Stem Cell Differentiation and Chemical Compounds

Abstract

Disclosed herein are compositions and methods for differentiation of stem cells into pancreatic cells.In some aspects, the methods provided herein relate to the production of pancreatic β cells, α cells, δ cells and EC cells in vitro in the presence of one or more chemical compounds that inhibit PI3K / Akt / mTOR signaling.In some aspects, the disclosure provides pharmaceutical compositions comprising the cells produced according to the methods disclosed herein, as well as methods of using them.

Description

[Technical Field]

[0001] cross reference

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 277,092, filed November 8, 2021, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] The production of stem cell-derived β-cells offers a potentially useful step toward the generation of pancreatic islets and pancreatic organs. One disease treatable by stem cell-derived tissues is diabetes. 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, may be cured by transplantation of new β-cells. While this strategy has enabled patients receiving transplants of cadaveric human islets to remain insulin-dependent for five or more years, 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, provided herein are in vitro compositions comprising Sox17-positive cells and an inhibitor of PI3K / Akt / mTOR signaling. In some cases, the composition further comprises stem cells. In some cases, the composition further comprises a growth factor from the TGF-β superfamily.

[0005]

[0005] In some aspects, provided herein is an in vitro composition comprising stem cells, an inhibitor of PI3K / Akt / mTOR signaling, and a growth factor from the TGF-β superfamily.

[0006] In some cases, the growth factor from the TGF-β superfamily is selected from the group consisting of inhibin, activin (e.g., activin A), 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). In some cases, the growth factor from the TGF-β superfamily includes activin A, GDF8, or both.

[0007] In some cases, the compositions provided herein contain at most about 100 ng / mL, at most about 80 ng / mL, at most about 60 ng / mL, at most about 50 ng / mL, at most about 25 ng / mL, at most about 20 ng / mL, at most about 15 ng / mL, at most about 10 ng / mL, at most about 5 ng / mL, or at most about 2 ng / mL of activin A. In some cases, the compositions contain 0.5 ng / mL to 500 ng / mL, 1 ng / mL to 250 ng / mL, 10 ng / mL to 200 ng / mL, 20 ng / mL to 150 ng / mL, 50 ng / mL to 120 ng / mL, 1 ng / mL to 50 ng / mL, 2 ng / mL to 25 ng / mL, or 5 ng / mL to 20 ng / mL of activin A. In some cases, the composition contains about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 12 ng / mL, about 14 ng / mL, about 15 ng / mL, about 18 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, or about 50 ng / mL of activin A.

[0008] In some aspects, provided herein is an in vitro composition comprising stem cells and an inhibitor of PI3K / Akt / mTOR signaling.

[0009] In some cases, the inhibitor of PI3K / Akt / mTOR signaling comprises an inhibitor of PI3K protein, an inhibitor of Akt protein, an inhibitor of mTOR, or any combination thereof. In some cases, the inhibitor of PI3K / Akt / mTOR signaling comprises one or more of GSK-690693, IPI-3063, AZD8055, omipalisib, GNE-477, VS-5584, BYL319, YM201636, PI4KIIIbeta-IN-10, nemiralisib, BYL719, FT113, or apitolisib, or any analog or derivative thereof. In some cases, the composition comprises an inhibitor of PI3K protein and an inhibitor of Akt protein. In some cases, the composition comprises GSK-690693, an analog or derivative thereof. In some cases, the composition comprises BYL719, an analog or derivative thereof. In some cases, the composition includes BYL319, an analog or derivative thereof. In some cases, the composition includes GSK-690693, an analog or derivative thereof, and BYL319, an analog or derivative thereof. In some cases, the composition includes GSK-690693, an analog or derivative thereof, and BYL719, an analog or derivative thereof. In some cases, the composition includes about 0.01 μM to about 1 μM, about 0.02 μM to about 0.8 μM, about 0.05 μM to about 0.5 μM, about 0.06 μM to about 0.2 μM, about 0.07 μM to about 0.15 μM, or about 0.08 μM to about 0.12 μM of GSK-690693 or an analog or derivative thereof. In some cases, the composition comprises about 0.01 μM, about 0.02 μM, about 0.04 μM, about 0.06 μM, about 0.08 μM, about 0.1 μM, about 0.12 μM, about 0.15 μM, about 0.2 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.8 μM, or about 1 μM of GSK-690693. In some instances, the composition comprises about 1 nM to about 500 nM, about 5 nM to about 250 nM, about 10 nM to about 200 nM, about 15 nM to about 150 nM, about 20 nM to about 100 nM, about 30 nM to about 80 nM, about 30 nM to about 60 nM, or about 35 nM to about 50 nM of BYL719, or an analog or derivative thereof.In some instances, the composition comprises about 1 nM, 4 nM, 8 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 80 nM, 90 nM, 100 nM, 200 nM, or 400 nM of BYL719. In some instances, the composition comprises about 0.08 μM to about 0.12 μM of GSK-690693 and about 35 nM to about 50 nM of BYL719. In some instances, the composition further comprises an activator of the WNT signaling pathway. In some cases, the activator of the WNT signaling pathway comprises one or more of Wnt3a, CHIR99021, 3F8, A 1070722, AR-A 014418, BIO, BIO-acetoxime, FRATide, 10Z-hymenialdisine, indirubin-3'oxime, kenpaullone, L803, L803-mts, lithium carbonate, NSC693868, SB 216763, SB 415286, TC-G 24, TCS 2002, TCS 21311, TWS 119, and analogs or derivatives thereof. In some cases, the composition further comprises a GSK3 inhibitor. In some cases, the composition further comprises 0.5 μM to 50 μM, 0.6 μM to 30 μM, 0.8 μM to 20 μM, 1 μM to 10 μM, or 2 μM to 5 μM of CHIR99021. In some cases, the composition further comprises about 0.5 μM, 0.6 μM, 0.8 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 8 μM, 10 μM, 15 μM, 20 μM, 25 μM, or 30 μM of CHIR99021.

[0009]

[0010] In some cases, the stem cells include embryonic stem cells. In some cases, the stem cells include induced pluripotent stem cells. In some cases, the stem cells are human cells. In some cases, the stem cells are genetically modified. In some cases, the composition includes a population of cells comprising Sox17-positive, Oct4-negative cells. In some cases, the population of cells includes at least about 50%, 60%, 65%, 70%, 75%, 80%, or 85% Sox17-positive, Oct4-negative cells. In some cases, the population of cells includes about 50% to about 90%, about 60% to about 90%, about 65% to about 90%, about 70% to about 90%, about 75% to about 90%, about 80% to about 90%, or about 75% to about 85% Sox17-positive, Oct4-negative cells.

[0010]

[0011] In some embodiments, provided herein is an in vitro composition comprising a plurality of FOXA2-positive, PDX1-negative cells and an inhibitor of PI3K / Akt / mTOR signaling. In some cases, the composition further comprises one or more agents selected from the group consisting of a protein kinase C activator, 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 cases, the composition comprises (a) phorbol 12,13-dibutyrate (PDBU), TPB, phorbol 12-myristate, or a combination thereof. (b) a protein kinase C activator selected from the group consisting of 13-acetate, and bryostatin 1; (b) a bone morphogenetic protein signaling pathway inhibitor, including LDN193189 or DMH-1; (c) 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; (d) SANT1, SANT2, SANT4, Cur6l4l4, forskolin, tomatidine, AY9944, triparanol, and (e) a sonic hedgehog pathway inhibitor selected from the group consisting of 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 cases, the composition further comprises a growth factor from the transforming growth factor beta (TGF-β) superfamily.In some cases, the growth factor from the TGF-β superfamily is selected from the group consisting of inhibin, activin (e.g., activin A), 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). In some cases, the growth factor from the TGF-β superfamily includes activin A, GDF8, or both. In some cases, the composition includes at most about 20 ng / mL of activin A. In some cases, the composition includes at most about 10 ng / mL, at most about 5 ng / mL, at most about 1 ng / mL, at most about 0.5 ng / mL, or at most about 0.1 ng / mL of activin A. In some cases, the composition comprises about 20 ng / mL, about 10 ng / mL, about 5 ng / mL, about 1 ng / mL, about 0.5 ng / mL, or about 0.1 ng / mL of activin A. In some cases, the composition further comprises PDX1-positive, NKX6.1-negative cells.

[0011]

[0012] In some embodiments, provided herein is an in vitro composition comprising a plurality of PDX1-positive, NKX6.1-negative cells and an inhibitor of PI3K / Akt / mTOR signaling. In some cases, the composition further comprises one or more agents selected from the group consisting of 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, a protein kinase C activator, and a sonic hedgehog (SHH) pathway inhibitor. In some instances, the composition 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 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. (f) a protein kinase C activator selected from the group consisting of SANT1, SANT2, SANT4, Cur61414, forskolin, tomatidine, AY9944, triparanol, and cyclopamine, and / or (g) a FoxO1 inhibitor, optionally AS1842856. In some cases, the composition further comprises a Notch signaling inhibitor, optionally the Notch signaling inhibitor is XXI or DAPI.In some cases, the composition further comprises a growth factor from the transforming growth factor beta (TGF-β) superfamily. In some cases, the growth factor from the TGF-β superfamily is selected from the group consisting of inhibin, activin (e.g., activin A), 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). In some cases, the growth factor from the TGF-β superfamily comprises activin A, GDF8, or both. In some cases, the composition comprises at most about 5 ng / mL of activin A. In some cases, the composition comprises at most about 2.5 ng / mL, at most about 1 ng / mL, at most about 0.5 ng / mL, at most about 0.1 ng / mL, or at most about 0.05 ng / mL of activin A. In some cases, the composition comprises about 5 ng / mL, about 2.5 ng / mL, about 1 ng / mL, about 0.5 ng / mL, about 0.1 ng / mL, or about 0.05 ng / mL of activin A. In some cases, the composition further comprises PDX1-positive, NKX6.1-positive cells. In some cases, the inhibitor of PI3K / Akt / mTOR signaling comprises an inhibitor of PI3K protein, an inhibitor of Akt protein, an inhibitor of mTOR, or any combination thereof. In some cases, the inhibitor of PI3K / Akt / mTOR signaling comprises one or more of GSK-690693, IPI-3063, AZD8055, omipalisib, GNE-477, VS-5584, BYL319, YM201636, PI4KIIIbeta-IN-10, nemiralisib, BYL719, FT113, apitolisib, or any analog or derivative thereof. In some cases, the composition comprises an inhibitor of a PI3K protein and an inhibitor of an Akt protein. In some cases, the composition comprises GSK-690693, an analog or derivative thereof. In some cases, the composition comprises BYL719, an analog or derivative thereof. In some cases, the composition comprises BYL319, an analog or derivative thereof.In some cases, the composition comprises GSK-690693, or an analog or derivative thereof, and BYL319, or an analog or derivative thereof. In some cases, the composition comprises GSK-690693, or an analog or derivative thereof, and BYL719, or an analog or derivative thereof.

[0012]

[0013] In some instances, the composition comprises about 0.01 μM to about 1 μM, about 0.02 μM to about 0.8 μM, about 0.05 μM to about 0.5 μM, about 0.06 μM to about 0.2 μM, about 0.07 μM to about 0.15 μM, or about 0.08 μM to about 0.12 μM of GSK-690693, or an analogue or derivative thereof. In some instances, the composition comprises about 0.01 μM, 0.02 μM, 0.04 μM, 0.06 μM, 0.08 μM, 0.1 μM, 0.12 μM, 0.15 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.8 μM, or 1 μM of GSK-690693. In some instances, the composition comprises about 1 nM to about 500 nM, about 5 nM to about 250 nM, about 10 nM to about 200 nM, about 15 nM to about 150 nM, about 20 nM to about 100 nM, about 30 nM to about 80 nM, about 30 nM to about 60 nM, or about 35 nM to about 50 nM of BYL719, or an analog or derivative thereof. In some instances, the composition comprises about 1 nM, 4 nM, 8 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 80 nM, 90 nM, 100 nM, 200 nM, or 400 nM of BYL719. In some instances, the composition comprises between about 0.08 μM and about 0.12 μM of GSK-690693 and between about 35 nM and about 50 nM of BYL719.

[0013]

[0014] In some cases, the compositions provided herein further comprise a water-soluble synthetic polymer. In some cases, the water-soluble synthetic polymer comprises polyvinyl alcohol, poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), PEG copolymer, poly(N-isopropylacrylamide), or polyacrylamide. In some cases, the water-soluble synthetic polymer comprises polyvinyl alcohol. In some cases, 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 cases, 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 cases, the water-soluble synthetic polymer is present in the medium at a concentration of about 0.05% (w / v). In some cases, the water-soluble synthetic polymer comprises polyvinyl alcohol that is less than 85% hydrolyzed. In some cases, the water-soluble synthetic polymer comprises polyvinyl alcohol that is about 80% hydrolyzed.

[0014]

[0015] In some embodiments, the compositions provided herein have a liquid volume of about 500 mL to about 50 L, about 1 L to about 10 L, about 2 L to about 5 L, about 3 L to about 4 L, about 2 L to about 30 L, or about 10 L to about 20 L. In some embodiments, the compositions provided herein have a liquid volume of about 10 mL to about 1000 mL, about 10 mL to about 100 mL, about 20 mL to about 50 mL, about 30 mL to about 40 mL, about 20 mL to about 30 mL, or about 10 mL to about 20 mL.

[0015]

[0016] In some aspects, methods are provided herein that include contacting a plurality of stem cells in vitro with an inhibitor of PI3K / Akt / mTOR signaling.

[0017] In some cases, the inhibitor of PI3K / Akt / mTOR signaling comprises an inhibitor of PI3K protein, an inhibitor of Akt protein, an inhibitor of mTOR, or any combination thereof. In some cases, the inhibitor of PI3K / Akt / mTOR signaling comprises one or more of GSK-690693, IPI-3063, AZD8055, omipalisib, GNE-477, VS-5584, BYL319, YM201636, PI4KIIIbeta-IN-10, nemiralisib, BYL719, FT113, apitolisib, or any analog or derivative thereof. In some cases, the inhibitor of PI3K / Akt / mTOR signaling comprises an inhibitor of PI3K protein and an inhibitor of Akt protein. In some cases, the inhibitor of PI3K / Akt / mTOR signaling comprises GSK-690693, an analog or derivative thereof. In some cases, the inhibitor of PI3K / Akt / mTOR signaling comprises BYL719, an analog or derivative thereof. In some cases, the inhibitor of PI3K / Akt / mTOR signaling comprises GSK-690693 and BYL719. In some cases, the contacting step comprises contacting the plurality of stem cells with about 0.01 μM to about 1 μM, about 0.02 μM to about 0.8 μM, about 0.05 μM to about 0.5 μM, about 0.06 μM to about 0.2 μM, about 0.07 μM to about 0.15 μM, or about 0.08 μM to about 0.12 μM GSK-690693. In some cases, the contacting step includes contacting the plurality of stem cells with about 0.01 μM, 0.02 μM, 0.04 μM, 0.06 μM, 0.08 μM, 0.1 μM, 0.12 μM, 0.15 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.8 μM, or 1 μM of GSK-690693. In some cases, the contacting step includes contacting the plurality of stem cells with about 1 nM to about 500 nM, about 5 nM to about 250 nM, about 10 nM to about 200 nM, about 15 nM to about 150 nM, about 20 nM to about 100 nM, about 30 nM to about 80 nM, about 30 nM to about 60 nM, or about 35 nM to about 50 nM of BYL719.In some cases, the contacting step includes contacting the plurality of stem cells with about 1 nM, 4 nM, 8 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 80 nM, 90 nM, 100 nM, 200 nM, or 400 nM of BYL719. In some cases, the contacting step includes contacting the plurality of stem cells with about 0.01 μM to about 1 μM, about 0.02 μM to about 0.8 μM, about 0.05 μM to about 0.5 μM, about 0.06 μM to about 0.2 μM, or about 0.07 μM to about 0.15 μM of GSK-690693 and about 1 nM to about 500 nM, about 5 nM to about 250 nM, about 10 nM to about 200 nM, about 15 nM to about 150 nM, about 20 nM to about 100 nM, about 30 nM to about 80 nM, about 30 nM to about 60 nM, or about 35 nM to about 50 nM of BYL719. In some instances, the contacting step includes contacting the plurality of stem cells with between about 0.08 μM and about 0.12 μM of GSK-690693 and between about 35 nM and about 50 nM of BYL719.

[0016]

[0018] In some cases, the method includes contacting a plurality of stem cells with an inhibitor of PI3K / Akt / mTOR signaling and a growth factor from the TGF-β superfamily. In some cases, the growth factor from the TGF-β superfamily includes activin A, GDF8, or both. In some cases, the method includes contacting the plurality of stem cells with about 0.5 ng / mL to about 500 ng / mL, about 1 ng / mL to about 250 ng / mL, about 10 ng / mL to about 200 ng / mL, about 20 ng / mL to about 150 ng / mL, about 50 ng / mL to about 120 ng / mL, about 1 ng / mL to about 50 ng / mL, about 2 ng / mL to about 25 ng / mL, or about 5 ng / mL to about 20 ng / mL of activin A. In some cases, the method includes contacting the plurality of stem cells with about 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 12 ng / mL, 14 ng / mL, 15 ng / mL, 18 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, or 50 ng / mL of activin A.

[0017]

[0019] In some cases, the method includes contacting a plurality of stem cells with an inhibitor of PI3K / Akt / mTOR signaling for about 24 hours to about 96 hours, about 36 hours to about 84 hours, about 48 hours to about 84 hours, about 60 hours to about 84 hours, or about 3 days.

[0018]

[0020] In some cases, the method also includes contacting the plurality of stem cells with an activator of the WNT signaling pathway. In some cases, the activator of the WNT signaling pathway includes one or more of Wnt3a, CHIR99021, 3F8, A 1070722, AR-A 014418, BIO, BIO-acetoxime, FRATide, 10Z-hymenialdisine, indirubin-3'oxime, kempaullone, L803, L803-mts, lithium carbonate, NSC693868, SB 216763, SB 415286, TC-G 24, TCS 2002, TCS 21311, TWS 119, and any analogs or derivatives thereof. In some cases, the activator of the WNT signaling pathway includes a GSK3 inhibitor. In some instances, the method includes contacting the plurality of stem cells with about 0.5 μM to about 50 μM, about 0.6 μM to about 30 μM, about 0.8 μM to about 20 μM, about 1 μM to about 10 μM, or 2 μM to about 5 μM CHIR99021. In some instances, the method includes contacting the plurality of stem cells with about 0.5 μM, 0.6 μM, 0.8 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 8 μM, 10 μM, 15 μM, 20 μM, 25 μM, or 30 μM CHIR99021. In some cases, the method includes culturing a plurality of stem cells in a first composition comprising an inhibitor of PI3K / Akt / mTOR signaling and an activator of the WNT signaling pathway for 12 to 48 hours, 12 to 36 hours, 18 to 30 hours, or about 1 day.

[0019]

[0021] In some cases, the method further includes culturing at least a portion of the resulting cells in a second composition comprising an inhibitor of PI3K / Akt / mTOR signaling for 12 to 72 hours, 24 to 72 hours, 36 to 72 hours, or about 2 days after culturing in the first composition. In some cases, the second composition does not include an activator of the WNT signaling pathway. In some cases, the second composition comprises the inhibitor of PI3K / Akt / mTOR signaling at the same concentration as the first composition.

[0020]

[0022] In some cases, the stem cells include embryonic stem cells. In some cases, the stem cells include induced pluripotent stem cells. In some cases, the stem cells are human cells. In some cases, the stem cells are genetically modified.

[0021]

[0023] In some cases, contacting a plurality of stem cells in vitro with an inhibitor of PI3K / Akt / mTOR signaling results in the production of a population of cells comprising Sox17-positive cells. In some cases, the population of cells comprises at least about 50%, 60%, 65%, 70%, 75%, 80%, or 85% Sox17-positive, Oct4-negative cells. In some cases, the population of cells comprises about 50% to about 90%, about 60% to about 90%, about 65% to about 90%, about 70% to about 90%, about 75% to about 90%, about 80% to about 90%, or about 75% to about 85% Sox17-positive, Oct4-negative cells.

[0022]

[0024] In some cases, the subject method results in the production of a population of cells comprising a percentage of Sox17-positive, Oct4-negative cells equivalent to the percentage of Sox17-positive, Oct4-negative cells in a population of cells produced by a reference method, the reference method including contacting a plurality of stem cells with about 100 ng / mL of activin A but not with an inhibitor of PI3K / Akt / mTOR signaling, and is otherwise identical to the subject method.

[0023]

[0025] In some cases, the method further includes differentiating the Sox17-positive cells into pancreatic beta cells; NKX6.1-positive, ISL1-positive cells; PDX1-positive, NKX6.1-positive cells; PDX1-positive, NKX6.1-negative cells; FOXA2-positive, PDX1-negative cells; or any combination thereof.

[0024]

[0026] In some cases, the method further includes contacting cells in the population of cells comprising Sox17-positive cells with a growth factor from the fibroblast growth factor (FGF) family. In some cases, 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 cases, the method includes culturing cells in the population of cells in a third composition comprising a growth factor from the fibroblast growth factor (FGF) family for 1 to 5 days, or 2 to 4 days, or about 1, 2, 3, 4, or 5 days. In some cases, contacting with a growth factor from the fibroblast growth factor (FGF) family results in the production of a population of cells comprising FOXA2-positive, PDX1-negative cells. In some cases, the population of cells comprising FOXA2-positive, PDX1-negative cells has a percentage of FOXA2-positive, PDX1-negative cells equivalent to the percentage of FOXA2-positive, PDX1-negative cells in a population of cells produced by a reference method, the reference method including contacting a plurality of stem cells with about 100 ng / mL of activin A but not with an inhibitor of PI3K / Akt / mTOR signaling, and is otherwise identical to the present method. In some cases, the present method further includes contacting cells in the population of cells comprising FOXA2-positive, PDX1-negative cells with 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 instances, the method includes treating cells in a population of cells comprising FOXA2-positive, PDX1-negative cells with (a) phorbol 12,13-dibutyrate (PDBU), TPB, or phorbol 12-myristate. (b) a protein kinase C activator selected from the group consisting of inhibin, activin (e.g., activin A), 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 (FGF) 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, Cur61414, 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 cases, the method includes culturing cells in the population of cells comprising FOXA2-positive, PDX1-negative cells in a fourth composition for 4 to 8 days, or 5 to 7 days, or about 4, 5, 6, 7, or 8 days, wherein the fourth composition 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.

