.1-negative pancreatic progenitor cells; PDX1-positive, NKX6.1-positive pancreatic progenitor cells; insulin-positive cells; and / or pancreatic beta cells), and a water-soluble polymer, wherein at least 20%, 30%, 40%, 50%, 60%, 90%, 95%, or 99% of the water-soluble polymer in the composition is a water-soluble synthetic polymer (e.g., any of the PVA molecules disclosed herein), and the remaining water-soluble polymer is a human serum albumin polypeptide. In some embodiments, the present disclosure provides a composition comprising a population of any of the cells disclosed herein (e.g., pluripotent stem cells; endoderm cells; gastrula cells; PDX1-positive, NKX6.1-negative pancreatic progenitor cells; PDX1-positive, NKX6.1-positive pancreatic progenitor cells; insulin-positive cells; and / or pancreatic beta cells), and a water-soluble polymer, wherein 1%, 2%, 3%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 50%, 50%, 60%, 80%, 90%, 95%, or 99% or less of the water-soluble polymer is a naturally occurring water-soluble polymer (e.g., HSA or BSA). In some embodiments, more than 90%, 95%, 99%, and up to 100% of the water-soluble polymer in the composition is a water-soluble synthetic polymer (e.g., PVA).

[0076]

[0091] In some examples, the water-soluble synthetic polymer applicable to the present compositions and methods includes polyvinyl alcohol (PVA). Polyvinyl alcohol, as described herein, can refer to a water-soluble synthetic polymer having the idealized formula [CHCH(OH)], which can be partially or completely hydrolyzed. In some examples, polyvinyl alcohol is produced by either partial or complete hydrolysis of polyvinyl acetate to remove acetate groups. In some examples, polyvinyl alcohol is at most 85% hydrolyzed, e.g., 80% hydrolyzed. The percentage of hydrolysis measures the approximate percentage (e.g., average percentage) of acetate residues hydrolyzed in the polyvinyl acetate precursor polymer. In some examples, polyvinyl alcohol is at least 85% hydrolyzed, e.g., 87-89% hydrolyzed, 87-90% hydrolyzed, or 99% hydrolyzed. In some embodiments, the polyvinyl alcohol is hydrolyzed to 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Without wishing to be bound by any particular theory, polyvinyl alcohol hydrolysis can be estimated by the function of a carrier molecule in the medium, typically serum or serum albumin, e.g., HSA. The percentage of hydrolysis of polyvinyl alcohol can be determined by the manufacturing method used to produce the polyvinyl alcohol, for example, how polyvinyl acetate precursor polymer is converted to polyvinyl alcohol, for example, by base-catalyzed transesterification with ethanol. In some examples, the water-soluble synthetic polymer preparation, e.g., polyvinyl alcohol, used in the method or present in the composition has a purity of at least 90%, e.g., at least 92%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or nearly 100%. Polyvinyl alcohol purity measures the percentage of synthetic polymer in the preparation having the idealized formula [CHCH(OH)], including any percentage of hydrolysis of polyvinyl alcohol.Impurities in polyvinyl alcohol preparations can include other polymeric materials that do not have the idealized formula [CH2CH(OH)]n, or other organic or inorganic materials.

[0077] Methods for generating pancreatic cells

[0092] In aspects, the present disclosure relates to compositions and methods for generating pancreatic cells (e.g., pancreatic endocrine cells) from pancreatic progenitor or precursor cells. Certain exemplary detailed protocols for generating endocrine cells to provide at least one SC-β cell and / or other pancreatic endocrine cells, e.g., SC-α cells and SC-δ cells, are described in International Application Publication No. WO2019 / 169351 and U.S. Patent Application Publication No. 200210198632, each of which is incorporated herein by reference in its entirety.

[0078]

[0093] In some examples, the methods disclosed herein include differentiating a plurality of pancreatic beta cell precursor cells in a serum- or serum albumin-free medium. Alternatively, in some examples, the medium does not contain albumin protein. In some examples, the medium does not contain human serum albumin (HSA). In some examples, the medium does not contain serum.

[0079]

[0094] In some of these cases, the culture medium comprises a water-soluble polymer, e.g., a water-soluble synthetic polymer. In some examples, the water-soluble polymer, e.g., a water-soluble synthetic polymer, acts as a substitute for serum or serum albumin in the culture medium, for example, a water-soluble polymer, e.g., a water-soluble synthetic polymer, and replaces serum or serum albumin for at least one of its functions, for example, to support and / or enhance cell survival, growth, differentiation, functional maturation, or stability in culture. In some examples, the method disclosed herein comprises differentiating a plurality of pancreatic beta cell precursor cells in a culture medium comprising a water-soluble polymer, e.g., a water-soluble synthetic polymer. In some of these cases, the water-soluble synthetic polymer comprises poloxamer, polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol (PEG), PEG copolymer, poly(N-isopropylacrylamide), or polyacrylamide. In some examples, the water-soluble synthetic polymer comprises polyvinyl alcohol.

[0080]

[0095] In some examples, the method includes differentiating a plurality of pancreatic beta cell precursor cells in a medium containing a water-soluble polymer, e.g., a water-soluble synthetic polymer, at a concentration of about 0.005% to about 0.5% (w / v), about 0.01% to about 0.2% (w / v), about 0.02% to about 0.1% (w / v), or about 0.03% to about 0.08% (w / v). In some examples, the method includes differentiating a plurality of pancreatic beta cell precursor cells in a medium containing a water-soluble polymer, e.g., a water-soluble synthetic polymer, at a concentration of about 0.04% to about 0.06% (w / v), e.g., about 0.05% (w / v) of the medium.

[0081]

[0096] In some embodiments, the in vitro compositions described herein further comprise a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol (PVA), poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), a PEG copolymer, poly(N-isopropylacrylamide), or polyacrylamide, and the water-soluble synthetic polymer is optionally polyvinyl alcohol. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol (PVA). In some embodiments, the water-soluble synthetic polymer is present in the medium at a concentration of about 0.005% to about 0.5% (w / v), about 0.01% to about 0.2% (w / v), about 0.02% to about 0.1% (w / v), or about 0.03% to about 0.08% (w / v). In some embodiments, the water-soluble synthetic polymer is present in the medium at a concentration of about 0.005% (w / v), 0.01% (w / v), 0.05% (w / v), 0.1% (w / v), 0.15% (w / v), 0.2% (w / v), 0.25% (w / v), 0.3% (w / v), 0.35% (w / v), 0.4% (w / v), 0.45% (w / v), or 0.5% (w / v). In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol (PVA), and the PVA is at most 85% (e.g., 75%-80%) hydrolyzed.

[0082]

[0097] In some embodiments, the water-soluble synthetic polymer is present in the medium at a concentration of about 0.005% to about 0.5% (w / v), about 0.01% to about 0.2% (w / v), about 0.02% to about 0.1% (w / v), or about 0.03% to about 0.08% (w / v). In some embodiments, the water-soluble synthetic polymer is present in the medium at a concentration of about 0.005% to about 0.5% (w / v), about 0.01% to about 0.2% (w / v), about 0.02% to about 0.1% (w / v), or about 0.03% to about 0.08% (w / v). In some embodiments, the water-soluble synthetic polymer is present in the medium at a concentration of about 0.005% (w / v), 0.01% (w / v), 0.05% (w / v), 0.1% (w / v), 0.15% (w / v), 0.2% (w / v), 0.25% (w / v), 0.3% (w / v), 0.35% (w / v), 0.4% (w / v), 0.45% (w / v), or 0.5% (w / v). In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol (PVA), and the PVA is at most 90% hydrolyzed. In some embodiments, the PVA is about 87% to 89% hydrolyzed.

[0083]

[0098] In some examples of the methods disclosed herein, the precursor cells of pancreatic beta cells include NKX6.1-positive, ISL1-positive cells (e.g., insulin-positive endocrine cells). In some examples, the method includes differentiating a plurality of NKX6.1-positive, ISL1-positive cells in a medium that does not contain serum or serum albumin. In some examples, the method includes differentiating a plurality of NKX6.1-positive, ISL1-positive cells in a medium that contains a water-soluble polymer, e.g., a water-soluble synthetic polymer. In some examples, the method results in the differentiation of the NKX6.1-positive, ISL1-positive cells into pancreatic beta cells. In some examples of methods for differentiating a plurality of NKX6.1-positive, ISL1-positive cells, the water-soluble synthetic polymer comprises polyvinyl alcohol that is about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% hydrolyzed. In some examples of methods for differentiating a plurality of NKX6.1-positive, ISL1-positive cells, the water-soluble synthetic polymer comprises polyvinyl alcohol that is greater than 85% hydrolyzed. In some examples, the water-soluble synthetic polymer comprises polyvinyl alcohol that is about 87% to about 89% hydrolyzed. In some examples of the method, the medium comprises a water-soluble polymer, e.g., a water-soluble synthetic polymer, and one or more agents selected from the group consisting of a transforming growth factor beta (TGF-β) signaling pathway inhibitor, a thyroid hormone signaling pathway activator, an epigenetic modifying compound, a growth factor from the epidermal growth factor (EGF) family, a retinoic acid (RA) signaling pathway activator, a sonic hedgehog (SHH) pathway inhibitor, a gamma-secretase inhibitor, a protein kinase inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a metabolite, a lipid, an amino acid, an MGLL inhibitor, a vitamin, zinc (e.g., ZnSO4), and a bone morphogenetic protein (BMP) signaling pathway inhibitor.In some examples, the method includes differentiating a plurality of NKX6.1-positive, ISL1-positive cells in a medium containing a water-soluble synthetic polymer, e.g., a water-soluble synthetic polymer, and one or more substances selected from the group consisting of a transforming growth factor beta (TGF-β) signaling pathway inhibitor, a thyroid hormone signaling pathway activator, an epigenetic modifying compound, a growth factor from the epidermal growth factor (EGF) family, a retinoic acid (RA) signaling pathway activator, a sonic hedgehog (SHH) pathway inhibitor, a gamma-secretase inhibitor, a protein kinase inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a metabolite, a lipid, an amino acid, an MGLL inhibitor, a vitamin, zinc (e.g., ZnSO), and a bone morphogenetic protein (BMP) signaling pathway inhibitor. In some examples, the method includes differentiating a plurality of NKX6.1-positive, ISL1-positive cells in a medium containing a water-soluble synthetic polymer, e.g., a water-soluble synthetic polymer, a transforming growth factor β (TGF-β) signaling pathway inhibitor, a thyroid hormone signaling pathway activator, an epigenetic modifying compound, a growth factor from the epidermal growth factor (EGF) family, a retinoic acid (RA) signaling pathway activator, a sonic hedgehog (SHH) pathway inhibitor, a gamma-secretase inhibitor, a protein kinase inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a metabolite, a lipid, an amino acid, a MGLL inhibitor, a vitamin, zinc (e.g., ZnSO), and a bone morphogenetic protein (BMP) signaling pathway inhibitor. In some examples of the method, the NKX6.1-positive, ISL1-positive cells are contacted with the water-soluble polymer, e.g., a water-soluble synthetic polymer, for about 7 to about 14 days.

[0084]

[0099] In some other examples of the methods disclosed herein, the differentiation of NKX6.1-positive, ISL1-positive cells into pancreatic beta cells is carried out in the presence of serum or serum albumin, such as human serum albumin (HSA), for example, about 0.1% HSA. In some examples, the differentiation of NKX6.1-positive, ISL1-positive cells into pancreatic beta cells is carried out in the absence of polyvinyl alcohol. In some examples, for example, a method of differentiating a plurality of NKX6.1-positive, ISL1-positive cells includes culturing the NKX6.1-positive, ISL1-positive cells in a medium containing, for example, serum or serum albumin, e.g., about 0.1% HSA, and one or more agents selected from the group consisting of a transforming growth factor beta (TGF-β) signaling pathway inhibitor, a thyroid hormone signaling pathway activator, an epigenetic modifying compound, a growth factor from the epidermal growth factor (EGF) family, a retinoic acid (RA) signaling pathway activator, a sonic hedgehog (SHH) pathway inhibitor, a gamma-secretase inhibitor, a protein kinase inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a metabolite, a lipid, an amino acid, an MGLL inhibitor, a vitamin, zinc (e.g., ZnSO4), and a bone morphogenetic protein (BMP) signaling pathway inhibitor. In some examples, the method can differentiate a plurality of NKX6.1-positive, ISL1-positive cells in a medium comprising HSA, e.g., about 0.1% HSA, and one or more agents selected from the group consisting of a transforming growth factor beta (TGF-β) signaling pathway inhibitor, a thyroid hormone signaling pathway activator, an epigenetic modifying compound, a growth factor from the epidermal growth factor (EGF) family, a retinoic acid (RA) signaling pathway activator, a sonic hedgehog (SHH) pathway inhibitor, a gamma-secretase inhibitor, a protein kinase inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a metabolite, a lipid, an amino acid, an MGLL inhibitor, a vitamin, zinc (e.g., ZnSO), and a bone morphogenetic protein (BMP) signaling pathway inhibitor.In some examples, the method includes differentiating a plurality of NKX6.1-positive, ISL1-positive cells in a medium comprising HSA, e.g., about 0.1% HSA, a transforming growth factor beta (TGF-β) signaling pathway inhibitor, a thyroid hormone signaling pathway activator, an epigenetic modifying compound, a growth factor from the epidermal growth factor (EGF) family, a retinoic acid (RA) signaling pathway activator, a sonic hedgehog (SHH) pathway inhibitor, a gamma-secretase inhibitor, a protein kinase inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a metabolite, a lipid, an amino acid, an MGLL inhibitor, a vitamin, zinc (e.g., ZnSO), and a bone morphogenetic protein (BMP) signaling pathway inhibitor.

[0085]

[0100] In some examples of the methods disclosed herein, the precursor cells of pancreatic beta cells include PDX1-positive, NKX6.1-positive cells (e.g., NKX6.1-positive pancreatic progenitor cells, e.g., PP2 cells). In some examples, the method includes differentiating a plurality of PDX1-positive, NKX6.1-positive cells in a medium that does not contain serum or serum albumin. In some examples, the method includes differentiating a plurality of PDX1-positive, NKX6.1-positive cells in a medium that contains a water-soluble polymer, e.g., a water-soluble synthetic polymer. In some examples, the method results in the differentiation of PDX1-positive, NKX6.1-positive cells into NKX6.1-positive, ISL1-positive cells. In some examples of the method, the medium further comprises one or more agents selected from the group consisting of a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, an epigenetic modifying compound, a growth factor from the epidermal growth factor (EGF) family, a retinoic acid (RA) signaling pathway activator, a sonic hedgehog (SHH) pathway inhibitor, a gamma-secretase inhibitor, a protein kinase inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a protein kinase activator, nicotinamide, and a bone morphogenetic protein (BMP) signaling pathway inhibitor. In some examples, the method includes differentiating a plurality of PDX1-positive, NKX6.1-positive cells in a medium containing a water-soluble polymer, e.g., a water-soluble synthetic polymer, and one or more agents selected from the group consisting of a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, an epigenetic modifying compound, a growth factor from the epidermal growth factor (EGF) family, a retinoic acid (RA) signaling pathway activator, a sonic hedgehog (SHH) pathway inhibitor, a gamma-secretase inhibitor, a protein kinase inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a protein kinase activator, nicotinamide, and a bone morphogenetic protein (BMP) signaling pathway inhibitor.In some examples, the method includes differentiating a plurality of PDX1-positive, NKX6.1-positive cells in a medium comprising a water-soluble polymer, e.g., a water-soluble synthetic polymer, a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, an epigenetic modifying compound, a growth factor from the epidermal growth factor (EGF) family, a retinoic acid (RA) signaling pathway activator, a sonic hedgehog (SHH) pathway inhibitor, a gamma-secretase inhibitor, a protein kinase inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a protein kinase activator, nicotinamide, and a bone morphogenetic protein (BMP) signaling pathway inhibitor.In some examples of the method, the medium comprises, in addition to the water-soluble polymer, (a) a TGF-β signaling pathway inhibitor selected from the group consisting of Alk5i II, A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB-525334, SD-208, and SB-505124; (b) a thyroid hormone signaling pathway activator, including T3 or GC-1; (c) 3-deazaneplanocin A (DZNep), GSK126, E (d) an epigenetic modifying compound selected from the group consisting of PZ6438, KD5170, MC1568, and TMP195; (d) a growth factor from the epidermal growth factor family, including betacellulin or EGF; (e) retinoic acid, CD1530, AM580, TTHRB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, and CD23. (f) a sonic hedgehog pathway inhibitor selected from the group consisting of SANT1, SANT2, SANT4, Cur6l4l4, forskolin, tomatidine, AY9944, triparanol, and cyclopamine; (g) a gamma-secretase inhibitor, including XXI or DAPT; (h) a protein kinase inhibitor, including staurosporine, Ro-31-8220, bicindolylmaleimide (Bis) compounds, 10'-{5"-[(methoxycarbonyl)amino]-2"-methyl}-phenylaminocarbonylstaurosporine, or stalalogues; (i) a ROCK inhibitor selected from the group consisting of thiazovivin, Y-27632, Fasudil / HA1077, and 14-1152; (j) a phorbol 12,13-dibutyrate (PDBU), TPB, or phorbol 12-myristate. 13-acetate, and bryostatin 1; and / or (k) a bone morphogenetic protein signaling pathway inhibitor, including LDN193189 or DMH-1.In some examples of the method for differentiating a plurality of PDX1-positive, NKX6.1-positive cells, the water-soluble synthetic polymer comprises 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% hydrolyzed polyvinyl alcohol. In some examples of the method for differentiating a plurality of PDX1-positive, NKX6.1-positive cells, the water-soluble synthetic polymer comprises more than 85% hydrolyzed polyvinyl alcohol. In some examples of the method, the water-soluble synthetic polymer comprises about 87% to 89% hydrolyzed polyvinyl alcohol. In some examples of the method, the PDX1-positive, NKX6.1-positive cells are contacted with the water-soluble synthetic polymer for about 5 to about 10 days or about 6 to about 9 days. In some examples, the PDX1-positive, NKX6.1-positive cells are contacted with the water-soluble polymer, eg, a water-soluble synthetic polymer, for about 5, 6, 7, 8, 9, or 10 days.

[0086]

[0101] In some examples of the methods disclosed herein, the precursor cells of pancreatic beta cells include PDX1-positive, NKX6.1-negative cells (e.g., pancreatic progenitor cells, e.g., PP1 cells). In some examples, the method includes differentiating a plurality of PDX1-positive, NKX6.1-negative cells in a medium that does not contain serum or serum albumin. In some examples, the method includes differentiating a plurality of PDX1-positive, NKX6.1-negative cells in a medium that contains a water-soluble polymer, e.g., a water-soluble synthetic polymer. In some examples, the method results in the differentiation of PDX1-positive, NKX6.1-negative cells into PDX1-positive, NKX6.1-positive cells. In some examples of the methods, the medium further includes one or more agents selected from the group consisting of a protein kinase C activator, a growth factor from the transforming growth factor beta (TGF-β) superfamily, a growth factor from the fibroblast growth factor (FGF) family, a retinoic acid (RA) signaling pathway activator, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a sonic hedgehog (SHH) pathway inhibitor. In some examples, the method includes differentiating a plurality of PDX1-positive, NKX6.1-negative cells in a medium containing a water-soluble polymer, e.g., a water-soluble synthetic polymer, and one or more drugs selected from the group consisting of a growth factor from the transforming growth factor beta (TGF-β) superfamily, a growth factor from the fibroblast growth factor (FGF) family, a retinoic acid (RA) signaling pathway activator, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a sonic hedgehog (SHH) pathway inhibitor. In some examples, the method includes differentiating a plurality of PDX1-positive, NKX6.1-negative cells in a medium containing a water-soluble polymer, e.g., a water-soluble synthetic polymer, a protein kinase C activator, a growth factor from the transforming growth factor beta (TGF-β) superfamily, a growth factor from the fibroblast growth factor (FGF) family, a retinoic acid (RA) signaling pathway activator, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a sonic hedgehog (SHH) pathway inhibitor.In some examples of the method, the medium comprises: (a) a growth factor from the transforming growth factor beta (TGF-β) superfamily selected from the group consisting of inhibin, activin, Müllerian inhibitory substance (MIS), bone morphogenetic protein (BMP), decapentaplegic (dpp), Vg-1, monoclonal nonspecific inhibitory factor (MNSF), growth differentiation factor 8 (GDF8), and / or growth differentiation factor 11 (GDF11); (b) a fibroblast growth factor (FGF) selected from the group consisting of keratinocyte growth factor (KGF), FGF2, FGF10, FGF21, and FGF8B; (c) a retinoic acid (RA) signaling pathway activator selected from the group consisting of retinoic acid, CD1530, AM580, TTHRB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, and CD2314; (d) a ROCK inhibitor selected from the group consisting of thiazovivin, Y-27632, Fasudil / HA1077, and 14-1152; (e) phorbol 12,13-dibutyrate (PDBU), TPB, phorbol 12-myristate. and / or (f) a protein kinase C activator selected from the group consisting of SANT1, SANT2, SANT4, Cur61414, forskolin, tomatidine, AY9944, triparanol, and cyclopamine. In some examples of the method for differentiating a plurality of PDX1-positive, NKX6.1-negative cells, the water-soluble synthetic polymer comprises 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% hydrolyzed polyvinyl alcohol. In some examples of the methods for differentiating a plurality of PDX1-positive, NKX6.1-negative cells, the water-soluble synthetic polymer comprises less than 85% hydrolyzed polyvinyl alcohol. In some examples, the water-soluble synthetic polymer comprises about 80% hydrolyzed polyvinyl alcohol.In some examples of the method, the PDX1-positive, NKX6.1-negative cells are contacted with the water-soluble polymer, e.g., a water-soluble synthetic polymer, for 4 to 8 days or 5 to 7 days. In some examples, the PDX1-positive, NKX6.1-negative cells are contacted with the water-soluble polymer, e.g., a water-soluble synthetic polymer, for about 4, 5, 6, 7, or 8 days.

[0087]

[0102] In some examples of the methods disclosed herein, the precursor cells of pancreatic beta cells include FOXA2-positive cells (e.g., primitive gut cells). In some examples, the method includes differentiating a plurality of FOXA2-positive cells in a medium that does not contain serum or serum albumin. In some examples, the method includes differentiating a plurality of FOXA2-positive cells in a medium that contains a water-soluble polymer, e.g., a water-soluble synthetic polymer. In some examples, the method results in the differentiation of the FOXA2-positive cells into PDX1-positive, NKX6.1-negative cells. In some examples of the methods, the medium further contains one or more agents selected from the group consisting of a protein kinase C activator, a growth factor from the transforming growth factor beta (TGF-β) superfamily, a bone morphogenetic protein signaling pathway inhibitor, a growth factor from the fibroblast growth factor (FGF) family, a retinoic acid (RA) signaling pathway activator, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a sonic hedgehog (SHH) pathway inhibitor. In some examples, the method includes differentiating a plurality of FOXA2-positive cells in a medium containing a water-soluble polymer, e.g., a water-soluble synthetic polymer, and one or more agents selected from the group consisting of a protein kinase C activator, a growth factor from the transforming growth factor beta (TGF-β) superfamily, a bone morphogenetic protein signaling pathway inhibitor, a growth factor from the fibroblast growth factor (FGF) family, a retinoic acid (RA) signaling pathway activator, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a sonic hedgehog (SHH) pathway inhibitor. In some examples, the method includes differentiating a plurality of FOXA2-positive cells in a medium containing a water-soluble polymer, e.g., a water-soluble synthetic polymer, a protein kinase C activator, a growth factor from the transforming growth factor beta (TGF-β) superfamily, a bone morphogenetic protein signaling pathway inhibitor, a growth factor from the fibroblast growth factor (FGF) family, a retinoic acid (RA) signaling pathway activator, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a sonic hedgehog (SHH) pathway inhibitor.In some examples of the method, the medium comprises: (a) a protein kinase C activator selected from the group consisting of phorbol 12,13-dibutyrate (PDBU), TPB, phorbol 12-myristate 13-acetate, and bryostatin 1; (b) a growth factor from the transforming growth factor beta (TGF-β) superfamily selected from the group consisting of inhibin, activin, Müllerian inhibitory substance (MIS), bone morphogenetic protein (BMP), decapentaplegic (dpp), Vg-1, monoclonal nonspecific suppressor of factor (MNSF), growth differentiation factor 8 (GDF8), and growth differentiation factor 11 (GDF11); (c) a bone morphogenetic protein signaling pathway inhibitor, including LDN193189 or DMH-1; and (d) a fibroblast growth factor selected from the group consisting of keratinocyte growth factor (KGF), FGF2, FGF10, FGF21, and FGF8B. (e) a sonic hedgehog pathway inhibitor selected from the group consisting of SANT1, SANT2, SANT4, Cur6l4l4, forskolin, tomatidine, AY9944, triparanol, and cyclopamine; (f) a retinoic acid signaling pathway activator selected from the group consisting of retinoic acid, CD1530, AM580, TTHRB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, and CD2314; and / or (g) a ROCK inhibitor selected from the group consisting of thiazovivin, Y-27632, Fasudil / HA1077, and 14-1152. In some examples of the method for differentiating a plurality of FOXA2-positive cells, the water-soluble synthetic polymer comprises 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% hydrolyzed polyvinyl alcohol. In some examples of the method for differentiating a plurality of FOXA2-positive cells, the water-soluble synthetic polymer comprises less than 85% hydrolyzed polyvinyl alcohol. In some examples, the water-soluble synthetic polymer comprises about 80% hydrolyzed polyvinyl alcohol.In some examples, the FOXA2-positive cells are contacted with the water-soluble polymer, e.g., a water-soluble synthetic polymer, for 1 to 3 days. In some examples, the FOXA2-positive cells are contacted with the water-soluble polymer, e.g., a water-soluble synthetic polymer, for about 1, 2, or 3 days.

[0088]

[0103] In some examples of the methods disclosed herein, the precursor cells of pancreatic beta cells include SOX17-positive cells (e.g., definitive endoderm cells). In some examples, the method includes differentiating a plurality of SOX17-positive cells in a medium free of serum or serum albumin. In some examples, the method includes differentiating a plurality of SOX17-positive cells in a medium containing a water-soluble polymer, e.g., a water-soluble synthetic polymer. In some examples, the method results in the differentiation of the SOX17-positive cells into FOXA2-positive cells. In some examples of the method, the medium further contains a growth factor from the fibroblast growth factor (FGF) family. In some examples, the method includes differentiating a plurality of SOX17-positive cells in a medium containing a water-soluble polymer, e.g., a water-soluble synthetic polymer, and a growth factor from the FGF family. In some examples of the method, the growth factor from the fibroblast growth factor (FGF) family is selected from the group consisting of keratinocyte growth factor (KGF), FGF2, FGF10, FGF21, and FGF8B. In some examples of the method for differentiating a plurality of SOX17-positive cells, the water-soluble synthetic polymer comprises 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% hydrolyzed polyvinyl alcohol. In some examples of the method for differentiating a plurality of SOX17-positive cells, the water-soluble synthetic polymer comprises less than 85% hydrolyzed polyvinyl alcohol. In some examples, the water-soluble synthetic polymer comprises about 80% hydrolyzed polyvinyl alcohol. In some examples of the method, the SOX17-positive cells are contacted with the water-soluble polymer, e.g., the water-soluble synthetic polymer, for 1 to 5 days or 2 to 4 days. In some examples, the SOX17-positive cells are contacted with the water-soluble polymer, e.g., the water-soluble synthetic polymer, for about 1, 2, 3, 4, or 5 days.

[0089]

[0104] In some examples of the methods disclosed herein, the precursor cells of pancreatic beta cells include stem cells (e.g., human stem cells). In some examples, the method includes differentiating a plurality of stem cells in a medium that does not contain serum or serum albumin. In some examples, the method includes differentiating a plurality of stem cells in a medium that includes a water-soluble polymer, e.g., a water-soluble synthetic polymer. In some examples, the method results in the differentiation of the stem cells into SOX17-positive cells. In some examples, the stem cells include embryonic stem cells or induced pluripotent stem cells. In some examples of the method, the medium further includes a growth factor from the transforming growth factor beta (TGF-β) superfamily, a WNT signaling pathway activator, or both. In some examples, the method includes differentiating a plurality of stem cells in a medium that includes a water-soluble polymer, e.g., a water-soluble synthetic polymer, a growth factor from the transforming growth factor beta (TGF-β) superfamily, and a WNT signaling pathway activator. In some examples of the method, the medium comprises (a) a growth factor from the transforming growth factor beta (TGF-β) superfamily selected from the group consisting of inhibin, activin, Müllerian inhibitory substance (MIS), bone morphogenetic protein (BMP), decapentaplegic (dpp), Vg-1, monoclonal nonspecific inhibitory factor (MNSF), growth differentiation factor 8 (GDF8), and growth differentiation factor 11 (GDF11); and / or (b) a growth factor from the transforming growth factor beta (TGF-β) superfamily selected from the group consisting of CHIR99021, 3F8, A 1070722, AR-A 014418, BIO, BIO-acetoxime, FRATide, 10Z-hymenialdisine, indirubin-3'oxime, Kenpaullone, L803, L803-mts, lithium carbonate, NSC 693868, SB 216763, SB 415286, TC-G 24, TCS 2002, TCS 21311, and TWS 119. In some examples of the method of differentiating a plurality of stem cells, the water soluble synthetic polymer comprises 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% hydrolyzed polyvinyl alcohol.In some examples of the method for differentiating a plurality of stem cells, the water-soluble synthetic polymer comprises less than 85% hydrolyzed polyvinyl alcohol. In some examples, the water-soluble synthetic polymer comprises about 80% hydrolyzed polyvinyl alcohol. In some examples of the method, the stem cells are contacted with the water-soluble polymer, e.g., the water-soluble synthetic polymer, for 1-5 days or 2-4 days. In some examples, the stem cells are contacted with the water-soluble polymer, e.g., the water-soluble synthetic polymer, for about 1, 2, 3, 4, or 5 days.

[0090]

[0105] In some examples, the methods disclosed herein include (i) differentiating a plurality of PDX1-positive, NKX6.1-negative pancreatic progenitor cells or precursor cells thereof in a medium comprising less than 85% hydrolyzed polyvinyl alcohol, thereby generating a plurality of PDX1-positive, NKX6.1-positive pancreatic progenitor cells; and (ii) culturing a plurality of PDX1-positive, NKX6.1-positive pancreatic progenitor cells in a composition comprising more than 85% hydrolyzed polyvinyl alcohol. In some examples, the method comprises the steps of differentiating stem cells into SOX17-positive cells (such as embryonic gut cells), differentiating SOX17-positive cells into FOXA2-positive cells (such as primitive gut cells), differentiating FOXA2-positive cells into PDX1-positive, NKX6.1-negative cells (such as PP1 cells), and differentiating PDX1-positive, NKX6.1-negative cells into PDX1-positive, NKX6.1-positive cells (such as PP2 cells), all of which are carried out in a medium comprising less than 85% hydrolyzed polyvinyl alcohol, for example, about 80% hydrolyzed polyvinyl alcohol.In some examples of the method, the step of differentiating PDX1-positive, NKX6.1-positive cells into NKX6.1-positive, ISL1-positive cells (such as insulin-positive endocrine cells) in a medium comprising more than 85% hydrolyzed polyvinyl alcohol.In some examples of the method, the polyvinyl alcohol that is less than 85% hydrolyzed is about 80% hydrolyzed. In some cases, polyvinyl alcohol that is more than 85% hydrolyzed is about 87% to about 89% hydrolyzed. In some cases, the culture differentiates a plurality of PDX1-positive, NKX6.1-positive pancreatic progenitor cells to produce NKX1-positive, ISL1-positive endocrine cells. In some cases, the culture degrades a plurality of PDX1-positive, NKX6.1-positive pancreatic progenitor cells to produce pancreatic beta cells. In some cases, the differentiation of NKX6.1-positive, ISL1-positive cells into pancreatic beta cells is performed in the presence of serum or serum albumin, e.g., human serum albumin (HSA), e.g., 0.1% HSA. In some cases, the differentiation of NKX6.1-positive, ISL1-positive cells into pancreatic beta cells is performed in the absence of polyvinyl alcohol.

[0091]

[0106] In some examples, the composition comprising greater than 85% hydrolyzed polyvinyl alcohol further comprises one or more agents selected from the group consisting of a protein kinase C activator, a growth factor from the transforming growth factor beta (TGF-β) superfamily, a bone morphogenetic protein signaling pathway inhibitor, a growth factor from the fibroblast growth factor (FGF) family, a retinoic acid (RA) signaling pathway activator, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a sonic hedgehog (SHH) pathway inhibitor. In some embodiments, the water-soluble synthetic polymer comprises 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% hydrolyzed polyvinyl alcohol.In some examples, the composition comprising greater than 85% hydrolyzed polyvinyl alcohol is selected from the group consisting of: (a) a protein kinase C activator selected from the group consisting of phorbol 12,13-dibutyrate (PDBU), TPB, phorbol 12-myristate 13-acetate, and bryostatin 1; (b) a growth factor from the transforming growth factor beta (TGF-β) superfamily selected from the group consisting of inhibin, activin, Müllerian inhibitory substance (MIS), bone morphogenetic protein (BMP), decapentaplegic (dpp), Vg-1, monoclonal nonspecific suppressor of factor (MNSF), growth differentiation factor 8 (GDF8), and growth differentiation factor 11 (GDF11); (c) a bone morphogenetic protein signaling pathway inhibitor, including LDN193189 or DMH-1; (d) a fibroblast growth factor selected from the group consisting of keratinocyte growth factor (KGF), FGF2, FGF10, FGF21, and FGF8B. (e) a sonic hedgehog pathway inhibitor selected from the group consisting of SANT1, SANT2, SANT4, Cur6l4l4, forskolin, tomatidine, AY9944, triparanol, and cyclopamine; (f) a retinoic acid signaling pathway activator selected from the group consisting of retinoic acid, CD1530, AM580, TTHRB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, and CD2314; and / or (g) a ROCK inhibitor selected from the group consisting of thiazovivin, Y-27632, Fasudil / HA1077, and 14-1152.

[0092]

[0107] In some embodiments, the methods disclosed herein relate to the use of nicotinamide and a growth factor from the EGF family instead of betacellulin during in vitro differentiation of PDX1-positive, NKX6.1-positive cells. In some examples, the methods include contacting a plurality of PDX1-positive, NKX6.1-positive cells with a composition comprising nicotinamide and a growth factor from the EGF family. In some examples, the composition does not include betacellulin.

[0093]

[0108] In some examples of the method for differentiating PDX1-positive, NKX6.1-positive cells, the growth factor from the EGF family includes EGF. In some examples, the method involves the use of a composition, for example, a medium, containing EGF at about 1 ng / mL to about 100 ng / mL, about 2 ng / mL to about 50 ng / mL, about 5 ng / mL to about 20 ng / mL, or about 7.5 ng / mL to about 15 ng / mL. In some examples, the composition contains about 10 ng / mL of EGF. In some examples, the composition contains about 1 mM to about 100 mM, about 2 mM to about 50 mM, about 5 mM to about 20 mM, or about 7.5 mM to about 15 mM of nicotinamide. In some examples, the composition contains about 10 mM of nicotinamide.

[0094]

[0109] In some examples of the methods for differentiating PDX1-positive, NKX6.1-positive cells, the composition contacted with the PDX1-positive, NKX6.1-positive cells comprises nicotinamide, a growth factor from the EGF family, and one or more agents selected from the group consisting of a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, an epigenetic modifying compound, a retinoic acid (RA) signaling pathway activator, a sonic hedgehog (SHH) pathway inhibitor, a gamma-secretase inhibitor, a protein kinase inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a bone morphogenetic protein (BMP) signaling pathway inhibitor. In some examples, the composition comprises nicotinamide, a growth factor from the EGF family, a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, an epigenetic modifying compound, a retinoic acid (RA) signaling pathway activator, a sonic hedgehog (SHH) pathway inhibitor, a gamma-secretase inhibitor, a protein kinase inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a bone morphogenetic protein (BMP) signaling pathway inhibitor.In some examples, the composition for differentiating PDX6.1-positive, NKX6.1-positive cells comprises: (a) a TGF-β signaling pathway inhibitor selected from the group consisting of Alk5i II, A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB-525334, SD-208, and SB-505124; (b) a thyroid hormone signaling pathway activator including T3 or GC-1; (c) 3-deazaneplanocin A (DZNep), (d) an epigenetic modifying compound selected from the group consisting of retinoic acid, CD1530, AM580, TTHRB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, and CD2314; (e) a pathway activator; (f) a sonic hedgehog pathway inhibitor selected from the group consisting of SANT1, SANT2, SANT4, Cur61414, forskolin, tomatidine, AY9944, triparanol, and cyclopamine; (f) a gamma-secretase inhibitor, including XI or DAPT; (g) a protein kinase inhibitor, including staurosporine, Ro-31-8220, a bicindolylmaleimide (Bis) compound, 10'-{5"-[(methoxycarbonyl)amino]-2"-methyl}-phenylaminocarbonylstaurosporine, or a stalalogue; (h) a ROCK inhibitor selected from the group consisting of thiazovivin, Y-27632, Fasudil / HA1077, and 14-1152; or (i) a bone morphogenetic protein signaling pathway inhibitor, including LDN193189 or DMH-1. In some embodiments of the method, the contacting is for 1 to 3 days. In some embodiments of the method, the contacting occurs for about 1, 2, or 3 days.In some examples, the method includes removing nicotinamide and growth factors from the EGF family from the plurality of PDX1-positive, NKX6.1-positive cells after 1 to 3 days of contact; and, after removal, contacting the plurality of PDX1-positive, NKX6.1-positive cells with a composition that does not contain nicotinamide or growth factors from the EGF family. In some examples, the method results in differentiation of the plurality of PDX1-positive, NKX6.1-positive cells into NKX6.1-positive, ISL1-positive cells.

[0095] cell composition

[0110] In some aspects, provided herein are cell compositions for the in vitro generation of pancreatic endocrine cells, such as pancreatic beta cells, pancreatic alpha cells, or pancreatic delta cells. In some aspects, provided herein are compositions comprising the cell compositions in combination with media and / or agents that may contribute to cell differentiation, growth, survival, and / or stability.

[0096]

[0111] In some examples, the composition (e.g., an in vitro composition) comprises a plurality of pancreatic beta cells or precursor cells thereof in a medium comprising a water-soluble polymer, e.g., a water-soluble synthetic polymer. In some examples, the medium in the compositions provided herein does not comprise albumin protein. In some examples, the medium does not comprise human serum albumin (HSA). In some examples, the medium does not comprise serum. Precursor cells of pancreatic beta cells can comprise, for example, Sox17-positive cells; FOXA2-positive cells, PDX1-positive cells, NKX6.1-positive cells, ISL1-positive cells, or insulin-positive endocrine cells. In some examples, Sox17-positive cells comprise definitive endoderm cells. In some examples, FOXA2-positive cells comprise primitive gut cells. In some examples, PDX1-positive cells comprise PDX1-positive, NKX6.1-negative cells (e.g., PP1 cells) and / or PDX1-positive, NKX6.1-positive cells (e.g., PP2 cells). In some examples, ISL1-positive cells comprise NKX6.1-positive, ISL1-positive cells (e.g., insulin-positive endocrine cells). In some instances, the insulin-positive endocrine cells include pancreatic beta cells.

[0097]

[0112] In some examples, the water-soluble synthetic polymer comprises polyvinyl alcohol, poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), PEG copolymer, poly(N-isopropylacrylamide), or polyacrylamide. In some examples, the water-soluble synthetic polymer comprises polyvinyl alcohol. In some examples of the compositions disclosed herein, the water-soluble synthetic polymer is present in the medium at a concentration of about 0.005% to about 0.5% (w / v), about 0.01% to about 0.2% (w / v), about 0.02% to about 0.1% (w / v), or about 0.03% to about 0.08% (w / v). In some of these examples, the water-soluble synthetic polymer is present in the medium at a concentration of about 0.04% to about 0.06% (w / v). In some examples, the water-soluble synthetic polymer is present in the medium at a concentration of about 0.05% (w / v).

[0098]

[0113] In some examples, the compositions disclosed herein comprise a plurality of insulin-positive cells. In some examples, the compositions disclosed herein comprise a plurality of non-native pancreatic beta cells. In some embodiments, any of the compositions disclosed herein comprises a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer comprises 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% hydrolyzed polyvinyl alcohol. In some of these examples, the water-soluble synthetic polymer comprises more than 85% hydrolyzed polyvinyl alcohol, e.g., about 87% to 89% hydrolyzed polyvinyl alcohol. In some of these examples, the composition further comprises NKX6.1-positive, ISL1-positive cells, e.g., insulin-positive endocrine cells. In some examples, the composition further comprises pancreatic alpha cells, pancreatic delta cells, pancreatic F cells, pancreatic epsilon cells, enterochromaffin cells, or any combination thereof. In some examples, the composition comprises a combination of pancreatic beta cells, pancreatic alpha cells, and pancreatic delta cells. In some of these examples, the medium further comprises one or more agents selected from the group consisting of a transforming growth factor beta (TGF-beta) signaling pathway inhibitor, a thyroid hormone signaling pathway activator, an epigenetic modifying compound, a protein kinase inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a bone morphogenetic protein (BMP) signaling pathway inhibitor. In some of these examples, the medium further comprises one or more agents selected from the group consisting of a transforming growth factor beta (TGF-β) signaling pathway inhibitor, a thyroid hormone signaling pathway activator, an epigenetic modifying compound, a growth factor from the epidermal growth factor (EGF) family, a retinoic acid (RA) signaling pathway activator, a sonic hedgehog (SHH) pathway inhibitor, a gamma-secretase inhibitor, a protein kinase inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a bone morphogenetic protein (BMP) signaling pathway inhibitor.

[0099]

[0114] In some examples, the composition comprises a plurality of PDX1-positive, NKX6.1-positive cells (e.g., PP2 cells) in a medium containing a water-soluble polymer. In some of these examples, the medium further comprises one or more agents selected from the group consisting of a protein kinase C activator, a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, an epigenetic modifying compound, a growth factor from the epidermal growth factor (EGF) family, a retinoic acid (RA) signaling pathway activator, a sonic hedgehog (SHH) pathway inhibitor, a gamma-secretase inhibitor, a protein kinase inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a bone morphogenetic protein (BMP) signaling pathway inhibitor.In some examples, the medium comprises a water-soluble polymer and (a) a TGF-β signaling pathway inhibitor selected from the group consisting of Alk5i II, A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB-525334, SD-208, and SB-505124; (b) a thyroid hormone signaling pathway activator, including T3 or GC-1; (c) 3-deazaneplanocin A (DZNep), GSK126, E (d) an epigenetic modifying compound selected from the group consisting of PZ6438, KD5170, MC1568, and TMP195; (d) a growth factor from the epidermal growth factor family, including betacellulin or EGF; (e) retinoic acid, CD1530, AM580, TTHRB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, and CD23. (f) a sonic hedgehog pathway inhibitor selected from the group consisting of SANT1, SANT2, SANT4, Cur6l4l4, forskolin, tomatidine, AY9944, triparanol, and cyclopamine; (g) a gamma-secretase inhibitor, including XXI or DAPT; (h) a protein kinase inhibitor, including staurosporine, Ro-31-8220, bicindolylmaleimide (Bis) compounds, 10'-{5"-[(methoxycarbonyl)amino]-2"-methyl}-phenylaminocarbonylstaurosporine, or stalalogues; (i) a ROCK inhibitor selected from the group consisting of thiazovivin, Y-27632, Fasudil / HA1077, and 14-1152; (j) a phorbol 12,13-dibutyrate (PDBU), TPB, or phorbol 12-myristate. 13-acetate, and bryostatin 1; and / or (k) a bone morphogenetic protein signaling pathway inhibitor, including LDN193189 or DMH-1.In some embodiments, the water-soluble synthetic polymer comprises 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% hydrolyzed polyvinyl alcohol. In some of these examples, the water-soluble synthetic polymer comprises more than 85% hydrolyzed polyvinyl alcohol, e.g., about 87% to 89% hydrolyzed polyvinyl alcohol. In some examples, the composition further comprises NKX6.1-positive, ISL1-positive cells, e.g., insulin-positive endocrine cells.

[0100]

[0115] In some examples, the composition comprises a plurality of PDX1-positive, NKX6.1-negative cells (e.g., PP1 cells) in a medium containing a water-soluble polymer. In some of these examples, the medium further comprises one or more agents selected from the group consisting of a protein kinase C activator, a growth factor from the transforming growth factor beta (TGF-β) superfamily, a growth factor from the fibroblast growth factor (FGF) family, a retinoic acid (RA) signaling pathway activator, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a sonic hedgehog (SHH) pathway inhibitor. In some examples, the medium comprises a water-soluble polymer and (a) a growth factor from the transforming growth factor beta (TGF-β) superfamily selected from the group consisting of inhibin, activin, Müllerian inhibitory substance (MIS), bone morphogenetic protein (BMP), decapentaplegic (dpp), Vg-1, monoclonal nonspecific inhibitory factor (MNSF), growth differentiation factor 8 (GDF8), and growth differentiation factor 11 (GDF11); (b) a fibroblast growth factor (FGF) selected from the group consisting of keratinocyte growth factor (KGF), FGF2, FGF10, FGF21, and FGF8B. (F) growth factors from the family; (c) retinoic acid (RA) signaling pathway activators selected from the group consisting of retinoic acid, CD1530, AM580, TTHRB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, and CD2314; (d) ROCK inhibitors selected from the group consisting of thiazovivin, Y-27632, Fasudil / HA1077, and 14-1152; (e) phorbol 12,13-dibutyrate (PDBU), TPB, phorbol 12-myristate 13-acetate, and bryostatin 1; and / or (f) a protein kinase C activator selected from the group consisting of SANT1, SANT2, SANT4, Cur6l4l4, forskolin, tomatidine, AY9944, triparanol, and cyclopamine.In some of these examples, the water-soluble synthetic polymer comprises less than 85% hydrolyzed polyvinyl alcohol, for example, about 80% hydrolyzed polyvinyl alcohol. In some examples, the composition further comprises PDX1-positive, NKX6.1-positive cells (e.g., PP2 cells).