[0025]

[0027] In some cases, contacting the cells in the population of cells comprising FOXA2-positive, PDX1-negative cells results in the production of a population of cells comprising PDX1-positive, NKX6.1-negative cells.

[0026]

[0028] In some cases, the population of cells comprising PDX1-positive, NKX6.1-negative cells has a percentage of PDX1-positive, NKX6.1-negative cells equivalent to the percentage of PDX1-positive, NKX6.1-negative cells in a population of cells produced by a reference method, the reference method including a step of contacting a plurality of stem cells with about 100 ng / mL of activin A but not with an inhibitor of PI3K / Akt / mTOR signaling, and is otherwise identical to the present method.

[0027]

[0029] In some cases, the method further includes contacting cells in the population of cells comprising PDX1-positive, NKX6.1-negative cells with one or more agents 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, a protein kinase C activator, a FoxO1 inhibitor, a sonic hedgehog (SHH) pathway inhibitor, and a Notch signaling inhibitor.In some instances, the method includes treating cells in a population of cells comprising PDX1-positive, NKX6.1-negative cells with (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 transforming growth factor beta (TGF-β) superfamily selected from the group consisting of keratinocyte growth factor (KGF), FGF2, FGF10, FGF21, and FGF8B. (c) a growth factor from the fibroblast growth factor (FGF) family, which is a marker for RA signaling, 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. (f) a protein kinase C activator selected from the group consisting of 13-acetate, and bryostatin 1; (g) a FoxO1 inhibitor, optionally AS1842856; (h) a Sonic hedgehog (SHH) pathway inhibitor selected from the group consisting of SANT1, SANT2, SANT4, Cur6l4l4, forskolin, tomatidine, AY9944, triparanol, and cyclopamine; and / or (i) a Notch signaling inhibitor, optionally XXI or DAPI.

[0028]

[0030] In some cases, the method includes culturing cells in the population of cells comprising PDX1-positive, NKX6.1-negative cells in a fifth composition for 4 to 8 days, or 5 to 7 days, or about 4, 5, 6, 7, or 8 days, wherein the fifth composition comprises one or more agents 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, a protein kinase C activator, a FoxO1 inhibitor, a sonic hedgehog (SHH) pathway inhibitor, and a Notch signaling inhibitor.

[0029]

[0031] In some cases, the step of contacting cells in a population of cells comprising PDX1-positive, NKX6.1-negative cells results in differentiation of the PDX1-positive, NKX6.1-negative cells into PDX1-positive, NKX6.1-positive cells, thereby producing a population of cells comprising PDX1-positive, NKX6.1-positive cells.

[0030]

[0032] In some cases, the population of cells comprising PDX1-positive, NKX6.1-positive cells has a percentage of PDX1-positive, NKX6.1-positive cells equivalent to the percentage of PDX1-positive, NKX6.1-positive cells in a population of cells produced by a reference method, the reference method including a step of contacting a plurality of stem cells with about 100 ng / mL of activin A but not with an inhibitor of PI3K / Akt / mTOR signaling, and is otherwise identical to the present method.

[0031]

[0033] In some cases, the first composition, the second composition, the third composition, the fourth composition, or the fifth composition further comprises a water-soluble synthetic polymer. In some cases, the water-soluble synthetic polymer comprises polyvinyl alcohol, poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), a PEG copolymer, poly(N-isopropylacrylamide), or polyacrylamide. In some cases, the water-soluble synthetic polymer comprises polyvinyl alcohol. In some cases, the water-soluble synthetic polymer is present 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 cases, the water-soluble synthetic polymer is present in the medium at a concentration of about 0.05% (w / v). In some cases, the water-soluble synthetic polymer comprises polyvinyl alcohol that is less than 85% hydrolyzed. In some cases, the water-soluble synthetic polymer comprises polyvinyl alcohol that is about 80% hydrolyzed.

[0032]

[0034] In some cases, the method further includes contacting cells in the population of cells comprising PDX1-positive, NKX6.1-positive cells with 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, a bone morphogenetic protein (BMP) signaling pathway inhibitor, and a Wnt signaling pathway inhibitor.In some instances, the method includes treating cells in a population of cells comprising PDX1-positive, NKX6.1-positive cells with (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, or SB-505124; (b) a thyroid hormone signaling pathway activator, including T3 or GC-1; or (c) a thyroid hormone signaling pathway activator, including 3-deazaneplanocin A (DZNep), GSK126, EP (d) an epigenetic modifying compound selected from the group consisting of Z6438, KD5170, MC1568, and TMP195; (e) a growth factor from the epidermal growth factor family, including betacellulin or EGF; (f) a compound selected from the group consisting of retinoic acid, CD1530, AM580, TTHRB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, and CD2314. (f) a retinoic acid signaling pathway activator 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, phorbol 12-myristate. 13-acetate, and bryostatin 1; (k) a protein kinase C activator selected from the group consisting of a bone morphogenetic protein signaling pathway inhibitor, including LDN193189 or DMH-1; and / or (l) a Wnt signaling pathway inhibitor, including NVP-TNKS656.

[0033]

[0035] In some cases, the method includes culturing cells in the population of cells comprising PDX1-positive, NKX6.1-positive cells in a sixth composition for 5 to 10 days, or 6 to 9 days, or about 5, 6, 7, 8, 9, or 10 days, wherein the sixth composition 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, a bone morphogenetic protein (BMP) signaling pathway inhibitor, and a Wnt signaling pathway inhibitor.

[0034]

[0036] In some cases, the sixth composition further comprises one or more of an acetyl-CoA-related metabolite (e.g., acetate), a vitamin (e.g., biotin), a histone deacetylase inhibitor (HDACi) (e.g., β-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), a one-carbon metabolic pathway intermediate (e.g., formate), and / or glutamine (e.g., L-glutamine).

[0035]

[0037] In some cases, contacting the cells in the population of cells results in producing a population of cells comprising NKX6.1-positive, ISL1-positive cells.

[0038] In some cases, the population of cells comprising NKX6.1-positive, ISL1-positive cells has a percentage of NKX6.1-positive, ISL1-positive cells equivalent to the percentage of NKX6.1-positive, ISL1-positive cells in a population of cells produced by a reference method, which includes contacting a plurality of stem cells with approximately 100 ng / mL of activin A but not with an inhibitor of PI3K / Akt / mTOR signaling, and is otherwise identical to the present method.

[0036]

[0039] In some cases, the method further includes differentiating the NKX6.1-positive, ISL1-positive cells into a population of cells comprising pancreatic beta cells. In some cases, the method further includes contacting cells in the population of cells comprising the NKX6.1-positive, ISL1-positive cells with a seventh composition comprising 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.

[0037]

[0040] In some cases, the population of cells comprising pancreatic beta cells has a percentage of pancreatic beta cells equivalent to the percentage of pancreatic beta cells in a population of cells produced by a reference method, the reference method including contacting a plurality of stem cells with about 100 ng / mL of activin A but not with an inhibitor of PI3K / Akt / mTOR signaling, and is otherwise identical to the present method.

[0038]

[0041] In some cases, the sixth or seventh composition further comprises a water-soluble synthetic polymer. In some cases, the water-soluble synthetic polymer comprises polyvinyl alcohol, poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), PEG copolymer, poly(N-isopropylacrylamide), or polyacrylamide. In some cases, the water-soluble synthetic polymer comprises polyvinyl alcohol. In some cases, the water-soluble synthetic polymer is present 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 cases, the water-soluble synthetic polymer is present in the medium at a concentration of about 0.05% (w / v). In some cases, the water-soluble synthetic polymer comprises polyvinyl alcohol that is greater than 85% hydrolyzed. In some cases, the water-soluble synthetic polymer comprises polyvinyl alcohol that is about 87% to 89% hydrolyzed.

[0039]

[0042] In some aspects, provided herein is a device comprising a population of cells obtained from the composition or the composition disclosed herein, or cells produced by the method disclosed herein. In some cases, the device is configured to produce and release insulin upon implantation into a subject. In some cases, the cells are encapsulated. In some cases, the device further comprises a semipermeable membrane configured to retain the cells within the device and allow insulin to pass through.

[0040]

[0043] In some aspects, provided herein are methods of treating a subject having a disease characterized by prolonged high blood glucose levels, comprising administering to the subject the composition or a population of cells obtained from a composition disclosed herein, or cells produced by a method disclosed herein, or implanting a device disclosed herein. In some cases, the disease is diabetes, optionally type 1 diabetes.

[0041]

[0044] 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]

[0042] [Figure 1A]

[0045] Figure 1A shows the effect of treatment of stem cells with various concentrations of activin A (stage 1 differentiation) on pancreatic β-cell differentiation. Figure 1A shows photographs (bottom) of cell clusters obtained at the completion of stage 1 ("S1C") by treatment with 100 ng / mL activin A ("100% AA"), 10 ng / mL activin A ("10% AA"), or 5 ng / mL activin A ("5% AA"), as well as the results of flow cytometry analysis of Oct4 and Sox17 expression in cells at S1C. Figure 1B shows photographs (bottom) of cell clusters obtained at the completion of stage 3 ("S3C") by treatment with 100% AA, 10% AA, or 5% AA, as well as the results of flow cytometry analysis of Pdx1 and Cdx2 expression in cells at S3C. Figure 1C shows the results of flow cytometry analysis of Pdx1 and Sox2 expression in cells at S3C. Figure 1D shows photographs of cell clusters obtained at the completion of stage 5 ("S5C") with 100% AA, 10% AA, or 5% AA (bottom) and the results of flow cytometry analysis of Nkx6.1 and Isl1 expression in cells at S5C (top). Flow cytometry analysis revealed 41.1% Nkx6.1-positive, Isl1-negative cells under 100% AA and 49.5% Nkx6.1-positive, Isl1-negative cells under 10% AA. Figure 1E shows a bar graph and table summarizing the percentage of Sox7-negative, Oct4-positive cells ("Sox17- / Oct4+") and Sox7-negative, Oct4-negative cells ("double negative") at S1C following treatment with 100 ng / mL (100%), 20 ng / mL (20%), 10 ng / mL (10%), 5 ng / mL (5%), or 0 ng / mL (0%) activin A at stage 1. [Figure 1B] Figure 1A shows the effect of treatment of stem cells with various concentrations of activin A (stage 1 differentiation) on pancreatic β-cell differentiation. Figure 1A shows photographs (bottom) of cell clusters obtained at the completion of stage 1 ("S1C") by treatment with 100 ng / mL activin A ("100% AA"), 10 ng / mL activin A ("10% AA"), or 5 ng / mL activin A ("5% AA"), as well as the results of flow cytometry analysis of Oct4 and Sox17 expression in cells at S1C. Figure 1B shows photographs (bottom) of cell clusters obtained at the completion of stage 3 ("S3C") by treatment with 100% AA, 10% AA, or 5% AA, as well as the results of flow cytometry analysis of Pdx1 and Cdx2 expression in cells at S3C. Figure 1C shows the results of flow cytometry analysis of Pdx1 and Sox2 expression in cells at S3C. Figure 1D shows photographs of cell clusters obtained at the completion of stage 5 ("S5C") with 100% AA, 10% AA, or 5% AA (bottom) and the results of flow cytometry analysis of Nkx6.1 and Isl1 expression in cells at S5C (top). Flow cytometry analysis revealed 41.1% Nkx6.1-positive, Isl1-negative cells under 100% AA and 49.5% Nkx6.1-positive, Isl1-negative cells under 10% AA. Figure 1E shows a bar graph and table summarizing the percentage of Sox7-negative, Oct4-positive cells ("Sox17- / Oct4+") and Sox7-negative, Oct4-negative cells ("double negative") at S1C following treatment with 100 ng / mL (100%), 20 ng / mL (20%), 10 ng / mL (10%), 5 ng / mL (5%), or 0 ng / mL (0%) activin A at stage 1. [Figure 1C]Figure 1A shows the effect of treatment of stem cells with various concentrations of activin A (stage 1 differentiation) on pancreatic β-cell differentiation. Figure 1A shows photographs (bottom) of cell clusters obtained at the completion of stage 1 ("S1C") by treatment with 100 ng / mL activin A ("100% AA"), 10 ng / mL activin A ("10% AA"), or 5 ng / mL activin A ("5% AA"), as well as the results of flow cytometry analysis of Oct4 and Sox17 expression in cells at S1C. Figure 1B shows photographs (bottom) of cell clusters obtained at the completion of stage 3 ("S3C") by treatment with 100% AA, 10% AA, or 5% AA, as well as the results of flow cytometry analysis of Pdx1 and Cdx2 expression in cells at S3C. Figure 1C shows the results of flow cytometry analysis of Pdx1 and Sox2 expression in cells at S3C. Figure 1D shows photographs of cell clusters obtained at the completion of stage 5 ("S5C") with 100% AA, 10% AA, or 5% AA (bottom) and the results of flow cytometry analysis of Nkx6.1 and Isl1 expression in cells at S5C (top). Flow cytometry analysis revealed 41.1% Nkx6.1-positive, Isl1-negative cells under 100% AA and 49.5% Nkx6.1-positive, Isl1-negative cells under 10% AA. Figure 1E shows a bar graph and table summarizing the percentage of Sox7-negative, Oct4-positive cells ("Sox17- / Oct4+") and Sox7-negative, Oct4-negative cells ("double negative") at S1C following treatment with 100 ng / mL (100%), 20 ng / mL (20%), 10 ng / mL (10%), 5 ng / mL (5%), or 0 ng / mL (0%) activin A at stage 1. [Figure 1D]Figure 1A shows the effect of treatment of stem cells with various concentrations of activin A (stage 1 differentiation) on pancreatic β-cell differentiation. Figure 1A shows photographs (bottom) of cell clusters obtained at the completion of stage 1 ("S1C") by treatment with 100 ng / mL activin A ("100% AA"), 10 ng / mL activin A ("10% AA"), or 5 ng / mL activin A ("5% AA"), as well as the results of flow cytometry analysis of Oct4 and Sox17 expression in cells at S1C. Figure 1B shows photographs (bottom) of cell clusters obtained at the completion of stage 3 ("S3C") by treatment with 100% AA, 10% AA, or 5% AA, as well as the results of flow cytometry analysis of Pdx1 and Cdx2 expression in cells at S3C. Figure 1C shows the results of flow cytometry analysis of Pdx1 and Sox2 expression in cells at S3C. Figure 1D shows photographs of cell clusters obtained at the completion of stage 5 ("S5C") with 100% AA, 10% AA, or 5% AA (bottom) and the results of flow cytometry analysis of Nkx6.1 and Isl1 expression in cells at S5C (top). Flow cytometry analysis revealed 41.1% Nkx6.1-positive, Isl1-negative cells under 100% AA and 49.5% Nkx6.1-positive, Isl1-negative cells under 10% AA. Figure 1E shows a bar graph and table summarizing the percentage of Sox7-negative, Oct4-positive cells ("Sox17- / Oct4+") and Sox7-negative, Oct4-negative cells ("double negative") at S1C following treatment with 100 ng / mL (100%), 20 ng / mL (20%), 10 ng / mL (10%), 5 ng / mL (5%), or 0 ng / mL (0%) activin A at stage 1. [Figure 1E]Figure 1A shows the effect of treatment of stem cells with various concentrations of activin A (stage 1 differentiation) on pancreatic β-cell differentiation. Figure 1A shows photographs (bottom) of cell clusters obtained at the completion of stage 1 ("S1C") by treatment with 100 ng / mL activin A ("100% AA"), 10 ng / mL activin A ("10% AA"), or 5 ng / mL activin A ("5% AA"), as well as the results of flow cytometry analysis of Oct4 and Sox17 expression in cells at S1C. Figure 1B shows photographs (bottom) of cell clusters obtained at the completion of stage 3 ("S3C") by treatment with 100% AA, 10% AA, or 5% AA, as well as the results of flow cytometry analysis of Pdx1 and Cdx2 expression in cells at S3C. Figure 1C shows the results of flow cytometry analysis of Pdx1 and Sox2 expression in cells at S3C. Figure 1D shows photographs of cell clusters obtained at the completion of stage 5 ("S5C") with 100% AA, 10% AA, or 5% AA (bottom) and the results of flow cytometry analysis of Nkx6.1 and Isl1 expression in cells at S5C (top). Flow cytometry analysis revealed 41.1% Nkx6.1-positive, Isl1-negative cells under 100% AA and 49.5% Nkx6.1-positive, Isl1-negative cells under 10% AA. Figure 1E shows a bar graph and table summarizing the percentage of Sox7-negative, Oct4-positive cells ("Sox17- / Oct4+") and Sox7-negative, Oct4-negative cells ("double negative") at S1C following treatment with 100 ng / mL (100%), 20 ng / mL (20%), 10 ng / mL (10%), 5 ng / mL (5%), or 0 ng / mL (0%) activin A at stage 1. [Figure 2A]

[0046]

[0023] Figure 2A shows the effect of treating stem cells with exemplary compounds at the completion of stage 1 differentiation. Figure 2A shows photographs of cell clusters obtained at S1C upon treatment with 100% AA, 10% AA, 10% activin A and GSK-690693, 10% activin A and BYL719, 10% activin A, GSK-690693 and BYL719, or 100% activin A, GSK-690693 and BYL719 at stage 1. Figure 2B shows the results of flow cytometry analysis of Oct4 and Sox17 expression in cells at S1C, and Figure 2C shows a bar graph summarizing these results. [Figure 2B]

[0023] Figure 2A shows the effect of treating stem cells with exemplary compounds at the completion of stage 1 differentiation. Figure 2A shows photographs of cell clusters obtained at S1C upon treatment with 100% AA, 10% AA, 10% activin A and GSK-690693, 10% activin A and BYL719, 10% activin A, GSK-690693 and BYL719, or 100% activin A, GSK-690693 and BYL719 at stage 1. Figure 2B shows the results of flow cytometry analysis of Oct4 and Sox17 expression in cells at S1C, and Figure 2C shows a bar graph summarizing these results. [Figure 2C]

[0023] Figure 2A shows the effect of treating stem cells with exemplary compounds at the completion of stage 1 differentiation. Figure 2A shows photographs of cell clusters obtained at S1C upon treatment with 100% AA, 10% AA, 10% activin A and GSK-690693, 10% activin A and BYL719, 10% activin A, GSK-690693 and BYL719, or 100% activin A, GSK-690693 and BYL719 at stage 1. Figure 2B shows the results of flow cytometry analysis of Oct4 and Sox17 expression in cells at S1C, and Figure 2C shows a bar graph summarizing these results. [Figure 3A]

[0047] Figure 3A shows the effect of treating stem cells with exemplary compounds at stage 1 upon completion of stage 3 differentiation. Figure 3A shows photographs of cell clusters obtained at S3C after stage 1 treatment with 100% AA, 10% AA, 10% activin A and GSK-690693, 10% activin A and BYL719, 10% activin A, GSK-690693 and BYL719, or 100% activin A, GSK-690693 and BYL719. Figure 3B shows the results of flow cytometry analysis of Pdx1 and Cdx2 expression in cells at S3C, which are summarized in the bar graphs in Figure 3C. Figure 3D shows the results of flow cytometry analysis of Pdx1 and Sox2 expression in cells at S3C, which are summarized in the bar graphs in Figure 3E. [Figure 3B] Figure 3A shows the effect of treating stem cells with exemplary compounds at stage 1 upon completion of stage 3 differentiation. Figure 3A shows photographs of cell clusters obtained at S3C after stage 1 treatment with 100% AA, 10% AA, 10% activin A and GSK-690693, 10% activin A and BYL719, 10% activin A, GSK-690693 and BYL719, or 100% activin A, GSK-690693 and BYL719. Figure 3B shows the results of flow cytometry analysis of Pdx1 and Cdx2 expression in cells at S3C, which are summarized in the bar graphs in Figure 3C. Figure 3D shows the results of flow cytometry analysis of Pdx1 and Sox2 expression in cells at S3C, which are summarized in the bar graphs in Figure 3E. [Figure 3C]Figure 3A shows the effect of treating stem cells with exemplary compounds at stage 1 upon completion of stage 3 differentiation. Figure 3A shows photographs of cell clusters obtained at S3C after stage 1 treatment with 100% AA, 10% AA, 10% activin A and GSK-690693, 10% activin A and BYL719, 10% activin A, GSK-690693 and BYL719, or 100% activin A, GSK-690693 and BYL719. Figure 3B shows the results of flow cytometry analysis of Pdx1 and Cdx2 expression in cells at S3C, which are summarized in the bar graphs in Figure 3C. Figure 3D shows the results of flow cytometry analysis of Pdx1 and Sox2 expression in cells at S3C, which are summarized in the bar graphs in Figure 3E. [Figure 3D] Figure 3A shows the effect of treating stem cells with exemplary compounds at stage 1 upon completion of stage 3 differentiation. Figure 3A shows photographs of cell clusters obtained at S3C after stage 1 treatment with 100% AA, 10% AA, 10% activin A and GSK-690693, 10% activin A and BYL719, 10% activin A, GSK-690693 and BYL719, or 100% activin A, GSK-690693 and BYL719. Figure 3B shows the results of flow cytometry analysis of Pdx1 and Cdx2 expression in cells at S3C, which are summarized in the bar graphs in Figure 3C. Figure 3D shows the results of flow cytometry analysis of Pdx1 and Sox2 expression in cells at S3C, which are summarized in the bar graphs in Figure 3E. [Figure 3E]Figure 3A shows the effect of treating stem cells with exemplary compounds at stage 1 upon completion of stage 3 differentiation. Figure 3A shows photographs of cell clusters obtained at S3C after stage 1 treatment with 100% AA, 10% AA, 10% activin A and GSK-690693, 10% activin A and BYL719, 10% activin A, GSK-690693 and BYL719, or 100% activin A, GSK-690693 and BYL719. Figure 3B shows the results of flow cytometry analysis of Pdx1 and Cdx2 expression in cells at S3C, which are summarized in the bar graphs in Figure 3C. Figure 3D shows the results of flow cytometry analysis of Pdx1 and Sox2 expression in cells at S3C, which are summarized in the bar graphs in Figure 3E. [Figure 4A]