[0101]

[0116] In some examples, the composition comprises a plurality of FOXA2-positive cells, e.g., gastrula cells, in a medium containing a water-soluble polymer. In some of these examples, the medium further comprises one or more agents selected from the group consisting of a protein kinase C activator, a growth factor from the transforming growth factor beta (TGF-β) superfamily, a bone morphogenetic protein signaling pathway inhibitor, a growth factor from the fibroblast growth factor (FGF) family, a retinoic acid (RA) signaling pathway activator, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a sonic hedgehog (SHH) pathway inhibitor. In some examples, the medium comprises a water-soluble polymer and (a) phorbol 12,13-dibutyrate (PDBU), TPB, phorbol 12-myristate, or (b) phorbol 12-myristate. (b) a protein kinase C activator selected from the group consisting of inhibin, activin, Müllerian inhibitory substance (MIS), bone morphogenetic protein (BMP), decapentaplegic (dpp), Vg-1, monoclonal nonspecific inhibitor of growth factor (MNSF), growth differentiation factor 8 (GDF8), and growth differentiation factor 11 (GDF11); (c) a bone morphogenetic protein signaling pathway inhibitor, including LDN193189 or DMH-1; (d) a fibroblast growth factor inhibitor selected from the group consisting of keratinocyte growth factor (KGF), FGF2, FGF10, FGF21, and FGF8B. (e) a sonic hedgehog pathway inhibitor selected from the group consisting of SANT1, SANT2, SANT4, Cur6l4l4, forskolin, tomatidine, AY9944, triparanol, and cyclopamine; (f) a retinoic acid signaling pathway activator selected from the group consisting of retinoic acid, CD1530, AM580, TTHRB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, and CD2314; and / or (g) a ROCK inhibitor selected from the group consisting of thiazovivin, Y-27632, Fasudil / HA1077, and 14-1152.In some embodiments, the water-soluble synthetic polymer comprises 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99% or 100% hydrolyzed polyvinyl alcohol. In some of these examples, the water-soluble synthetic polymer comprises less than 85% hydrolyzed polyvinyl alcohol, for example, about 80% hydrolyzed polyvinyl alcohol. In some of these examples, the composition further comprises PDX1-positive, NKX6.1-negative cells (e.g., PP1 cells).

[0102]

[0117] In some examples, the composition comprises a plurality of SOX17-positive cells (e.g., definitive endoderm cells) in a medium containing a water-soluble polymer. In some of these examples, the medium further comprises a growth factor from the fibroblast growth factor (FGF) family. In some examples, the growth factor from the fibroblast growth factor (FGF) family is selected from the group consisting of keratinocyte growth factor (KGF), FGF2, FGF10, FGF21, and FGF8B. In some embodiments, the water-soluble synthetic polymer comprises 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% hydrolyzed polyvinyl alcohol. In some of these examples, the water-soluble synthetic polymer comprises less than 85% hydrolyzed polyvinyl alcohol, for example, about 80% hydrolyzed polyvinyl alcohol. In some examples, the composition further comprises FOXA2-positive cells, eg, gastrula cells.

[0103]

[0118] In some examples, the composition comprises a plurality of pluripotent stem cells (e.g., human embryonic stem cells or induced pluripotent stem cells) in a medium comprising a water-soluble polymer. In some of these examples, the medium further comprises a growth factor from the transforming growth factor beta (TGF-β) superfamily, a WNT signaling pathway activator, or both. In some examples, the medium comprises a water-soluble polymer, (a) a growth factor from the transforming growth factor beta (TGF-β) superfamily selected from the group consisting of inhibin, activin, Müllerian inhibitory substance (MIS), bone morphogenetic protein (BMP), decapentaplegic (dpp), Vg-1, monoclonal nonspecific inhibitory factor (MNSF), growth differentiation factor 8 (GDF8), and / or growth differentiation factor 11 (GDF11); and / or (b) a compound selected from the group consisting of CHIR99021, 3F8, A 1070722, AR-A 014418, BIO, BIO-acetoxime, FRATide, 10Z-hymenialdisine, indirubin-3'oxime, Kenpaullone, L803, L803-mts, lithium carbonate, NSC 693868, SB 216763, SB 415286, TC-G 24, TCS 2002, TCS 21311, and TWS 119. In some embodiments, the water-soluble synthetic polymer comprises 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% hydrolyzed polyvinyl alcohol. In some of these examples, the water-soluble synthetic polymer comprises less than 85% hydrolyzed polyvinyl alcohol, for example, about 80% hydrolyzed polyvinyl alcohol. In some examples, the composition further comprises SOX17-positive cells (e.g., definitive endoderm cells).

[0104]

[0119] In some examples, a composition (e.g., an in vitro composition) comprises a plurality of PDX1-positive, NKX6.1-positive cells (e.g., PP2 cells), nicotinamide, and a growth factor from the epidermal growth factor (EGF) family. In some examples, the composition does not comprise betacellulin. The growth factor from the EGF family can comprise EGF. In some examples, the composition comprises about 1 ng / mL to about 100 ng / mL, about 2 ng / mL to about 50 ng / mL, about 5 ng / mL to about 20 ng / mL, or about 7.5 ng / mL to about 15 ng / mL of EGF, for example, about 2 ng / mL, about 5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 40 ng / mL, or about 50 ng / mL of EGF. In some examples, the composition comprises about 10 ng / mL of EGF. In some examples, the composition contains about 1 mM to about 100 mM, about 2 mM to about 50 mM, about 5 mM to about 20 mM, or about 7.5 mM to about 15 mM nicotinamide, for example, about 2 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 40 mM, or about 50 mM nicotinamide. In some examples, the composition contains about 10 mM nicotinamide. In some of these examples, the composition further contains one or more agents selected from the group consisting of a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, an epigenetic modifying compound, a retinoic acid (RA) signaling pathway activator, a sonic hedgehog (SHH) pathway inhibitor, a gamma-secretase inhibitor, a protein kinase C activator, a protein kinase inhibitor, a Rho-associated protein kinase inhibitor, a coiled-coil-containing protein kinase (ROCK) inhibitor, and a bone morphogenetic protein (BMP) signaling pathway inhibitor.In some examples, the composition comprises a plurality of PDX1-positive, NKX6.1-positive cells (e.g., PP2 cells), nicotinamide, and a growth factor from the epidermal growth factor (EGF) family, and (a) a TGF-β signaling pathway inhibitor selected from the group consisting of Alk5i II, A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB-525334, SD-208, and SB-505124; (b) a thyroid hormone signaling pathway activator, including T3 or GC-1; (c) 3-deazaneplanocin A (DZNep (d) an epigenetic modifying compound selected from the group consisting of retinoic acid, CD1530, AM580, TTHRB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, and CD2314; (e) sonic hedgehog pathway inhibitors selected from the group consisting of SANT1, SANT2, SANT4, Cur61414, forskolin, tomatidine, AY9944, triparanol, and cyclopamine; (f) gamma-secretase inhibitors, including XXI or DAPT; (g) protein kinase inhibitors, including staurosporine, Ro-31-8220, bicindolylmaleimide (Bis) compounds, 10'-{5"-[(methoxycarbonyl)amino]-2"-methyl}-phenylaminocarbonylstaurosporine, or stalalogues; (h) ROCK inhibitors, selected from the group consisting of thiazovivin, Y-27632, Fasudil / HA1077, and 14-1152; (i) phorbol 12,13-dibutyrate (PDBU), TPB, phorbol 12-myristate 13-acetate, and protein kinase C activators, including bryostatin 1; and / or (j) bone morphogenetic protein signaling pathway inhibitors, including LDN193189 or DMH-1.

[0105]

[0120] In some embodiments, the present disclosure provides a composition comprising any of the water-soluble polymers disclosed herein and pluripotent stem cells, in some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, or 30-40%, 40-50%, 50-95%, 50-75%, 70-95%, 70-80%, 80-95%, 85-95%, or 90-95% of the cells in the composition are pluripotent stem cells.

[0106]

[0121] In some embodiments, the present disclosure provides a composition comprising any of the water-soluble polymers disclosed herein and embryonic somatic cells, in which at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, or 30-40%, 40-50%, 50-95%, 50-75%, 70-95%, 70-80%, 80-95%, 85-95%, or 90-95% of the cells in the composition are definitive endoderm cells.

[0107]

[0122] In some embodiments, the present disclosure provides a composition comprising any of the water-soluble polymers disclosed herein and any of the gastrula cells, in which at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, or 30-40%, 40-50%, 50-95%, 50-75%, 70-95%, 70-80%, 80-95%, 85-95%, or 90-95% of the cells in the composition are gastrula cells.

[0108]

[0123] In some embodiments, the present disclosure provides a composition comprising any of the water-soluble polymers disclosed herein and PDX1-positive, NKX6.1-negative cells, in which at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, or 30-40%, 40-50%, 50-95%, 50-75%, 70-95%, 70-80%, 80-95%, 85-95%, or 90-95% of the cells in the composition are PDX1-positive, NKX6.1-negative cells.

[0109]

[0124] In some embodiments, the present disclosure provides a composition comprising any of the water-soluble polymers disclosed herein and PDX1-positive, NKX6.1-positive cells. In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, or 30-40%, 40-50%, 50-95%, 50-75%, 70-95%, 70-80%, 80-95%, 85-95%, or 90-95% of the cells in the composition are PDX1-positive, NKX6.1-positive cells. In some embodiments, 70-80%, 70-85%, or 75-85% of the cells in the composition are PDX1-positive, NKX6.1-positive cells.

[0110]

[0125] In some embodiments, the present disclosure provides a composition comprising any of the water-soluble polymers disclosed herein and NKX6.1-positive, ISL1-positive cells. In some embodiments, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, or 5-10%, 10-20%, 20-30%, 30-30%, 40-40%, 50-95%, 50-75%, 70-95%, 70-80%, 80-95%, 85-95%, or 90-95% of the cells in the composition are NKX6.1-positive, ISL1-positive cells. In some embodiments, the present disclosure provides a composition comprising any of the water-soluble polymers disclosed herein and NKX6.1-positive, ISL1-negative cells. In some embodiments, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, or 5-10%, 10-20%, 20-30%, 30-30%, 40-40%, 50-95%, 50-75%, 70-95%, 70-80%, 80-95%, 85-95%, or 90-95% of the cells in the composition are NKX6.1-positive, ISL1-negative cells. In some embodiments, the present disclosure provides a composition comprising any of the water-soluble polymers disclosed herein and NKX6.1-negative, ISL1-positive cells. In some embodiments, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 5-10%, 10-20%, 20-30%, 30-30%, 40-40%, 50-95%, 50-75%, 70-95%, 70-80%, 80-95%, 85-95%, or 90-95% of the cells in the composition are NKX6.1 negative, ISL1 negative. In some embodiments, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, or 5-10%, 10-20%, 20-30%, 30-30%, 40-40%, 50-95%, 50-75%, 70-95%, 70-80%, 80-95%, 85-95%, or 90-95% of the cells in the composition are NKX6.1-negative, ISL1-negative. In some embodiments, 30-35% of the cells in the composition are NKX6.1-positive, ISL1-positive cells.

[0111] Stem Cells and Reprogramming

[0126] Provided herein is the use of stem cells to generate SC-β cells (e.g., mature pancreatic β cells or β-like cells) or their precursors. In one embodiment, embryonic cells can be used to provide at least one SC-β cell, either in place of or in addition to stem cells, using similar protocols described in U.S. Patent Application Publication Nos. 2015 / 0240212 and 2015 / 0218522, or U.S. Patent Application Nos. 14 / 684,129; 14 / 684,101; and 17 / 390,839 (the U.S. counterparts of International Application Publication No. WO2022 / 026932), each of which is incorporated herein by reference in its entirety. Suitable embryonic cells can be prepared, for example, from primordial embryonic cells present in human fetal material harvested approximately 8-11 weeks after the last menstrual cycle. Illustrative methods for preparing embryonic cells are described, for example, in Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998 and US Pat. No. 6,090,622.

[0112]

[0127] Provided herein are compositions and methods for producing SC-β cells (e.g., pancreatic β cells), as well as pancreatic α cells and / or pancreatic δ cells. In some embodiments, the present disclosure provides a method for producing a cell population enriched for pancreatic α cells. In some embodiments, the present disclosure provides a method for producing a cell population enriched for pancreatic δ cells.

[0113]

[0128] Generally, at least one SC-β cell or precursor thereof, e.g., a pancreatic progenitor cell produced by the methods disclosed herein, can comprise a mixture or combination of various cells, such as primitive gut cells, PDX1-positive pancreatic progenitor cells, PDX1-positive, NKX6-1-positive pancreatic progenitor cells, Ngn3-positive endocrine progenitor cells, insulin-positive endocrine cells (e.g., NKX6.1-positive, ISL1-positive cells, or β-like cells), and / or other pluripotent or stem cells.

[0114]

[0129] Any suitable culture protocol can be followed to generate at least one pancreatic alpha, beta, and / or delta cell, or a precursor thereof, for differentiating stem cells or pluripotent cells to a desired stage of differentiation. In some embodiments, at least one pancreatic alpha, beta, and / or delta cell, or a precursor thereof, is generated by culturing at least one pluripotent cell for a period of time and under conditions suitable for the at least one pluripotent cell to differentiate into at least one pancreatic alpha, beta, and / or delta cell, or a precursor thereof.

[0115]

[0130] In some embodiments, the at least one pancreatic alpha, beta and / or delta cell or precursor thereof is a substantially pure population of pancreatic alpha, beta and / or delta cells or precursors thereof, hi some embodiments, the population of pancreatic alpha, beta and / or delta cells or precursors thereof is substantially free of or devoid of embryonic stem cells or pluripotent cells or iPS cells.

[0116]

[0131] In some embodiments, somatic cells, e.g., fibroblasts, can be isolated from a subject, e.g., as a tissue biopsy, e.g., a skin biopsy, and reprogrammed into induced pluripotent stem cells for further differentiation to generate at least one pancreatic alpha, beta, and / or delta cell, or precursor thereof, for use in the compositions and methods described herein. In some embodiments, the somatic cells, e.g., fibroblasts, are maintained in culture by methods known to those of skill in the art, and in some embodiments, expanded before being converted into pancreatic alpha, beta, and / or delta cells by the methods disclosed herein.

[0117]

[0132] In some embodiments, at least one pancreatic alpha, beta and / or delta cell or precursor thereof is maintained in culture by methods known to those of skill in the art, and in some embodiments, expanded before being converted into a pancreatic alpha, beta and / or delta cell by methods described herein.

[0118]

[0133] Furthermore, the at least one pancreatic alpha, beta, and / or delta cell or precursor thereof, e.g., pancreatic progenitor, can be from any mammalian species, non-limiting examples include murine, bovine, primate, porcine, equine, ovine, or human cells. In some embodiments, the at least one pancreatic alpha, beta, and / or delta cell or precursor thereof is derived from a human individual.

[0119] stem cells

[0134] Embodiments of the present disclosure relate to the use of stem cells for the production of pancreatic α, β, and / or δ cells or their precursors. As used herein, the term "stem cell" can refer to a cell (e.g., plant stem cell, vertebrate stem cell) that has the ability to self-renew and produce differentiated cell types (e.g., plant stem cell, vertebrate stem cell) (Morrison et al., (1997) Cell 88:287-298). In the context of cytogenetics, the adjectives "differentiated" or "differentiating" are relative terms. A "differentiated cell" can be a cell that has progressed further downstream in the developmental pathway than the cell it is being compared to. Thus, pluripotent stem cells can differentiate into lineage-restricted progenitor cells (e.g., definitive endoderm cells), which can then differentiate into further restricted cells (e.g., pancreatic progenitor cells), which can play characteristic roles in certain tissue types, and into end-stage cells (e.g., terminally differentiated cells, e.g., β cells) that may or may not retain the ability to further proliferate. Stem cells can be characterized by the presence and absence of certain markers (e.g., proteins, RNA, etc.). Stem cells can also be identified by functional assays, both in vitro and in vivo, particularly assays related to the ability of stem cells to give rise to multiple differentiated progeny. In one embodiment, the host cells are adult stem cells, somatic stem cells, non-embryonic stem cells, embryonic stem cells, hematopoietic stem cells, including pluripotent stem cells, and trophoblast stem cells.

[0120]

[0135] Stem cells of interest, such as those that can be used in the methods provided herein, can include pluripotent stem cells (PSCs). As used herein, the term "pluripotent stem cells" or "PSCs" refers to stem cells that can generate all cell types in an organism. Thus, PSCs can give rise to cells of all germ layers in an organism (e.g., endoderm, mesoderm, and ectoderm in vertebrates). Pluripotent cells can form teratomas and contribute to ectodermal, mesodermal, or endodermal tissues in an organism. Plant pluripotent stem cells can give rise to all cell types in the plant (e.g., roots, stems, leaves, and other cells).

[0121]

[0136] Embodiments of the present disclosure relate to the use of PSCs for the production of pancreatic beta cells or their precursors. Animal PSCs can be derived in several different ways. For example, embryonic stem cells (ESCs) can be derived from the inner cell mass of an embryo (Thomson et al., Science. 1998 Nov. 6; 282(5391):1145-7). Meanwhile, induced pluripotent stem cells (iPSCs) can be derived from somatic cells (Takahashi et al., Cell. 2007 Nov. 30; 131(5):861-72; Takahashi et al., Nat. Protoc. 2007; 2(12):3081-9; Yu et al., Science. 2007 Dec. 21; 318(5858):1917-20. Epub 2007 Nov. 20). The term PSC can refer to pluripotent stem cells regardless of their origin, and therefore can encompass the terms ESC and iPSC, as well as the term fetal embryonic stem cells (EGSC), which is another example of a PSC. PSCs can be in the form of established cell lines, which can be obtained directly from primary embryonic tissue or can be derived from somatic cells.

[0122]

[0137] Embodiments of the present disclosure relate to the use of ESCs for the production of pancreatic alpha, beta, and / or delta cells or their precursors. "Embryonic stem cells (ESCs)" may refer to PSCs isolated from an embryo, typically from the inner cell mass of a blastocyst. ESC lines include, for example, hESBGN-01, hESBGN-02, hESBGN-03, hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3, HES-4, HES-5, HES-6 (ES Cell International); Miz-hES1 (MizMedi Hospital-Seoul National University); HSF-1, HSF-6 (University of California at San Francisco); and H1, H7, H9, H13, H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute) NIH Human Embryonic Stem Cell Stem cells of interest also include embryonic stem cells from other primates, such as rhesus monkey stem cells and marmoset stem cells. Stem cells can be obtained from any mammalian species, such as humans, horses, cows, pigs, dogs, cats, rodents, such as mice, rats, hamsters, and primates (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 nucleocytoplasmic ratio, distinct borders, and prominent nuclei. Furthermore, ESCs can express SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase, but not SSEA-1. Examples of methods for 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 in its entirety.Methods for expanding undifferentiated forms of hESCs are described in WO 99 / 20741, WO 01 / 51616, and WO 03 / 020920, each of which is incorporated herein in its entirety.

[0123]

[0138] "Fetal embryonic stem cells (EGSCs) or fetal embryonic cells" or "EG cells" can refer to embryonic cells and / or embryonic cell precursor cells, such as PSCs derived from primordial embryonic cells, e.g., embryonic cells that can become sperm and eggs. Fetal embryonic cells (EG cells) are believed to have similar properties to the above-mentioned fetal stem cells. Examples of methods for producing and characterizing EG cells can be found, for example, in U.S. Patent No. 7,153,684; Matsui, Y. et al., (1992) Cell 70:841; Shamblott, M. et al., (2001) Proc. Natl. Acad. Sci. USA 98: 113; Shamblott, M. et al., (1998) Proc. Natl. Acad. Sci. USA, 95:13726; and Koshimizu, U. et al., (1996) Development, 122:1235, each of which is incorporated herein in its entirety.

[0124]

[0139] Embodiments of the present disclosure relate to the use of iPSCs for the production of pancreatic alpha, beta, and / or delta cells or their precursors. "Induced pluripotent stem cells" or "iPSCs" can refer to PSCs derived from non-PSC cells (e.g., from cells differentiated into PSCs). iPSCs can be derived from a variety of different cell types, including terminally differentiated cells. iPSCs have an embryonic stem cell-like morphology and can grow as flat colonies with a large nuclear-cytoplasmic ratio, distinct borders, and prominent nuclei. Furthermore, iPSCs can express one or more key pluripotency markers known to those skilled in the art, including, but not limited to, alkaline phosphatase, SSEA3, SSEA4, Sox2, Oct3 / 4, Nanog, TRA160, TRA181, TDGF1, Dnmt3b, FoxD3, GDF3, Cyp26a1, TERT, and zfp42. Examples of methods for generating and characterizing iPSCs can be found, for example, in U.S. Patent Publication Nos. 2009 / 0047263, 2009 / 0068742, 2009 / 0191159, 2009 / 0227032, 2009 / 0246875, and 2009 / 0304646, each of which is incorporated herein in its entirety. Generally, to generate iPSCs, somatic cells are provided with reprogramming factors known in the art (e.g., Oct4, SOX2, KLF4, MYC, Nanog, Lin28, etc.) that reprogram somatic cells to pluripotent stem cells.

[0125]

[0140] Embodiments of the present disclosure relate to the use of somatic cells for the production of pancreatic α, β, and / or δ cells or their precursors. "Somatic cells" can refer to any cell in an organism that does not normally give rise to all cell types in the organism in the absence of experimental manipulation. In other words, somatic cells can be cells that are fully differentiated and therefore do not naturally give rise to cells of all three germ layers of an organism, such as ectoderm, mesoderm, and endoderm. For example, somatic cells include neurons and neuronal 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 mesodermal or endodermal lineages.

[0126]

[0141] In certain instances, stem cells may be undifferentiated (e.g., cells not committed to a particular lineage) before exposure to at least one differentiation factor or composition according to the methods disclosed herein, while in other instances, it may be desirable to differentiate stem cells into one or more intermediate cell types before exposure to at least one differentiation factor or composition described herein. For example, stem cells may exhibit morphological, biological, or physical characteristics of undifferentiated cells, which can be used to distinguish them from differentiated cells of fetal or adult origin. In some instances, undifferentiated cells may appear microscopically in two dimensions in colonies of cells with a high nucleus / cytoplasm ratio and prominent nuclei. Stem cells may be used by themselves (e.g., substantially free of any undifferentiated cells) or in the presence of differentiated cells. In certain instances, stem cells may be cultured in the presence of suitable nutrients and, optionally, other cells, to grow and optionally differentiate. For example, fetal fibroblasts or fibroblast-like cells may be present in the culture to support stem cell growth. Fibroblasts may be present during one stage of stem cell development, but not necessarily at all stages. For example, fibroblasts may be added to a culture of stem cells at an initial culture stage and not be added to the culture of stem cells at one or more subsequent culture stages.

[0127]

[0142] Stem cells used in all aspects of the invention can be any cells derived from any type of tissue (e.g., embryonic tissue, such as fetal or pre-fetal tissue, or adult tissue), and these stem cells can be characterized by their ability, under appropriate conditions, to generate progeny consisting of various cell types, e.g., derivatives of all or at least one of the three germ layers (endoderm, mesoderm, and ectoderm). These cell types can be provided in the form of established cell lines, or can be obtained directly from primary embryonic tissue and immediately used for differentiation. Cells listed in the NIH Human Embryonic Stem Cell Registry include hESBGN-01, hESBGN-02, hESBGN-03, and hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3, HES-4, HES-5, and HES-6 (ES Cell International); Miz-hES1 (MizMedi Hospital-Seoul National University); HSF-1 and FISF-6 (University of California at San Francisco); and H1, H7, H9, H13, and H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute)). In some embodiments, the source of human stem cells or pluripotent stem cells used for chemically induced differentiation into mature insulin-positive cells did not involve the destruction of a human embryo. In some embodiments, the source of human stem cells or pluripotent stem cells used for chemically induced differentiation into mature insulin-positive cells did not involve the destruction of a human embryo.

[0128]

[0143] In another example, stem cells can be isolated from tissues, including solid tissues. In some embodiments, the tissue is skin, adipose tissue (e.g., adipose tissue), muscle tissue, heart or cardiac tissue. In other embodiments, the tissue is, for example, but not limited to, umbilical cord blood, placenta, bone marrow, or cartilage.

[0129]

[0144] Stem cells that can be used in the methods provided herein can also include various types of embryonic cells exemplified by human embryonic stem (hES) cells described by Thomson et al. (1998) Science 282:1145, embryonic stem cells from other primates such as rhesus monkey stem cells (Thomson et al. (1995) Proc. Natl. Acad. Sci. USA 92:7844), marmoset stem cells (Thomson et al. (1996) Biol. Reprod. 55:254), and human fetal embryo (hEG) cells (Shambloft et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). The methods provided herein are also applicable to stem cells committed to lineages such as mesodermal stem cells and other early cardiac developmental cells (see, e.g., Reyes et al., (2001) Blood 98:2615-2625; Eisenberg and Bader, (1996) Circ Res. 78(2):205-16). Stem cells can be obtained from any mammalian species, e.g., human, horse, cow, pig, dog, cat, rodent, e.g., mouse, rat, hamster, primate, etc. In some embodiments, human embryos have not been destroyed for the source of pluripotent cells used in the methods and compositions disclosed herein. In some embodiments, human embryos have not been destroyed for the source of pluripotent cells used in the methods and compositions disclosed herein.

[0130]

[0145] A mixture of cells from a suitable source of endothelial, muscle, and / or neuronal stem cells can be harvested from a mammalian donor for the purposes of the present disclosure. A suitable source is the hematopoietic microenvironment. For example, preferably fixed (e.g., mobilized) circulating peripheral blood can be removed from a subject. In one embodiment, the stem cells can be reprogrammed stem cells, such as stem cells derived from somatic cells or differentiated cells. In such embodiments, the dedifferentiated stem cells can be, for example, but not limited to, neoplastic cells, tumor cells, and cancer cells, or induced reprogrammed cells, such as induced pluripotent stem cells or iPS cells.

[0131]

[0146] In some embodiments, the pancreatic alpha, beta and / or delta cells described herein are selected from the group consisting of hair cells, keratinocytes, gonadotrophs, corticotrophs, thyrotrophs, somatotrophs, lactotrophs, chromaffin cells, parafollicular cells, glomus cells, melanocytes, nevus cells, Merkel cells, odontoblasts, cementoblasts, corneal keratinocytes, retinal müller cells, retinal pigment epithelial cells, neurons, glial cells (e.g., oligodendrocytes, astrocytes), ependymal cells, pineal cells, lung cells (e.g., type I pneumocytes and type II pneumocytes), Clara cells, goblet cells, G cells, D cells, ECL cells, gastric chief cells, parietal cells, pituitary cells, K cells, D cells, I cells, goblet cells, Paneth cells, enterocytes, microfold cells, hepatocytes, hepatic stellate cells (e.g., Kupffer cells from mesoderm), gallbladder cells, atrial cells, pancreatic stellate cells, pancreatic α cells, pancreatic β cells, pancreatic δ cells, pancreatic F cells (e.g., PP cells), pancreatic ε cells, thyroid (e.g., follicular cells), parathyroid (e.g., parathyroid chief cells), eosinophilic cells, urothelial cells, osteoblasts, osteocytes, chondroblasts, chondrocytes, fibroblasts, fibrocytes, myoblasts, myocytes, myosatellite cells, tenocytes, cardiac myocytes, lipoblasts, adipocytes, interstitial cells of Cajal, angioblasts, endothelial cells, mesangial cells (e.g., intraglomerular mesangial cells and extraglomerular mesangial cells), juxtaglomerular cells, macula densa cells, interstitial cells cells), interstitial cells, telocyte simple epithelial cells, podocytes, renal proximal tubule brush border cells, Sertoli cells, Leydig cells, granulosa cells, Pegg cells, germ layer cells, sperm, eggs, lymphocytes, myeloid cells, endothelial progenitor cells, endothelial stem cells, hemangioblasts, mesoangioblasts, pericyte mural cells, splenocytes (e.g., T lymphocytes, B lymphocytes, dendritic cells, microphages, leukocytes), trophoblast stem cells, or any combination thereof.

[0132]

[0147] As used herein, the term "reprogramming" can refer to the process of altering or reversing the differentiation state of a somatic cell. Cells can be partially or terminally differentiated before reprogramming. Reprogramming can also include the complete reversal of the differentiation state of a somatic cell to a pluripotent cell. Such complete reversal of differentiation can generate induced pluripotent (iPS) cells. As used herein, reprogramming can also include the partial reversal of the differentiation state of a cell, e.g., to a pluripotent state cell or somatic cell, which is a cell that is neither pluripotent nor pluripotent but has lost one or more specific characteristics of the differentiated cell from which it originated, e.g., the direct reprogramming of a differentiated cell to various somatic cell types. Reprogramming can also include the alteration, e.g., reversal, of at least some of the heritable patterns of nucleic acid modification (e.g., methylation), chromatin condensation, epigenetics, genomic imprinting, etc., that occur during cell differentiation as a zygote develops into an adult.

[0133]

[0148] As used herein, the term "reprogramming factor" can refer to a molecule associated with the "reprogramming" of a cell, i.e., differentiation, and / or dedifferentiation, and / or transdifferentiation, whereby a cell is converted into a different cell type or phenotype. Reprogramming factors generally affect the expression of genes associated with cell differentiation, dedifferentiation, and / or transdifferentiation. Transcription factors are examples of reprogramming factors.

[0134]

[0149] As used herein, the term "differentiation" and its grammatical equivalents refer to the process by which a less specialized cell (e.g., a more primitive cell with greater cellular potential) becomes a more specialized cell type (e.g., a less primitive cell with less cellular potential); the term "dedifferentiation" refers to the process by which a more specialized cell becomes a less specialized cell type (e.g., a more primitive cell with greater cellular potential); and the term "transdifferentiation" can refer to the process by which a cell of a particular cell type is converted into another cell type without significantly changing its level of "cellular potential" or "primitiveness." Without wishing to be bound by theory, a cell is considered to "transdifferentiate" if it is converted from one lineage-committed or terminally differentiated cell type to another lineage-committed or terminally differentiated cell type without significantly changing its level of "cellular potential" or "primitiveness."

[0135]

[0150] As used herein, the term "cell differentiation potential" should be understood to mean the ability of a cell to differentiate into cells of different lineages. For example, pluripotent cells (e.g., stem cells) have the potential to differentiate into cells of any of the three germ layers, i.e., endoderm (the inner lining of the stomach, the gastrointestinal tract, and the lungs), mesoderm (muscle, bone, blood, and urogenital tract), or ectoderm (epithelial tissue and the nervous system), and therefore have high cell differentiation potential. Pluripotent cells (e.g., stem cells or certain types of induced stem cells) have the ability to give rise to cells from a large but limited number of lineages (e.g., hematopoietic stem cells, cardiac stem cells, or neural stem cells, etc.) and have relatively lower cell differentiation potential than pluripotent cells. Cells committed to a specific lineage or terminally differentiated may have even lower cell differentiation potential. Specific examples of transdifferentiation known in the art include, for example, the conversion of pancreatic exocrine cells to beta cells.

[0136]

[0151] Thus, cells can be differentiated into more primitive cells (e.g., terminally differentiated cells can be differentiated to pluripotent or multipotent), or cells can be dedifferentiated into less primitive cells (e.g., pluripotent or multipotent cells can be differentiated into lineage-committed or terminally differentiated cells). However, in one embodiment, cells can be converted or transdifferentiated from one cell type (or phenotype) to another cell type (or phenotype), e.g., at a similar level of cellular potential. Thus, in one embodiment of the present disclosure, the inducing step of the present disclosure can reprogram cells of the present disclosure to differentiate, dedifferentiate, and / or transdifferentiate. In one embodiment of the present disclosure, the inducing step of the present disclosure can reprogram cells to transdifferentiate.

[0137]

[0152] Those skilled in the art are aware of methods for using one or more exogenous polynucleotide or polypeptide reprogramming factors to reprogram or induce cells of a particular type to become cells of another type, e.g., by differentiation, dedifferentiation, and / or transdifferentiation. Such methods may rely on the introduction of genetic material encoding one or more transcription factors or other polypeptides associated with cellular reprogramming. For example, PDX1, Ngn3, and MafA, or functional fragments thereof, are all known to encode peptides capable of inducing cellular differentiation, dedifferentiation, and / or transdifferentiation of 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 those described above) is contacted with a cell to, for example, induce the cells of the present disclosure. Those skilled in the art will recognize that other genes are also involved in cellular reprogramming, and that exogenous molecules encoding such genes (or functional fragments thereof) and the encoded polypeptides are also considered to be polynucleotide or polypeptide reprogramming factors (e.g., polynucleotides or polypeptides that subsequently affect the expression levels of other genes associated with cellular reprogramming). For example, it has been shown that the introduction of exogenous polynucleotide or polypeptide epigenetic gene silencer that reduces the inactivation of p53 can increase the efficiency of inducing induced pluripotent stem cells (iPSCs).Therefore, the exogenous polynucleotide or polypeptide that encodes epigenetic silencer and other genes or proteins that are thought to be directly or indirectly involved in cell reprogramming or increase cell programming efficiency can be considered to constitute exogenous polynucleotide or polypeptide reprogramming factors.Those skilled in the art will recognize that there are other methods that affect cell reprogramming, such as the introduction of RNAi molecules (or genetic material that encodes RNAi molecules) that can knock down the expression of genes that are involved in inhibiting cell reprogramming.Thus, any exogenous polynucleotide or polypeptide molecule that is associated with or promotes cellular reprogramming should be understood to be an exogenous polynucleotide or polypeptide reprogramming factor as described herein.

[0138]

[0153] It is recognized that the present methods can utilize "routine" tissue culture components, such as media, sera, serum substitutes, supplements, antibiotics, and the like, such as, for example, RPMI, renal epithelial cell basal medium (REBM), Dulbecco's modified Eagle's medium (DMEM), MCDB131 medium, CMRL 1066 medium, F12, fetal calf serum (FCS), fetal bovine serum (FBS), bovine serum albumin (BSA), D-glucose, L-glutamine, GlutaMAX™-1 (dipeptide, L-alanine-L-glutamine), B27, heparin, progesterone, putrescine, laminin, nicotinamide, insulin, transferrin, sodium selenite, selenium, ethanolamine, human epidermal growth factor (hEGF), basic fibroblast growth factor (bFGF), hydrocortisone, epinephrine, normacin, penicillin, streptomycin, gentamicin, and amphotericin. It should be understood 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 this disclosure: these components are neither small molecules as defined herein nor reprogramming factors as defined herein.

[0139]

[0154] Thus, in one embodiment, the present disclosure does not include culturing cells with one or more exogenous polynucleotide or polypeptide reprogramming factors. Thus, in one embodiment, the methods of the present disclosure do not include the introduction of one or more exogenous polynucleotide or polypeptide reprogramming factors, e.g., by introducing a transposon, a viral transgenic vector (such as a retroviral vector), a plasmid, mRNA, miRNA, a peptide, or a fragment of any of these molecules, that are involved in producing induced α, β, and / or δ cells or otherwise inducing the cells of the present disclosure to differentiate, dedifferentiate, and / or transdifferentiate.

[0140]

[0155] That is, in one embodiment, the method is carried out in the absence of one or more exogenous polynucleotide or polypeptide reprogramming factors.Therefore, it should be understood that in one embodiment, the method of the present disclosure utilizes small molecules (for example, HDAC inhibitors) to reprogram cells without adding polypeptide transcription factors, other polypeptide factors specifically associated with inducing differentiation, dedifferentiation, and / or transdifferentiation, polynucleotide sequences encoding polypeptide transcription factors, polynucleotide sequences encoding other polypeptide factors specifically associated with inducing differentiation, dedifferentiation, and / or transdifferentiation, mRNA, interfering RNA, microRNA, and fragments thereof.

[0141] Methods for producing induced beta cells from stem cells

[0156] Methods for producing SC-β cells (e.g., non-native pancreatic β cells) are provided herein. Detailed exemplary protocols for generating endocrine cells from stem cells to provide at least one SC-β cell are described in U.S. Patent Application Publication Nos. 2015 / 0240212, 2015 / 0218522, 2021 / 0198632, and International Application Publication No. WO2019 / 169351, each of which is incorporated herein by reference in its entirety.

[0142]

[0157] The endoderm can give rise to the digestive and respiratory tracts, thyroid gland, liver, and pancreas. A representative disease of the endodermal lineage is type 1 diabetes, which results from the destruction of insulin-producing beta cells. The in vitro generation of functional beta cells from human pluripotent stem cells (hPSCs) could provide a practical and renewable cell source for replacement cell therapy of type 1 diabetes. Embryonic stem (ES) cells, generated from the inner cell mass of blast-stage embryos, represent a promising cell source for transplantation of any damaged cells or cell-based therapies. They can be maintained in culture, self-renew, and proliferate indefinitely as undifferentiated ES cells. ES cells can differentiate into all cell types in the body, including ectodermal, mesodermal, and endodermal lineage cells or tissues. The major advantage of ES cells is their potential for stable self-renewal and differentiation in culture.

[0143]

[0158] Definitive endoderm is produced in vivo from the inner cell mass by the embryonic process of gastrulation, during which epiblast cells are directed to form three germ layers. Definitive endoderm can give rise to a variety of cells and tissues that contribute to vital organs, such as pancreatic beta cells, hepatocytes of the liver, alveolar cells of the lung, thyroid gland, thymus, and the epithelial linings of the nutrient and respiratory tracts. This differs from the primitive endoderm, which is an extraembryonic tissue that can give rise to visceral and parietal endoderm. Definitive endoderm derived from ES cells can theoretically become any endodermal derivative, and directing ES cells toward the endodermal lineage is essential for generating therapeutic endodermal derivatives.

[0144]

[0159] The precise patterning of the definitive endoderm along the anterior-posterior axis ultimately leads to the formation of the primitive gut. The primitive gut, derived from the definitive endoderm, gives rise to the pharynx, esophagus, stomach, duodenum, small intestine, and large intestine, as well as related organs such as the pancreas, lungs, thyroid gland, thymus, parathyroid gland, and liver along the anterior-posterior axis. The anterior portion of the foregut of the primitive gut gives rise to the lungs, thyroid gland, esophagus, and stomach. The pancreas, liver, and duodenum originate from the posterior portion of the foregut. The midgut and hindgut of the primitive gut give rise to the small intestine and large intestine. The anterior foregut expresses developmental markers NK2 homeobox (NKX2-1) and SRY (sex-determining region Y)-box 2 (SOX2); the posterior foregut expresses hematopoietically expressed homeobox (HHEX), pancreatic and duodenal homeobox 1 (PDX1), onecut homeobox 1 (ONECUT1, also known as HNF6), and hepatocyte nuclear factor 4 alpha (HNF4A); and the midgut / hindgut expresses caudal homeobox 1 (CDX1), caudal homeobox 2 (CDX2), and motor neuron and pancreatic homeobox 1 (MNX1) (3, 19, 20).

[0145]

[0160] Successful differentiation of pancreatic α, β, and / or δ cells requires that the differentiated cells synthesize and secrete physiologically appropriate amounts of insulin. An exemplary stepwise protocol for directing hPSC cell differentiation has been developed, involving a differentiation process that recapitulates key stages of normal pancreatic endocrine development. Differentiation of hPSC cells into hormone-expressing pancreatic endocrine cells is achieved by transitioning hPSC cells through key stages of embryonic development: differentiation into mesoderm and definitive endoderm, establishment of archenteron endoderm, patterning of the posterior foregut, and specification and maturation of pancreatic endoderm and endocrine progenitor cells. Through these stages, hPSC cells acquire a pancreatic endocrine phenotype and the ability to secrete insulin in response to glucose in vitro.

[0146]

[0161] In some embodiments, an in vitro composition described herein (e.g., an in vitro Stage 1, Stage 2, Stage 3, Stage 4, or Stage 5 composition) further comprises a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol (PVA), poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), a PEG copolymer, poly(N-isopropylacrylamide), or polyacrylamide, and optionally, the water-soluble synthetic polymer is polyvinyl alcohol. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol (PVA). In some embodiments, the water-soluble synthetic polymer is present in the medium at a concentration of about 0.005% to about 0.5% (w / v), about 0.01% to about 0.2% (w / v), about 0.02% to about 0.1% (w / v), or about 0.03% to about 0.08% (w / v). In some embodiments, the water-soluble synthetic polymer is present in the medium at a concentration of about 0.005% (w / v), 0.01% (w / v), 0.05% (w / v), 0.1% (w / v), 0.15% (w / v), 0.2% (w / v), 0.25% (w / v), 0.3% (w / v), 0.35% (w / v), 0.4% (w / v), 0.45% (w / v), or 0.5% (w / v). Polyvinyl alcohol as described herein can refer to a water-soluble synthetic polymer having the idealized formula [CHCH(OH)] that can be partially or fully hydrolyzed. In some examples, polyvinyl alcohol is produced by either partial or complete hydrolysis of polyvinyl acetate to remove the acetate groups. In some examples, the polyvinyl alcohol is at most 85% hydrolyzed, for example, 80% hydrolyzed. The percentage of hydrolysis measures the approximate percentage (e.g., average percentage) of acetate residues that are hydrolyzed in the polyvinyl acetate precursor polymer. In some examples, the polyvinyl alcohol is at least 85% hydrolyzed, e.g., 87-89% hydrolyzed, 87-90% hydrolyzed, or 99% hydrolyzed.In some embodiments, the polyvinyl alcohol is 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% hydrolyzed. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol (PVA), and the PVA is at most 90% hydrolyzed (e.g., 87%-89%). In some embodiments, the PVA is 80% hydrolyzed (e.g., in Stages 1-4). In some embodiments, the PVA is 89% hydrolyzed (e.g., in Stage 5). In some embodiments, the Stage 6 composition specifically excludes the water-soluble synthetic polymer (e.g., PVA). In some embodiments, the Stage 6 composition includes serum albumin (e.g., HSA) in place of the water-soluble synthetic polymer.

[0147]

[0162] Generally, at least one pancreatic alpha, beta and / or delta cell or precursor thereof, e.g., the pancreatic cells produced according to the methods disclosed herein, may comprise a mixture or combination of different cells, e.g., PDX1-positive, NKX6.1-negative pancreatic progenitor cells, pancreatic progenitor cells that co-express PDX1 and NKX6-1, Ngn3-positive endocrine progenitor cells, insulin-positive endocrine cells (e.g., NKX6.1-positive, ISL1-positive cells, or beta-like cells), and / or other pluripotent or stem cell-like cells.

[0148]

[0163] The at least one pancreatic alpha, beta, and / or delta cell, or precursor thereof, can be produced according to any suitable culture protocol that differentiates stem cells or pluripotent cells to a desired differentiation stage. In some embodiments, the at least one pancreatic alpha, beta, and / or delta cell, or precursor thereof, is produced by culturing at least one pluripotent cell for a time and under conditions suitable for differentiating the at least one pluripotent cell into at least one pancreatic alpha, beta, and / or delta cell, or precursor thereof.

[0149]

[0164] In some embodiments, the at least one pancreatic alpha, beta, and / or delta cell or precursor thereof is a substantially pure population of pancreatic alpha, beta, and / or delta cells or precursors thereof. In some embodiments, the population of pancreatic alpha, beta, and / or delta cells or precursors thereof comprises a mixture of pluripotent cells or differentiated cells. In some embodiments, the population of pancreatic alpha, beta, and / or delta cells or precursors thereof is substantially free of or devoid of embryonic stem cells or pluripotent cells or iPS cells.

[0150]

[0165] In some embodiments, somatic cells, e.g., fibroblasts, can be isolated from a subject, e.g., as a tissue biopsy, e.g., a skin biopsy, and reprogrammed into induced pluripotent stem cells for further differentiation to generate at least one SC-β cell or precursor thereof for use in the compositions and methods described herein. In some embodiments, the somatic cells, e.g., fibroblasts, are maintained in culture by methods known to those of skill in the art, and in some embodiments, expanded before being converted into pancreatic α, β, and / or δ cells by the methods disclosed herein.

[0151]

[0166] In some embodiments, at least one pancreatic alpha, beta and / or delta cell or precursor thereof is maintained in culture by methods known to those of skill in the art, and in some embodiments expanded before being converted to a pancreatic alpha, beta and / or delta cell by the methods disclosed herein.

[0152]

[0167] Furthermore, the at least one pancreatic α, β, and / or δ cell or precursor thereof, e.g., pancreatic progenitor, may be from any mammalian species, non-limiting examples of which include murine, bovine, simian, porcine, equine, ovine, or human cells. For clarity and simplicity, the methods described herein refer to at least one mammalian pancreatic α, β, and / or δ cell or precursor thereof, but it should be understood that all of the methods described herein can be readily applied to other cell types of at least one pancreatic α, β, and / or δ cell or precursor thereof. In some embodiments, the at least one pancreatic α, β, and / or δ cell or precursor thereof is derived from a human individual.

[0153] Definitive endoderm cells

[0168] Aspects of the present disclosure include definitive endoderm cells. As used herein, definitive endoderm cells can be derived from any source or produced according to any suitable protocol. In some aspects, pluripotent stem cells, such as iPSCs or hESCs, are differentiated into endoderm cells. In some aspects, endoderm cells (stage 1) are further differentiated into, for example, primitive gut cells (stage 2), PDX1-positive, NKX6.1-negative pancreatic progenitor cells (stage 3), PDX1-positive, NKX6.1-positive pancreatic progenitor cells (stage 4), or Ngn3-positive endocrine precursor cells or insulin-positive endocrine cells (stage 5), and then induced or matured into SC-β cells (stage 6). In some embodiments, stage 4 cells (e.g., cells at stage 4, day 5) are contacted with a PKC activator (e.g., PDBU) for 1, 2, 3, 4, or 5 days. In some embodiments, stage 4 cells (e.g., stage 4, day 5 cells) are contacted with a PKC activator (e.g., PDBU) to become stage 5 (e.g., stage 5, day 2 cells). See, e.g., International Application Publication No. WO2020 / 033879, incorporated herein by reference in its entirety.

[0154]

[0169] In some examples, definitive endoderm cells can be obtained by differentiating at least some pluripotent cells in a population into definitive endoderm cells, for example, by contacting a population of pluripotent cells with i) at least one growth factor from the TGF-β superfamily and ii) a WNT signaling pathway activator to induce differentiation of at least some of the pluripotent cells into definitive endoderm cells, wherein the definitive endoderm cells express at least one marker characteristic of definitive endoderm.

[0155]

[0170] Any growth factor from the TGF-β superfamily that can induce pluripotent stem cells (e.g., alone or in combination with a WNT signaling pathway activator) to differentiate into definitive endoderm cells can be used in the methods provided herein. In some examples, the growth factor from the TGF-β superfamily comprises activin A. In some examples, the growth factor from the TGF-β superfamily comprises growth differentiation factor 8 (GDF8). Any WNT signaling pathway activator that can induce pluripotent stem cells (e.g., alone or in combination with a growth factor from the TGF-β superfamily) to differentiate into definitive endoderm cells can be used in the methods provided herein. In some examples, the WNT signaling pathway activator comprises CHIR99021, 3F8, A 1070722, AR-A 014418, BIO, BIO-acetoxime, FRATide, 10Z-hymenialdisine, indirubin-3'oxime, kenpaullone, L803, L803-mts, lithium carbonate, NSC 693868, SB216763, SB 415286, TC-G 24, TCS 2002, TCS 21311, or TWS 119. In some embodiments, the WNT signaling pathway activator comprises CHIR99021. In some examples, the WNT signaling pathway activator comprises Wnt3a recombinant protein.

[0156]

[0171] In some examples, differentiating at least some of the pluripotent cells in the population into definitive endoderm cells is accomplished by a process of contacting the population of pluripotent cells with i) activin A, and ii) CHIR99021 for a suitable period of time, for example, about 2 days, about 3 days, about 4 days, or about 5 days, to induce differentiation of at least some of the pluripotent cells in the population into definitive endoderm cells, wherein the definitive endoderm cells express at least one marker characteristic of definitive endoderm. In some examples, differentiating at least some of the pluripotent cells in the population into definitive endoderm cells is accomplished by a process of contacting the population of pluripotent cells with i) activin A, and ii) CHIR99021 for 1 day, followed by contacting the population with activin A (in the absence of CHIR99021) for 2 days.