[0048] Figure 4A shows the effect of treating stem cells with exemplary compounds at stage 1 upon completion of stage 5 differentiation. Figure 4A shows photographs of cell clusters obtained at S5C upon treatment with 100% AA, 10% AA, 10% activin A and GSK-690693, 10% activin A and BYL719, 10% activin A, GSK-690693 and BYL719, or 100% activin A, GSK-690693 and BYL719 at stage 1. Figure 4B shows the results of flow cytometry analysis of Nkx6.1 and Isl1 expression in cells at S5C, which are summarized in the bar graphs in Figures 4C-4D. Figures 4E and 4F show bar graphs summarizing the total cell yield (Figure 4E) and the yield of Isl1-positive, Nkx6.1-positive cells (Isl1+ / Nkx6.1+) and Isl1-positive, Nkx6.1-negative cells (Isl1+ / Nkx6.1-) at S5C, respectively (Figure 4F). [Figure 4B]Figure 4A shows the effect of treating stem cells with exemplary compounds at stage 1 upon completion of stage 5 differentiation. Figure 4A shows photographs of cell clusters obtained at S5C upon treatment with 100% AA, 10% AA, 10% activin A and GSK-690693, 10% activin A and BYL719, 10% activin A, GSK-690693 and BYL719, or 100% activin A, GSK-690693 and BYL719 at stage 1. Figure 4B shows the results of flow cytometry analysis of Nkx6.1 and Isl1 expression in cells at S5C, which are summarized in the bar graphs in Figures 4C-4D. Figures 4E and 4F show bar graphs summarizing the total cell yield (Figure 4E) and the yield of Isl1-positive, Nkx6.1-positive cells (Isl1+ / Nkx6.1+) and Isl1-positive, Nkx6.1-negative cells (Isl1+ / Nkx6.1-) at S5C, respectively (Figure 4F). [Figure 4C] Figure 4A shows the effect of treating stem cells with exemplary compounds at stage 1 upon completion of stage 5 differentiation. Figure 4A shows photographs of cell clusters obtained at S5C upon treatment with 100% AA, 10% AA, 10% activin A and GSK-690693, 10% activin A and BYL719, 10% activin A, GSK-690693 and BYL719, or 100% activin A, GSK-690693 and BYL719 at stage 1. Figure 4B shows the results of flow cytometry analysis of Nkx6.1 and Isl1 expression in cells at S5C, which are summarized in the bar graphs in Figures 4C-4D. Figures 4E and 4F show bar graphs summarizing the total cell yield (Figure 4E) and the yield of Isl1-positive, Nkx6.1-positive cells (Isl1+ / Nkx6.1+) and Isl1-positive, Nkx6.1-negative cells (Isl1+ / Nkx6.1-) at S5C, respectively (Figure 4F). [Figure 4D]Figure 4A shows the effect of treating stem cells with exemplary compounds at stage 1 upon completion of stage 5 differentiation. Figure 4A shows photographs of cell clusters obtained at S5C upon treatment with 100% AA, 10% AA, 10% activin A and GSK-690693, 10% activin A and BYL719, 10% activin A, GSK-690693 and BYL719, or 100% activin A, GSK-690693 and BYL719 at stage 1. Figure 4B shows the results of flow cytometry analysis of Nkx6.1 and Isl1 expression in cells at S5C, which are summarized in the bar graphs in Figures 4C-4D. Figures 4E and 4F show bar graphs summarizing the total cell yield (Figure 4E) and the yield of Isl1-positive, Nkx6.1-positive cells (Isl1+ / Nkx6.1+) and Isl1-positive, Nkx6.1-negative cells (Isl1+ / Nkx6.1-) at S5C, respectively (Figure 4F). [Figure 4E] Figure 4A shows the effect of treating stem cells with exemplary compounds at stage 1 upon completion of stage 5 differentiation. Figure 4A shows photographs of cell clusters obtained at S5C upon treatment with 100% AA, 10% AA, 10% activin A and GSK-690693, 10% activin A and BYL719, 10% activin A, GSK-690693 and BYL719, or 100% activin A, GSK-690693 and BYL719 at stage 1. Figure 4B shows the results of flow cytometry analysis of Nkx6.1 and Isl1 expression in cells at S5C, which are summarized in the bar graphs in Figures 4C-4D. Figures 4E and 4F show bar graphs summarizing the total cell yield (Figure 4E) and the yield of Isl1-positive, Nkx6.1-positive cells (Isl1+ / Nkx6.1+) and Isl1-positive, Nkx6.1-negative cells (Isl1+ / Nkx6.1-) at S5C, respectively (Figure 4F). [Figure 4F]Figure 4A shows the effect of treating stem cells with exemplary compounds at stage 1 upon completion of stage 5 differentiation. Figure 4A shows photographs of cell clusters obtained at S5C upon treatment with 100% AA, 10% AA, 10% activin A and GSK-690693, 10% activin A and BYL719, 10% activin A, GSK-690693 and BYL719, or 100% activin A, GSK-690693 and BYL719 at stage 1. Figure 4B shows the results of flow cytometry analysis of Nkx6.1 and Isl1 expression in cells at S5C, which are summarized in the bar graphs in Figures 4C-4D. Figures 4E and 4F show bar graphs summarizing the total cell yield (Figure 4E) and the yield of Isl1-positive, Nkx6.1-positive cells (Isl1+ / Nkx6.1+) and Isl1-positive, Nkx6.1-negative cells (Isl1+ / Nkx6.1-) at S5C, respectively (Figure 4F). [Figure 5A]

[0049] Figure 5A shows the effect of treatment of stem cells with exemplary compounds at stage 1 on cells at the completion of 7 days of stage 6 differentiation according to an exemplary stage 6 media regimen ("Regimen 1"). Figure 5A shows the results of flow cytometry analysis of Nkx6.1 and Isl1 expression at stage 6 day 7 ("S6d7") with 100% AA, 10% AA, 10% activin A and GSK-690693, 10% activin A and BYL719, 10% activin A, GSK-690693 and BYL719 treatments at stage 1. Figure 5B shows a bar graph summarizing the results of Figure 5A. Figure 5C shows the results of flow cytometry analysis of C-peptide ("C-pep") and glucagon ("GCG") expression in cells at S6d7. Figure 5D shows the results of flow cytometry analysis of somatostatin ("SST") and GCG expression in cells at S6d7. Figure 5E shows the results of flow cytometry analysis of SST and C-pep expression in cells at S6d7. [Figure 5B]Figure 5A shows the effect of treatment of stem cells with exemplary compounds at stage 1 on cells at the completion of 7 days of stage 6 differentiation according to an exemplary stage 6 media regimen ("Regimen 1"). Figure 5A shows the results of flow cytometry analysis of Nkx6.1 and Isl1 expression at stage 6 day 7 ("S6d7") with 100% AA, 10% AA, 10% activin A and GSK-690693, 10% activin A and BYL719, 10% activin A, GSK-690693 and BYL719 treatments at stage 1. Figure 5B shows a bar graph summarizing the results of Figure 5A. Figure 5C shows the results of flow cytometry analysis of C-peptide ("C-pep") and glucagon ("GCG") expression in cells at S6d7. Figure 5D shows the results of flow cytometry analysis of somatostatin ("SST") and GCG expression in cells at S6d7. Figure 5E shows the results of flow cytometry analysis of SST and C-pep expression in cells at S6d7. [Figure 5C] Figure 5A shows the effect of treatment of stem cells with exemplary compounds at stage 1 on cells at the completion of 7 days of stage 6 differentiation according to an exemplary stage 6 media regimen ("Regimen 1"). Figure 5A shows the results of flow cytometry analysis of Nkx6.1 and Isl1 expression at stage 6 day 7 ("S6d7") with 100% AA, 10% AA, 10% activin A and GSK-690693, 10% activin A and BYL719, 10% activin A, GSK-690693 and BYL719 treatments at stage 1. Figure 5B shows a bar graph summarizing the results of Figure 5A. Figure 5C shows the results of flow cytometry analysis of C-peptide ("C-pep") and glucagon ("GCG") expression in cells at S6d7. Figure 5D shows the results of flow cytometry analysis of somatostatin ("SST") and GCG expression in cells at S6d7. Figure 5E shows the results of flow cytometry analysis of SST and C-pep expression in cells at S6d7. [Figure 5D] Figure 5A shows the effect of treatment of stem cells with exemplary compounds at stage 1 on cells at the completion of 7 days of stage 6 differentiation according to an exemplary stage 6 media regimen ("Regimen 1"). Figure 5A shows the results of flow cytometry analysis of Nkx6.1 and Isl1 expression at stage 6 day 7 ("S6d7") with 100% AA, 10% AA, 10% activin A and GSK-690693, 10% activin A and BYL719, 10% activin A, GSK-690693 and BYL719 treatments at stage 1. Figure 5B shows a bar graph summarizing the results of Figure 5A. Figure 5C shows the results of flow cytometry analysis of C-peptide ("C-pep") and glucagon ("GCG") expression in cells at S6d7. Figure 5D shows the results of flow cytometry analysis of somatostatin ("SST") and GCG expression in cells at S6d7. Figure 5E shows the results of flow cytometry analysis of SST and C-pep expression in cells at S6d7. [Figure 5E] Figure 5A shows the effect of treatment of stem cells with exemplary compounds at stage 1 on cells at the completion of 7 days of stage 6 differentiation according to an exemplary stage 6 media regimen ("Regimen 1"). Figure 5A shows the results of flow cytometry analysis of Nkx6.1 and Isl1 expression at stage 6 day 7 ("S6d7") with 100% AA, 10% AA, 10% activin A and GSK-690693, 10% activin A and BYL719, 10% activin A, GSK-690693 and BYL719 treatments at stage 1. Figure 5B shows a bar graph summarizing the results of Figure 5A. Figure 5C shows the results of flow cytometry analysis of C-peptide ("C-pep") and glucagon ("GCG") expression in cells at S6d7. Figure 5D shows the results of flow cytometry analysis of somatostatin ("SST") and GCG expression in cells at S6d7. Figure 5E shows the results of flow cytometry analysis of SST and C-pep expression in cells at S6d7. [Figure 6A]

[0050] Figure 6A shows the effect of treatment of stem cells with exemplary compounds at stage 1 on cells at the completion of 7 days of stage 6 differentiation according to different exemplary stage 6 media regimens ("Regimen 2"). Figure 6A shows the results of flow cytometry analysis of Nkx6.1 and Isl1 expression at stage 6 day 7 ("S6d7") with 100% AA, 10% AA, 10% activin A and GSK-690693, 10% activin A and BYL719, 10% activin A, GSK-690693 and BYL719 treatments at stage 1. Figure 6B shows a bar graph summarizing the results of Figure 6A. Figure 6C shows the results of flow cytometry analysis of C-peptide ("C-pep") and glucagon ("GCG") expression in cells at S6d7. Figure 6D shows the results of flow cytometry analysis of somatostatin ("SST") and GCG expression in cells at S6d7. Figure 6E shows the results of flow cytometry analysis of SST and C-pep expression in cells at S6d7. [Figure 6B]Figure 6A shows the effect of treatment of stem cells with exemplary compounds at stage 1 on cells at the completion of 7 days of stage 6 differentiation according to different exemplary stage 6 media regimens ("Regimen 2"). Figure 6A shows the results of flow cytometry analysis of Nkx6.1 and Isl1 expression at stage 6 day 7 ("S6d7") with 100% AA, 10% AA, 10% activin A and GSK-690693, 10% activin A and BYL719, 10% activin A, GSK-690693 and BYL719 treatments at stage 1. Figure 6B shows a bar graph summarizing the results of Figure 6A. Figure 6C shows the results of flow cytometry analysis of C-peptide ("C-pep") and glucagon ("GCG") expression in cells at S6d7. Figure 6D shows the results of flow cytometry analysis of somatostatin ("SST") and GCG expression in cells at S6d7. Figure 6E shows the results of flow cytometry analysis of SST and C-pep expression in cells at S6d7. [Figure 6C] Figure 6A shows the effect of treatment of stem cells with exemplary compounds at stage 1 on cells at the completion of 7 days of stage 6 differentiation according to different exemplary stage 6 media regimens ("Regimen 2"). Figure 6A shows the results of flow cytometry analysis of Nkx6.1 and Isl1 expression at stage 6 day 7 ("S6d7") with 100% AA, 10% AA, 10% activin A and GSK-690693, 10% activin A and BYL719, 10% activin A, GSK-690693 and BYL719 treatments at stage 1. Figure 6B shows a bar graph summarizing the results of Figure 6A. Figure 6C shows the results of flow cytometry analysis of C-peptide ("C-pep") and glucagon ("GCG") expression in cells at S6d7. Figure 6D shows the results of flow cytometry analysis of somatostatin ("SST") and GCG expression in cells at S6d7. Figure 6E shows the results of flow cytometry analysis of SST and C-pep expression in cells at S6d7. [Figure 6D] Figure 6A shows the effect of treatment of stem cells with exemplary compounds at stage 1 on cells at the completion of 7 days of stage 6 differentiation according to different exemplary stage 6 media regimens ("Regimen 2"). Figure 6A shows the results of flow cytometry analysis of Nkx6.1 and Isl1 expression at stage 6 day 7 ("S6d7") with 100% AA, 10% AA, 10% activin A and GSK-690693, 10% activin A and BYL719, 10% activin A, GSK-690693 and BYL719 treatments at stage 1. Figure 6B shows a bar graph summarizing the results of Figure 6A. Figure 6C shows the results of flow cytometry analysis of C-peptide ("C-pep") and glucagon ("GCG") expression in cells at S6d7. Figure 6D shows the results of flow cytometry analysis of somatostatin ("SST") and GCG expression in cells at S6d7. Figure 6E shows the results of flow cytometry analysis of SST and C-pep expression in cells at S6d7. [Figure 6E] Figure 6A shows the effect of treatment of stem cells with exemplary compounds at stage 1 on cells at the completion of 7 days of stage 6 differentiation according to different exemplary stage 6 media regimens ("Regimen 2"). Figure 6A shows the results of flow cytometry analysis of Nkx6.1 and Isl1 expression at stage 6 day 7 ("S6d7") with 100% AA, 10% AA, 10% activin A and GSK-690693, 10% activin A and BYL719, 10% activin A, GSK-690693 and BYL719 treatments at stage 1. Figure 6B shows a bar graph summarizing the results of Figure 6A. Figure 6C shows the results of flow cytometry analysis of C-peptide ("C-pep") and glucagon ("GCG") expression in cells at S6d7. Figure 6D shows the results of flow cytometry analysis of somatostatin ("SST") and GCG expression in cells at S6d7. Figure 6E shows the results of flow cytometry analysis of SST and C-pep expression in cells at S6d7. [Figure 7A]

[0051] Figure 7A shows a graph summarizing the percentage of SC-islet beta cells and the percentage of SC-islet non-beta cells in the cell composition at completion of stage 5 differentiation ("S5C") under various culture conditions with either (a) 100 ng / mL activin A or (b) 0.1 μM GSK-690693 (also referred to as "GSK690693") and 10 ng / ml activin A at stage 1. Figure 7B shows a graph summarizing the percentage of SC-islet beta cells and the percentage of SC-islet non-beta cells in the cell composition at day 7 of stage 6 ("S6d7") under various culture conditions with either (a) 100 ng / mL activin A or (b) 0.1 μM GSK-690693 (also referred to as "GSK690693") and 10 ng / ml activin A at stage 1. [Figure 7B] Figure 7A shows a graph summarizing the percentage of SC-islet beta cells and the percentage of SC-islet non-beta cells in the cell composition at completion of stage 5 differentiation ("S5C") under various culture conditions with either (a) 100 ng / mL activin A or (b) 0.1 μM GSK-690693 (also referred to as "GSK690693") and 10 ng / ml activin A at stage 1. Figure 7B shows a graph summarizing the percentage of SC-islet beta cells and the percentage of SC-islet non-beta cells in the cell composition at day 7 of stage 6 ("S6d7") under various culture conditions with either (a) 100 ng / mL activin A or (b) 0.1 μM GSK-690693 (also referred to as "GSK690693") and 10 ng / ml activin A at stage 1. DETAILED DESCRIPTION OF THE INVENTION

[0043]

[0052] Pancreatic islet transplantation is a promising therapy that can achieve significant clinical benefits for diabetic subjects, such as subjects with type I diabetes.Because the supply of pancreatic islet sources from donor pancreatic tissue is limited, improved techniques are needed for producing transplantable pancreatic islets from alternative sources, such as stem cells.Improved methods for producing pancreatic islet components (e.g., SC-β cells) can lead to more effective therapeutic products (e.g., SC-β cells with improved functionality), improved methods for producing SC-islets for human therapeutic use (e.g., lower production costs and / or higher cell yields), or a combination thereof.

[0044]

[0053] In particular, compositions and methods for improved in vitro production of SC-β cells are provided herein. Certain compositions and combinations of agents disclosed herein facilitate cost-effective, large-scale in vitro production of SC-β cells. For example, the present disclosure provides novel formulations and differentiation methods that utilize small molecule compounds instead of certain growth factors while maintaining comparable or even improved cell yields for the relative percentages of desired cell populations (e.g., on-target differentiated cells and resulting SC-β cells at various differentiation stages), in vitro SC-β cell function, and post-transplant viability, function, and immunogenicity. The disclosed compositions and methods may be particularly advantageous for large-scale production of SC-islets for human therapeutic use. Small molecule compounds in the compositions and methods disclosed herein include, for example, inhibitors of PI3K / Akt / mTOR signaling.

[0045]

[0054] While various embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous changes, modifications, and substitutions may occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed.

[0046] definition

[0055] 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.

[0047]

[0056] 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.

[0048]

[0057] Furthermore, use of the term "including" and other forms such as "include," "includes," and "included" is not limiting.

[0058] 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.

[0049]

[0059] 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.

[0050]

[0060] When used herein, the term "about" and its grammatical equivalents in connection with a reference numerical value can include the numerical value itself and a range of values ​​plus or minus 10% of that numerical value.

[0051]

[0061] 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.

[0052]

[0062] 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.

[0053]

[0063] 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.

[0054]

[0064] 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.

[0055]

[0065] The term "progenitor thereof" in reference to a stem cell-derived pancreatic cell (e.g., an SC-β cell) can refer to 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 be differentiated into an SC-β cell when cultured under conditions suitable for differentiating the progenitor cell into an insulin-positive endocrine cell.

[0056]

[0066] The term "exocrine cells," as used herein, may refer to cells of an exocrine gland, i.e., a gland that excretes its secretions through a duct. In certain embodiments, exocrine cells may refer to pancreatic secretory cells, which are pancreatic cells capable of producing enzymes secreted into the small intestine. These enzymes can help digest food as it passes through the digestive tract. Pancreatic exocrine cells, also known as the islets of Langerhans, can secrete two hormones: insulin and glucagon. Pancreatic exocrine cells can be one of several cell types: alpha-2 cells (which can produce the hormone glucagon), or beta cells (which can manufacture the hormone insulin), and alpha-1 cells (which can produce the regulator somatostatin). Non-insulin-producing exocrine cells, as the term is used herein, may refer to alpha-2 cells or alpha-1 cells.

[0057]

[0067] The terms "stem cell-derived beta cells," "SC-beta cells," "functional beta cells," "functional pancreatic beta cells," "mature SC-beta cells," "SC-pancreatic islet beta cells," and their grammatical equivalents may 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 display 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" are interchangeable when used herein. In some embodiments, "SC-beta cells" include mature pancreatic cells. It should be understood that SC-β cells need not be derived (e.g., directly) from stem cells, as the methods of the present disclosure can use any cell as a starting point to derive SC-β cells from any insulin-positive endocrine cell or its precursor (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, trans-differentiated versions of any of the foregoing, etc., can be used, and the invention is not intended to be limited in this manner). In some embodiments, SC-β cells exhibit a response to multiple glucose challenges (e.g., a series of at least one, at least two, or at least three or more glucose challenges). In some embodiments, the response resembles the response of endogenous pancreatic islets (e.g., human pancreatic islets) to multiple glucose challenges. In some embodiments, the morphology of SC-β cells resembles that of endogenous β cells. In some embodiments, 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 two weeks of 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 drugs (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.

[0058]

[0068] 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 adult 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 an otherwise cell-like phenotype).

[0059]

[0069] The terms "insulin-positive β-like cells," "insulin-positive endocrine cells," and their grammatical equivalents may 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.

[0060]

[0070] The term "beta cell marker" refers, without limitation, to proteins, peptides, nucleic acids, protein and nucleic acid polymorphisms, splice variants, protein or nucleic acid fragments, elements, and other analytes expressed or present in pancreatic beta cells. Exemplary beta cell markers include, but are not limited to, pancreatic and duodenal homeobox 1 (Pdx1, 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 beta cell marker is a nuclear 3 cell marker. In some embodiments, the beta cell marker is Pdx1 or PH3.

[0061]

[0071] 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 expressed or present in pancreatic endocrine cells. Exemplary pancreatic endocrine cell markers include, but are not limited to, Ngn-3, NeuroD, and Islet-1.

[0062]

[0072] The terms "pancreatic progenitor cells," "pancreatic endocrine precursor cells," "pancreatic precursors," "pancreatic endocrine precursors," and their grammatical equivalents are used interchangeably herein and can refer to stem cells that can become pancreatic hormone-expressing cells that can form pancreatic endocrine cells, pancreatic exocrine cells, or pancreatic duct cells. These cells are committed to differentiation toward at least one type of pancreatic cell, such as insulin-producing beta cells, glucagon-producing alpha cells, somatostatin-producing delta cells (or D cells), and / or pancreatic polypeptide-producing F cells. Such cells can express at least one of the following markers: NGN3, NKX2.2, NeuroD, ISL-1, Pax4, Pax6, or ARX.

[0063]

[0073] As used herein, the term "Pdx1-positive pancreatic progenitor cells" can 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 expression of NKX6.1 (or Nkx6.1). Because Pdx1-positive pancreatic progenitor cells lack expression of NKX6.1, they can also be referred to as Pdx1-positive, NKX6.1-negative pancreatic progenitor cells. In some cases, Pdx1-positive pancreatic progenitor cells can also be referred to as "pancreatic foregut endoderm cells." As used herein, the terms "PDX1," "Pdx1," and "PDX-1" are equivalent and interchangeable.

[0064]

[0074] The term "Pdx1-positive, NKX6-1-positive pancreatic progenitor cells" as used herein may refer to cells that are pancreatic endoderm (PE) cells capable of differentiating 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 may 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 using an anti-NKX6-1 antibody or quantitative RT-PCR. As used herein, the terms "Nkx6.1," "NKX6.1," and "NKX6-1" are equivalent and interchangeable. In some cases, Pdx1-positive, NKX6-1-positive pancreatic progenitor cells may also be referred to as "pancreatic foregut precursor cells."

[0065]

[0075] The term "Ngn3-positive endocrine precursor cells" as used herein refers to precursors of pancreatic endocrine cells that express the transcription factor neurogenin-3 (Ngn3). Precursor cells are more differentiated than pluripotent stem cells and can differentiate into only a few cell types. In particular, Ngn3-positive endocrine precursor cells have the ability to differentiate into five pancreatic endocrine cell types (α, β, δ, ε, and PP). Ngn3 expression can be assessed by any method known to those skilled in the art, such as immunohistochemistry using anti-Ngn3 antibodies or quantitative RT-PCR.

[0066]

[0076] The terms "NeuroD" and "NeuroD1" are used interchangeably to identify proteins and their encoding genes expressed in pancreatic endocrine precursor cells.

[0077] 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 genomic modifications. 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 epigenetic changes can persist through cell divisions during the lifespan of a cell and can also persist for multiple generations, even if they do not involve changes in the organism's underlying DNA sequence. One example of epigenetic changes 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.

[0067]

[0078] The term "epigenetic modifying compound" refers to a chemical compound that induces epigenetic changes in genes, i.e., alters the expression of genes without altering their DNA sequence. Epigenetic changes help determine whether genes are turned on or off and can affect the production of proteins 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 thus may contribute to or be the result of environmental modifications of phenotypes or pathological phenotypes. Importantly, epigenetic modifications play a key role in the regulation of pluripotency genes, which are inactivated during differentiation. Non-limiting exemplary epigenetic modifying compounds include DNA methylation inhibitors, histone acetyltransferase inhibitors, histone deacetylase inhibitors, histone methyltransferase inhibitors, bromodomain inhibitors, or any combination thereof.

[0068]

[0079] 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, in which approximately 98% of 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, and thymus.

[0069]

[0080] 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).

[0070]

[0081] As used herein, the term "adult cell" may refer to cells found throughout the body after embryonic development.

[0082] As used herein, the term "endodermal cell" can refer to a cell that is from one of the three primary germ cell layers in a very early embryo (the other two being mesoderm and ectoderm). The endoderm is the innermost of the three layers. Endodermal cells differentiate to give rise first to the fetal gut, then to the lining of the respiratory and digestive tract (including the intestine), liver, and pancreas.

[0071]

[0083] As used herein, the term "cells of endodermal origin" refers to any cell that develops or differentiates from endodermal cells. For example, cells of endodermal origin include cells of the liver, lung, pancreas, thymus, intestine, stomach, and thyroid. Without wishing to be bound by theory, liver and pancreatic progenitor cells (also referred to as pancreatic progenitor cells) arise from endodermal cells of the embryonic foregut. Shortly after their identification, liver and pancreatic progenitors rapidly acquire distinct cellular functions and regenerative capabilities. These changes are driven by inductive signals and genetic regulatory factors that are highly conserved among vertebrates. Interest in organ development and regeneration has been fueled by the strong need for hepatocytes and pancreatic β cells in the therapeutic treatment of liver failure and type 1 diabetes. Studies in various model organisms and humans have revealed a network of evolutionarily conserved inductive signals and transcription factors that trigger hepatocyte and pancreatic cell differentiation and provide guidance on how to promote hepatocyte and β cell differentiation from diverse stem and progenitor cell types.

[0072]

[0084] 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.

[0073]

[0085] 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.

[0086] As used herein, the term "primitive gut cells" 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. Primitive gut cells have the ability 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.

[0074]

[0087] The term "stem cell," as used herein, 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 also 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 by 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 are themselves 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 either 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.

[0075]

[0088] The term "pluripotent," as used herein, may 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.

[0076]

[0089] 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.

[0077]

[0090] 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.

[0078]

[0091] 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 domestic animal, or cows, sheep, pigs, 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.

[0079]

[0092] As used herein, the term "administering" may refer to 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. In some embodiments, any of the compositions can be administered via the hepatic portal vein. One or more such routes can be employed. Parenteral administration can be, for example, by 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.

[0080]

[0093] The terms "treat," "treating," "treatment," and their grammatical equivalents, when applied to isolated cells, include subjecting the cells to any kind of process or condition, or performing any kind of operation or procedure on the cells. When applied to a subject, the term refers to providing medical or surgical attention, care, or management to an individual. The individual is typically ill or injured, or at increased risk of developing a disease compared to the average member of the population, and is in need of such attention, care, or management.

[0081]

[0094] As used herein, the terms "treating" and "treatment" can refer to administering an effective amount of a composition to a subject so that the subject experiences a reduction in at least one symptom of a disease or an improvement in the disease, e.g., a beneficial or desired clinical result. For purposes of this invention, a beneficial or desired clinical result includes, but is not limited to, alleviation of one or more symptoms, whether detectable or undetectable, attenuation of the extent of the disease, stabilization of the disease state (e.g., not worsening), delay or slowing of disease progression, improvement or relief of the disease state, and remission (e.g., partial or total), whether detectable or undetectable. Treating can also refer to prolonging survival compared to expected survival in the absence of treatment. Thus, those skilled in the art will understand that treatment can improve the disease state, but may not be a complete cure for the disease. As used herein, the term "treatment" includes prophylaxis. Alternatively, treatment is "effective" if the progression of the disease is reduced or halted. "Treatment" can also mean prolonging survival compared to expected survival in the absence of treatment. Those in need of treatment include those already diagnosed with heart disease, as well as those who are at high risk of developing heart disease due to genetic susceptibility or other factors such as weight, diet and health.

[0082]

[0095] The terms "therapeutically effective amount," "therapeutic amount," or their grammatical equivalents may refer to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount may vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the ability of the compositions described herein to induce a desired response in one or more subjects. The exact amount of the composition of the present disclosure to be administered may be determined by a physician, taking into account individual differences in age, weight, tumor size, extent of infection or metastasis, and the patient's (subject's) condition.

[0083]

[0096] Alternatively, the pharmacological and / or physiological effect of administering one or more compositions described herein to a patient or subject may be "prophylactic," e.g., the effect completely or partially prevents a disease or its symptoms. A "prophylactically effective amount" may refer to an amount effective, at dosages, and for periods of time necessary, to achieve a desired prophylactic result (e.g., prevention of disease onset).

[0084]

[0097] 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]," and the numerical value is inclusive of the number itself.

[0085]

[0098] 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.

[0086]

[0099] The generally disclosed ranges may be referred to as, for example, "X is administered on or about days 1-2, or on or about days 2-3 [or any range of values]," and the ranges include the numbers themselves (e.g., the endpoints of the ranges) and all individual numbers within the recited ranges.

[0087]

[0100] 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.

[0088] Inhibitors of PI3K / Akt / mTOR signaling

[0101] In some embodiments, the compositions and methods disclosed herein relate to the use of one or more inhibitors of PI3K / Akt / mTOR signaling during the differentiation of cells in the pancreatic lineage (e.g., Sox17-positive definitive endoderm cells, FOXA2-positive gastrula cells, PDX1-positive, NKX6.1-negative pancreatic progenitor 1 cells, PDX1-positive, NKX6.1-negative pancreatic progenitor 2 cells, insulin-positive pancreatic endocrine cells, or SC-pancreatic beta cells).

[0089]

[0102] Without wishing to be bound by any particular theory, activation of phosphoinositide 3-kinase (PI3K) phosphorylates and activates protein kinase B (PKB, also known as Akt). Phosphorylation and activation of Akt can have many downstream effects, such as activation of cAMP response element-binding protein (CREB), inhibition of p27, localization of forkhead box O (FOXO) in the cytoplasm, activation of PtdIns-3ps, and activation of the mechanistic target of rapamycin (mTOR, also known as mammalian target of rapamycin). As used herein, the term "inhibitor of PI3K / Akt / mTOR signaling" refers to a drug that inhibits the activity of PI3K protein, Akt protein, mTOR protein, or any combination thereof. The term "inhibitor of PI3K / Akt / mTOR signaling" is not intended to be limited to drugs that have an inhibitory effect on the signaling cascade that occurs inside cells from PI3K to Akt and mTOR. Alternatively, in some cases, the inhibitors of PI3K / Akt / mTOR signaling disclosed herein have an inhibitory effect on the activity of PI3K protein without any immediate effect on the activation of Akt or mTOR, or on the activity of Akt protein without any immediate effect on the activation of mTOR or the activity of PI3K protein. In some cases, the inhibitors of PI3K / Akt / mTOR signaling disclosed herein directly inhibit the activity of mTOR protein without any immediate effect on the activity of Akt protein or the activity of PI3K protein. In some cases, the inhibitors of PI3K / Akt / mTOR signaling directly inhibit the activity of PI3K protein, the activity of Akt protein, the activity of mTOR protein, or any combination thereof. In some cases, the inhibitors of PI3K / Akt / mTOR signaling indirectly inhibit the activity of PI3K protein, the activity of Akt protein, the activity of mTOR protein, or any combination thereof.

[0090]

[0103] In some embodiments, the inhibitors of PI3K / Akt / mTOR signaling disclosed herein include inhibitors of PI3K proteins, such as class I PI3K, class II PI3K, or class III PI3K. In some embodiments, the inhibitors of PI3K / Akt / mTOR signaling disclosed herein inhibit more than one PI3K protein, for example, more than one class I, II, or III PI3K, or PI3K in more than one class. In some embodiments, the inhibitors of PI3K / Akt / mTOR signaling disclosed herein include pan-PI3K inhibitors, for example, agents that inhibit virtually all types of PI3K. Class I PI3Ks discussed herein may include PIK3CA, PIK3CB, PIK3CG, PIK3CD, PIK3R1, PIK3R2, PIK3R3, PIK3R4, PIK3R5, and PIK3R6. Class II PI3Ks discussed herein may include PIK3C2A, PIK3C2B, PIK3C2G. Class III PI3Ks discussed herein may include PIK3C3.

[0091]

[0104] In some embodiments, the inhibitors of PI3K / Akt / mTOR signaling disclosed herein include inhibitors of Akt proteins, such as Akt1, Akt2, or Akt3. In some embodiments, the inhibitors of PI3K / Akt / mTOR signaling disclosed herein inhibit more than one Akt protein, for example, Akt1 and Akt2, Akt1 and Akt3, or Akt2 and Akt3. In some embodiments, the inhibitors of PI3K / Akt / mTOR signaling disclosed herein inhibit Akt1 but have minimal or no effect on Akt2 or Akt3. In some embodiments, the inhibitors of PI3K / Akt / mTOR signaling disclosed herein inhibit Akt2 but have minimal or no effect on Akt1 or Akt3. In some embodiments, inhibitors of PI3K / Akt / mTOR signaling disclosed herein inhibit Akt3 but have minimal or no effect on Akt1 or Akt2. In some embodiments, inhibitors of PI3K / Akt / mTOR signaling disclosed herein include pan-Akt inhibitors, e.g., agents that inhibit substantially all three types of Akt proteins.

[0092]

[0105] In some embodiments, the inhibitors of PI3K / Akt / mTOR signaling disclosed herein include inhibitors of mTOR. Without wishing to be bound by any particular theory, activated mTOR protein binds with other proteins and functions as core components of two different protein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), which have different downstream cell signaling pathways and can regulate different cellular processes. In some embodiments, the inhibitors of PI3K / Akt / mTOR signaling disclosed herein inhibit the formation of mTORC1. In some embodiments, the inhibitors of PI3K / Akt / mTOR signaling disclosed herein inhibit the activity of mTORC1 and, therefore, at least some of the target proteins and / or cell signaling pathways activated by mTORC1. In some embodiments, the inhibitors of PI3K / Akt / mTOR signaling disclosed herein inhibit the formation of mTORC2. In some embodiments, inhibitors of PI3K / Akt / mTOR signaling disclosed herein inhibit the activity of mTORC2, and thus at least a portion of a target protein and / or cell signaling pathway activated by mTORC2. In some embodiments, inhibitors of PI3K / Akt / mTOR signaling disclosed herein inhibit the formation of both mTORC1 and mTORC2. In some embodiments, inhibitors of PI3K / Akt / mTOR signaling disclosed herein inhibit both mTORC1 and mTORC2, and thus at least a portion of a target protein and / or cell signaling pathway activated by both mTORC1 or mTORC2.

[0093]

[0106] In some embodiments, inhibitors of PI3K / Akt / mTOR signaling disclosed herein include, but are not limited to, GSK-690693, IPI-3063, AZD8055, omipalisib, GNE-477, VS-5584, BYL319, YM201636, PI4KIIIbeta-IN-10, nemiralisib, BYL719, FT113, apitolisib, and any analogs or derivatives thereof. In some embodiments, the inhibitor of PI3K / Akt / mTOR signaling disclosed herein is one or more of GSK-690693, IPI-3063, AZD8055, omipalisib, GNE-477, VS-5584, BYL319, YM201636, PI4KIIIbeta-IN-10, nemiralisib, BYL719, FT113, apitolisib, or any analog or derivative thereof.

[0094]

[0107] In some embodiments, the inhibitors of PI3K / Akt / mTOR signaling disclosed herein have Formula (I):

[0095] [ka]

[0096] or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, prodrug, composition, or mixture thereof.

[0108] In some embodiments, the present disclosure provides an inhibitor of PI3K / Akt / mTOR, wherein the inhibitor is any of the molecules disclosed in U.S. Patent Application Publication No. 2008076763, which is incorporated herein in its entirety. For example, in some embodiments, the present disclosure provides a compound of formula (II):

[0097] [ka]

[0098] wherein X is absent or is selected from the group consisting of O, S and CR 20 R 21where R 20 R 21 are independently selected from hydrogen, fluorine, cyclopropyl, substituted cyclopropyl, cyclobutyl, substituted cyclobutyl, cyclopentyl, substituted cyclopentyl, -C1-C4 alkyl, and substituted -C1-C4 alkyl; or R 20 R 21 together with the carbon to which they are attached form cyclopropyl, substituted cyclopropyl, cyclobutyl, substituted cyclobutyl, cyclopentyl or substituted cyclopentyl; R 2 R 2’ are independently selected from hydrogen, fluorine, cyclopropyl, substituted cyclopropyl, cyclobutyl, substituted cyclobutyl, cyclopentyl, substituted cyclopentyl, -C1-C4 alkyl, and substituted -C1-C4 alkyl; Or, R 2 R 2’ together with the carbon to which they are attached form cyclopropyl, substituted cyclopropyl, cyclobutyl, substituted cyclobutyl, cyclopentyl or substituted cyclopentyl; R 3 is selected from the group consisting of hydrogen, cyclopropyl, substituted cyclopropyl, cyclobutyl, substituted cyclobutyl, cyclopentyl, substituted cyclopentyl, cyclopropylmethyl, substituted cyclopropylmethyl, -C1-C4 alkyl, and substituted -C1-C4 alkyl; R 4 R 4’ are independently selected from hydrogen, fluorine, cyclopropyl, substituted cyclopropyl, cyclobutyl, substituted cyclobutyl, cyclopentyl, substituted cyclopentyl, -C1-C4 alkyl, and substituted -C1-C4 alkyl, or R 4 R 4’ together with the carbon to which they are attached form cyclopropyl, substituted cyclopropyl, cyclobutyl, substituted cyclobutyl, cyclopentyl or substituted cyclopentyl; R 5 R 5’are independently selected from hydrogen, fluorine, cyclopropyl, substituted cyclopropyl, cyclobutyl, substituted cyclobutyl, cyclopentyl, substituted cyclopentyl, -C1-C4 alkyl, and substituted -C1-C4 alkyl; or R 5 R 5’ together with the carbon to which they are attached form cyclopropyl, substituted cyclopropyl, cyclobutyl, substituted cyclobutyl, cyclopentyl or substituted cyclopentyl; R 1 is selected from the group consisting of hydrogen, -C1-C4 alkyl, and substituted -C1-C4 alkyl; X is not present or R 20 R 21 If R 1 may further be fluorine) and / or pharmaceutically acceptable salts, hydrates, solvates and prodrugs thereof.

[0099]

[0109] The term "-C1-C4 alkyl," as used herein, means a straight or branched, saturated or unsaturated hydrocarbon chain containing from 1 to 4 carbon atoms. Examples of -C1-C4 alkyl, as used herein, include -CH3, -CH2-CH3, -CH2-CH2-CH3, -CH(CH3)2, -CH2-CF3, -C(CH3), -(CH2)3-CH3, -CH2-CH(CH3)2, -CH(CH3)-CH2-CH3, -CH-CH2, and -C[same]C-CH3.

[0100]

[0110] In some embodiments, the inhibitors of PI3K / Akt / mTOR signaling disclosed herein have formula (III):

[0101] [ka]

[0102] or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, prodrug, composition, or mixture thereof.

[0111] In some embodiments, the present disclosure provides an inhibitor of PI3K / Akt / mTOR, wherein the inhibitor is any of the molecules disclosed in U.S. Patent Application Publication No. 2010105711, the entirety of which is incorporated herein. For example, in some embodiments, the present disclosure provides a compound of formula (IV):

[0103] [ka]

[0104] (Wherein A represents heteroaryl; R 1 represents (1) optionally substituted alkyl, (2) optionally substituted cycloalkyl, (3) optionally substituted aryl, (4) optionally substituted amine, (5) optionally substituted sulfonyl, (6) halo; R 2 is hydrogen, deuterium or R 1 represents a substituent as defined for R 3 represents hydrogen, halo, or optionally substituted alkyl, except for (S)-pyrrolidine-1,2-dicarboxylic acid 2-amide 1-({5-[2-(tert-butyl)-pyrimidin-4-yl]-4-methylthiazol-2-yl}-amide) The present invention provides a PI3K / Akt / mTOR inhibitor comprising the structure:

[0105]

[0112] The prefix "C1-C7" refers to a group having up to and including 7, especially up to and including 4, carbon atoms, which group is either linear or branched with single or multiple branches.

[0106]

[0113] "Alkyl" refers to a straight or branched chain alkyl group, preferably a straight or branched chain C 1~12 represents alkyl, and particularly preferably represents straight or branched C1~7 Alkyl represents, for example, methyl, ethyl, n- or isopropyl, n-, iso-, sec- or tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, or n-dodecyl, with methyl, ethyl, n-propyl, isopropyl, and n-butyl and isobutyl being particularly preferred. Alkyl may be unsubstituted or substituted. Exemplary substituents include, but are not limited to, deuterium, hydroxy, alkoxy, halo, and amino. An example of a substituted alkyl is trifluoromethyl. Cycloalkyl may also be a substituent of alkyl. Examples of such cases are the moieties (alkyl)-cyclopropyl or alkanediyl-cycloproyl, such as -CH2-cyclopropyl. C1-C7-Alkyl is preferably alkyl having from 1 up to 7 (inclusive), preferably from 1 up to 4 (inclusive), linear or branched, preferably lower alkyl is butyl, such as n-butyl, sec-butyl, isobutyl, tert-butyl, propyl, such as n-propyl or isopropyl, ethyl or preferably methyl.

[0107]

[0114] Each alkyl portion of other groups such as "alkoxy," "alkoxyalkyl," "alkoxycarbonyl," "alkoxycarbonylalkyl," "alkylsulfonyl," "alkylsulfoxyl," "alkylamino," and "haloalkyl" has the same meaning as described in the above definition of "alkyl."

[0108]

[0115] "Alkanediyl" refers to a straight-chain or branched-chain alkanediyl group bonded to the moiety by two different carbon atoms, and preferably represents a straight-chain or branched-chain C1-12 alkanediyl, and particularly preferably represents a straight-chain or branched-chain C1-6 alkanediyl, such as methanediyl (-CH2-), 1,2-ethanediyl (-CH2-CH2-), 1,1-ethanediyl (-CH(CH3)-), 1,1-, 1,2-, 1,3-propanediyl, and 1,1-, 1,2-, 1,3-, 1,4-butanediyl, with methanediyl, 1,1-ethanediyl, 1,2-ethanediyl, 1,3-propanediyl, and 1,4-butanediyl being particularly preferred.

[0109]

[0116] "Alkenediyl" refers to a straight-chain or branched-chain alkenediyl group bonded to a molecule by two different carbon atoms, preferably a straight-chain or branched-chain C2-6 alkanediyl, such as -CH=CH-, -CH=C(CH3)-, -CH=CH-CH2-, -C(CH3)=CH-CH2-, -CH=C(CH3)-CH2-, -CH=CH-C(CH3)H-, -CH=CH-CH=CH-, -C(CH3)=CH-CH=CH-, -CH=C(CH3)-CH=CH-, with -CH=CH-CH2- and -CH=CH-CH=CH- being particularly preferred. The alkenediyl may be substituted or unsubstituted.