[0157]

[0172] In some examples, the method includes contacting a population of pluripotent cells with a suitable concentration of a growth factor from the TGF-β superfamily (e.g., activin A), such as about 10 ng / mL, about 20 ng / mL, about 50 ng / mL, about 75 ng / mL, about 80 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 110 ng / mL, about 120 ng / mL, about 130 ng / mL, about 140 ng / mL, about 150 ng / mL, about 175 ng / mL, about 180 ng / mL, about 200 ng / mL, about 250 ng / mL, or about 300 ng / mL, thereby differentiating the pluripotent cells into definitive endoderm cells. In some examples, the method includes using about 100 ng / mL of activin A to differentiate the pluripotent cells into definitive endoderm cells. In some examples, the methods include the use of about 200 ng / mL of activin A for the differentiation of pluripotent cells to definitive endoderm cells.

[0158]

[0173] In some examples, the method includes contacting a population of pluripotent cells with a suitable concentration of a WNT signaling pathway activator (e.g., CHIR99021), 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, thereby differentiating the pluripotent cells into definitive endoderm cells. In some examples, the method includes using about 2 μM CHIR99021 to differentiate the pluripotent cells into definitive endoderm cells. In some examples, the methods include the use of about 5 μM CHIR99021 for the differentiation of pluripotent cells to definitive endoderm cells.

[0159]

[0174] In some examples, the definitive endoderm cells generated by the methods disclosed herein express at least one marker selected from the group consisting of Nodal, Tmprss2, Tmem30b, St14, Spink3, Sh3gl2, Ripk4, Rab1S, Npnt, Clic6, Cldn5, Cacna1b, Bnip1, Anxa4, Emb, FoxA1, Sox17, and Rbm35a, and the expression of the at least one marker is upregulated at a statistically significant amount in the definitive endoderm cells relative to the original pluripotent stem cells from which it was derived. In some examples, the definitive endoderm cells generated by the methods disclosed herein do not express at least one marker selected from the group consisting of Gata4, SPARC, AFP, and Dab2 at a statistically significant amount relative to the original pluripotent stem cells from which it was derived. In some examples, definitive endoderm cells generated by the methods disclosed herein do not express at least one marker selected from the group consisting of Zic1, Pax6, Flk1, and CD31 at a statistically significant level relative to the original pluripotent stem cells from which they were derived. In some examples, definitive endoderm cells generated by the methods disclosed herein have a statistically significant higher level of Smad2 phosphorylation relative to the original pluripotent stem cells from which they were derived. In some examples, definitive endoderm cells generated by the methods disclosed herein have the ability to form a gut tube in vivo. In some examples, definitive endoderm cells generated by the methods disclosed herein can be differentiated into cells with morphology characteristic of intestinal cells, and the cells with morphology characteristic of intestinal cells express FoxA2 and / or Claudin6. In some examples, definitive endoderm cells generated by the methods disclosed herein can be further differentiated into cells of endodermal origin.

[0160]

[0175] In some examples, the population of pluripotent stem cells is cultured in the presence of at least one beta cell differentiation factor before any differentiation or during the first stage of differentiation. Any pluripotent stem cells can be used, such as human pluripotent stem cells or human iPS cells, or any pluripotent stem cells discussed herein or other suitable pluripotent stem cells. In some examples, the beta cell differentiation factors described herein can be present in the culture medium of the population of pluripotent stem cells, or can be added once or periodically during the growth (e.g., replication or expansion) of the population of pluripotent stem cells. In certain examples, the population of pluripotent stem cells can be exposed to at least one beta cell differentiation factor before any differentiation. In other examples, the population of pluripotent stem cells can be exposed to at least one beta cell differentiation factor during the first stage of differentiation.

[0161] gastrula cells

[0176] The embodiments of the present disclosure include primitive gut cells.The primitive gut cells used herein can be derived from any source or produced according to any suitable protocol.In some embodiments, definitive endoderm cells are differentiated into primitive gut cells.In some embodiments, primitive gut cells are further differentiated into, for example, PDX1 positive, NKX6.1 negative pancreatic progenitor cells, PDX1 positive, NKX6.1 positive pancreatic progenitor cells, Ngn3 positive endocrine progenitor cells, insulin positive endocrine cells, and then induce or mature into SC-β cells.

[0162]

[0177] In some examples, primitive gut cells can be obtained by differentiating at least some of the definitive endoderm cells in the population into primitive gut cells, for example, by contacting the definitive endoderm cells with at least one growth factor from the fibroblast growth factor (FGF) family to induce differentiation of at least some of the definitive endoderm cells into primitive gut cells, wherein the primitive gut cells express at least one marker characteristic of primitive gut cells.

[0163]

[0178] Any growth factor from the FGF family that can induce definitive endoderm cells (e.g., alone or in combination with other factors) to differentiate into primitive gut cells 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.

[0164]

[0179] In some examples, primitive gut cells can be obtained by differentiating at least some of the definitive endoderm cells in the population into primitive gut cells, for example, by contacting the definitive endoderm cells with KGF for a period of time, for example, about 1 day, about 2 days, about 3 days, or about 4 days, to induce differentiation of at least some of the definitive endoderm cells into primitive gut cells.

[0165]

[0180] In some examples, the method includes differentiating definitive endoderm cells into primitive gut cells by contacting the cells with a growth factor from the FGF family (e.g., KGF) at a suitable concentration, for example, about 10 ng / mL, about 20 ng / mL, about 50 ng / mL, about 75 ng / mL, about 80 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 110 ng / mL, about 120 ng / mL, about 130 ng / mL, about 140 ng / mL, about 150 ng / mL, about 175 ng / mL, about 180 ng / mL, about 200 ng / mL, about 250 ng / mL, or about 300 ng / mL. In some examples, the method includes using about 50 ng / mL of KGF to differentiate the definitive endoderm cells into primitive gut cells. In some examples, the method includes using about 100 ng / mL of KGF to differentiate the definitive endoderm cells into primitive gut cells.

[0166] PDX1-positive pancreatic progenitor cells

[0181] The embodiments of the present disclosure include PDX1 positive, NKX6.1 negative pancreatic progenitor cells.The PDX1 positive, NKX6.1 negative pancreatic progenitor cells used herein can be derived from any source or produced according to any suitable protocol.In some embodiments, primitive gut cells are differentiated into PDX1 positive, NKX6.1 negative pancreatic progenitor cells.In some embodiments, PDX1 positive, NKX6.1 negative pancreatic progenitor cells are further differentiated into, for example, PDX1 positive, NKX6.1 positive pancreatic progenitor cells, Ngn3 positive endocrine progenitor cells, insulin positive endocrine cells, and then induced or matured into pancreatic α, β and / or δ cells.

[0167]

[0182] In some embodiments, PDX1-positive, NKX6.1-negative pancreatic progenitor cells can be obtained by differentiating at least a portion of the primitive gut cells in the population into PDX1-positive, NKX6.1-negative pancreatic progenitor cells, for example, by contacting the primitive gut cells with i) at least one BMP signaling pathway inhibitor, ii) a growth factor from the TGF-β superfamily, iii) at least one growth factor from the FGF family, iv) at least one SHH pathway inhibitor, v) at least one retinoic acid (RA) signaling pathway activator, vi) at least one protein kinase C activator, and vii) a ROCK inhibitor, to induce differentiation of at least a portion of the primitive gut cells into PDX1-positive, NKX6.1-negative pancreatic progenitor cells.

[0168]

[0183] In some embodiments, PDX1-positive, NKX6.1-negative pancreatic progenitor cells can be obtained by differentiating at least a portion of the primitive gut cells in the population into PDX1-positive, NKX6.1-negative pancreatic progenitor cells, for example, by contacting the primitive gut cells with i) at least one BMP signaling pathway inhibitor, ii) a growth factor from the TGF-β superfamily, iii) at least one growth factor from the FGF family, iv) at least one SHH pathway inhibitor, v) at least one retinoic acid (RA) signaling pathway activator, and vi) at least one protein kinase C activator, and inducing differentiation of at least a portion of the primitive gut cells into PDX1-positive, NKX6.1-negative pancreatic progenitor cells.

[0169]

[0184] In some examples, PDX1-positive, NKX6.1-negative pancreatic progenitor cells can be obtained by differentiating at least a portion of the primitive gut cells in the population into PDX1-positive, NKX6.1-negative pancreatic progenitor cells, for example, by contacting the primitive gut cells with i) at least one BMP signaling pathway inhibitor, ii) at least one growth factor from the FGF family, iii) at least one SHH pathway inhibitor, iv) at least one retinoic acid (RA) signaling pathway activator, and v) at least one protein kinase C activator, to induce differentiation of at least a portion of the primitive gut cells into PDX1-positive, NKX6.1-negative pancreatic progenitor cells.

[0170]

[0185] In some examples, PDX1-positive, NKX6.1-negative pancreatic progenitor cells can be obtained by differentiating at least a portion of the primitive gut cells in the population into PDX1-positive, NKX6.1-negative pancreatic progenitor cells, for example, by contacting the primitive gut cells with: i) at least one SHH pathway inhibitor; ii) at least one retinoic acid (RA) signaling pathway activator; and iii) at least one protein kinase C activator.

[0171]

[0186] In some examples, PDX1-positive, NKX6.1-negative pancreatic progenitor cells can be obtained by differentiating at least a portion of the primitive gut cells in the population into PDX1-positive, NKX6.1-negative pancreatic progenitor cells, for example, by contacting the primitive gut cells with i) at least one growth factor from the FGF family and ii) at least one activator of the retinoic acid (RA) signaling pathway to induce differentiation of at least a portion of the primitive gut cells into PDX1-positive, NKX6.1-negative pancreatic progenitor cells. In some examples, Pdx1-positive, NKX6.1-negative pancreatic progenitor cells can be obtained by differentiating at least some of the primitive gut cells in the population into Pdx1-positive pancreatic progenitor cells, for example, by contacting the primitive gut cells with DMH-1, activin A, retinoic acid, KGF, Sant1, LDN193189, and PdBU on day 1, and with activin A, retinoic acid, KGF, Sant1, LDN193189, and PdBU on day 2. In some embodiments, the cells are not contacted with DMH-1 on day 2.

[0172]

[0187] Any BMP signaling pathway inhibitor that can induce primitive gut cells to differentiate into PDX1-positive, NKX6.1-negative pancreatic progenitor cells (e.g., alone or in combination with a growth factor from the TGF-β superfamily, at least one growth factor from the FGF family, at least one SHH pathway inhibitor, at least one retinoic acid signaling pathway activator, at least one protein kinase C activator, and a ROCK inhibitor) can be used in the methods provided herein. In some examples, the BMP signaling pathway inhibitor includes LDN193189 or DMH-1. In some examples, the method includes contacting the primitive gut cells with a BMP signaling pathway inhibitor (e.g., LDN193189) at a concentration of, for example, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 110 nM, about 120 nM, about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about 200 nM, about 210 nM, about 220 nM, about 230 nM, about 240 nM, about 250 nM, about 280 nM, about 300 nM, about 400 nM, about 500 nM, or about 1 μM. In some examples, the method includes contacting the primitive gut cells with a BMP signaling pathway inhibitor (e.g., DMH-1) at a concentration of, for example, about 0.01 μM, about 0.02 μM, about 0.05 μM, about 0.1 μM, about 0.2 μM, about 0.5 μM, about 0.8 μM, about 1 μM, about 1.2 μM, about 1.5 μM, about 1.75 μM, about 2 μM, about 2.2 μM, about 2.5 μM, about 2.75 μM, about 3 μM, about 3.25 μM, about 3.5 μM, about 3.75 μM, about 4 μM, about 4.5 μM, about 5 μM, about 8 μM, about 10 μM, about 15 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, or about 100 μM.

[0173]

[0188] Any growth factor from the TGF-β superfamily that can induce primitive gut cells to differentiate into PDX1-positive, NKX6.1-negative pancreatic progenitor cells (e.g., alone or in combination with at least one BMP signaling pathway inhibitor, a growth factor from the FGF family, at least one SHH pathway inhibitor, at least one retinoic acid signaling pathway activator, at least one protein kinase C activator, and a ROCK inhibitor) can be used. In some examples, the growth factor from the TGF-β family includes activin A. In some examples, the growth factor from the TGF-β family includes activin A or GDF8. In some examples, the method includes contacting the primitive gut cells with a growth factor from the TGF-β superfamily (e.g., activin A) at a concentration of, for example, about 5 ng / mL, about 7.5 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 11 ng / mL, about 12 ng / mL, about 13 ng / mL, about 14 ng / mL, about 15 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, about 19 ng / mL, about 20 ng / mL, about 21 ng / mL, about 22 ng / mL, about 23 ng / mL, about 24 ng / mL, about 25 ng / mL, about 26 ng / mL, about 27 ng / mL, about 28 ng / mL, about 29 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, or about 100 ng / mL.

[0174]

[0189] Any growth factor from the FGF family that can induce primitive gut cells to differentiate into PDX1-positive, NKX6.1-negative pancreatic progenitor cells (e.g., alone or in combination with at least one BMP signaling pathway inhibitor, a growth factor from the TGF-β superfamily, at least one SHH pathway inhibitor, at least one retinoic acid signaling pathway activator, at least one protein kinase C activator, and a ROCK inhibitor) can be used. In some examples, the at least one growth factor from the FGF family includes keratinocyte growth factor (KGF). In some examples, the at least one growth factor from the FGF family is selected from the group consisting of FGF2, FGF8B, FGF10, and FGF21. In some examples, the method includes contacting the primitive gut cells with a growth factor from the FGF family (e.g., KGF) at a concentration of, for example, about 10 ng / mL, about 20 ng / mL, about 50 ng / mL, about 75 ng / mL, about 80 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 110 ng / mL, about 120 ng / mL, about 130 ng / mL, about 140 ng / mL, about 150 ng / mL, about 175 ng / mL, about 180 ng / mL, about 200 ng / mL, about 250 ng / mL, or about 300 ng / mL.

[0175]

[0190] Any SHH pathway inhibitor that can induce primitive gut cells to differentiate into PDX1-positive, NKX6.1-negative pancreatic progenitor cells (e.g., alone or in combination with at least one BMP signaling pathway inhibitor, at least one growth factor from the FGF family, a growth factor from the TGF-β superfamily, at least one retinoic acid signaling pathway activator, at least one protein kinase C activator, and a ROCK inhibitor) can be used. In some examples, the SHH pathway inhibitor includes Sant1. In some examples, the method comprises administering to the primitive gut cells at a concentration of, for example, about 0.001 μM, about 0.002 μM, about 0.005 μM, about 0.01 μM, about 0.02 μM, about 0.03 μM, about 0.05 μM, about 0.08 μM, about 0.1 μM, about 0.12 μM, about 0.13 μM, about 0.14 μM, about 0.15 μM, about 0.16 μM, about 0.17 μM, about 0.18 μM, about 0.19 μM, about 0.2 μM, about 0.21 μM, about 0.22 μM, about 0.23 μM, about 0.24 μM, about 0.25 μM, about 0.26 μM, about 0.27 μM, about 0.28 μM, about 0.29 μM, about 0.30 μM, about 0.31 μM, about 0.32 μM, about 0.33 μM, about 0.34 μM, about 0.35 μM, about 0.36 μM, about 0.37 μM, about 0.38 μM, about 0.39 μM, about 10 ... The method includes contacting the cells with an SHH pathway inhibitor (e.g., Sant1) at a concentration of about 0.24 μM, about 0.25 μM, about 0.26 μM, about 0.27 μM, about 0.28 μM, about 0.29 μM, about 0.3 μM, about 0.31 μM, about 0.32 μM, about 0.33 μM, about 0.34 μM, about 0.35 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about 0.6 μM, about 0.8 μM, about 1 μM, about 2 μM, or about 5 μM.

[0176]

[0191] Any RA signaling pathway activator that can induce primitive gut cells to differentiate into PDX1-positive, NKX6.1-negative pancreatic progenitor cells (e.g., alone or in combination with at least one BMP signaling pathway inhibitor, at least one growth factor from the FGF family, at least one SHH pathway inhibitor, at least one protein kinase C activator, and a ROCK inhibitor) can be used. In some examples, the RA signaling pathway activator comprises retinoic acid. In some examples, the method comprises administering to the primitive gut cells at a concentration of, for example, about 0.02 μM, about 0.1 μM, about 0.2 μM, about 0.25 μM, about 0.3 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about 0.55 μM, about 0.6 μM, about 0.65 μM, about 0.7 μM, about 0.75 μM, about 0.8 μM, about 0.85 μM, about 0.9 μM, about 1 μM, about 1.1 μM, about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM, about 1.8 μM, about 1.9 μM, about 2 μM, about 2.1 μM, about 2.2 μM, about 2.3 μM, about 2.4 μM, or 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 of a RA signaling pathway activator (e.g., retinoic acid).

[0177]

[0192] Any PKC activator that can induce primitive gut cells to differentiate into PDX1-positive, NKX6.1-negative pancreatic progenitor cells (e.g., alone or in combination with at least one BMP signaling pathway inhibitor, at least one growth factor from the FGF family, at least one SHH pathway inhibitor, at least one RA signaling pathway activator, and a ROCK inhibitor) can be used. In some examples, the PKC activator includes PdBU. In some examples, the PKC activator includes TPB. In some examples, the method includes administering to the primitive gut cells 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 includes contacting the primary gut cells with a PKC activator (e.g., PdBU) at a concentration of, for example, 10 nM to 1 mM, 10 nM to 500 μM, 10 nM to 1 μM, 10 to 800 nM, 100 to 900 nM, 300 to 800 nM, 300 to 600 nM, 400 to 600 nM, 450 to 550 nM, or about 500 nM. In some embodiments, the primary gut cells are not treated with a PKC activator (e.g., PDBU).

[0178]

[0193] Any ROCK inhibitor that can induce primitive gut cells to differentiate into PDX1-positive, NKX6.1-negative pancreatic progenitor cells (for example, alone or in combination with at least one BMP signaling pathway inhibitor, at least one growth factor from the FGF family, at least one SHH pathway inhibitor, a PKC activator, and at least one RA signaling pathway activator) can be used. In some examples, the ROCK inhibitor comprises thiazovivin, Y-27632, Fasudil / HA1077, or H-1152. In some examples, the ROCK inhibitor comprises Y-27632. In some examples, the ROCK inhibitor comprises thiazovivin. In some examples, the method further comprises administering to the primitive gut cells a therapeutically effective amount of HCl 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, The method includes contacting the cells with a ROCK inhibitor (e.g., Y-27632 or Thiazovivin) at a concentration of 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.

[0179]

[0194] In some examples, PDX1-positive, NKX6.1-negative pancreatic progenitor cells can be obtained by differentiating at least a portion of the primitive gut cells in the population into PDX1-positive, NKX6.1-negative pancreatic progenitor cells, for example, by contacting the primitive gut cells with retinoic acid, KGF, Sant1, DMH-1, PdBU, thiazovivin, and activin A for a suitable period of time, for example, about 1 day, about 2 days, about 3 days, or about 4 days. In some examples, PDX1-positive, NKX6.1-negative pancreatic progenitor cells can be obtained by differentiating at least a portion of the primitive gut cells in the population into PDX1-positive, NKX6.1-negative pancreatic progenitor cells, for example, by contacting the primitive gut cells with retinoic acid, KGF, Sant1, DMH-1, PdBU, thiazovivin, and activin A for about 2 days. In some examples, PDX1-positive, NKX6.1-negative pancreatic progenitor cells can be obtained by differentiating at least a portion of primitive gut cells in S3 medium.

[0180] NKX6.1-positive pancreatic progenitor cells

[0195] The embodiments of the present disclosure include PDX1 positive, NKX6.1 positive pancreatic progenitor cells.The PDX1 positive, NKX6.1 positive pancreatic progenitor cells used herein can be derived from any source or produced according to any suitable protocol.In some embodiments, PDX1 positive, NKX6.1 negative pancreatic progenitor cells are differentiated into PDX1 positive, NKX6.1 positive pancreatic progenitor cells.In some embodiments, PDX1 positive, NKX6.1 positive pancreatic progenitor cells are further differentiated into, for example, Ngn3 positive endocrine progenitor cells or insulin positive endocrine cells, and then induce or mature into pancreatic alpha, beta and / or delta cells.

[0181]

[0196] In some aspects, a method for generating PDX1-positive, NKX6.1-positive pancreatic progenitor cells from PDX1-positive, NKX6.1-negative pancreatic progenitor cells includes contacting a population of cells comprising PDX1-positive, NKX6.1-negative pancreatic progenitor cells with at least two beta cell differentiation factors (e.g., under conditions that promote cell clustering and / or promote cell survival) comprising: a) at least one growth factor from the fibroblast growth factor (FGF) family; b) a sonic hedgehog pathway inhibitor; and, optionally, c) a low concentration of a retinoic acid (RA) signaling pathway activator to induce differentiation of at least one PDX1-positive, NKX6.1-negative pancreatic progenitor cell in the population into a PDX1-positive, NKX6.1-positive pancreatic progenitor cell.

[0182]

[0197] In some examples, the PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive, NKX6.1-negative pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) a low concentration of an RA signaling pathway activator to induce differentiation of at least some of the PDX1-positive, NKX6.1-negative pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells.

[0183]

[0198] In some examples, PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive, NKX6.1-negative pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) an RA signaling pathway activator, iv) a ROCK inhibitor, and v) at least one growth factor from the TGF-β superfamily to induce differentiation of at least some PDX1-positive, NKX6.1-negative pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells. In some examples, PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive, NKX6.1-negative pancreatic progenitor cells with at least one growth factor from the FGF family under conditions that promote cell clustering. In some embodiments, PDX1-positive, NKX6.1-negative pancreatic progenitor cells are administered with a PKC activator (e.g., PDBU). See, for example, International Application Publication No. WO2020 / 033879, which is incorporated by reference herein in its entirety.

[0184]

[0199] Any growth factor from the FGF family that can induce PDX1-positive, NKX6.1-negative pancreatic progenitor cells to differentiate into PDX1-positive, NKX6.1-positive pancreatic progenitor cells (for example, alone or in combination with at least one SHH pathway inhibitor, ROCK inhibitor, growth factor from the TGF-β superfamily, and at least one retinoic acid signaling pathway activator) can be used in the methods provided herein. In some examples, the at least one growth factor from the FGF family includes keratinocyte growth factor (KGF). In some examples, the at least one growth factor from the FGF family is selected from the group consisting of FGF2, FGF8B, FGF10, and FGF21. In some examples, the method includes contacting PDX1-positive, NKX6.1-negative pancreatic progenitor cells with a growth factor from the FGF family (e.g., KGF, FGF2, or both) 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.

[0185]

[0200] Any SHH pathway inhibitor that can induce PDX1-positive, NKX6.1-negative pancreatic progenitor cells to differentiate into PDX1-positive, 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 retinoic acid signaling pathway activator, a ROCK inhibitor, and at least one growth factor from the TGF-β superfamily) can be used in the methods provided herein. In some examples, the SHH pathway inhibitor includes Sant1. In some examples, the method comprises administering to a subject a subject a subject a subject's PDX1-positive, NKX6.1-negative pancreatic progenitor cells at a concentration of, for example, about 0.001 μM, about 0.002 μM, about 0.005 μM, about 0.01 μM, about 0.02 μM, about 0.03 μM, about 0.05 μM, about 0.08 μM, about 0.1 μM, about 0.12 μM, about 0.13 μM, about 0.14 μM, about 0.15 μM, about 0.16 μM, about 0.17 μM, about 0.18 μM, about 0.19 μM, about 0.2 μM, about 0.21 μM, about 0.22 μM, about 0.23 μM, about 0.24 μM, about 0.25 μM, about 0.26 μM, about 0.27 μM, about 0.28 μM, about 0.29 μM, about 0.30 μM, about 0.31 μM, about 0.32 μM, about 0.33 μM, about 0.34 μM, about 0.35 μM, about 0.36 μM, about 0.37 μM, about 0.38 μM, about 0.39 μM, about 10 ... The method includes contacting the cells with an SHH pathway inhibitor (e.g., Sant1) at a concentration of about 0.23 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.