[0110]

[0117] "Cycloalkyl" refers to a saturated or partially saturated monocyclic, fused polycyclic, or spiropolycyclic carbocyclic ring having 3 to 12 ring atoms per carbocyclic ring. Illustrative cycloalkyl groups include the following moieties: cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Cycloalkyl can be unsubstituted or substituted; exemplary substituents are provided in the definition of alkyl, including alkyl itself (e.g., methyl). A moiety such as (CH3)cyclopropyl is considered a substituted cycloalkyl.

[0111]

[0118] "Aryl" refers to an aromatic homocyclic ring system (i.e., only carbon as ring-forming atoms) having six or more carbon atoms, and aryl is preferably an aromatic moiety having 6 to 14 ring carbon atoms, more preferably 6 to 10 ring carbon atoms, such as phenyl or naphthyl, preferably phenyl. Aryl may be unsubstituted or unsubstituted or substituted heterocyclyl as described below, in particular pyrrolidinyl, e.g. pyrrolidino, oxopyrrolidinyl, e.g. oxopyrrolidino, C1-C7-alkyl-pyrrolidinyl, 2,5-di(C1-C7 alkyl)pyrrolidinyl, e.g. 2,5-di-(C1-C7 alkyl)-pyrrolidino, tetrahydrofuranyl, thiophenyl, C1-C7-alkylpyrazolidinyl, pyridinyl, C1-C7-alkylpiperidinyl, piperidino, piperidino substituted with amino or N-mono- or N,N-di-(lower alkyl, phenyl, C1-C7-alkanoyl and / or phenyl lower alkyl)-amino, unsubstituted or N-lower alkyl-substituted piperidinyl bonded via a ring carbon atom, piperazino, lower alkylpiperazino, morpholino, thiomorpholino, S-oxothiomo morpholino or S,S-dioxothiomorpholino; C1-C7-alkyl, amino-C1-C7-alkyl, N-C1-C7-alkanoylamino-C1-C7-alkyl, N-C1-C7-alkanesulfonyl-amino-C1-C7-alkyl, carbamoyl-C1-C7-alkyl, [N-mono- or N,N-di-(C1-C7-alkyl)-carbamoyl]C1-C7-alkyl, C1-C7-alkanesulfonyl phenyl-C1-C7-alkyl, C1-C7-alkanesulfonyl-C1-C7-alkyl, phenyl, naphthyl, mono- to tri-[C1-C7-alkyl, halo and / or cyano]-phenyl or mono- to tri-[C1-C7-alkyl, halo and / or cyano]-naphthyl; C3-C8-cycloalkyl, mono- to tri-[C1-C7-alkyl and / or hydroxy]-C3-C8-cycloalkyl;Halo, hydroxy, lower alkoxy, lower-alkoxy-lower alkoxy, (lower alkoxy)-lower alkoxy-lower alkoxy, halo-C1-C7-alkoxy, phenoxy, naphthyloxy, phenyl or naphthyl-lower alkoxy; amino-C1-C7-alkoxy, lower alkanoyloxy, benzoyloxy, naphthoyloxy, formyl (CHO), amino, N-mono- or N,N-di-(C1-C7-alkyl)-amino, C1-C7-alkanoylamino, C1-C7-alkanesulfonylamino, carboxy, lower alkoxycarbonyl, for example, phenyl or naphthyl-lower alkoxycarbonyl, for example, benzyloxycarbonyl; C1-C7-alkanoyl, for example, acetyl, benzoyl, naphthoyl , carbamoyl, N-mono- or N,N-disubstituted carbamoyl, for example N-mono- or N,N-disubstituted carbamoyl, wherein the substituents are selected from lower alkyl, (lower alkoxy)-lower alkyl and hydroxy-lower alkyl; amidino, guanidino, ureido, mercapto, lower alkylthio, phenyl or naphthylthio, phenyl or naphthyl-lower alkylthio, lower alkyl-phenylthio, lower alkyl-naphthylthio, halo-lower alkylmercapto, sulfo (—SOH), lower alkanesulfonyl, phenyl or naphthylsulfonyl, phenyl or naphthyl-lower alkylsulfonyl, alkylphenylsulfonyl, halo-lower alkylsulfonyl, for example trifluoromethanesulfonyl;and optionally substituted with one or more, preferably up to three, more preferably up to two substituents independently selected from the group consisting of sulfonamido, benzosulfonamido, azide, azido-C1-C7-alkyl, in particular azidomethyl, C1-C7-alkanesulfonyl, sulfamoyl, N-mono- or N,N-di-(C1-C7-alkyl)-sulfamoyl, morpholinosulfonyl, thiomorpholinosulfonyl, cyano and nitro, wherein the substituted alkyl (or substituted aryl, heterocyclyl, etc. as described herein) substituent or part of a substituent is optionally substituted with one or more, preferably up to three, more preferably up to two, substituents independently selected from the group consisting of substituted alkyl (or substituted aryl, heterocyclyl, etc. as described herein). Each phenyl or naphthyl (as in phenoxy or naphthoxy) as mentioned above is itself unsubstituted or substituted with one or more, for example up to three, preferably one or two, substituents independently selected from halo, halo-lower alkyl, for example trifluoromethyl, hydroxy, lower alkoxy, azido, amino, N-mono- or N,N-di-(lower alkyl and / or C1-C7-alkanoyl)-amino, nitro, carboxy, lower alkoxycarbonyl, carbamoyl, cyano and / or sulfamoyl;

[0112]

[0119] "Heterocyclyl" refers to a heterocyclic group that is unsaturated (= possessing the maximum possible number of conjugated double bonds in the ring(s)), saturated, or partially saturated, preferably monocyclic, or in a broader aspect of the invention, bicyclic, tricyclic, or spirocyclic ring, and has 3 to 24, more preferably 4 to 16, most preferably 5 to 10, and most preferably 5 or 6 ring atoms, where one or more, preferably 1 to 4, especially 1 or 2, ring atoms are heteroatoms (and therefore the remaining ring atoms are carbon). The bound ring (i.e., the ring that is attached to the molecule) preferably has 4 to 12, especially 5 to 7 ring atoms. The term heterocyclyl also includes heteroaryl. Heterocyclic groups (heterocyclyl) may be unsubstituted or substituted with one or more, in particular 1 to 3, substituents independently selected from the group consisting of the substituents defined above for substituted alkyl and / or from one or more of the following substituents: oxo (=O), thiocarbonylimino (=NH), imino-lower alkyl.Furthermore, heterocyclyl is in particular oxiranyl, azirinyl, aziridinyl, 1,2-oxathiolanyl, thienyl (=thiophenyl), furanyl, tetrahydrofuryl, pyranyl, thiopyranyl, thianthrenyl, isobenzofuranyl, benzofuranyl, chromenyl, 2H-pyrrolyl, pyrrolyl, pyrrolinyl, pyrrolidinyl, imidazolyl, imidazolidinyl, benzimidazolyl, pyrazolyl, pyrazinyl, pyrazolidinyl, thiazolyl, isothiazolyl, dithiazolyl, oxazolyl. , isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, piperidinyl, piperazinyl, pyridazinyl, morpholinyl, thiomorpholinyl, (S-oxo or S,S-dioxo)-thiomorpholino, indolizinyl, azepanyl, diazepanyl, especially 1,4-diazepanyl, isoindolyl, 3H-indolyl, indolyl, benzimidazolyl, coumaryl, indazolyl, triazolyl, tetrazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, tetrahydroquinolyl quinolyl, tetrahydroisoquinolyl, decahydroquinolyl, octahydroisoquinolyl, benzofuranyl, dibenzofuranyl, benzothiophenyl, dibenzothiophenyl, phthalazinyl, naphthyridinyl, quinoxalyl, quinazolinyl, quinazolinyl, cinnolinyl, pteridinyl, carbazolyl, beta-carbolinyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, furazanyl, phenazinyl, phenothiazinyl, phenoxazinyl, chromenyl, isochromanyl and heterocyclyl groups selected from the group consisting of aryl, chromanyl, benzo[1,3]dioxol-5-yl and 2,3-dihydrobenzo[1,4]dioxin-6-yl, each of which groups is unsubstituted or substituted with one or more, preferably up to three, substituents selected from those described above for substituted aryl and / or one or more of the following substituents: oxo (O), thiocarbonyl (S), imino (=NH), imino-lower alkyl.

[0113]

[0120] "Arylalkyl" refers to an aryl group bonded to the molecule via an alkyl group such as a methyl or ethyl group, preferably phenethyl or benzyl, especially benzyl. Similarly, cycloalkyl-alkyl and heterocyclyl-alkyl refer to a cycloalkyl group bonded to the molecule via an alkyl group or a heterocyclyl group bonded to the molecule via an alkyl group. In each case, the aryl, heterocyclyl, cycloalkyl, and alkyl may be substituted as defined above.

[0114]

[0121] In some embodiments, compositions comprising inhibitors of PI3K / Akt / mTOR signaling disclosed herein include GSK-690693 and BYL719, or derivatives, analogs, or variants thereof.

[0115] Differentiation stages

[0122] In some embodiments, pancreatic differentiation as disclosed herein is carried out in a stepwise manner. In the stepwise progression, "Stage 1" or "S1" refers to the first stage of the differentiation process, where pluripotent stem cells are differentiated into cells expressing markers characteristic of definitive endoderm cells ("DE," "Stage 1 cells," or "S1 cells"). "Stage 2" refers to the second stage, where cells expressing markers characteristic of definitive endoderm cells are differentiated into cells expressing markers characteristic of intestinal cells ("GT," "Stage 2 cells," or "S2 cells"). "Stage 3" refers to the third stage, where cells expressing markers characteristic of intestinal cells are differentiated into cells expressing markers characteristic of pancreatic progenitor 1 cells ("PP1," "Stage 3 cells," or "S3 cells"). "Stage 4" refers to the fourth stage, differentiation of cells expressing markers characteristic of pancreatic progenitor 1 cells into cells expressing markers characteristic of pancreatic progenitor 2 cells ("PP2," "Stage 4 cells," or "S4 cells"). "Stage 5" refers to the fifth stage, differentiation of cells expressing markers characteristic of pancreatic progenitor 2 cells (e.g., PDX.1+, NKX6.1+) into cells expressing markers characteristic of pancreatic endoderm cells and / or pancreatic endocrine progenitor cells (e.g., insulin+) ("EN," "Stage 5 cells," or "S5 cells"). "Stage 6" refers to the differentiation of cells expressing markers characteristic of pancreatic endocrine progenitor cells (e.g., insulin) into cells expressing markers characteristic of pancreatic endocrine beta cells ("SC-β cells") or pancreatic endocrine alpha cells ("SC-α cells"). However, it should be understood that not all cells within a particular population progress through these stages at the same rate, i.e., some cells may progress less or more along the differentiation pathway than the majority of cells present in that population. For example, in some embodiments, SC-β cells can be identified during stage 5, at the end of stage 5, at the beginning of stage 6, etc.Examples of methods for producing cells at any one of stages 1 to 6 are provided in, for example, U.S. Patent No. 10,030,229, U.S. Patent No. 10,443,042, U.S. Patent Publication Nos. 20200332262, 20210198632A1, and 20220090020, each of which is incorporated by reference in its entirety.

[0116] Reprogramming

[0123] The term "reprogramming," as used herein, refers to the process of altering or reversing the differentiation state of a somatic cell. Cells can be partially or terminally differentiated before reprogramming. Reprogramming encompasses the complete reversal of the differentiation state of a somatic cell to a pluripotent cell. Such complete reversal of differentiation generates induced pluripotent (iPS) cells. As used herein, reprogramming also encompasses the partial reversal of the differentiation state of a cell, e.g., to a pluripotent state or somatic cell, which is a cell that is neither pluripotent nor pluripotent but has lost one or more specific characteristics of the differentiated cell from which it originated, e.g., the direct reprogramming of a differentiated cell into various somatic cell types. Reprogramming generally involves altering, e.g., reversing, at least some of the heritable patterns of nucleic acid modification (e.g., methylation), chromatin condensation, epigenetic changes, genomic imprinting, etc., that occur during cell differentiation as a zygote develops into an adult.

[0117]

[0124] As used herein, the term "reprogramming factor" is intended to 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.

[0118]

[0125] The term "differentiation" and its grammatical equivalents, as used herein, refer to the process by which a less specialized cell (i.e., a more naive cell with greater cellular potential) becomes a more specialized cell type (i.e., a less naive cell with less cellular potential); the term "dedifferentiation" refers to the process by which a more specialized cell becomes a less specialized cell type (i.e., a more naive cell with greater cellular potential); and the term "transdifferentiation" refers 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 "naivety." 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 "naivety."

[0119]

[0126] As used herein, the term "cytopotency" refers to 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 cytopotency. 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), and therefore have relatively lower cytopotency than pluripotent cells. Cells committed to a specific lineage or terminally differentiated may have even lower cytopotency. Specific examples of transdifferentiation known in the art include, for example, the conversion of fibroblastic beta cells or exocrine pancreatic cells to beta cells.

[0120]

[0127] Thus, cells can be differentiated into more naive cells (e.g., terminally differentiated cells can be differentiated to pluripotent or multipotent), or cells can be dedifferentiated into less naive 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 reprograms 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 reprograms cells to transdifferentiate.

[0121]

[0128] 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 induce, for example, a cell of the present disclosure. Those skilled in the art will recognize that other genes are also involved in cellular reprogramming, and that exogenous molecules 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 level of another gene 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 can directly or indirectly participate in cell reprogramming or increase cell programming efficiency are 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.

[0122]

[0129] In some embodiments of the present disclosure, the methods exclude the use of reprogramming factor(s) that are not small molecules. However, it will be recognized that the method may utilize tissue culture components, such as media, serum, 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 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. Indeed, these components are not small molecules as defined herein and / or reprogramming factors as defined herein. However, the cell culture components and metabolites disclosed herein can be used to enhance the cell reprogramming and differentiation methods disclosed herein. For example, the combination of cell culture components / additives and metabolites disclosed herein can improve the efficiency of SC-β cell production and their function.

[0123]

[0130] 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 beta cells or otherwise inducing the cells of the present disclosure to differentiate, dedifferentiate, and / or transdifferentiate.

[0124]

[0131] That is, in one embodiment, the method is carried out in the absence of one or more exogenous polynucleotide or polypeptide reprogramming factors (e.g., activin A). Thus, it should be understood that in one embodiment, the disclosed method utilizes small molecules to reprogram cells without the addition of 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.

[0125]

[0132] In some embodiments, the present disclosure provides methods in which one or more small molecules supplement, replace, and / or reduce the use of activin A in differentiation protocols.

[0126] stem cells

[0133] The term "stem cell" is used herein to refer to cells (e.g., plant stem cells, vertebrate stem cells) that have the capacity to self-renew and generate differentiated cell types (Morrison et al., (1997) Cell 88:287-298). In the context of cellular ontogeny, the adjectives "differentiated" or "differentiating" are relative terms. A "differentiated cell" is a cell that has progressed further along the developmental pathway than the cell to which it is being compared. Thus, pluripotent stem cells can differentiate into lineage-restricted progenitor cells (e.g., mesodermal stem cells), which can differentiate into further lineage-restricted cells (e.g., beta cell progenitors), which can differentiate into terminal cells (i.e., terminally differentiated cells, e.g., beta cells) that play characteristic roles in a tissue type and may or may not retain the ability to proliferate further. Stem cells can be characterized by the presence of certain markers (e.g., proteins, RNA, etc.) and the absence of certain markers. 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.

[0127]

[0134] Stem cells of interest include pluripotent stem cells (PSCs). The terms "pluripotent stem cells" or "PSCs" are used herein to refer to stem cells that can generate all cell types in an organism. Thus, PSCs can give rise to cells of all germ cell layers of an organism (e.g., endoderm, mesoderm, and ectoderm in vertebrates). Pluripotent cells can form teratomas and contribute to the ectodermal, mesodermal, and endodermal tissues of an organism. Plant pluripotent stem cells can give rise to all cell types of the plant (e.g., roots, stems, leaves, and other cells).

[0128]

[0135] Animal PSCs can be derived in several different ways. For example, embryonic stem cells (ESCs) are derived from the inner cell mass of an embryo (Thomson et al., Science. 1998 Nov. 6; 282(5391):1145-7), while induced pluripotent stem cells (iPSCs) are 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 refers to pluripotent stem cells regardless of their origin, and therefore encompasses 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.

[0129]

[0136] "Embryonic stem cells (ESCs)" refers 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, 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 HSF-6 (University of California at San Francisco); and H1, H7, H9, H13, and H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute)). The stem cells of interest are listed in the Registry. Stem cells of interest also include embryonic stem cells from other primates, such as rhesus monkey stem cells and marmoset stem cells. Stem cells can be obtained from any mammalian species, 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 typically grow as flat colonies with a large nucleus-to-cytoplasm ratio, distinct borders, and prominent nuclei. Furthermore, ESCs 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.

[0130]

[0137] "Fetal embryonic stem cells" (EGSCs) or "fetal embryonic cells" or "EG cells" refer to PSCs derived from embryonic cells and / or embryonic cell precursors, such as primordial embryonic cells, i.e., embryonic cells capable of developing into sperm and eggs. Fetal embryonic cells (EG cells) are believed to have similar properties to the above-described fetal stem cells. Examples of methods for producing and characterizing EG cells can be found, for example, in U.S. Patent No. 7,153,684; Matsui, Y. et al., (1992) Cell 70:841; Shamblott, M. et al., (2001) Proc. Natl. Acad. Sci. USA 98:113; Shamblott, M. et al., (1998) Proc. Natl. Acad. Sci. USA, 95:13726; and Koshimizu, U. et al., (1996) Development, 122:1235, each of which is incorporated herein in its entirety.

[0131]

[0138] "Induced pluripotent stem cells" or "iPSCs" refer to PSCs derived from non-PSC cells (i.e., from cells differentiated into PSCs). iPSCs can be derived from a variety of different cell types, including terminally differentiated cells. iPSCs have an ES cell-like morphology and grow as flat colonies with a large nuclear-cytoplasmic ratio, distinct borders, and prominent nuclei. Furthermore, iPSCs 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.

[0132]

[0139] In some embodiments, the population of cells is obtained in vitro from stem cells. In some embodiments, the stem cells are genetically modified. In some embodiments, the stem cells have reduced expression of one or more of beta-2 microglobulin, ABO, FUT1, CXCL10, renalase, CIITA, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR relative to non-genetically modified stem cells. In some embodiments, the stem cells have increased expression of one or more of CD47, PDL1, HLA-G, CD46, CD55, CD59, and / or CTLA relative to non-genetically modified stem cells.

[0133]

[0140] In certain instances, stem cells may be undifferentiated (e.g., cells not committed to a particular lineage) before exposure to at least one beta cell maturation factor 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 cell maturation factor(s) 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.

[0134]

[0141] 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 are 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.

[0135]

[0142] In another embodiment, stem cells can be isolated from tissues, including solid tissues.In some embodiments, the tissue is skin, adipose tissue (for example, 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.

[0136]

[0143] Stem cells of interest include various types of embryonic cells, such as human embryonic stem cells (hES), as described in Thomson et al. (1998) Science 282:1145; embryonic stem cells from other primates, such as rhesus stem cells (Thomson et al. (1995) Proc. Natl. Acad. Sci. USA 92:7844); marmoset stem cells (Thomson et al. (1996) Biol. Reprod. 55:254); and human embryonic germ (hEG) cells (Shambloft et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). Also applicable are 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 & Bader (1996) Circ Res. 78(2):205-16). Stem cells can be obtained from any mammalian species, e.g., human, bovine, porcine, canine, feline, rodent, e.g., mouse, rat, hamster, primate, etc. In some embodiments, human embryos were not destroyed for the source of pluripotent cells used in the methods and compositions disclosed herein.

[0137]

[0144] A mixture of cells from a suitable source of endothelial, muscle, and / or neural stem cells can be collected from a mammalian donor by methods known in the art. A suitable source is the hematopoietic microenvironment. For example, preferably fixed (i.e., 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.

[0138]

[0145] In some embodiments, SC-beta cells are selected from the group consisting of hair cells, keratinocytes, gonadotropes, corticotropes, thyrotropes, somatotropes, mammary glands, chromaffin cells, parafollicular cells, glomus cells, melanocytes, nevus cells, Merkel cells, odontoblasts, cementoblasts, corneal keratinocytes, retinal muller 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, Cells include: peritoneal septal cells, pancreatic stellate cells, pancreatic α cells, pancreatic β cells, pancreatic δ cells, pancreatic F cells (e.g., PP cells), pancreatic ε cells, thyroid gland (e.g., follicular cells), parathyroid gland (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, and 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.

[0139] Use of small molecule compounds for beta cell differentiation

[0146] Provided herein are compositions and methods related to the differentiation of stem cells into pancreatic lineage cells, including small molecule compounds, such as those that can replace or supplement the use of growth factor(s) from the TGF-β superfamily.In some cases, the compositions and methods disclosed herein relate to the differentiation of stem cells into, for example, Sox17-positive cells (e.g., definitive endoderm cells), FOXA2-positive cells (e.g., primitive gut cells), Pdx1-positive cells (pancreatic progenitor cells, e.g., Pdx1-positive, Nkx6.1-negative pancreatic progenitor 1 cells, or Pdx1-positive, Nkx6.1-positive pancreatic progenitor 2 cells), insulin-positive pancreatic endocrine cells, or β cells (e.g., stem cell-derived β cells, or non-native pancreatic β cells).In some embodiments, the small molecule compound comprises an inhibitor of PI3K / Akt / mTOR signaling, such as GSK690693 or its analogue.

[0140] Stem cell differentiation

[0147] In some embodiments, the methods provided herein relate to differentiation of stem cells (e.g., pluripotent stem cells, e.g., iPSCs or hESCs) by contacting the stem cells with an inhibitor of PI3K / Akt / mTOR signaling. In some embodiments, contacting the stem cells with one or more inhibitors of PI3K / Akt / mTOR signaling results in the production of a population of cells comprising Sox17-positive cells (e.g., definitive endoderm cells).