[0186]

[0201] Any RA signaling pathway activator that can induce PDX1-positive, NKX6.1-negative pancreatic progenitor cells to differentiate into PDX1-positive, 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 administering to a subject a subject a subject a subject's PDX1-positive, NKX6.1-negative pancreatic progenitor cells at a concentration of, for example, about 0.02 μM, about 0.1 μM, about 0.2 μM, about 0.25 μM, about 0.3 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about 0.55 μM, about 0.6 μM, about 0.65 μM, about 0.7 μM, about 0.75 μM, about 0.8 μM, about 0.85 μM, about 0.9 μM, about 1 μM, about 1.1 μM, about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM, about 1.8 μM, about 1.9 μM, about 2 μM, about 2.1 μM, about 2.2 μM, about 2.3 μM, about 2.4 μM, The method includes contacting the subject with a RA signaling pathway activator (e.g., retinoic acid) at a concentration of about 1 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.

[0187]

[0202] Any ROCK inhibitor that can induce PDX1-positive, NKX6.1-negative pancreatic progenitor cells to differentiate into PDX1-positive, NKX6.1-positive pancreatic progenitor cells (for example, alone or in combination with at least one growth factor from the FGF family, at least one SHH pathway inhibitor, an RA signaling pathway activator, and at least one growth factor from the TGF-β superfamily) can be used. In some examples, ROCK inhibitors include Thiazovivin, Y-27632, Fasudil / HA1077, and 14-1152. In some examples, the method comprises administering to the patient PDX1-positive, NKX6.1-negative pancreatic progenitor cells at a concentration of, for example, about 0.2 μM, about 0.5 μM, about 0.75 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 7.5 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, or about 20 μM. The method includes contacting the subject with a ROCK inhibitor (e.g., Y-27632 or Thiazovivin) at a concentration of about 1 μM, about 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.

[0188]

[0203] Any activator from the TGF-β superfamily can be used to induce PDX1-positive, NKX6.1-negative pancreatic progenitor cells to differentiate into PDX1-positive, NKX6.1-positive pancreatic progenitor cells (for example, alone or in combination with at least one growth factor from the FGF family, at least one SHH pathway inhibitor, RA signaling pathway activator, and a ROCK inhibitor).In some examples, the activator from the TGF-β superfamily includes activin A or GDF8. In some examples, the method involves treating PDX1-positive, NKX6.1-negative pancreatic progenitor cells with a growth factor from the TGF-β superfamily (e.g., activin A) 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, The method includes contacting the antibody with about 3.2ng / mL, about 3.4ng / mL, about 3.6ng / mL, about 3.8ng / mL, about 4ng / mL, about 4.2ng / mL, about 4.4ng / mL, about 4.6ng / mL, about 4.8ng / mL, about 5ng / mL, about 5.2ng / mL, about 5.4ng / mL, about 5.6ng / mL, about 5.8ng / mL, about 6ng / mL, about 6.2ng / mL, about 6.4ng / mL, about 6.6ng / mL, about 6.8ng / mL, about 7ng / mL, about 8ng / mL, about 9ng / mL, about 10ng / mL, about 20ng / mL, about 30ng / mL, or about 50ng / mL. In some examples, the method includes contacting PDX1-positive, NKX6.1-negative pancreatic progenitor cells with a concentration of a growth factor from the TGF-β superfamily (e.g., activin A), for example, about 5 ng / mL.

[0189]

[0204] In some examples, PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive, NKX6.1-negative 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, NKX6.1-negative 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, NKX6.1-negative pancreatic progenitor cells with KGF 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, NKX6.1-negative pancreatic progenitor cells in S3 medium.

[0190]

[0205] In some examples, PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive, NKX6.1-negative pancreatic progenitor cells for 5 or 6 days under conditions that promote cell cluster formation with KGF, Sant1, RA, thiazovivin, and / or activin A. In some embodiments, PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by a) contacting PDX1-positive, NKX6.1-negative 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.

[0191] Insulin-positive endocrine cells

[0206] The embodiments of the present disclosure include insulin-positive endocrine cells (e.g., NKX6.1-positive, ISL1-positive, or β-like cells).The insulin-positive endocrine cells used herein can be derived from any source or produced according to any suitable protocol.In some embodiments, PDX1-positive, NKX6.1-positive pancreatic progenitor cells are differentiated into insulin-positive endocrine cells (e.g., NKX6.1-positive, ISL1-positive, or β-like cells).In some embodiments, insulin-positive endocrine cells are further differentiated, for example, by induction or maturation into SC-β cells.

[0192]

[0207] In some embodiments, a method for generating insulin-positive endocrine cells from PDX1-positive, NKX6.1-positive pancreatic progenitor cells includes contacting a cell population (e.g., under conditions that promote cell cluster formation) containing PDX1-positive, NKX6-1-positive pancreatic progenitor cells with a) a TGF-β signaling pathway inhibitor, b) a thyroid hormone signaling pathway activator, and c) a protein kinase inhibitor and / or a sonic hedgehog inhibitor to induce differentiation of at least one PDX1-positive, NKX6.1-positive pancreatic progenitor cell in the population into an insulin-positive endocrine cell, wherein the insulin-positive endocrine cell (ceil) expresses insulin. In some examples, the insulin-positive endocrine cell expresses PDX1, NKX6.1, ISL1, NKX2.2, Mafb, glis3, Sur1, Kir6.2, Znt8, SLC2A1, SLC2A3, and / or insulin.

[0193]

[0208] Any TGF-β signaling pathway inhibitor that can induce the differentiation of PDX1-positive, NKX6.1-positive pancreatic progenitor cells into insulin-positive endocrine cells (e.g., alone or in combination with other β cell differentiation factors, such as thyroid hormone signaling pathway activators) 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.

[0194]

[0209] Any thyroid hormone signaling pathway activator that can induce differentiation of PDX1-positive, NKX6.1-positive pancreatic progenitor cells into insulin-positive endocrine cells (e.g., alone or in combination with other beta cell differentiation factors, such as TGF-β signaling pathway inhibitors) can be used. In some examples, the thyroid hormone signaling pathway activator comprises triiodothyronine (T3). In some examples, the thyroid hormone signaling pathway activator comprises GC-1.

[0195]

[0210] In some examples, the method includes contacting a cell population (e.g., PDX1-positive, NKX6.1-positive pancreatic progenitor cells) with at least one additional factor. In some examples, the method includes contacting the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with at least one of: i) an SHH pathway inhibitor, ii) an RA signaling pathway activator, iii) a gamma-secretase inhibitor, iv) at least one growth factor from the epidermal growth factor (EGF) family, v) a protein kinase inhibitor, vi) a TGF-β signaling pathway inhibitor, or vii) a thyroid hormone signaling pathway activator. In some embodiments, the method includes contacting a cell population (e.g., PDX1-positive, 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) an RA signaling pathway activator, iii) a gamma-secretase inhibitor, iv) at least one growth factor from the epidermal growth factor (EGF) family, v) a protein kinase inhibitor, vi) a TGF-β signaling pathway inhibitor, vii) a thyroid hormone signaling pathway activator, or viii) a PKC activator. In some embodiments, the PDX1-positive, NKX6.1-positive pancreatic progenitor cells are administered a PKC activator (e.g., PDBU) for 1, 2, or 3 days. See, for example, International Application Publication No. WO2020 / 033879 or U.S. Patent Application Publication No. 2021 / 0238553, each of which is incorporated herein by reference in its entirety.

[0196]

[0211] In some examples, the method includes contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells with at least one of: i) an SHH pathway inhibitor, ii) an RA signaling pathway activator, iii) a gamma-secretase inhibitor, iv) at least one growth factor from the epidermal growth factor (EGF) family, v) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, vi) a TGF-β signaling pathway inhibitor, vii) a thyroid hormone signaling pathway activator, viii) a protein kinase inhibitor (e.g., staurosporine), or ix) a ROCK inhibitor.

[0197]

[0212] In some examples, the method includes contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells with at least one of: i) an SHH pathway inhibitor, ii) an RA signaling pathway activator, iii) a gamma-secretase inhibitor, iv) at least one growth factor from the epidermal growth factor (EGF) family, v) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, vi) a TGF-β signaling pathway inhibitor, vii) a thyroid hormone signaling pathway activator, viii) an epigenetic modifying compound, ix) a protein kinase inhibitor, or x) a ROCK inhibitor.

[0198]

[0213] In some examples, the method includes contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells with at least one of: i) an SHH pathway inhibitor, ii) an RA signaling pathway activator, iii) a gamma-secretase inhibitor, iv) at least one growth factor from the epidermal growth factor (EGF) family, v) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, vi) a TGF-β signaling pathway inhibitor, vii) a thyroid hormone signaling pathway activator, viii) an epigenetic modifying compound, ix) a protein kinase inhibitor (e.g., staurosporine), x) a ROCK inhibitor, and / or xi) nicotinamide.

[0199]

[0214] In some embodiments, in the method for generating insulin-positive endocrine cells from PDX1-positive, NKX6.1-positive pancreatic progenitor cells, some of the differentiation factors are present only for the first 1, 2, 3, 4, or 5 days during the differentiation step. In some examples, some of the differentiation factors, such as an SHH pathway inhibitor, a PKC activator, an RA signaling pathway activator, and at least one growth factor from the EGF family, are removed from the medium and / or are not present in the medium after the first 1, 2, or 3 days of incubation.

[0200]

[0215] In some examples, the method includes treating PDX1-positive, NKX6.1-positive pancreatic progenitor cells with at least one of: i) an SHH pathway inhibitor, ii) an RA signaling pathway activator, iii) a gamma-secretase inhibitor, iv) a growth factor from the epidermal growth factor (EGF) family, v) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, vi) a TGF-β signaling pathway inhibitor, vii) a thyroid hormone signaling pathway activator, viii) an epigenetic modifying compound, ix) a protein kinase inhibitor (e.g., staurosporine), and / or x) a ROCK inhibitor for 1, 2, 3, or 4 days. (e.g., for 3 days), and then contacting the cells with at least one of i) a gamma-secretase inhibitor, ii) a bone morphogenetic protein (BMP) signaling pathway inhibitor, iii) a TGF-β signaling pathway inhibitor, iv) a thyroid hormone signaling pathway activator, v) an epigenetic modifying compound, vi) a protein kinase inhibitor, and / or vii) a ROCK inhibitor, in the absence of i) an SHH pathway inhibitor, ii) an RA signaling pathway activator, and / or a growth factor from the epidermal growth factor (EGF) family, for 2, 3, 4, 5, or 6 days (e.g., 4 days).

[0201]

[0216] Any gamma-secretase inhibitor can induce the differentiation of PDX1-positive, NKX6.1-positive pancreatic progenitor cells in a population into insulin-positive endocrine cells (for example, alone or in combination with either a TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator). In some examples, the gamma-secretase inhibitor comprises XXI. In some examples, the gamma-secretase inhibitor comprises DAPT. In some examples, the method comprises administering to the patient a dose of PDX1-positive, NKX6.1-positive pancreatic progenitor cells at a concentration of, for example, about 0.01 μM, about 0.02 μM, about 0.05 μM, about 0.075 μM, about 0.1 μM, about 0.2 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1 μM, about 1.1 μM, about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM, about 1.8 μM, about 1.9 μM, about 2 μM, about 2.1 μM, about 2.2 μM, about 2.3 μM, about 2.4 μM, about 2.5 μM, 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 of a gamma secretase inhibitor (e.g., XXI).

[0202]

[0217] Any growth factor from the EGF family that can induce differentiation of PDX1-positive, NKX6.1-positive pancreatic progenitor cells in the population into insulin-positive endocrine cells (e.g., alone or in combination with any TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway activator) can be used. In some examples, at least one growth factor from the EGF family includes betacellulin. In some examples, at least one growth factor from the EGF family includes EGF. In some examples, the methods include contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a growth factor from the EGF family (e.g., betacellulin) at a concentration of, for example, about 1 ng / mL, about 2 ng / mL, about 4 ng / mL, about 6 ng / mL, about 8 ng / mL, about 10 ng / mL, about 12 ng / mL, about 14 ng / mL, about 16 ng / mL, about 18 ng / mL, about 20 ng / mL, about 22 ng / mL, about 24 ng / mL, about 26 ng / mL, about 28 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 75 ng / mL, about 80 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 150 ng / mL, about 200 ng / mL, about 250 ng / mL, or about 300 ng / mL.

[0203]

[0218] In some embodiments, the method includes contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a nicotinamide concentration of about 1 mM to about 100 mM, about 2 mM to about 50 mM, about 5 mM to about 20 mM, or about 7.5 mM to about 15 mM. In some examples, the composition includes about 10 mM nicotinamide.

[0204]

[0219] Any RA signaling pathway activator that can induce the differentiation of PDX1-positive, NKX6.1-positive pancreatic progenitor cells into insulin-positive endocrine cells (e.g., alone or in combination with a TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator) can be used. In some examples, the RA signaling pathway activator includes RA. In some examples, the method comprises administering to the patient a dose of PDX1-positive, NKX6.1-positive pancreatic progenitor cells at a concentration of, for example, about 0.02 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, The method includes contacting the subject with a RA signaling pathway activator (e.g., retinoic acid) at a concentration of about 1 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.

[0205]

[0220] Any SHH pathway inhibitor that can induce differentiation of PDX1-positive, NKX6.1-positive pancreatic progenitor cells into insulin-positive endocrine cells (e.g., alone or in combination with either a TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator) can be used in the methods provided herein. In some examples, the SHH pathway inhibitor includes Sant1. In some examples, the method comprises administering to a subject ... The method includes contacting the cells with an SHH pathway inhibitor (e.g., Sant1) at a concentration of about 0.23 μM, about 0.24 μM, about 0.25 μM, about 0.26 μM, about 0.27 μM, about 0.28 μM, about 0.29 μM, about 0.3 μM, about 0.31 μM, about 0.32 μM, about 0.33 μM, about 0.34 μM, about 0.35 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about 0.6 μM, about 0.8 μM, about 1 μM, about 2 μM, or about 5 μM.

[0206]

[0221] Any BMP signaling pathway inhibitor that can induce the differentiation of PDX1-positive, NKX6.1-positive pancreatic progenitor cells into insulin-positive endocrine cells (e.g., alone or in combination with a TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator) can be used. In some examples, the BMP signaling pathway inhibitor includes LDN193189 or DMH-1. In some examples, the method includes contacting PDX1-positive, 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.

[0207]

[0222] Any ROCK inhibitor that can induce the differentiation of PDX1-positive, NKX6.1-positive pancreatic progenitor cells in the population into insulin-positive endocrine cells (for example, alone or in combination with any TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway activator) can be used.In some examples, the ROCK inhibitor comprises Thiazovivin, Y-27632, Fasudil / HA1077, or H-1152.In some examples, the ROCK inhibitor comprises Y-27632.In some examples, the ROCK inhibitor comprises Thiazovivin. In some examples, the method comprises administering to the patient a dose of PDX1-positive, NKX6.1-positive pancreatic progenitor cells at a concentration of, for example, about 0.2 μM, about 0.5 μM, about 0.75 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 7.5 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, or about 18 μM. The method includes contacting the subject with a ROCK inhibitor (e.g., Y-27632 or Thiazovivin) at a concentration of about 1 μM, about 18 μM, about 19 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 35 μM, about 40 μM, about 50 μM, or about 100 μM.

[0208]

[0223] Any epigenetic modification compound can be used that can induce the differentiation of PDX1-positive, NKX6.1-positive pancreatic progenitor cells in the population into insulin-positive endocrine cells (for example, alone or in combination with any TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway activator).In some examples, the epigenetic modification compound comprises a histone methyltransferase inhibitor or an HDAC inhibitor.In some examples, the epigenetic modification compound comprises a histone methyltransferase inhibitor, such as DZNep.In some examples, the epigenetic modification compound comprises an HDAC inhibitor, such as KD5170. In some examples, the method includes contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells with an epigenetic modifying compound (e.g., DZNep or KD5170) at a concentration of, for example, about 0.01 μM, about 0.025 μM, about 0.05 μM, about 0.075 μM, about 0.1 μM, about 0.15 μM, about 0.2 μM, about 0.5 μM, about 0.75 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 7.5 μM, about 8 μM, about 9 μM, about 10 μM, about 15 μM, about 20 μM, about 25 μM, about 30 μM, about 35 μM, about 40 μM, about 50 μM, or about 100 μM.

[0209]

[0224] 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 PDX1-positive, NKX6.1-positive pancreatic progenitor cells in the population into insulin-positive endocrine cells (for example, alone or in combination with any TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway activator) can be used. In some examples, the protein kinase inhibitor comprises staurosporine.

[0210]

[0225] In some examples, the method includes contacting a population of cells (e.g., PDX1-positive, NKX6.1-positive pancreatic progenitor cells) with XXI, Alk5i, T3 or GC-1, RA, Sant1, and betacellulin for 7 days to induce differentiation of at least one PDX1-positive, NKX6.1-positive pancreatic progenitor cell in the population into an insulin-positive endocrine cell, wherein the insulin-positive endocrine cell expresses insulin. In some examples, the method includes contacting a population of cells (e.g., PDX1-positive, NKX6.1-positive pancreatic progenitor cells) with XXI, Alk5i, T3 or GC-1, RA, Sant1, betacellulin, and LDN193189 for 7 days to induce differentiation of at least one PDX1-positive, NKX6.1-positive pancreatic progenitor cell in the population into an insulin-positive endocrine cell, wherein the insulin-positive endocrine cell expresses insulin. In some embodiments, one or more differentiation factors are added as part of Stage 5, e.g., only on the first 1, 2, 3, 4, 5, or 6 days of Stage 5, or only on the last 1, 2, 3, 4, 5, or 6 days of Stage 5. In one example, cells are contacted with an SHH signaling pathway inhibitor only on the first 2, 3, 4, or 5 days of Stage 5, after which the SHH signaling pathway inhibitor is removed from the medium. In another example, cells are contacted with a BMP signaling pathway inhibitor only on the first 1, 2, or 3 days of Stage 5, after which the BMP signaling pathway inhibitor is removed from the medium.

[0211]

[0226] In some examples, the method includes culturing a population of cells (e.g., PDX1-positive, NKX6.1-positive pancreatic progenitor cells) in BE5 medium to induce differentiation of at least one NKX6.1-positive pancreatic progenitor cell in the population into an insulin-positive endocrine cell, wherein the insulin-positive endocrine cell expresses insulin.

[0212] Pancreatic beta cells

[0227] Aspects of the present disclosure include producing pancreatic β cells (e.g., non-native pancreatic β cells), which in some instances are similar in morphology and function to endogenous mature β cells, but are nevertheless distinct from native β cells.

[0213]

[0228] In some examples, the insulin-positive pancreatic endocrine cells produced using the methods provided herein can form cell clusters, either alone or together with other types of cells, such as their precursors, such as stem cells, definitive endoderm cells, primitive gut cells, PDX1-positive, NKX6.1-negative pancreatic progenitor cells, or PDX1-positive, NKX6.1-positive pancreatic progenitor cells.

[0214]

[0229] In some examples, a cell population comprising insulin-positive endocrine cells can be directly induced to mature into SC-β cells without the addition of any exogenous differentiation factors (e.g., a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, a PKC activator, a growth factor from the TGF-β superfamily, FGF family, or EGF family, an SHH signaling pathway inhibitor, a γ-secretase inhibitor, a ROCK inhibitor, or a BMP signaling pathway inhibitor). In some embodiments, the methods provided herein include contacting a cell population comprising NKX6.1-positive, ISL1-positive endocrine cells with a water-soluble polymer (e.g., serum albumin protein or PVA), a TGF-β signaling pathway inhibitor, an SHH pathway inhibitor, a thyroid hormone signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and / or an epigenetic modifying compound. In some embodiments, the methods provided herein include contacting a cell population comprising NKX6.1-positive, ISL1-positive endocrine cells with human serum albumin protein. In some embodiments, the methods provided herein include contacting a cell population comprising NKX6.1-positive, ISL1-positive endocrine cells with any of the polyvinyl alcohol molecules described herein. In some embodiments, the methods provided herein include contacting a cell population comprising NKX6.1-positive, ISL1-positive endocrine cells with a PKC activator.

[0215]

[0230] In some examples, a cell population containing insulin-positive endocrine cells can be induced to mature into SC-β cells by contacting insulin-positive endocrine cells with a differentiation factor. The differentiation factor can include at least one inhibitor of the TGF-β signaling pathway and an activator of the thyroid hormone signaling pathway described herein. In some examples, SC-β cells can be obtained by contacting a cell population containing insulin-positive endocrine cells with Alk5i and T3 or GC-1.

[0216]

[0231] In some examples, insulin-positive endocrine cells can be matured in NS-GFs medium, MCDB131 medium, DMEM medium, or CMRL medium. In some examples, insulin-positive endocrine cells can be matured in CMRL medium supplemented with 10% FBS. In some examples, insulin-positive endocrine cells can be matured in DMEM / F12 medium supplemented with 1% HSA. In other examples, SC-β cells can be obtained by culturing a population of cells containing insulin-positive endocrine cells in MCDB131 medium, which may be supplemented with 2% BSA. In some examples, MCDB131 medium supplemented with 2% BSA for maturing insulin-positive endocrine cells into SC-β cells may not contain the small molecule factors described herein. In some examples, MCDB131 medium supplemented with 2% BSA for maturing insulin-positive endocrine cells into SC-β cells may not contain serum (e.g., FBS). In other examples, SC-β cells can be obtained by culturing a cell population containing insulin-positive endocrine cells in MCDB131 medium, which can be supplemented with 0.05% HSA and vitamin C. In some examples, SC-β cells can be obtained by culturing a cell population containing insulin-positive endocrine cells in MCDB131 medium, which can be supplemented with 0.05% HSA, ITS-X, vitamin C, and glutamine (Gln, e.g., 4 mM). In some examples, the type of medium can be changed during S6. For example, S6 cells are cultured in MCDB131 medium, which can be supplemented with 0.05% HSA and vitamin C, for the first 2-4 days, and then cultured in DMEM / F12 medium supplemented with 1% HSA. In some examples, additional factors are introduced into the medium. For example, S6 cells can be cultured in MCDB131 medium, which can be supplemented with 0.05% HSA, ITS-X, vitamin C, and glutamine (Gln, e.g., 4 mM), for 10–12 days, during which ZnSO is introduced from day 4 of S6 culture.

[0217]

[0232] In some aspects, the disclosure provides a method of producing SC-β cells from pluripotent cells, the method comprising: a) differentiating pluripotent stem cells in a population into definitive endoderm cells by contacting the pluripotent stem cells with at least one factor from the TGFβ superfamily and a WNT signaling pathway activator for three days; b) differentiating at least a portion of the definitive endoderm cells into primitive gut cells by a process of contacting the definitive endoderm cells with at least one factor from the FGF family for three days; c) differentiating at least a portion of the primitive gut cells into PDX1-positive pancreatic progenitor cells by a process of contacting the PDX1-positive pancreatic progenitor cells with i) a retinoic acid signaling pathway activator, ii) at least one factor from the FGF family, iii) an SHH pathway inhibitor, iv) a BMP signaling pathway inhibitor (e.g., DMH-1 or LDN193189), v) a PKC activator, and vi) a ROCK inhibitor, under conditions that promote cell clustering. and e) differentiating at least a portion of the PDX1-positive pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells by a process of contacting the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with i) a TGF-β signaling pathway inhibitor, ii) at least one SHH pathway inhibitor, and optionally iii) an RA signaling pathway activator, and optionally iv) a ROCK inhibitor, and v) at least one factor from the TGF-β superfamily for 5 days. and f) differentiating at least a portion of the PDX1-positive, NKX6.1-positive pancreatic progenitor cells into PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells by a process of contacting the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a PDX1-positive, NKX6.1-positive, insulin-positive endocrine cell line, the PDX1-positive, NKX6.1-positive, pancreatic progenitor cells with a PDX1-positive, NKX6.1-positive, insulin-positive endocrine cell line, the NKX6.1-positive, insulin-positive, ...and differentiating at least a portion of the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells by a process of culturing the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells in a medium that does not contain exogenous differentiation factors (e.g., NS-GFs medium, MCDB medium supplemented with BSA, MCDB131 medium, or DMEM / F12 medium) for 7 to 14 days and inducing in vitro maturation of at least a portion of the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells, wherein the SC-β cells exhibit a GSIS response in vitro and / or in vivo. In some examples, the GSIS response resembles the GSIS response of endogenous mature β cells.