[0141]

[0148] In some cases, the methods disclosed herein include contacting a plurality of stem cells with an inhibitor of PI3K / Akt / mTOR signaling and a growth factor from the TGF-β superfamily (e.g., activin A). In some cases, the methods disclosed herein also include contacting the stem cells with an activator of the WNT signaling pathway in addition to the inhibitor of PI3K / Akt / mTOR signaling. In some cases, the methods disclosed herein include contacting the stem cells with an activator of the WNT signaling pathway, an inhibitor of PI3K / Akt / mTOR signaling, and a growth factor from the TGF-β superfamily.

[0142]

[0149] The method disclosed herein can utilize a reduced amount of growth factor from the TGF-β superfamily when compared with a reference method that does not include an inhibitor of PI3K / Akt / mTOR signaling.For example, in the presence of an inhibitor of PI3K / Akt / mTOR signaling, a growth factor from the TGF-β superfamily (e.g., activin A) can be applied at a concentration of at most 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or 5% of the concentration applied in the absence of an inhibitor of PI3K / Akt / mTOR signaling for the differentiation of at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45% or 40% of stem cells into Sox17-positive cells (e.g., definitive endoderm cells). In some cases, a growth factor from the TGF-β superfamily (e.g., activin A) can be applied at a concentration of about 50%, 40%, 30%, 20%, 10%, 5%, 1%, or 0% of the concentration applied in the absence of an inhibitor of PI3K / Akt / mTOR signaling for differentiation of at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40% of stem cells into Sox17-positive cells (e.g., definitive endoderm cells).

[0143]

[0150] Contacting stem cells with an inhibitor of PI3K / Akt / mTOR signaling according to the present disclosure can result in the production of a population of cells having cellular components comparable to a population of cells produced by a reference method, which includes contacting stem cells with about 100 ng / mL of activin A but not with an inhibitor of PI3K / Akt / mTOR signaling, but is otherwise identical to the present method. For example, a population of cells produced according to the present disclosure can have a percentage of Sox17-positive cells equivalent to the percentage of Sox17-positive cells in a population of cells produced by the reference method (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%). In some cases, a population of cells produced according to the present disclosure can have a percentage of Sox17-positive, Oct4-negative cells equivalent to the percentage of Sox17-positive, Oct4-negative cells in a population of cells produced by the reference method.

[0144]

[0151] In some cases, contacting stem cells with an inhibitor of PI3K / Akt / mTOR signaling according to the present disclosure can result in the production of a population of cells comprising at least about 50%, 60%, 65%, 70%, 75%, 80%, or 85% Sox17-positive, Oct4-negative cells. In some cases, contacting stem cells with an inhibitor of PI3K / Akt / mTOR signaling can result in the production of a population of cells comprising about 50% to about 90%, about 60% to about 90%, about 65% to about 90%, about 70% to about 90%, about 75% to about 90%, about 80% to about 90%, or about 75% to about 85% Sox17-positive, Oct4-negative cells. In some embodiments, contacting stem cells with an inhibitor of PI3K / Akt / mTOR signaling in accordance with the present disclosure may result in the production of a population of cells comprising 50-90%, 50-85%, 50-75%, 50-65%, 60-90%, 60-85%, 60-75%, 70-90%, 70-85%, 80-85%, or 80-90% Sox17-positive, Oct4-negative cells.

[0145]

[0152] In some cases, Sox17-positive cells (e.g., definitive endoderm cells) can be obtained by contacting a population of stem cells with any one or more of: i) at least one growth factor from the TGF-β superfamily, ii) a WNT signaling pathway activator, and optionally, iii) any one or more of any of the inhibitors of PI3K / Akt / mTOR signaling disclosed herein (e.g., GSK-690693 and / or BYL719) to induce differentiation of at least a portion of the stem cells into definitive endoderm cells, wherein the definitive endoderm cells express at least one marker characteristic of definitive endoderm, e.g., Sox17.

[0146]

[0153] PI3K / Akt / mTOR signaling inhibitors that can be used in differentiating stem cells into Sox17-positive cells include PI3K inhibitors, Akt protein inhibitors, mTOR inhibitors, or any combination thereof. For example, small molecule compounds such as GSK-690693, IPI-3063, AZD8055, omipalisib, GNE-477, VS-5584, BYL319, YM201636, PI4KIII beta-IN-10, nemiralisib, BYL719, FT113, apitolisib, or any analogs or derivatives thereof can be used in differentiating stem cells into Sox17-positive cells. In some cases, the method includes contacting stem cells with both a PI3K protein inhibitor and an Akt protein inhibitor, for example, BYL719 and GSK-690693. In some cases, the method includes contacting the stem cells with an inhibitor of an Akt protein (e.g., GSK-690693) and a growth factor from the TGF-β superfamily (e.g., activin A).

[0147]

[0154] In some embodiments, the method includes differentiating the stem cells into Sox17-positive cells (e.g., definitive endoderm cells) by contacting the stem cell population with an inhibitor of PI3K / Akt / mTOR signaling at a suitable concentration. For example, in some cases, the method includes differentiating the stem cells into Sox17-positive cells (e.g., definitive endoderm cells) by contacting the stem cell population with about 0.01 μM to about 1 μM, about 0.02 μM to about 0.8 μM, about 0.05 μM to about 0.5 μM, about 0.06 μM to about 0.2 μM, about 0.07 μM to about 0.15 μM, or about 0.08 μM to about 0.12 μM of GSK-690693, an analog, or a derivative thereof. In some cases, the method includes differentiating the stem cells into Sox17-positive cells (e.g., definitive endoderm cells) by contacting the population of stem cells with about 0.01 μM, 0.02 μM, 0.04 μM, 0.06 μM, 0.08 μM, 0.1 μM, 0.12 μM, 0.15 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.8 μM, or 1 μM of GSK-690693, an analog or derivative thereof.

[0148]

[0155] In some instances, the method includes differentiating the stem cells into Sox17-positive cells (e.g., definitive endoderm cells) by contacting the population of stem cells with about 1 nM to about 500 nM, about 5 nM to about 250 nM, about 10 nM to about 200 nM, about 15 nM to about 150 nM, about 20 nM to about 100 nM, about 30 nM to about 80 nM, about 30 nM to about 60 nM, or about 35 nM to about 50 nM of BYL719, an analog, or a derivative thereof. In some cases, the method includes differentiating the stem cells into Sox17-positive cells (e.g., definitive endoderm cells) by contacting the population of stem cells with about 1 nM, 4 nM, 8 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 80 nM, 90 nM, 100 nM, 200 nM, or 400 nM of BYL719, an analog, or a derivative thereof.

[0149]

[0156] In some instances, the method includes treating the population of stem cells with between about 0.01 μM and about 1 μM, between about 0.02 μM and about 0.8 μM, between about 0.05 μM and about 0.5 μM, between about 0.06 μM and about 0.2 μM, or between about 0.07 μM and about 0.15 μM of GSK-690693, and between about 1 nM and about 500 nM, between about 5 nM and about 250 nM, between about 10 nM and about 200 nM, between about 15 nM and about 150 nM, between about 20 nM and about 250 nM, between about 20 nM and about 250 nM, between about 20 nM and about 250 nM, between about 25 ... The method includes a step of differentiating stem cells into Sox17-positive cells (e.g., definitive endoderm cells) by contacting the stem cells with 0 nM to about 100 nM, about 30 nM to about 80 nM, about 30 nM to about 60 nM, or about 35 nM to about 50 nM of BYL719, for example, with about 0.08 μM to about 0.12 μM of GSK-690693 and about 35 nM to about 50 nM of BYL719.

[0150]

[0157] In some cases, the method includes differentiating the stem cells into Sox17-positive cells (e.g., definitive endoderm cells) by contacting a population of stem cells with a WNT signaling pathway activator (e.g., CHIR99021) at an appropriate concentration, for example, about 0.01 μM, about 0.05 μM, about 0.1 μM, about 0.2 μM, about 0.5 μM, about 0.8 μM, about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM, about 5 μM, about 8 μM, about 10 μM, about 12 μM, about 15 μM, about 20 μM, about 30 μM, about 50 μM, about 100 μM, or about 200 μM. In some cases, the method includes using about 2 μM CHIR99021 to differentiate the stem cells into definitive endoderm cells. In some cases, the method includes using about 3 μM CHIR99021 for differentiating stem cells into definitive endoderm cells. In some cases, the method includes differentiating the stem cells into Sox17-positive cells (e.g., definitive endoderm cells) by contacting the population of stem cells with a WNT signaling pathway activator (e.g., CHIR99021) at an appropriate concentration of 0.5-10 μM, 1-10 μM, 1-7 μM, 1-5 μM, 2-4 μM, or 2.5-3.5 μM.

[0151]

[0158] Any growth factor from the TGF-β superfamily that can induce stem cells to differentiate into definitive endoderm cells (e.g., alone or in combination with a WNT signaling pathway activator and / or an inhibitor of PI3K / Akt / mTOR signaling) can be used in the methods provided herein. In some cases, a growth factor from the TGF-β superfamily comprises activin A. In some cases, a growth factor from the TGF-β superfamily comprises growth differentiation factor 8 (GDF8). Any WNT signaling pathway activator that can induce pluripotent stem cells to differentiate into definitive endoderm cells (e.g., alone or in combination with a growth factor from the TGF-β superfamily and / or an inhibitor of PI3K / Akt / mTOR signaling) can be used in the methods provided herein. In some cases, 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 cases, the WNT signaling pathway activator comprises Wnt3a recombinant protein or a functional variant thereof.

[0152]

[0159] In some examples, the method includes differentiating the stem cells into definitive endoderm cells by contacting the population of stem cells with an appropriate concentration, for example, about 5 ng / mL, 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, or about 100 ng / mL, or about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 12 ng / mL, about 14 ng / mL, about 15 ng / mL, about 18 ng / mL, or about 25 ng / mL of a growth factor from the TGF-β superfamily (e.g., activin A). In some cases, the method includes using about 10 ng / mL of activin A to differentiate the stem cells into definitive endoderm cells. In some instances, the methods involve using about 100 ng / mL of activin A to differentiate stem cells into definitive endoderm cells. In some instances, the methods involve using 10-200 ng / mL, 10-400 ng / mL, 10-150 ng / mL, 10-120 ng / mL, 90-120 ng / mL, 95-105 ng / mL, 1-20 ng / mL, 5-25 ng / mL, 5-50 ng / mL, 10-50 ng / mL, 5-15 ng / mL, or 8-12 ng / mL of activin A to differentiate stem cells into definitive endoderm cells.

[0153]

[0160] In some cases, Sox17-positive cells (e.g., definitive endoderm cells) can be obtained by culturing stem cells in a composition comprising an inhibitor of PI3K / Akt / mTOR signaling for about 24 hours to about 96 hours, about 36 hours to about 84 hours, about 48 hours to about 84 hours, about 60 hours to about 84 hours, for example, about 1 day, about 2 days, or about 3 days. In some cases, Sox17-positive cells (e.g., definitive endoderm cells) can be obtained by culturing stem cells in a composition comprising an inhibitor of PI3K / Akt / mTOR and a growth factor from the TGF-β superfamily (e.g., activin A) for about 24 hours to about 96 hours, about 36 hours to about 84 hours, about 48 hours to about 84 hours, about 60 hours to about 84 hours, for example, about 1 day, about 2 days, or about 3 days.

[0154]

[0161] In some cases, the method includes a two-step protocol for treating stem cells. For example, the method may include culturing stem cells in a first composition comprising an inhibitor of PI3K / Akt / mTOR signaling and an activator of the WNT signaling pathway for 12 to 48 hours, 12 to 36 hours, 18 to 30 hours, or about 1 day. Following the culturing in the first composition, the method may further include culturing the resulting cell population in a second composition comprising an inhibitor of PI3K / Akt / mTOR signaling for 12 to 72 hours, 24 to 72 hours, or 36 to 72 hours, e.g., about 1 day, or about 2 days.

[0155]

[0162] In some cases, differentiating at least some of the stem cells in the population into definitive endoderm cells is achieved by a process of contacting the population of stem cells with i) an inhibitor of PI3K / Akt / mTOR and ii) CHIR99021 for a suitable period of time, e.g., about 1 day, and then contacting the resulting population of cells with an inhibitor of PI3K / Akt / mTOR for a suitable period of time, e.g., about 1 day, about 2 days, about 3 days, about 4 days, or about 5 days, to induce differentiation of at least some of the stem cells in the population into definitive endoderm cells, wherein the definitive endoderm cells express at least one marker characteristic of definitive endoderm, e.g., Sox17.

[0156]

[0163] In some cases, differentiating at least some of the stem cells in the population into definitive endoderm cells is achieved by a process of contacting the population of stem cells with i) an inhibitor of PI3K / Akt / mTOR, ii) CHIR99021, and iii) activin A for a suitable period of time, for example, about 1 day, and then contacting the resulting population of cells with i) an inhibitor of PI3K / Akt / mTOR and ii) activin A for a suitable period of time, for example, about 1 day, about 2 days, about 3 days, about 4 days, or about 5 days, to induce differentiation of at least some of the stem cells in the population into definitive endoderm cells, wherein the definitive endoderm cells express at least one marker characteristic of definitive endoderm, for example, Sox17.

[0157]

[0164] In some cases, 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 pluripotent stem cells from which they are derived. In some cases, 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 pluripotent stem cells from which they are derived. In some cases, the definitive endoderm cells generated by the methods disclosed herein do not express at least one marker selected from the group consisting of Zic1, Pax6, Flk1, and CD31 at a statistically significant amount relative to the pluripotent stem cells from which they are derived. In some cases, the definitive endoderm cells generated by the methods disclosed herein have a statistically significantly higher level of Smad2 phosphorylation relative to the pluripotent stem cells from which they are derived. In some cases, the definitive endoderm cells generated by the methods disclosed herein have the ability to form a gut tube in vivo. In some cases, the definitive endoderm cells generated by the methods disclosed herein can differentiate into cells having morphology characteristic of intestinal cells, and the cells having morphology characteristic of intestinal cells express FoxA2 and / or Claudin6. In some cases, the definitive endoderm cells generated by the methods disclosed herein can further differentiate into cells of endodermal origin.

[0158]

[0165] In some cases, 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, such as human pluripotent stem cells or human iPS cells, or any pluripotent stem cells discussed herein or other suitable pluripotent stem cells, can be used. In some cases, 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.

[0159] Differentiation of FOXA2-positive, PDX1-negative cells

[0166] In some embodiments, the methods provided herein relate to differentiation of FOXA2-positive, PDX1-negative cells (e.g., primitive gut cells) by contacting a plurality of FOXA2-positive, PDX1-negative cells with an inhibitor of PI3K / Akt / mTOR signaling. In some embodiments, contacting the FOXA2-positive, PDX1-negative cells with the inhibitor of PI3K / Akt / mTOR signaling results in the production of a population of cells comprising PDX1-positive cells (e.g., PDX1-positive cells, NKX6.1-negative cells, e.g., pancreatic progenitor 1 cells).

[0160]

[0167] In some cases, the methods disclosed herein include contacting a plurality of FOXA2-positive, PDX1-negative cells with an inhibitor of PI3K / Akt / mTOR signaling and 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, v) at least one protein kinase C activator, vi) a ROCK inhibitor, and vii) a growth factor from the TGF-β superfamily. In some cases, the methods disclosed herein include contacting a plurality of FOXA2-positive, PDX1-negative cells with an inhibitor of PI3K / Akt / mTOR signaling and 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, v) at least one protein kinase C activator, and vi) a ROCK inhibitor, without a growth factor from the TGF-β superfamily.

[0161]

[0168] In some aspects, 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.

[0162]

[0169] 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.

[0163]

[0170] In some cases, the 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.

[0164]

[0171] In some cases, the 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.

[0165]

[0172] In some cases, 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.

[0166]

[0173] In some embodiments, the methods disclosed herein utilize reduced amounts of growth factors from the TGF-β superfamily when compared to a reference method that does not include an inhibitor of PI3K / Akt / mTOR signaling for the differentiation of FOXA2-positive, PDX1-negative cells (e.g., primitive gut cells) into PDX1-positive cells. For example, in the presence of an inhibitor of PI3K / Akt / mTOR signaling, a growth factor from the TGF-β superfamily (e.g., activin A) can be applied at a concentration of at most 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% of the concentration applied in the absence of an inhibitor of PI3K / Akt / mTOR signaling to differentiate at least 50%, 60%, 70%, 80%, 90%, or 95% (e.g., 50-90%, 50-80%, 50-70%, or 50-60%) of FOXA2-positive, PDX1-negative cells (e.g., gastrula cells) in culture into PDX1-positive cells. In some cases, a growth factor from the TGF-β superfamily (e.g., activin A) can be applied at a concentration of about 50%, 40%, 30%, 20%, 10%, 5%, 1%, or 0% of the concentration applied in the absence of an inhibitor of PI3K / Akt / mTOR signaling to differentiate at least 50%, 60%, 70%, 80%, 90%, or 95% (e.g., 50-90%, 50-80%, 50-70%, or 50-60%) of FOXA2-positive, PDX1-negative cells (e.g., gastrula cells) in culture into PDX1-positive cells.

[0167]

[0174] Any growth factor from the TGF-β superfamily (e.g., 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, alone or in any combination) that can induce primitive gut cells to differentiate into PDX1-positive, NKX6.1-negative pancreatic progenitor cells can be used. In some cases, the growth factor from the TGF-β family includes activin A. In some cases, 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 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. In some embodiments, the concentration of a growth factor from the TGF-β superfamily (e.g., activin A) is 5-50 ng / mL, 15-30 ng / mL, 12-28 ng / mL, 15-25 ng / mL, or 18-22 ng / mL.

[0168]

[0175] In some embodiments, the method includes contacting the gastrula cells with a reduced concentration of a growth factor from the TGF-β superfamily (e.g., activin A), e.g., at most about 5 ng / mL, at most about 2.5 ng / mL, 1 ng / mL, 0.5 ng / mL, 0.1 ng / mL, or 0.05 ng / mL, e.g., at about 2.5 ng / mL, 1 ng / mL, 0.5 ng / mL, 0.1 ng / mL, or 0.05 ng / mL, in the presence of an inhibitor of the PI3K / Akt / mTOR pathway. In some embodiments, the concentration of the growth factor from the TGF-β superfamily (e.g., activin A) is 0.5-5 ng / mL, 1.5-3 ng / mL, 1.2-2.8 ng / mL, 1.5-2.5 ng / mL, or 1.8-2.2 ng / mL.

[0169]

[0176] Any BMP signaling pathway inhibitor (e.g., 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, alone or in any combination) that can induce primitive gut cells to differentiate into PDX1-positive, NKX6.1-negative pancreatic progenitor cells can be used in the methods provided herein. In some cases, the BMP signaling pathway inhibitor comprises LDN193189 or DMH-1. In some examples, the method includes contacting the primitive gut 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. 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. In some examples, the method includes contacting the gastrula cells with a BMP signaling pathway inhibitor (e.g., DMH-1) at a concentration of, for example, 50-1000 nM, 50-500 nM, 50-300 nM, 100-300 nM, 200-300 nM, 200-500 nM, or 225-275 nM.

[0170]

[0177] Any growth factor from the FGF family (e.g., 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, alone or in any combination) that can induce primitive gut cells to differentiate into PDX1-positive, NKX6.1-negative pancreatic progenitor cells can be used. In some cases, the at least one growth factor from the FGF family includes keratinocyte growth factor (KGF). In some cases, 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. 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, 10-200 ng / mL, 10-150 ng / mL, 10-100 ng / mL, 25-75 ng / mL, 40-60 ng / mL, or 45-55 ng / mL.

[0171]

[0178] Any SHH pathway inhibitor (e.g., 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 any combination thereof) capable of inducing differentiation of primitive gut cells into PDX1-positive, NKX6.1-negative pancreatic progenitor cells can be used. In some cases, the SHH pathway inhibitor comprises Sant1. In some examples, the method includes injecting primitive gut cells with a soluble ATP containing, 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 μM, about 10 μM, about 110 μM, about 111 μM, about 112 μM, about 113 μM, about 114 μM, about 115 μM, about 116 μM, about 117 μM, about 118 μM, about 119 μM, about 120 μM, about 121 μM, about 122 μM, about 123 μM, about 124 μM, about 125 μM, about 126 μM, about 1 The method includes contacting the cells with an SHH pathway inhibitor (e.g., Sant1) at a concentration of 3 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. In some examples, the method includes contacting the gastrula cells with an SHH pathway inhibitor (e.g., Sant1) at a concentration of 50-1000 nM, 50-500 nM, 50-300 nM, 100-300 nM, 200-300 nM, 200-500 μM, or 225-275 nM.

[0172]

[0179] 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 cases, 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). In some examples, the method includes contacting the primitive gut cells with an RA signaling pathway activator (e.g., retinoic acid) at a concentration of, for example, 0.2-5 μM, 0.8-3 μM, 0.8-2.5 μM, 1-2.5 μM, 1.5-2.2 μM, 1.8-2.2 μM, or 1.9-2.1 μM.

[0173]

[0180] Any PKC activator capable of inducing primitive gut cells to differentiate into PDX1-positive, NKX6.1-negative pancreatic progenitor cells (e.g., 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, alone or in any combination) can be used. In some cases, the PKC activator comprises PdBU. In some cases, the PKC activator comprises TPB. In some examples, the method includes treating primitive gut cells with a soluble ATP containing ATP at a concentration of, for example, about 10 nM, 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 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 270 μM, about 280 μM, about 290 μM, about 300 μM, about 310 μM, about 320 μM, about 330 μM, about 340 μM, about 350 μM, about 360 μM, about 370 μM, about 380 μM, about 390 μM, about 400 μM, about 410 μM, about 420 μM, about 430 μM, about 440 μM, about 450 μM, about 460 μM, about 470 μM, about 480 μM, about 490 μM, about 500 μM, about 510 μM, about 520 μM, about 530 μM, The method includes contacting the PKC activator (e.g., PdBU) at a concentration of 60 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 primitive gut cells with a PKC activator (e.g., PdBU) at a concentration of 10 nM to 1 mM, 10 nM to 500 μM, 10 nM to 1 μM, 10 to 800 nM, 100 to 900 nM, 300 to 800 nM, 300 to 600 nM, 400 to 600 nM, 450 to 550 nM, or about 500 nM. In some embodiments, the primitive gut cells are not treated with a PKC activator (e.g., PDBU).

[0174]

[0181] Any ROCK 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, at least one SHH pathway inhibitor, a PKC activator, and at least one RA signaling pathway activator) can be used. In some cases, the ROCK inhibitor comprises Thiazovivin, Y-27632, Fasudil / HA1077, or H-1152. In some cases, the ROCK inhibitor comprises Y-27632. In some cases, 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 primary intestinal 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. In some examples, the method includes contacting the primary intestinal cells with a ROCK inhibitor (e.g., Y-27632 or Thiazovivin) at a concentration of 0.2-5 μM, 0.8-3 μM, 1-4 μM, 1.5-4 μM, 1.8-3.5 μM, 2-3 μM, or 2.4-2.6 μM.

[0175]

[0182] In some cases, 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, e.g., about 1 day, about 2 days, about 3 days, about 4 days, 18-72 hours, 36-60 hours, 40-54 hours, or 44-52 hours. In some cases, the 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, 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 two days. In some cases, the PDX1-positive, NKX6.1-negative pancreatic progenitor cells can be obtained by differentiating at least some of the primitive gut cells in S3 medium.

[0176] Differentiation of PDX1-positive, NKX6.1-negative pancreatic progenitor cells

[0183] In some embodiments, the methods provided herein relate to the differentiation of PDX1-positive, NKX6.1-negative cells (e.g., pancreatic progenitor 1 cells) by contacting a plurality of PDX1-positive, NKX6.1-negative cells with an inhibitor of PI3K / Akt / mTOR signaling. In some embodiments, contacting the PDX1-positive, NKX6.1-negative cells with an inhibitor of PI3K / Akt / mTOR signaling results in the production of a population of cells comprising PDX1-positive, NKX6.1-positive cells (e.g., pancreatic progenitor 2 cells).

[0177]

[0184] In some cases, the methods disclosed herein include contacting a plurality of PDX1-positive, NKX6.1-negative cells (e.g., pancreatic progenitor 1 cells) with an inhibitor of PI3K / Akt / mTOR signaling and 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, and optionally, vi) a protein kinase C activator. In some cases, the methods disclosed herein include contacting a plurality of PDX1-positive, NKX6.1-negative cells (e.g., pancreatic progenitor 1 cells) with an inhibitor of PI3K / Akt / mTOR signaling and 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 optionally, v) a protein kinase C activator, without a growth factor from the TGF-β superfamily.

[0178]

[0185] In some embodiments, the methods disclosed herein utilize a reduced amount of a growth factor from the TGF-β superfamily when compared to a reference method that does not include an inhibitor of PI3K / Akt / mTOR signaling for the differentiation of PDX1-positive, NKX6.1-negative cells (e.g., pancreatic progenitor 1 cells) into PDX1-positive, NKX6.1-positive cells (e.g., pancreatic progenitor 2 cells). For example, in the presence of an inhibitor of PI3K / Akt / mTOR signaling, a growth factor from the TGF-β superfamily (e.g., activin A) reduces the differentiation of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% (e.g., 40-90%, 40-80%, 40-70%, 40-80%) of PDX1-positive, NKX6.1-negative cells (e.g., pancreatic progenitor 1 cells) in culture. It can be applied at a concentration of at most 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% of the concentration applied in the absence of inhibitors of PI3K / Akt / mTOR signaling for differentiation into PDX1-positive, NKX6.1-positive cells (e.g., pancreatic progenitor 2 cells) of ~60%, 40-50%, 60-90%, 60-80%, or 70-90% of the total. In some cases, a growth factor from the TGF-β superfamily (e.g., activin A) can be applied at a concentration of about 50%, 40%, 30%, 20%, 10%, 5%, 1%, or 0% of the concentration applied in the absence of an inhibitor of PI3K / Akt / mTOR signaling to differentiate at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% (e.g., 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 60-90%, 60-80%, or 70-90%) of PDX1-positive, NKX6.1-negative cells (e.g., pancreatic progenitor 1 cells) in culture into PDX1-positive, NKX6.1-positive cells (e.g., pancreatic progenitor 2 cells).

[0179]

[0186] 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 (e.g., under conditions that promote cell clustering and / or promote cell survival) with at least two beta cell differentiation factors comprising: a) at least one growth factor from the fibroblast growth factor (FGF) family, b) a sonic hedgehog pathway inhibitor, and, optionally, c) a 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.

[0180]

[0187] In some cases, 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) an RA signaling pathway activator to induce differentiation of the PDX1-positive, NKX6.1-negative pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells.

[0181]

[0188] In some cases, the PDX1-positive, NKX6.1-positive pancreatic progenitor cells are cultured 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 SHH pathway inhibitor, and optionally, iii) an activator of the RA signaling pathway, iv) a ROCK inhibitor, and v) at least one growth factor from the TGF-β superfamily to induce differentiation of at least a portion (e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% (e.g., 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 60-90%, 60-80%) of the PDX1-positive, NKX6.1-negative pancreatic progenitor cells in the culture. The PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by inducing differentiation of 0%, or 70-90%, of PDX1-positive, NKX6.1-positive pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells. In some cases, the PDX1-positive, NKX6.1-negative pancreatic progenitor cells are obtained by contacting the 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, the PDX1-positive, NKX6.1-negative pancreatic progenitor cells are contacted with a PKC activator (e.g., PDBU). See, for example, U.S. Patent Application Publication Nos. 20210238553A1 and 20220143374A1, which are incorporated by reference in their entireties.

[0182]

[0189] Any growth factor from the FGF family (for example, alone or in any 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) that can induce PDX1 positive, NKX6.1 negative pancreatic progenitor cells to differentiate into PDX1 positive, NKX6.1 positive pancreatic progenitor cells can be used in the methods provided herein.In some cases, the at least one growth factor from the FGF family comprises keratinocyte growth factor (KGF).In some cases, 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) 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. 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) at a concentration of, for example, 10-200 ng / mL, 10-150 ng / mL, 10-100 ng / mL, 25-75 ng / mL, 40-60 ng / mL, or 45-55 ng / mL.

[0183]

[0190] Any SHH pathway inhibitor (for example, alone or in any 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) that can induce PDX1-positive, NKX6.1-negative pancreatic progenitor cells to differentiate into PDX1-positive, NKX6.1-positive pancreatic progenitor cells can be used in the methods provided herein. In some cases, the SHH pathway inhibitor comprises Sant1. In some examples, the methods include administering to the PDX1-positive, NKX6.1-negative pancreatic progenitor cells 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 cell culture medium 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. In some examples, the method includes contacting PDX1-positive, NKX6.1-negative pancreatic progenitor cells with an SHH pathway inhibitor (e.g., Sant1) at a concentration of, for example, 50-1000 nM, 50-500 nM, 50-300 nM, 100-300 nM, 200-300 nM, 200-500 nM, or 225-275 nM.

[0184]

[0191] 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 cases, 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. In some examples, the method includes contacting PDX1-positive, NKX6.1-negative pancreatic progenitor cells with a RA signaling pathway activator (e.g., retinoic acid) at a concentration of 1-500 nM, 50-400 nM, 50-250 nM, 50-150 nM, 80-200 nM, 75-125 nM, or 90-110 nM, or the like.

[0185]

[0192] 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 (e.g., alone or in combination with at least one growth factor from the FGF family, at least one SHH pathway inhibitor, an RA signaling pathway activator, and at least one growth factor from the TGF-β superfamily) can be used. In some cases, the ROCK inhibitor includes Thiazovivin, Y-27632, Fasudil / HA1077, or 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 PDX1-positive, NKX6.1-negative pancreatic progenitor cells with a ROCK inhibitor (e.g., Y-27632 or Thiazovivin) at a concentration of 0.2-5 μM, 0.8-3 μM, 1-4 μM, 1.5-4 μM, 1.8-3.5 μM, 2-3 μM, or 2.4-2.6 μM.

[0186]

[0193] Any activator from the TGF-β superfamily 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, an RA signaling pathway activator, and a ROCK inhibitor) can be used. In some cases, 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 growth factor from the TGF-β superfamily (e.g., activin A) at a concentration of about 5 ng / mL, or in some embodiments, the concentration of the growth factor from the TGF-β superfamily (e.g., activin A) is 1-15 ng / mL, 3-12 ng / mL, 5-12 ng / mL, 5-20 ng / mL, 8-20 ng / mL, 8-15 ng / mL, 9-11 ng / mL, or 8-12 ng / mL.

[0187]

[0194] In some embodiments, in the presence of an inhibitor of the PI3K / Akt / mTOR pathway, the method includes contacting the primitive gut cells with a reduced concentration of a growth factor from the TGF-β superfamily (e.g., activin A), e.g., at most about 20 ng / mL, at most about 10 ng / mL, 5 ng / mL, 1 ng / mL, 0.5 ng / mL, or 0.1 ng / mL, e.g., at most about 10 ng / mL, 5 ng / mL, 1 ng / mL, 0.5 ng / mL, or 0.1 ng / mL. In some embodiments, the concentration of a growth factor from the TGF-β superfamily (e.g., activin A) is 0.1-1.5 ng / mL, 0.3-1.2 ng / mL, 0.5-1.2 ng / mL, 0.5-2.0 ng / mL, 0.8-2.0 ng / mL, 0.8-1.5 ng / mL, 0.9-1.1 ng / mL, or 0.8-1.2 ng / mL.

[0188]

[0195] In some cases, the 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, and optionally, an inhibitor of PI3K / Akt / mTOR signaling, under conditions that promote cell clustering, for 5 or 6 days or 96-170 hours, 120-170 hours, 130-160 hours, or 140-150 hours. In some cases, the 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, and optionally, an inhibitor of PI3K / Akt / mTOR signaling, under conditions that promote cell clustering, for 5 or 6 days or 96-170 hours, 120-170 hours, 130-160 hours, or 140-150 hours. In some cases, the PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting the PDX1-positive, NKX6.1-negative pancreatic progenitor cells with KGF for 5 or 6 days, or for 96-170 hours, 120-170 hours, 130-160 hours, or 140-150 hours under conditions that promote cell clustering. In some cases, the PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting the PDX1-positive, NKX6.1-negative pancreatic progenitor cells in S4 medium.

[0189]

[0196] In some cases, the PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting the PDX1-positive, NKX6.1-negative pancreatic progenitor cells with KGF, Sant1, RA, thiazovivin, and activin A, and optionally, an inhibitor of PI3K / Akt / mTOR signaling, under conditions that promote cell clustering, for 5 or 6 days or 96-170 hours, 120-170 hours, 130-160 hours, or 140-150 hours.

[0190] Methods for producing stem cell-derived beta cells

[0197] 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. 20150240212, 20150218522, 20210198632A1, 20210238553A1, and 20220143374A1, each of which is incorporated by reference in its entirety.

[0191]

[0198] 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.

[0192]

[0199] 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.

[0193]

[0200] Precise patterning of the definitive endoderm along the anterior-posterior axis ultimately leads to the formation of the primitive gut. The definitive endoderm-derived primitive gut 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).

[0194]

[0201] Successful differentiation of pancreatic beta cells requires that the differentiated cells synthesize and secrete physiologically appropriate amounts of insulin. An exemplary stepwise protocol for directing hPSC differentiation has been developed, involving a differentiation process that recapitulates key stages of normal pancreatic endocrine development. Differentiation of hPSCs into hormone-expressing pancreatic endocrine cells is achieved by transitioning hPSCs 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, hPSCs can acquire a pancreatic endocrine phenotype and the ability to secrete insulin in response to glucose in vitro.

[0195]

[0202] Generally, at least one pancreatic alpha, beta and / or delta cell or precursor thereof, e.g., pancreatic progenitor cells generated 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 co-expressing 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.

[0196]

[0203] The at least one pancreatic α, β, and / or δ 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 α, β, and / or δ cell, or precursor thereof, is produced by culturing at least one pluripotent cell for a time and under conditions suitable for the at least one pluripotent cell to differentiate into at least one pancreatic α, β, and / or δ cell, or precursor thereof.

[0197]

[0204] 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.

[0198]

[0205] 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.

[0199]

[0206] 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.

[0200]

[0207] 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.

[0201] Definitive endoderm cells

[0208] Embodiments of the present disclosure include definitive endoderm cells. As used herein, definitive endoderm cells may be obtained from any source or produced according to any suitable protocol, including methods disclosed herein that involve the use of small molecule compounds, such as inhibitors of PI3K / Akt / mTOR signaling.

[0202]

[0209] Definitive endoderm is produced in vivo from the inner cell mass by the embryonic process of gastrulation, in 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 lining 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.

[0203]

[0210] 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).

[0204]

[0211] As described herein, the definitive endoderm cell used herein can be derived from any source or produced according to any suitable protocol.In some embodiments, pluripotent stem cells, such as iPSCs or hESCs, are differentiated into endoderm cells.In some embodiments, 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 progenitor cells or insulin positive endocrine cells (stage 5), and then are induced into SC-β cells or matured (stage 6).

[0205]

[0212] In some embodiments, definitive endoderm cells can be obtained by differentiating at least some of the pluripotent cells in the population into definitive endoderm cells, for example, by contacting the 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.

[0206]

[0213] Any growth factor from the TGF-β superfamily (e.g., alone or in combination with a WNT signaling pathway activator) capable of inducing pluripotent stem cells to differentiate into definitive endoderm cells can be used in the methods provided herein. In some embodiments, the growth factor from the TGF-β superfamily comprises activin A. In some embodiments, the growth factor from the TGF-β superfamily comprises growth differentiation factor 8 (GDF8). Any WNT signaling pathway activator (e.g., alone or in combination with a growth factor from the TGF-β superfamily) capable of inducing pluripotent stem cells to differentiate into definitive endoderm cells can be used in the methods provided herein. In some embodiments, the WNT signaling pathway activator comprises CHIR99021. In some embodiments, the WNT signaling pathway activator comprises Wnt3a recombinant protein.

[0207]

[0214] In some embodiments, 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 embodiments, the process comprises contacting the population of pluripotent cells with activin A and CHIR99021 for 1 day, followed by activin A (in the absence of CHIR99021) for an additional 1 or 2 days.

[0208]

[0215] In some examples, the method includes differentiating the pluripotent cells into definitive endoderm cells by contacting the pluripotent cells with an appropriate 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 of a growth factor from the TGF-β superfamily (e.g., activin A). 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 embodiments, the methods involve the use of about 70-130 ng / ml, 80-120 ng / ml, or 90-110 ng / ml of activin A for the differentiation of pluripotent cells to definitive endoderm cells. In some embodiments, the methods involve the use of about 200 ng / mL of activin A for the differentiation of pluripotent cells to definitive endoderm cells.

[0209]

[0216] In some examples, the methods include differentiating pluripotent cells into definitive endoderm cells by contacting the pluripotent cells with a WNT signaling pathway activator (e.g., CHIR99021) at an appropriate concentration, e.g., about 0.01 μM, about 0.05 μM, about 0.1 μM, about 0.2 μM, about 0.5 μM, about 0.8 μM, about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM, about 5 μM, about 8 μM, about 10 μM, about 12 μM, about 15 μM, about 20 μM, about 30 μM, about 50 μM, about 100 μM, or about 200 μM. In some embodiments, the methods include using about 1-5 μM or 2-4 μM CHIR99021 for the differentiation of pluripotent cells into definitive endoderm cells. In some embodiments, the method comprises the use of about 2 μM CHIR99021 for the differentiation of pluripotent cells to definitive endoderm cells. In some embodiments, the method comprises the use of about 3 μM CHIR99021 for the differentiation of pluripotent cells to definitive endoderm cells. In some embodiments, the method comprises the use of about 5 μM CHIR99021 for the differentiation of pluripotent cells to definitive endoderm cells.

[0210]

[0217] In some embodiments, the cells are further contacted with a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol. In some cases, the polyvinyl alcohol is at least 78% hydrolyzed, e.g., 79-81% hydrolyzed, 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 PVA is 80% hydrolyzed.

[0211]

[0218] In some embodiments, 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 pluripotent stem cells from which they were derived. In some embodiments, 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 pluripotent stem cells from which they were derived. In some embodiments, the definitive endoderm cells generated by the methods disclosed herein do not express a statistically significant amount of at least one marker selected from the group consisting of Zic1, Pax6, Flk1, and CD31 compared to the pluripotent stem cells from which they were derived. In some embodiments, the definitive endoderm cells generated by the methods disclosed herein have a statistically significant higher level of Smad2 phosphorylation relative to the pluripotent stem cells from which they were derived. In some embodiments, the definitive endoderm cells generated by the methods disclosed herein have the ability to form a gut tube in vivo. In some embodiments, the definitive endoderm cells generated by the methods disclosed herein can be differentiated into cells having morphology characteristic of intestinal cells, and the cells having morphology characteristic of intestinal cells express FoxA2 and / or Claudin6. In some embodiments, the definitive endoderm cells generated by the methods disclosed herein can be further differentiated into cells of endodermal origin.

[0212]

[0219] In some embodiments, 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 of the pluripotent stem cells discussed herein or other suitable pluripotent stem cells. In some embodiments, 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.

[0213] gastrula cells

[0220] 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 be induced into SC-β cells or mature.

[0214]

[0221] In some embodiments, the primitive gut cells can be obtained by differentiating at least some of the definitive endoderm cells in the population into primitive gut cells, e.g., 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.

[0215]

[0222] Any growth factor from the FGF family that can induce definitive endoderm cells to differentiate into primitive gut cells (e.g., alone or in combination with other factors) can be used in the methods provided herein. In some embodiments, at least one growth factor from the FGF family comprises keratinocyte growth factor (KGF). In some embodiments, at least one growth factor from the FGF family comprises FGF2. In some embodiments, at least one growth factor from the FGF family comprises FGF8B. In some embodiments, at least one growth factor from the FGF family comprises FGF10. In some embodiments, at least one growth factor from the FGF family comprises FGF21.

[0216]

[0223] In some embodiments, primitive gut tube cells can be obtained by differentiating at least some of the definitive endoderm cells in the population into primitive gut tube cells, for example, by contacting the definitive endoderm cells with KGF for a certain period of time, e.g., about 1 day, about 2 days, about 3 days, or about 4 days, 24-96 hours, 50-80 hours, 60-80 hours, or 65-75 hours, to induce differentiation of at least some of the definitive endoderm cells into primitive gut tube cells.

[0217]

[0224] In some embodiments, the method comprises differentiating the definitive endoderm cells into primitive gut cells by contacting the definitive endoderm cells with a growth factor from the FGF family (e.g., KGF) at an appropriate concentration, e.g., 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 embodiments, the method comprises using about 50 ng / mL of KGF for the differentiation of the definitive endoderm cells into primitive gut cells. In some embodiments, the methods comprise the use of about 100 ng / mL of KGF for the differentiation of definitive endoderm cells into primitive gut cells.

[0218]

[0225] In some embodiments, the cells are further contacted with a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol. In some examples, the polyvinyl alcohol is at least 78% hydrolyzed, e.g., 79-81% hydrolyzed, 87-89% hydrolyzed, 87-90% hydrolyzed, or 99% hydrolyzed. In some embodiments, the polyvinyl alcohol (PVA) 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 PVA is 80% hydrolyzed.

[0219]

[0226] In some embodiments, the cells are contacted with any of the inhibitors of PI3K / Akt / mTOR signaling as disclosed herein. PDX1-positive pancreatic progenitor cells

[0227] Embodiments of the present disclosure include PDX1-positive, NKX6.1-negative pancreatic progenitor cells. As used herein, PDX1-positive, NKX6.1-negative pancreatic progenitor cells can be obtained from any source or produced according to any suitable protocol, including the methods disclosed herein, including the use of small molecule compounds such as inhibitors of PI3K / Akt / mTOR signaling.

[0220]

[0228] In some embodiments, the primitive gut cells are differentiated into PDX1-positive pancreatic progenitor cells (e.g., PDX1-positive, NKX6.1-negative cells). In some embodiments, the PDX1-positive pancreatic progenitor cells are NKX6.1-negative and can be further differentiated into, for example, NKX6.1-positive pancreatic progenitor cells, Ngn3-positive endocrine progenitor cells, or insulin-positive endocrine cells, followed by induction or maturation into SC-β cells.

[0221]

[0229] In some aspects, PDX1-positive pancreatic progenitor cells can be obtained by differentiating at least a portion of the primitive gut cells in the population into PDX1-positive pancreatic progenitor cells, for example, by contacting the primitive gut cells with i) at least one BMP signaling pathway inhibitor, ii) a growth factor from the TGF-β superfamily, iii) at least one growth factor from the FGF family, iv) at least one SHH pathway inhibitor, v) at least one retinoic acid (RA) signaling pathway activator, vi) at least one protein kinase C activator, and vii) a ROCK inhibitor, to induce differentiation of at least a portion of the primitive gut cells into PDX1-positive pancreatic progenitor cells, wherein the PDX1-positive pancreatic progenitor cells express PDX1.

[0222]

[0230] In some aspects, PDX1-positive pancreatic progenitor cells can be obtained by differentiating at least a portion of the primitive gut cells in the population into PDX1-positive pancreatic progenitor cells, for example, by contacting the primitive gut cells with i) at least one BMP signaling pathway inhibitor, ii) a growth factor from the TGF-β superfamily, iii) at least one growth factor from the FGF family, iv) at least one SHH pathway inhibitor, v) at least one retinoic acid (RA) signaling pathway activator, and vi) at least one protein kinase C activator, to induce differentiation of at least a portion of the primitive gut cells into PDX1-positive pancreatic progenitor cells, wherein the PDX1-positive pancreatic progenitor cells express PDX1.