[0218]

[0233] In some aspects, the present disclosure provides a method for producing SC-β cells from pluripotent cells, the method comprising: a) differentiating pluripotent stem cells in a population into definitive endoderm cells by contacting the pluripotent stem cells with at least one factor from the TGFβ superfamily and a WNT signaling pathway activator for three days; b) differentiating at least a portion of the definitive endoderm cells into primitive endoderm cells by a process of contacting the definitive endoderm cells with at least one factor from the FGF family for three days; and c) differentiating the primitive endoderm cells into: i) retinoids; and (d) differentiating at least a portion of the primitive gut cells into PDX1-positive, NKX6.1-negative pancreatic progenitor cells by a process of contacting the PDX1-positive, NKX6.1-negative pancreatic progenitor cells with i) an activator of the phosphate signaling pathway, ii) at least one factor from the FGF family, iii) an inhibitor of the SHH pathway, iv) an inhibitor of the BMP signaling pathway, v) a PKC activator, vi) a ROCK inhibitor, and vii) a growth factor from the TGFβ superfamily for two days under conditions that promote cell clustering. and e) differentiating at least a portion of the PDX1-positive, NKX6.1-negative pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells by a process of contacting the PDX1-positive, NKX6.1-negative pancreatic progenitor cells with i) at least one growth factor, ii) at least one SHH pathway inhibitor, and optionally iii) an RA signaling pathway activator, and optionally iv) a ROCK inhibitor, and v) at least one factor from the TGF-β superfamily for 5 days; and f) differentiating at least a portion of the PDX1-positive, NKX6.1-positive pancreatic progenitor cells into PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells by a process of contacting the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with iii) at least one SHH pathway inhibitor, iv) an 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.and differentiating at least a portion of the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells by a process of culturing the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells in a medium that does not contain exogenous differentiation factors (e.g., NS-GFs medium, MCDB medium supplemented with BSA, MCDB131 medium, or DMEM / F12 medium) for 7 to 14 days and inducing in vitro maturation of at least a portion of the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells, wherein the SC-β cells exhibit a GSIS response in vitro and / or in vivo. In some examples, the GSIS response resembles the GSIS response of endogenous mature β cells.

[0219]

[0234] In some aspects, the disclosure provides a method for producing SC-β cells from pluripotent cells, the method comprising: a) differentiating pluripotent stem cells in a population into definitive endoderm cells by contacting the pluripotent stem cells with at least one factor from the TGFβ superfamily and a WNT signaling pathway activator for three days; b) differentiating at least a portion of the definitive endoderm cells into primitive gut cells by a process of contacting the definitive endoderm cells with at least one factor from the FGF family for three days; and c) differentiating the primitive gut cells into SC-β cells by: i) activating a retinoic acid signaling pathway; d) differentiating at least a portion of the primitive gut cells into PDX1-positive, NKX6.1-negative pancreatic progenitor cells by a process of contacting the PDX1-positive, NKX6.1-negative pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) an RA signaling pathway activator, and any other suitable activator of the RA signaling pathway, under conditions that promote cell clustering. and e) differentiating at least a portion of the PDX1-positive, NKX6.1-negative pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells by a process of contacting the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with i) a TGF-β signaling pathway inhibitor, ii) a TH signaling pathway activator, iii) at least one SHH pathway inhibitor, iv) an RA signaling pathway activator, v) a γ-secretase inhibitor, and optionally vi) differentiating at least a portion of the PDX1-positive, NKX6.1-positive pancreatic progenitor cells into PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells by contacting them with at least one growth factor from the epidermal growth factor (EGF) family for 5 to 7 days; and f) culturing the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells in a medium that does not contain exogenous differentiation factors (e.g., NS-GFs medium, MCDB medium supplemented with BSA, MCDB131 medium, or DMEM / F12 medium) for 7 to 14 days to differentiate them into PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells.and differentiating at least a portion of the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells by a process of inducing in vitro maturation of at least a portion of the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells, wherein the SC-β cells exhibit a GSIS response in vitro and / or in vivo. In some examples, the GSIS response resembles the GSIS response of endogenous mature β cells.

[0220]

[0235] In some aspects, the disclosure provides a method for producing SC-β cells from pluripotent cells, the method comprising: a) differentiating pluripotent stem cells in a population into definitive endoderm cells by contacting the pluripotent stem cells with at least one factor from the TGFβ superfamily and a WNT signaling pathway activator for three days; b) differentiating at least a portion of the definitive endoderm cells into primitive gut cells by a process of contacting the definitive endoderm cells with at least one factor from the FGF family for three days; and c) differentiating the primitive gut cells into definitive endoderm cells by: i) inducing a retinoic acid signal; and (d) differentiating at least a portion of the primitive gut cells into PDX1-positive, NKX6.1-negative pancreatic progenitor cells by contacting the PDX1-positive, NKX6.1-negative pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one factor from the FGF family, iii) an SHH pathway inhibitor, iv) a BMP signaling pathway inhibitor (e.g., DMH-1 or LDN193189), v) a PKC activator, and vi) a ROCK inhibitor under conditions that promote cell clustering. and e) differentiating at least a portion of the PDX1-positive, NKX6.1-negative 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 at least one SHH pathway inhibitor, and optionally iii) an RA signaling pathway activator, and optionally iv) a ROCK inhibitor, and v) at least one factor from the TGFβ superfamily for 5 or 6 days; and e) differentiating 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) an SHH pathway inhibitor, ii) an RA signaling pathway activator, iii) a γ-secretase inhibitor. , iv) at least one growth factor from the epidermal growth factor (EGF) family, v) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, vi) a TGF-β signaling pathway inhibitor, vii) a thyroid hormone signaling pathway activator, viii) an epigenetic modifying compound (e.g., DZNep or KD5170), ix) a protein kinase inhibitor, and x) a ROCK inhibitor, for 5 to 7 days to purify at least a portion of the PDX1-positive, NKX6.1-positive pancreatic progenitor cells.and f) differentiating at least a portion of the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells by a process of culturing the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells in a medium that does not contain exogenous differentiation factors (e.g., NS-GFs medium, MCDB medium supplemented with BSA, MCDB131 medium, or DMEM / F12 medium) for 7 to 14 days to induce in vitro maturation of at least a portion of the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells, where the SC-β cells exhibit a GSIS response in vitro and / or in vivo. In some examples, the GSIS response resembles the GSIS response of endogenous mature β cells.

[0221]

[0236] In some aspects, the present disclosure provides a method for producing SC-β cells from pluripotent cells, the method comprising: a) differentiating pluripotent stem cells in a population into definitive endoderm cells by contacting the pluripotent stem cells with at least one factor from the TGFβ superfamily and a WNT signaling pathway activator for three days; b) differentiating at least a portion of the definitive endoderm cells into primitive gut tube cells by a process of contacting the definitive endoderm cells with at least one factor from the FGF family for three days; and c) differentiating 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 three days. and (d) differentiating at least a portion of the primitive gut duct cells into PDX1-positive, NKX6.1-negative pancreatic progenitor cells by contacting the duct cells with: i) a retinoic acid signaling pathway activator; ii) at least one factor from the FGF family; iii) an SHH pathway inhibitor; iv) a BMP signaling pathway inhibitor (e.g., DMH-1 or LDN193189); v) a PKC activator; and vi) a ROCK inhibitor; and d) differentiating the PDX1-positive, NKX6.1-negative pancreatic progenitor cells under conditions that promote cell clustering. and e) differentiating at least a portion of the PDX1-positive, NKX6.1-negative pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells by a process of contacting the PDX1-positive, NKX6.1-negative pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) an RA signaling pathway activator, and optionally iv) a ROCK inhibitor, and v) at least one factor from the TGFβ superfamily for 5 or 6 days. Pancreatic progenitor cells were contacted with i) a γ-secretase inhibitor, ii) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, iii) a TGF-β signaling pathway inhibitor, iv) a thyroid hormone signaling pathway activator, v) an epigenetic modifying compound (e.g., DZNep or KD5170), vi) a protein kinase inhibitor, and vii) a ROCK inhibitor for 5–7 days, and within the first 3 days of the 5–7 days, PDX1-positive, NKX6.1-positive pancreatic progenitor cells were later identified as PDX1-positive, NKX6-positive.f) differentiating at least a portion of the PDX1-positive, NKX6.1-positive pancreatic progenitor cells into PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells by a process of contacting the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with an SHH pathway inhibitor, an RA signaling pathway, and at least one growth factor from the EGF family; and f) differentiating at least a portion of the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells by a process of culturing the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells in a medium that does not contain exogenous differentiation factors (e.g., NS-GFs medium, MCDB medium supplemented with BSA, MCDB131 medium, or DMEM / F12 medium) for 7 to 14 days to induce in vitro maturation of at least a portion of the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells, wherein the SC-β cells exhibit a GSIS response in vitro and / or in vivo. In some instances, the GSIS response resembles that of endogenous mature beta cells.

[0222]

[0237] In some aspects, the present disclosure provides a method for producing SC-β cells from pluripotent cells, the method comprising: a) differentiating pluripotent stem cells in a population into definitive endoderm cells by contacting the pluripotent stem cells with at least one factor from the TGFβ superfamily and a WNT signaling pathway activator for three days; b) differentiating at least a portion of the definitive endoderm cells into primitive endoderm cells by a process of contacting the definitive endoderm cells with at least one factor from the FGF family for three days; and c) differentiating the primitive endoderm cells into: i) retinoblastoma cells; and (d) differentiating at least a portion of the primitive gut cells into PDX1-positive, NKX6.1-negative pancreatic progenitor cells by a process of contacting the PDX1-positive, NKX6.1-negative pancreatic progenitor cells with i) an activator of the insulin signaling pathway, ii) at least one factor from the FGF family, iii) an SHH pathway inhibitor, iv) a BMP signaling pathway inhibitor (e.g., DMH-1 or LDN193189), v) a PKC activator, and vi) a ROCK inhibitor under conditions that promote cell clustering. and e) differentiating at least a portion of the PDX1-positive, NKX6.1-negative pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells by a process of contacting the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with i) an SHH pathway inhibitor, ii) an RA signaling pathway activator, iii) a γ-secretase inhibitor, iv) an epithelial cell line, and optionally ii) an RA signaling pathway activator, and optionally iii) a ROCK inhibitor, and v) at least one factor from the TGFβ superfamily for 5 or 6 days. At least a portion of the PDX1-positive, NKX6.1-positive pancreatic progenitor cells are induced to be PDX1-positive and NKX6-positive by a process of contacting the cells with at least one growth factor from the epidermal growth factor (EGF) family, v) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, vi) a TGF-β signaling pathway inhibitor, vii) a thyroid hormone signaling pathway activator, viii) an epigenetic modifying compound (e.g., DZNep or KD5170), ix) a protein kinase inhibitor, and x) a ROCK inhibitor for 5 to 7 days.and f) differentiating at least a portion of the PDX1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells.

[0223]

[0238] The medium used to culture the cells separated from the first cell cluster can be xeno-free. Xeno-free medium for culturing cells and / or cell clusters of animal origin cannot contain any other animal-derived products. In some cases, xeno-free medium for culturing human cells and / or cell clusters cannot contain any non-human animal-derived products. For example, xeno-free medium for culturing human cells and / or cell clusters can contain human platelet lysate (PLT) instead of fetal bovine serum (FBS). For example, the medium can contain about 1% to about 20%, about 5% to about 15%, about 8% to about 12%, or about 9 to about 11% serum. In some cases, the medium can contain about 10% serum. In some cases, the medium cannot contain small molecules and / or FBS. For example, the medium can contain MCDB131 basal medium supplemented with 2% BSA. In some cases, the medium is serum-free. In some examples, the medium may contain exogenous small molecules or signaling pathway agonists or antagonists, such as growth factors from the fibroblast growth factor family (FGF, e.g., FGF2, FGF8B, FGF10, or FGF21), sonic hedgehog antagonists (e.g., Sant1, Sant2, Sant4, Sant4, Cur61414, forskolin, tomatidine, AY9944, triparanol, cyclopamine, or derivatives thereof), retinoic acid signaling inhibitors, or the like. Signaling agonists (e.g., retinoic acid, CD1530, AM580, TTHPB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, or CD2314), inhibitors of Rho-associated coiled-coil-containing protein kinase (ROCK) (e.g., thiazovivin, Y-27632, fasudil / HA1077, and 14-1152), activators of protein kinase C (PKC) (e.g., phorbol 12,13-dibutyrate (PDBU), TPB, phorbol 12-myristate 13-acetate, bryostatin 1, or their derivatives), antagonists of the TGFβ superfamily (e.g., Alk5 inhibitor II (CAS 446859-33-2), A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB-525334, SD-208, SB-505124, or derivatives thereof), inhibitors of bone morphogenetic protein (BMP) type 1 receptor (e.g., LDN193189 or derivatives thereof), thyroid hormone signaling pathway activators (e.g., T3, GC-1, or derivatives thereof), gamma secretase inhibitors (e.g., XXI, DAPT, or derivatives thereof), activators of the TGF-β signaling pathway (e.g., WNT3a or activin A) The reaggregation medium may not include growth factors from the epidermal growth factor (EGF) family (e.g., betacellulin or EGF), a wide range of kinases (e.g., staurosporine or derivatives thereof), non-essential amino acids, vitamins or antioxidants (e.g., cyclopamine, vitamin D, vitamin C, vitamin A, or derivatives thereof), or other additives such as N-acetylcysteine, zinc sulfate, or heparin. In some cases, the reaggregation medium may not include exogenous extracellular matrix molecules. In some cases, the reaggregation medium does not include Matrigel™. In some cases, the reaggregation medium does not include 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.

[0224]

[0239] Those skilled in the art will recognize that the concentration of serum albumin supplemented in the medium can vary. For example, the medium (e.g., MCDB131) can contain about 0.01%, 0.05%, 0.1%, 1%, about 2%, about 3%, about 4%, about 5%, about 10%, or about 15% BSA. In other cases, the medium can contain about 0.01%, 0.05%, 0.1%, 1%, about 2%, about 3%, about 4%, about 5%, about 10%, or about 15% HSA. The medium used (e.g., MCDB131 medium) can contain components not found in traditional basal media, such as trace elements, putrescine, adenine, thymidine, and high levels of some amino acids and vitamins. These additives can allow the medium to be supplemented with very low levels of serum or defined components. The medium may be protein and / or growth factor-free and may be supplemented with EGF, hydrocortisone, and / or glutamine. The medium may contain one or more extracellular matrix molecules (e.g., extracellular proteins). Non-limiting exemplary extracellular matrix molecules used in the medium may include collagen, placental matrix, fibronectin, laminin, merosin, tenascin, heparin, heparin sulfate, chondroitin sulfate, dermatan sulfate, aggrecan, biglycan, thrombospondin, vitronectin, and decorin. In some cases, the medium includes laminin, such as LN-332. In some cases, the medium includes heparin.

[0225]

[0240] For example, medium can be periodically changed during culturing, so as to provide the optimal environment for cells in the medium.When culturing the cells separated from the first cell cluster for reaggregation, medium can be changed at least or about every 4 hours, every 12 hours, every 24 hours, every 48 hours, every 3 days or every 4 days.For example, medium can be changed about every 48 hours.

[0226]

[0241] In some cases, cells can be cultured under dynamic conditions (e.g., conditions in which cells are subjected to constant movement or agitation while in suspension culture). To dynamically culture cells, cells can be cultured in a vessel (e.g., a non-adherent vessel such as a spinner flask (e.g., 200 ml to 3000 ml, e.g., 250 ml; 100 ml; or 125 ml Erlenmeyer)) that can be connected to a control unit and thus provide a controlled culture system. In some cases, cells can be cultured under non-dynamic conditions (e.g., static culture) while maintaining their proliferation capacity. To non-dynamically culture cells, cells can be cultured in an adherent culture vessel. The adherent culture vessel may be coated with any substrate for cell adhesion, such as an extracellular matrix (ECM), to improve cell adhesion to the vessel surface. The substrate for cell adhesion can be any material intended to attach stem cells or feeder cells (if used). 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™, and lysed cell membrane preparations.

[0227]

[0242] The medium in the dynamic cell culture vessel (e.g., spinner flask) can be agitated (e.g., by a stirrer). The spin speed can be correlated with the size of the reaggregated second cell clusters. The spin speed can be controlled so that the size of the second cell clusters can resemble endogenous pancreatic islets. In some cases, the spin speed is controlled so that the size of the second cell clusters can be about 75 μm to about 250 μm. The spin speed of the dynamic cell culture vessel (e.g., spinner flask) can be about 20 revolutions per minute (rpm) to about 100 rpm, for example, about 30 rpm to about 90 rpm, about 40 rpm to about 60 rpm, or about 45 rpm to about 50 rpm. In some cases, the spin speed can be about 50 rpm.

[0228]

[0243] The stage 6 cells provided herein may or may not be subjected to the separation and reaggregation process described herein. In some cases, cell clusters containing insulin-positive endocrine cells may be reaggregated. Reaggregation of cell clusters can enrich insulin-positive endocrine cells. In some cases, insulin-positive endocrine cells in the cell clusters may be further matured into pancreatic beta cells. For example, after reaggregation, the second cell cluster may exhibit in vitro GSIS similar to native pancreatic islets. For example, after reaggregation, the second cell cluster may contain non-native pancreatic beta cells exhibiting in vitro GSIS. In some embodiments, the reaggregation process may be performed according to the disclosures of PCT Application Publication No. WO2019 / 018818 or U.S. Patent Application Publication No. 2020 / 0332262, each of which is incorporated herein by reference in its entirety.

[0229]

[0244] The stage 6 cells obtained according to the methods provided herein can have a high recovery rate after cryopreservation and reaggregation procedures. In some cases, stage 6 cells obtained in a differentiation process involving the treatment of a BMP signaling pathway inhibitor (e.g., DMH-1 or LDN) and a growth factor from the TGF-β superfamily (e.g., activin A) in stage 3 and the treatment of an epigenetic modification compound (e.g., a histone methyltransferase inhibitor, e.g., an EZH2 inhibitor, e.g., DZNep) in stage 5 can have a higher recovery rate after cryopreservation after stage 5 compared to the corresponding cell population without such treatment. In some cases, stage 6 cells 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 modifying compound (e.g., a histone methyltransferase inhibitor, e.g., an EZH2 inhibitor, e.g., DZNep) at stage 5 may have a higher recovery rate after cryopreservation after stage 5 compared to a 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, stage 6 cells 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) in stage 3 and treatment with an epigenetic modifying compound (e.g., a histone methyltransferase inhibitor, e.g., an EZH2 inhibitor, e.g., DZNep) in stage 5 may have a recovery rate after cryopreservation after stage 5 that is at least about 35%, 37.5%, 40%, 42.5%, 45%, 47.5%, 48%, 49%, or 50%. The recovery rate can be calculated as the percentage of cells that survive and form reaggregated cell clusters after the cryopreservation, thawing, recovery, and reaggregation procedures compared to the cells before cryopreservation.

[0230]

[0245] In some embodiments, the present disclosure relates to cryopreservation of non-native pancreatic beta cells or their precursors obtained using the methods provided herein. In some embodiments, a cell population comprising non-native pancreatic beta cells can be preserved by cryopreservation. For example, a cell population comprising non-native beta cells, e.g., stage 6 cells, can in some cases be separated into a cell suspension, e.g., a suspension of single cells, and the cell suspension can be cryopreserved, e.g., frozen in a cryopreservation solution. Cell separation can be performed by any of the techniques provided herein, e.g., enzymatic treatment. Cells can be frozen at temperatures 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 up to -200°C. In some cases, cells are frozen at a temperature of about -80°C. In some cases, cells are frozen at a temperature of about -195°C. Any cooling method can be used to produce the low temperatures required for cryopreservation, including, but not limited to, electric freezers, solid carbon dioxide, and liquid nitrogen. In some cases, any cryopreservation solution available to one skilled in the art, including both custom-made and commercially available solutions, can be used to incubate cells for cryogenic storage. For example, a solution containing a cryoprotectant can be used. Cryoprotectants can be agents that are designed to protect cells from freezing damage.For example, cryoprotectants can be substances that can lower the glass transition temperature of cryopreservation solutions.Exemplary cryoprotectants that can be used include DMSO (dimethyl sulfoxide), glycols (e.g., ethylene glycol, propylene glycol, and glycerol), dextran (e.g., dextran-40), and trehalose.Additional agents can be added to cryopreservation solutions for other purposes.In some cases, commercially available cryopreservation solutions, such as FrostaLife™, pZerve™, Prime-XV®, Gibco Synth-a-Freeze Cryopreservation Medium, STEM-CELLBANKER®, CryoStor® Freezing Media, HypoThermosol® FRS Preservation Media, and CryoDefend® Stem Cells Media, can be used in the methods provided herein. In some embodiments, the present disclosure provides a composition comprising a plurality of isolated cells (e.g., isolated insulin-positive endocrine precursor cells) and DMEM / F12. In some embodiments, the present disclosure provides a composition comprising a plurality of isolated cells (e.g., isolated insulin-positive endocrine precursor cells) and zinc (e.g., ZnSO4). In some embodiments, the present disclosure provides a composition comprising a plurality of isolated cells (e.g., isolated insulin-positive endocrine precursor cells) and zinc-human serum albumin (HSA).

[0231]

[0246] During the differentiation process, cells can be subjected to the irradiation treatment provided herein. In some cases, a cell population at stage 6, for example, a cell population or cell cluster having cells differentiated from insulin-positive endocrine cells to pancreatic β cells, is irradiated for a certain period of time. In some cases, a cell population at stage 6 after reaggregation after recovery from cryopreservation is irradiated for a certain period of time. In some cases, cryopreserved cells (e.g., cells cryopreserved at the end of stage 5) are irradiated for a certain period of time before thawing and recovery for the next differentiation process.

[0232]

[0247] In some embodiments, the stage 6 cells comprise NKX6.1-positive, insulin-positive cells. In some embodiments, the stage 6 cells comprise NKX6.1-positive, insulin-negative cells. In some embodiments, the stage 6 cells comprise C-peptide-positive cells. In some embodiments, the stage 6 cells or cells having characteristics of stage 6 cells are incubated in NS-GF medium, MCDB131 medium, DMEM medium, or CMRL medium. In some embodiments, stage 6 cells or cells having characteristics of stage 6 cells are contacted with any one or more of a vitamin or antioxidant (e.g., vitamin C), a water-soluble polymer (e.g., any of the human serum albumin proteins or polyvinyl alcohol molecules disclosed herein), a TGF-beta pathway inhibitor (e.g., ALK5 inhibitor II), a bone morphogenetic protein (BMP) type 1 receptor inhibitor (e.g., LDN193189), a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor (e.g., thiazovivin), a histone methyltransferase inhibitor (e.g., DZNEP), and a protein kinase inhibitor (e.g., staurosporine). In some embodiments, stage 6 cells are contacted with a PKC activator (see, e.g., WO2019217487, incorporated herein by reference in its entirety). In some embodiments, stage 6 cells are not contacted with a PKC activator. In some embodiments, stage 6 cells are not contacted with a water-soluble synthetic polymer. In some embodiments, stage 6 cells are contacted with serum albumin (eg, HSA) instead of a water-soluble synthetic polymer.

[0233]

[0248] In some embodiments, the present disclosure provides a composition comprising a population of insulin-positive cells and a lipid. In some embodiments, the present disclosure provides a method of contacting a population of insulin-positive cells with a lipid. In some embodiments, the lipid is a saturated fatty acid. In some embodiments, the saturated fatty acid is palmitic acid. In some embodiments, the lipid is an unsaturated fatty acid. In some embodiments, the unsaturated fatty acid is oleic acid, linoleic acid, or palmitoleic acid.

[0234]

[0249] In some embodiments, the present disclosure provides a composition comprising a population of insulin-positive cells and MCDB 131. In some embodiments, the present disclosure provides a method of contacting a population of insulin-positive cells with MCDB 131. In some embodiments, the present disclosure provides a composition comprising a population of insulin-positive cells and DMEM / F12. In some embodiments, the present disclosure provides a method of contacting a population of insulin-positive cells with DMEM / F12. In some embodiments, the present disclosure provides a composition comprising a population of insulin-positive cells and zinc. In some embodiments, the present disclosure provides a method of contacting a population of insulin-positive cells with zinc. In some embodiments, the present disclosure provides a composition comprising a population of insulin-positive cells and ZnSO4. In some embodiments, the present disclosure provides a method of contacting a population of insulin-positive cells with ZnSO4.

[0235]

[0250] In some embodiments, the present disclosure provides a composition comprising a population of insulin-positive cells and at least one metabolite. In some embodiments, the present disclosure provides a method of contacting a population of insulin-positive cells with at least one metabolite. In some embodiments, the at least one metabolite is glutamate, acetate, b-hydroxybutarate, L-carnitine, taurine, formate, or biotin. In some embodiments, the present disclosure provides a composition comprising a population of insulin-positive cells and one, two, three, four, five, six, or seven of glutamate, acetate, b-hydroxybutarate, L-carnitine, taurine, formate, or biotin. In some embodiments, the present disclosure provides a method of contacting a population of insulin-positive cells with one, two, three, four, five, six, or seven of glutamate, acetate, b-hydroxybutarate, L-carnitine, taurine, formate, or biotin. In some embodiments, the composition comprises DMEM / F12. In some embodiments, the composition comprises zinc (e.g., ZnSO4). In some embodiments, the composition comprises human serum albumin.

[0236]

[0251] In some embodiments, the present disclosure provides a composition comprising a population of insulin-positive cells and at least one amino acid. In some embodiments, the present disclosure provides a method of contacting a population of insulin-positive cells with at least one amino acid. In some embodiments, the at least one amino acid is alanine, glutamate, glycine, proline, threonine, or tryptophan. In some embodiments, the at least one amino acid is arginine, histidine, lysine, aspartic acid, glutamic acid, serine, asparagine, glutamine, cysteine, selenocysteine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, glutamate, glycine, proline, threonine, or tryptophan. In some embodiments, the present disclosure provides a composition comprising a population of insulin-positive cells and at least one vitamin. In some embodiments, the present disclosure provides a method of contacting a population of insulin-positive cells with at least one vitamin. In some embodiments, the at least one vitamin is biotin or riboflavin.

[0237]

[0252] In some embodiments, the present disclosure provides a composition comprising a population of insulin-positive cells and a monoglyceride lipase (MGLL) inhibitor. In some embodiments, the present disclosure provides a method of contacting a population of insulin-positive cells with at least one vitamin. In some embodiments, the MGLL inhibitor is JJKK048, KML29, NF1819, JW642, JZL184, JZL195, JZP361, pristimerin, or URB602, or any of their derivatives.

[0238]

[0253] In some embodiments, any of the cells disclosed herein (e.g., an SC-derived beta cell or any of the cells in any of the clusters disclosed herein) comprises a genomic disruption in at least one (e.g., 1, 2, or 3) gene sequence, wherein the disruption reduces or eliminates expression of a protein encoded by the gene sequence. In some embodiments, the at least one gene sequence encodes an MHC-class I gene. In some embodiments, the MHC-class I gene encodes beta-2 microglobulin (B2M), HLA-A, HLA-B, or HLA-C. In some embodiments, the at least one gene sequence encodes CIITA. In some embodiments, the cell comprises a genomic disruption in a gene encoding HLA-A and HLA-B, but not in a gene encoding HLA-C. In some embodiments, the cell comprises a genomic disruption in a natural killer cell-activating ligand gene. In some embodiments, the natural killer cell-activating ligand gene encodes intercellular adhesion molecule 1 (ICAM1), CD58, CD155, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), intercellular adhesion molecule 1 (CADM1), MHC class I polypeptide-related sequence A (MICA), or MHC class I polypeptide-related sequence B (MICB). In some embodiments, the cells have decreased expression of one or more of beta-2 microglobulin, CIITA, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLADR compared to ungenetically modified cells. In some embodiments, the cells have increased expression of CD47, PDL1, HLA-G, CD46, CD55, CD59, and CTLA compared to ungenetically modified cells. In certain embodiments, the pancreatic islet cells (e.g., SC-beta cells) disclosed herein have increased expression of PDL1 compared to endogenous pancreatic islet cells from a healthy control subject. In certain embodiments, the pancreatic islet cells (eg, SC-beta cells) disclosed herein have increased expression of CD47 compared to endogenous pancreatic islet cells from a healthy control subject.In some embodiments, the genome disruption is induced by the use of a gene editing system, for example, CRISPR Cas technology.

[0239] differentiation factor

[0254] Aspects of the present disclosure relate to contacting progenitor cells (e.g., stem cells, e.g., iPS cells, definitive endoderm cells, primitive gut cells, PDX1-positive, NKX6.1-negative pancreatic progenitor cells, PDX1-positive, NKX6.1-positive pancreatic progenitor cells, insulin-positive endocrine cells) with beta cell differentiation factors, e.g., to induce differentiation of mature insulin-positive endocrine cells or other progenitor cells into SC-beta cells (e.g., mature pancreatic beta cells). In some embodiments, the differentiation factors are capable of inducing differentiation of pluripotent cells (e.g., iPSCs or hESCs) into definitive endoderm cells, e.g., according to the methods described herein. In some embodiments, the differentiation factors are capable of inducing differentiation of definitive endoderm cells into primitive gut cells, e.g., according to the methods described herein. In some embodiments, the differentiation factors are capable of inducing differentiation of primitive gut cells into PDX1-positive, NKX6.1-negative pancreatic progenitor cells, e.g., according to the methods described herein. In some embodiments, the differentiation factor can induce the differentiation of PDX1-positive, NKX6.1-negative pancreatic progenitor cells into NKX6-1-positive pancreatic progenitor cells, for example, according to the methods described herein. In some embodiments, the differentiation factor can induce the differentiation of NKX6-1-positive pancreatic progenitor cells into insulin-positive endocrine cells, for example, according to the methods described herein. In some embodiments, the differentiation factor can induce the maturation of insulin-positive endocrine cells into SC-β cells, for example, according to the methods described herein.

[0240]

[0255] At least one differentiation factor described herein can be used alone or in combination with other differentiation agents to produce SC-β cells according to the methods disclosed herein, hi some embodiments, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten differentiation factors described herein are used in the methods of producing SC-β cells.

[0241] Transforming growth factor-β (TGF-β) superfamily

[0256] Embodiments of the present disclosure relate to the use of growth factors from the transforming growth factor-β (TGF-β) superfamily as differentiation factors. The term "TGF-β superfamily" refers to proteins that share structural and functional characteristics of known TGFβ family members. The TGFβ family of proteins includes the TGFβ series of proteins, inhibins (including inhibin A and inhibin B), activins (including activin A, activin B, and activin AB), MIS (Müllerian inhibitory substance), BMP (bone morphogenetic protein), dpp (decapentaplegic), Vg-1, and MNSF (monoclonal nonspecific inhibitory factor). The activity of proteins in this family can be based on specific binding to specific receptors on various cell types. Members of this family can share regions of sequence identity, particularly in the C-terminus, that correlate with their function. The TGFβ family can include over 100 distinct proteins, all of which share at least one region of amino acid sequence identity. Family members that can be used in the methods disclosed herein include, but are not limited to, the following proteins, which are identified by GenBank accession numbers: P07995, P18331, P08476, Q04998, P03970, P43032, P55102, P27092, P42917, P09529, P27093, P04088, Q04999, P17491, P55104, Q9WUK5, P55103, O88959, O08717, P58166, O61643, P35621, P09534, P48970, Q9NR23, P25703, P30884, P12643, P49001, P21274, O46564, O19006, P22004, P20722, Q04 906, Q07104, P30886, P18075, P23359, P22003, P34821, P49003, Q90751, P21275, Q06826, P30885, P34820, Q29607, P12644, Q90752, O46576, P27539, P48969, Q26 974, P07713, P91706, P91699, P27091, O42222, Q24735, P20863, O18828, P55106,Q9PTQ2、O14793、O08689、O42221、O18830、O18831、O18836、O35312、O42220、P43026、P43027、P43029、O95390、Q9R229、O93449、Q9Z1W4、Q9BDW8、P43028、Q7Z4P5、P50414、P17246、P54831、P04202、P01137、P09533、P18341、O19011、Q9Z1Y6、P07200、Q9Z217、O95393、P55105、P30371、Q9MZE2、Q07258、Q96S42、P97737、AAA97415.1、NP-776788.1、NP-058824.1、EAL24001.1、1 S4Y、NP-001009856.1、NP-1-032406.1、NP-999193.1、XP-519...

Claims

1. An in vitro composition comprising a plurality of pancreatic beta cells or precursor cells thereof in a medium comprising a water-soluble synthetic polymer.

2. 2. The in vitro composition of claim 1, wherein the composition comprises precursor cells of pancreatic beta cells, and the precursor cells of pancreatic beta cells comprise Sox17-positive cells, FOXA2-positive cells, PDX1-positive cells, NKX6.1-positive cells, ISL1-positive cells, and / or insulin-positive endocrine cells.

3. The in vitro composition of claim 1 or 2, wherein the water-soluble synthetic polymer comprises polyvinyl alcohol, poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), PEG copolymer, poly(N-isopropylacrylamide), or polyacrylamide.

4. 3. The in vitro composition of claim 1 or 2, wherein the water-soluble synthetic polymer comprises polyvinyl alcohol.

5. 5. The in vitro composition of any one of claims 1 to 4, wherein the water-soluble synthetic polymer is present in the culture medium at a concentration of about 0.005% to about 0.5% (w / v), about 0.01% to about 0.2% (w / v), about 0.02% to about 0.1% (w / v), or about 0.03% to about 0.08% (w / v).

6. 5. The in vitro composition according to any one of claims 1 to 4, wherein the water-soluble synthetic polymer is present in the culture medium at a concentration of about 0.04% to about 0.06% (w / v).

7. 5. An in vitro composition according to any one of claims 1 to 4, wherein the water-soluble synthetic polymer is present in the culture medium at a concentration of about 0.05% (w / v).

8. 8. The in vitro composition of claim 1, wherein the composition comprises a plurality of NKX6.1 positive, insulin positive cells and / or non-native pancreatic beta cells.

9. 9. The in vitro composition of claim 8, wherein the water-soluble synthetic polymer comprises polyvinyl alcohol that is greater than 85% hydrolyzed.

10. 9. The in vitro composition of claim 8, wherein the water-soluble synthetic polymer comprises about 87% to 89% hydrolyzed polyvinyl alcohol.

11. The in vitro composition of claim 8 , wherein the composition further comprises NKX6.1-positive, ISL1-positive pancreatic endocrine cells.

12. 12. The in vitro composition of any one of claims 8 to 11, wherein the composition further comprises pancreatic alpha cells, pancreatic delta cells, pancreatic F cells, pancreatic epsilon cells, enterochromaffin cells, or any combination thereof.

13. 13. The in vitro composition of any one of claims 8 to 12, wherein the medium further comprises one or more agents selected from the group consisting of a transforming growth factor beta (TGF-β) signaling pathway inhibitor, a thyroid hormone signaling pathway activator, an epigenetic modifying compound, a growth factor from the epidermal growth factor (EGF) family, a retinoic acid (RA) signaling pathway activator, a sonic hedgehog (SHH) pathway inhibitor, a gamma secretase inhibitor, a protein kinase inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a bone morphogenetic protein (BMP) signaling pathway inhibitor.

14. 8. The in vitro composition of claim 1, wherein the composition comprises a precursor cell of a pancreatic beta cell, and the precursor cell of a pancreatic beta cell comprises a plurality of PDX1 positive, NKX6.1 positive cells.

15. 15. The in vitro composition of claim 14, wherein the medium further comprises one or more agents selected from the group consisting of a protein kinase C activator, a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, an epigenetic modifying compound, a growth factor from the epidermal growth factor (EGF) family, a retinoic acid (RA) signaling pathway activator, a sonic hedgehog (SHH) pathway inhibitor, a gamma secretase inhibitor, a protein kinase inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a bone morphogenetic protein (BMP) signaling pathway inhibitor.

16. The medium is (a) a TGF-β signaling pathway inhibitor selected from the group consisting of Alk5i II, A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB-525334, SD-208, and SB-505124; (b) thyroid hormone signaling pathway activators, including T3 or GC-1; (c) an epigenetic modifying compound selected from the group consisting of 3-deazaneplanocin A (DZNep), GSK126, EPZ6438, KD5170, MC1568, and TMP195; (d) a growth factor from the epidermal growth factor family, including betacellulin or EGF; (e) a retinoic acid signaling pathway activator selected from the group consisting of retinoic acid, CD1530, AM580, TTHRB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, and CD2314; (f) a sonic hedgehog pathway inhibitor selected from the group consisting of SANT1, SANT2, SANT4, Cur6l4l4, forskolin, tomatidine, AY9944, triparanol, and cyclopamine; (g) γ-secretase inhibitors, including XXI or DAPT; (h) protein kinase inhibitors, including staurosporine, Ro-31-8220, bicindolylmaleimide (Bis) compounds, 10'-{5"-[(methoxycarbonyl)amino]-2"-methyl}-phenylaminocarbonylstaurosporine, or stalalogues; (i) a ROCK inhibitor selected from the group consisting of thiazovivin, Y-27632, Fasudil / HA1077, and 14-1152; (j) a protein kinase C activator selected from the group consisting of phorbol 12,13-dibutyrate (PDBU), TPB, phorbol 12-myristate 13-acetate, and bryostatin 1; and / or (k) the in vitro composition of claim 14, further comprising a bone morphogenetic protein signaling pathway inhibitor, comprising LDN193189 or DMH-1.

17. 17. An in vitro composition according to any one of claims 14 to 16, wherein the water-soluble synthetic polymer comprises polyvinyl alcohol that is greater than 85% hydrolyzed.

18. 17. The in vitro composition of any one of claims 14 to 16, wherein the water-soluble synthetic polymer comprises about 87% to 89% hydrolyzed polyvinyl alcohol.

19. The in vitro composition of claim 14 , wherein the precursor cells of pancreatic beta cells further comprise NKX6.1-positive, ISL1-positive cells.

20. 8. The in vitro composition of claim 1, wherein the composition comprises a precursor cell of a pancreatic beta cell, and the precursor cell of a pancreatic beta cell comprises a plurality of PDX1 positive, NKX6.1 negative cells.

21. 21. The in vitro composition of claim 20, wherein the medium further comprises one or more agents selected from the group consisting of a protein kinase C activator, a growth factor from the transforming growth factor beta (TGF-β) superfamily, a growth factor from the fibroblast growth factor (FGF) family, a retinoic acid (RA) signaling pathway activator, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a sonic hedgehog (SHH) pathway inhibitor.

22. The medium is (a) a growth factor from the transforming growth factor beta (TGF-β) superfamily selected from the group consisting of inhibin, activin, Mullerian inhibitory substance (MIS), bone morphogenetic protein (BMP), decapentaplegic (dpp), Vg-1, monoclonal nonspecific suppressor factor (MNSF), growth differentiation factor 8 (GDF8), and growth differentiation factor 11 (GDF11); (b) a growth factor from the fibroblast growth factor (FGF) family selected from the group consisting of keratinocyte growth factor (KGF), FGF2, FGF10, FGF21, and FGF8B; (c) a retinoic acid (RA) signaling pathway activator selected from the group consisting of retinoic acid, CD1530, AM580, TTHRB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, and CD2314; (d) a ROCK inhibitor selected from the group consisting of thiazovivin, Y-27632, Fasudil / HA1077, and 14-1152; (e) a protein kinase C activator selected from the group consisting of phorbol 12,13-dibutyrate (PDBU), TPB, phorbol 12-myristate 13-acetate, and bryostatin 1; and / or 21. The in vitro composition of claim 20, further comprising: (f) a sonic hedgehog (SHH) pathway inhibitor selected from the group consisting of SANT1, SANT2, SANT4, Cur6l4l4, forskolin, tomatidine, AY9944, triparanol, and cyclopamine.

23. 23. An in vitro composition according to any one of claims 20 to 22, wherein the water-soluble synthetic polymer comprises polyvinyl alcohol that is less than 85% hydrolyzed.

24. 23. An in vitro composition according to any one of claims 20 to 22, wherein the water-soluble synthetic polymer comprises about 80% hydrolyzed polyvinyl alcohol.

25. 25. The in vitro composition of any one of claims 20 to 24, wherein the precursor cells of pancreatic beta cells further comprise PDX1 positive, NKX6.1 positive cells.

26. 8. The in vitro composition of claim 1, wherein the composition comprises a precursor cell of a pancreatic beta cell, the precursor cell of a pancreatic beta cell comprising a plurality of FOXA2-positive gastrula cells.

27. 27. The in vitro composition of claim 26, wherein the medium further comprises one or more agents selected from the group consisting of a protein kinase C activator, a growth factor from the transforming growth factor beta (TGF-β) superfamily, a bone morphogenetic protein signaling pathway inhibitor, a growth factor from the fibroblast growth factor (FGF) family, a retinoic acid (RA) signaling pathway activator, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a sonic hedgehog (SHH) pathway inhibitor.

28. The medium is (a) a protein kinase C activator selected from the group consisting of phorbol 12,13-dibutyrate (PDBU), TPB, phorbol 12-myristate 13-acetate, and bryostatin 1; (b) a growth factor from the transforming growth factor beta (TGF-β) superfamily selected from the group consisting of inhibin, activin, Mullerian inhibitory substance (MIS), bone morphogenetic protein (BMP), decapentaplegic (dpp), Vg-1, monoclonal nonspecific suppressor factor (MNSF), growth differentiation factor 8 (GDF8), and growth differentiation factor 11 (GDF11); (c) a bone morphogenetic protein signaling pathway inhibitor, including LDN193189 or DMH-1; (d) a growth factor from the fibroblast growth factor (FGF) family selected from the group consisting of keratinocyte growth factor (KGF), FGF2, FGF10, FGF21, and FGF8B; (e) a sonic hedgehog pathway inhibitor selected from the group consisting of SANT1, SANT2, SANT4, Cur6l4l4, forskolin, tomatidine, AY9944, triparanol, and cyclopamine; (f) a retinoic acid signaling pathway activator selected from the group consisting of retinoic acid, CD1530, AM580, TTHRB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, and CD2314; and / or The in vitro composition of claim 26, further comprising: (g) a ROCK inhibitor selected from the group consisting of thiazovivin, Y-27632, Fasudil / HA1077, and 14-1152.

29. 29. An in vitro composition according to any one of claims 26 to 28, wherein the water-soluble synthetic polymer comprises polyvinyl alcohol that is less than 85% hydrolyzed.

30. 29. An in vitro composition according to any one of claims 26 to 28, wherein the water-soluble synthetic polymer comprises about 80% hydrolyzed polyvinyl alcohol.

31. 31. The in vitro composition of any one of claims 26 to 30, wherein the precursor cells of pancreatic beta cells further comprise PDX1 positive, NKX6.1 negative cells.

32. 8. The in vitro composition of claim 1, wherein the composition comprises a precursor cell of a pancreatic beta cell, and the precursor cell of a pancreatic beta cell comprises a plurality of SOX17-positive definitive endoderm cells.

33. 33. The in vitro composition of claim 32, wherein the culture medium further comprises a growth factor from the fibroblast growth factor (FGF) family.

34. 33. The in vitro composition of claim 32, wherein the growth factor from the fibroblast growth factor (FGF) family is selected from the group consisting of keratinocyte growth factor (KGF), FGF2, FGF10, FGF21, and FGF8B.

35. 35. An in vitro composition according to any one of claims 32 to 34, wherein the water-soluble synthetic polymer comprises polyvinyl alcohol that is less than 85% hydrolyzed.

36. 35. An in vitro composition according to any one of claims 32 to 34, wherein the water-soluble synthetic polymer comprises about 80% hydrolyzed polyvinyl alcohol.

37. 37. The in vitro composition of any one of claims 32 to 36, wherein the precursor cells of pancreatic beta cells further comprise FOXA2 positive cells.

38. 8. The in vitro composition of claim 1, wherein the composition comprises precursor cells of pancreatic beta cells, and the precursor cells of pancreatic beta cells comprise a plurality of pluripotent stem cells.

39. 39. The in vitro composition of claim 38, wherein the medium further comprises a growth factor from the transforming growth factor beta (TGF-β) superfamily, a WNT signaling pathway activator, or both.

40. The medium is (a) a growth factor from the transforming growth factor beta (TGF-β) superfamily selected from the group consisting of inhibin, activin, Müllerian inhibitory substance (MIS), bone morphogenetic protein (BMP), decapentaplegic (dpp), Vg-1, monoclonal nonspecific inhibitory factor (MNSF), growth differentiation factor 8 (GDF8), and growth differentiation factor 11 (GDF11); and / or 39. The in vitro composition of claim 38, further comprising (b) a WNT signaling pathway activator selected from the group consisting of CHIR99021, 3F8, A 1070722, AR-A 014418, BIO, BIO-acetoxime, FRATide, 10Z-hymenialdisine, indirubin-3'oxime, kenpaullone, L803, L803-mts, lithium carbonate, NSC 693868, SB 216763, SB 415286, TC-G 24, TCS 2002, TCS 21311, and TWS 119.

41. 41. An in vitro composition according to any one of claims 38 to 40, wherein the water-soluble synthetic polymer comprises less than 85% hydrolyzed polyvinyl alcohol.

42. 41. An in vitro composition according to any one of claims 38 to 40, wherein the water-soluble synthetic polymer comprises about 80% hydrolyzed polyvinyl alcohol.

43. 43. The in vitro composition of any one of claims 38 to 42, wherein the composition further comprises SOX17 positive cells.

44. 44. The in vitro composition of any one of claims 38 to 43, wherein the pluripotent stem cells comprise human stem cells.

45. 45. The in vitro composition of any one of claims 38 to 44, wherein the pluripotent stem cells comprise embryonic stem cells or induced pluripotent stem cells.

46. 46. ​​An in vitro composition according to any one of claims 1 to 45, wherein the culture medium does not contain albumin protein.

47. 46. ​​The in vitro composition of any one of claims 1 to 45, wherein the culture medium does not contain human serum albumin (HSA).

48. 47. An in vitro composition according to any one of claims 1 to 46, wherein the culture medium is serum-free.

49. A method comprising differentiating a plurality of pancreatic beta cell precursor cells in a medium that does not contain serum or serum albumin.

50. 50. The method of claim 49, wherein the medium comprises a water-soluble synthetic polymer.

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