[0223]

[0231] In some embodiments, PDX1-positive pancreatic progenitor cells can be obtained by differentiating at least a portion of the primitive gut cells in the population into PDX1-positive pancreatic progenitor cells, for example, by contacting the primitive gut cells with i) at least one BMP signaling pathway inhibitor, ii) at least one growth factor from the FGF family, iii) at least one SHH pathway inhibitor, iv) at least one retinoic acid (RA) signaling pathway activator, and v) at least one protein kinase C activator, to induce differentiation of at least a portion of the primitive gut cells into PDX1-positive pancreatic progenitor cells, wherein the PDX1-positive pancreatic progenitor cells express PDX1.

[0224]

[0232] In some embodiments, PDX1-positive pancreatic progenitor cells can be obtained by differentiating at least a portion of the primitive gut cells in the population into PDX1-positive pancreatic progenitor cells, for example, by contacting the primitive gut cells with i) at least one SHH pathway inhibitor, ii) at least one retinoic acid (RA) signaling pathway activator, and iii) at least one protein kinase C activator, wherein the PDX1-positive pancreatic progenitor cells express PDX1.

[0225]

[0233] In some embodiments, PDX1-positive pancreatic progenitor cells can be obtained by differentiating at least a portion of the primitive gut cells in the population into PDX1-positive pancreatic progenitor cells, for example, by contacting the primitive gut cells with i) at least one growth factor from the FGF family and ii) at least one retinoic acid (RA) signaling pathway activator to induce differentiation of at least a portion of the primitive gut cells into PDX1-positive pancreatic progenitor cells, wherein the PDX1-positive pancreatic progenitor cells express PDX1.

[0226]

[0234] Any BMP signaling pathway inhibitor that can induce primitive gut cells to differentiate into PDX1-positive pancreatic progenitor cells (e.g., alone or in combination with a growth factor from the TGF-β superfamily, at least one growth factor from the FGF family, at least one SHH pathway inhibitor, at least one retinoic acid signaling pathway activator, at least one protein kinase C activator, and a ROCK inhibitor) can be used in the methods provided herein. In some embodiments, the BMP signaling pathway inhibitor comprises 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. In some examples, the method includes contacting the gastrula cells with a BMP signaling pathway inhibitor (e.g., DMH-1) at a concentration of about 220-280 nM, about 230-270 nM, about 240-260 nM, or about 245-255 nM, etc. In some examples, the method includes contacting the gastrula cells with a BMP signaling pathway inhibitor (e.g., DMH-1) at a concentration of about 250 nM.

[0227]

[0235] Any growth factor from the TGF-β superfamily (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) that can induce differentiation of primitive gut cells into PDX1-positive pancreatic progenitor cells can be used. In some embodiments, the growth factor from the TGF-β family includes activin A. In some embodiments, the growth factor from the TGF-β family includes 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. In some examples, the method includes contacting the gastrula cells with a growth factor from the TGF-β superfamily (e.g., activin A) at a concentration of, for example, about 17-23 ng / ml, about 18-22 ng / ml, or about 19-21 ng / ml. In some examples, the method includes contacting the gastrula cells with a growth factor from the TGF-β superfamily (e.g., activin A) at a concentration of about 20 ng / ml.

[0228]

[0236] Any growth factor from the FGF family that can induce primitive gut cells to differentiate into PDX1-positive pancreatic progenitor cells (e.g., alone or in combination with at least one BMP signaling pathway inhibitor, a growth factor from the TGF-β superfamily, at least one SHH pathway inhibitor, at least one retinoic acid signaling pathway activator, at least one protein kinase C activator, and a ROCK inhibitor) can be used. In some embodiments, the at least one growth factor from the FGF family comprises keratinocyte growth factor (KGF). In some embodiments, 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 primary 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. In some examples, the method includes contacting the primary gut cells with a growth factor from the FGF family (e.g., KGF) at a concentration of, for example, about 20-80 ng / mL, about 30-70 ng / mL, about 40-60 ng / mL, or about 45-55 ng / mL. In some examples, the method includes contacting the primitive gut cells with a growth factor from the FGF family (eg, KGF) at a concentration of about 50 ng / ml.

[0229]

[0237] Any SHH pathway inhibitor that can induce primitive gut cells to differentiate into PDX1-positive pancreatic progenitor cells (e.g., alone or in combination with at least one BMP signaling pathway inhibitor, at least one growth factor from the FGF family, a growth factor from the TGF-β superfamily, at least one retinoic acid signaling pathway activator, at least one protein kinase C activator, and a ROCK inhibitor) can be used. In some embodiments, the SHH pathway inhibitor comprises Sant1. In some examples, the method comprises administering to the primitive gut cells at a concentration of, for example, about 0.001 μM, about 0.002 μM, about 0.005 μM, about 0.01 μM, about 0.02 μM, about 0.03 μM, about 0.05 μM, about 0.08 μM, about 0.1 μM, about 0.12 μM, about 0.13 μM, about 0.14 μM, about 0.15 μM, about 0.16 μM, about 0.17 μM, about 0.18 μM, about 0.19 μM, about 0.2 μM, about 0.21 μM, about 0.22 μM, about 0.23 μM, about 0.24 μM, about 0.25 μM, about 0.26 μM, about 0.27 μM, about 0.28 μM, about 0.29 μM, about 0.30 μM, about 0.31 μM, about 0.32 μM, about 0.33 μM, about 0.34 μM, about 0.35 μM, about 0.36 μM, about 0.37 μM, about 0.38 μM, about 0.39 μM, about 10 ... The method includes contacting the primary gut cells with an SHH pathway inhibitor (e.g., Sant1) at a concentration of about 220-280 nM, about 230-270 nM, about 240-260 nM, or about 245-255 nM, or about 255 nM, or about 260 nM, or about 270 nM, or about 280 nM, or about 290 nM, or about 300 nM, or about 310 nM, or about 320 nM, or about 330 nM, or about 340 nM, or about 355 nM, or about 360 nM, or about 370 nM, or about 380 nM, or about 390 nM, or about 400 nM, or about 410 nM, or about 420 nM, or about 430 nM, or about 440 nM, or about 455 nM, or about 50 ... In some examples, the method includes contacting the primitive gut cells with an SHH pathway inhibitor (e.g., Sant1) at a concentration of about 250 nM.

[0230]

[0238] Any RA signaling pathway activator capable of inducing primitive gut cells to differentiate into PDX1-positive pancreatic progenitor cells (e.g., alone or in combination with at least one BMP signaling pathway inhibitor, at least one growth factor from the FGF family, at least one SHH pathway inhibitor, at least one protein kinase C activator, and a ROCK inhibitor) can be used. In some embodiments, the RA signaling pathway activator comprises retinoic acid. In some examples, the method includes administering to the primitive gut cells a soluble ATP-containing solution containing, for example, about 0.02 μM, about 0.1 μM, about 0.2 μM, about 0.25 μM, about 0.3 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about 0.55 μM, about 0.6 μM, about 0.65 μM, about 0.7 μM, about 0.75 μM, about 0.8 μM, about 0.85 μM, about 0.9 μM, about 1 μM, about 1.1 μM, about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM, about 1.8 μM, about 1.9 μM, about 2 μM, about 2.1 μM, about 2.2 μM, about 2.3 μM, about 2.4 μM, about 2.5 μM, The method includes contacting the subject with a RA signaling pathway activator (e.g., retinoic acid) at a concentration of about 1 μM, about 2.6 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. In some examples, the method includes contacting the gastrula cells with an RA signaling pathway activator (e.g., retinoic acid) at a concentration of, for example, about 1.7 to 2.3 μM, about 1.8 to 2.2 μM, or about 1.9 to 2.1 μM. In some examples, the method includes contacting the gastrula cells with an RA signaling pathway activator (e.g., retinoic acid) at a concentration of about 2 μM.

[0231]

[0239] Any PKC activator that can induce primitive gut cells to differentiate into PDX1-positive pancreatic progenitor cells (e.g., alone or in any 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 embodiments, the PKC activator comprises PdBU. In some embodiments, the PKC activator comprises TPPB. In some examples, the method comprises injecting primitive gut cells with at least one of the following: 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 The method includes contacting the cells with a PKC activator (e.g., PdBU or TPPB) at a concentration of about 1 mM, 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 gastrula cells with a PKC activator (e.g., PdBU or TPPB) at a concentration of, for example, 10 nM to 1 mM, 10 nM to 500 μM, 10 nM to 1 μM, 10 to 800 nM, 100 to 900 nM, 300 to 800 nM, 300 to 600 nM, 400 to 600 nM, 450 to 550 nM, or about 500 nM.In some examples, the method includes contacting the primary gut cells with a PKC activator (e.g., PdBU or TPPB) at a concentration of about 450-550 mM, about 475-525 nM, about 490-510 nM, or about 495-505 nM, etc. In some examples, the method includes contacting the primary gut cells with a PKC activator (e.g., PdBU or TPPB) at a concentration of about 500 nM. In some embodiments, the primary gut cells are not treated with a PKC activator (e.g., PDBU).

[0232]

[0240] Any ROCK inhibitor (e.g., alone or in combination with at least one BMP signaling pathway inhibitor, at least one growth factor from the FGF family, at least one SHH pathway inhibitor, a PKC activator, and at least one RA signaling pathway activator) that can induce primitive gut cells to differentiate into PDX1-positive pancreatic progenitor cells can be used. In some embodiments, the ROCK inhibitor comprises thiazovivin, Y-27632, fasudil / HA1077, or H-1152. In some embodiments, the ROCK inhibitor comprises Y-27632. In some embodiments, the ROCK inhibitor comprises thiazovivin. In some examples, the method includes injecting primitive gut cells with a concentration of, for example, about 0.2 μM, about 0.5 μM, about 0.75 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 7.5 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 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 31 μM, about 32 μM, about 33 μM, about 34 μM, about 35 μM, about 36 μM, about 37 μM, about 38 μM, about 39 μM, about 40 μM, about 41 μM, about 42 μM, about 43 μM, about 44 μM, about 45 μM, about 46 μM, about 47 μM, about 48 μM, about 49 μM, about 50 μM, about 51 μM, about 52 μM, about 53 μM, about 54 μM, about 55 μM, about 56 μM, about 57 μM, about 58 μM, about 59 μM, about 60 μM, about 61 μM, about 62 μM, about 63 μM, about 64 μM, about 65 μM, about 66 μM, about 67 μM, about 68 The method includes contacting the gastrula cells with a ROCK inhibitor (e.g., Y-27632 or thiazovivin) at a concentration of about 1 μM, about 19 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 35 μM, about 40 μM, about 50 μM, or about 100 μM. In some examples, the method includes contacting the gastrula cells with a ROCK inhibitor (e.g., Y-27632 or thiazovivin) at a concentration of, for example, about 2.2 to 2.8 μM, about 2.3 to 2.7 μM, or about 2.4 to 2.6 μM. In some examples, the method includes contacting the gastrula cells with a ROCK inhibitor (e.g., Y-27632 or thiazovivin) at a concentration of about 2.5 μM.

[0233]

[0241] In some embodiments, the cells are further contacted with a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol. In some cases, the polyvinyl alcohol is at least 78% hydrolyzed, e.g., 79-81% hydrolyzed, 87-89% hydrolyzed, 87-90% hydrolyzed, or 99% hydrolyzed. In some embodiments, the polyvinyl alcohol (PVA) 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 PVA is 80% hydrolyzed.

[0242] In some embodiments, PDX1-positive pancreatic progenitor cells can be obtained by differentiating at least some of the primitive gut cells in the population into PDX1-positive pancreatic progenitor cells, e.g., by contacting the primitive gut cells with retinoic acid, KGF, Sant1, DMH-1, PdBU, thiazovivin, and activin A for a suitable period of time, e.g., about 1 day, about 2 days, about 3 days, or about 4 days. In some embodiments, PDX1-positive pancreatic progenitor cells can be obtained by differentiating at least some of the primitive gut cells in the population into PDX1-positive pancreatic progenitor cells, e.g., by contacting the primitive gut cells with retinoic acid, KGF, Sant1, DMH-1, PdBU, thiazovivin, and activin A for about 2 days. In some embodiments, PDX1-positive pancreatic progenitor cells can be obtained by differentiating at least a portion of the primitive gut cells in the population into PDX1-positive pancreatic progenitor cells, for example, by contacting the primitive gut cells with retinoic acid, KGF, Sant1, DMH-1, PdBU, thiazovivin, and activin A for one day, followed by contacting the cells with retinoic acid, KGF, Sant1, PdBU, thiazovivin, and activin A for one day (in the absence of DMH-1).

[0234] NKX6.1-positive pancreatic progenitor cells

[0243] The embodiments of the present disclosure include NKX6.1-positive pancreatic progenitor cells.The NKX6.1-positive pancreatic progenitor cells used herein can be obtained from any source or produced according to any suitable protocol, including the method disclosed herein, which includes the use of small molecule compounds such as inhibitors of PI3K / Akt / mTOR signaling.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 induced or matured into SC-β cells.

[0235]

[0244] In some aspects, a method for generating NKX6.1-positive pancreatic progenitor cells from PDX1-positive pancreatic progenitor cells includes contacting a population of cells comprising PDX1-positive pancreatic progenitor cells (e.g., under conditions that promote cell clustering and / or promote cell survival) with at least two beta cell differentiation factors comprising: a) at least one growth factor from the fibroblast growth factor (FGF) family, b) a sonic hedgehog pathway inhibitor, and, optionally, c) a low concentration of a retinoic acid (RA) signaling pathway activator, to induce differentiation of at least one PDX1-positive pancreatic progenitor cell in the population into an NKX6.1-positive pancreatic progenitor cell.

[0236]

[0245] In some embodiments, the PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally, iii) an RA signaling pathway activator to induce differentiation of at least a portion of the PDX1-positive pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells, wherein the PDX1-positive, NKX6.1-positive pancreatic progenitor cells express PDX1 and NKX6.1.

[0237]

[0246] In some embodiments, the PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally, iii) 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 a portion of the PDX1-positive pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells. In some embodiments, following the 3, 4, or 5 day contacting step, the PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting the PDX1-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally, iii) a Rat signaling pathway activator, iv) a ROCK inhibitor, and v) at least one growth factor from the TGF-β superfamily, and then the cells are contacted with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally, iii) a Rat signaling pathway activator, iv) a ROCK inhibitor, and v) at least one growth factor from the TGF-β superfamily. In some embodiments, the PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting the PDX1-positive pancreatic progenitor cells with at least one growth factor from the FGF family under conditions that promote cell clustering. In some embodiments, the growth factor from the FGF family is KGF.

[0238]

[0247] In some embodiments, the present disclosure provides a method of culturing a first cell population comprising PDX1-positive, NKX6.1-negative cells in a medium comprising any one or combination of: i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, iii) an RA signaling pathway activator, iv) a ROCK inhibitor, and v) a growth factor from the TGF-β superfamily for about 1, 2, 3, 4, or 5 days (e.g., 2-4 days, 3-4 days, or 4-5 days), thereby producing a second cell population. In some embodiments, the second cell population is then incubated for about 1, 2, or 3 days (e.g., 1-2 days, 1-3 days, or 2-3 days) in a composition comprising any one or combination of: i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, iii) an RA signaling pathway activator, iv) a ROCK inhibitor, v) a growth factor from the TGF-β superfamily, vi) a PKC activator, vii) a FoxO1 inhibitor, and optionally viii) a Notch signaling inhibitor.

[0239]

[0248] In some embodiments, in the medium for culturing the first population of cells, the growth factor from the FGF family is present at a concentration of about 45-55 ng / ml, about 46-54 ng / ml, about 47-53 ng / ml, about 48-52 ng / ml, or about 49-51 ng / ml, the SHH pathway inhibitor is present at a concentration of about 200-300 nM, about 220-280 nM, or about 240-260 nM, and the RA syndrome inhibitor is present at a concentration of about 200-300 nM, about 220-280 nM, or about 240-260 nM. The signaling pathway activator is present at a concentration of about 1.7-2.3 μM, about 1.8-2.2 μM, or about 1.9-2.1 μM, the ROCK inhibitor is present at a concentration of about 2-3 μM, about 2.2-2.8 μM, or about 2.4-2.6 μM, and / or the growth factor from the TGF-β superfamily is present at a concentration of about 2-8 ng / ml, about 3-7 ng / ml, or about 4-6 ng / ml.

[0240]

[0249] In some embodiments, in the medium for culturing the second population of cells, the growth factor from the FGF family is present at a concentration of about 45-55 ng / ml, about 46-54 ng / ml, about 47-53 ng / ml, about 48-52 ng / ml, or about 49-51 ng / ml; the SHH pathway inhibitor is present at a concentration of about 200-300 nM, about 220-280 nM, or about 240-260 nM; the RA signaling pathway activator is present at a concentration of about 1.7-2.3 μM, about 1.8-2.2 μM, or about 1.9-2.1 μM; and the ROCK inhibitor is present at a concentration of about 2-3 μM, about 2.2-2.8 μM, or about 2.5-3.8 μM. or about 2.4-2.6 μM; the growth factor from the TGF-β superfamily is present at a concentration of about 2-8 ng / ml, about 3-7 ng / ml, or about 4-6 ng / ml; the PKC activator is present at a concentration of about 0.2-0.8 μM, about 0.3-0.7 μM, or about 0.4-0.6 μM; the FoxO1 inhibitor is present at a concentration of about 0.7-1.3 μM, about 0.8-1.2 μM, or about 0.9-1.1 μM; and optionally the Notch signaling inhibitor is present at a concentration of about 1.7-2.3 μM, about 1.8-2.2 μM, or about 1.9-2.1 μM.

[0241]

[0250] In some embodiments, the PDX1-positive pancreatic progenitor cells are generated from a pluripotent cell population. In some embodiments, the PDX1-positive pancreatic progenitor cells are generated from an iPS cell population. In some embodiments, the PDX1-positive pancreatic progenitor cells are generated from an ESC cell population. In some embodiments, the PDX1-positive pancreatic progenitor cells are generated from a definitive endoderm cell population. In some embodiments, the PDX1-positive pancreatic progenitor cells are generated from an primitive gut cell population.

[0242]

[0251] Any growth factor from the FGF family that can induce PDX1-positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (e.g., alone or in combination with at least one SHH pathway inhibitor, ROCK inhibitor, growth factor from the TGF-β superfamily, and at least one retinoic acid signaling pathway activator) can be used in the methods provided herein. In some embodiments, the at least one growth factor from the FGF family comprises keratinocyte growth factor (KGF). In some embodiments, the at least one growth factor from the FGF family is selected from the group consisting of FGF8B, FGF10, and FGF21. In some examples, the method includes contacting PDX1-positive pancreatic progenitor cells with a growth factor from the FGF family (e.g., KGF) at a concentration of, for example, about 10 ng / mL, about 20 ng / mL, about 50 ng / mL, about 75 ng / mL, about 80 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 110 ng / mL, about 120 ng / mL, about 130 ng / mL, about 140 ng / mL, about 150 ng / mL, about 175 ng / mL, about 180 ng / mL, about 200 ng / mL, about 250 ng / mL, or about 300 ng / mL. In some examples, the method includes contacting the PDX1-positive pancreatic progenitor cells with a growth factor from the FGF family (e.g., KGF) at a concentration of about 20-80 ng / ml, about 30-70 ng / ml, about 40-60 ng / ml, or about 45-55 ng / ml, etc. In some examples, the method includes contacting the PDX1-positive pancreatic progenitor cells with about 50 ng / ml of a growth factor from the FGF family (e.g., KGF).

[0243]

[0252] Any SHH pathway inhibitor that can induce PDX1-positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (e.g., alone or in combination with at least one growth factor from the FGF family, a 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 embodiments, the SHH pathway inhibitor comprises Sant1. In some examples, the method comprises administering to the patient a dose of PDX1-positive pancreatic progenitor cells at a concentration of, for example, about 0.001 μM, about 0.002 μM, about 0.005 μM, about 0.01 μM, about 0.02 μM, about 0.03 μM, about 0.05 μM, about 0.08 μM, about 0.1 μM, about 0.12 μM, about 0.13 μM, about 0.14 μM, about 0.15 μM, about 0.16 μM, about 0.17 μM, about 0.18 μM, about 0.19 μM, about 0.2 μM, about 0.21 μM, about 0.22 μM, about The method includes contacting the cells with an SHH pathway inhibitor (e.g., Sant1) at a concentration of 0.23 μM, about 0.24 μM, about 0.25 μM, about 0.26 μM, about 0.27 μM, about 0.28 μM, about 0.29 μM, about 0.3 μM, about 0.31 μM, about 0.32 μM, about 0.33 μM, about 0.34 μM, about 0.35 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about 0.6 μM, about 0.8 μM, about 1 μM, about 2 μM, or about 5 μM. In some examples, the method includes contacting PDX1-positive pancreatic progenitor cells with an SHH pathway inhibitor (e.g., Sant1) at a concentration of about 220-280 nM, about 230-270 nM, about 240-260 nM, or about 245-255 nM, etc. In some examples, the method includes contacting PDX1-positive pancreatic progenitor cells with an SHH pathway inhibitor (e.g., Sant1) at a concentration of about 250 nM.

[0244]

[0253] Any RA signaling pathway activator (for example, 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, alone or in any combination) that can induce PDX1-positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells can be used. In some embodiments, the RA signaling pathway activator comprises retinoic acid. In some examples, the methods include administering PDX1-positive pancreatic progenitor cells to a patient at a concentration of, for example, about 0.02 μM, about 0.1 μM, about 0.2 μM, about 0.25 μM, about 0.3 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about 0.55 μM, about 0.6 μM, about 0.65 μM, about 0.7 μM, about 0.75 μM, about 0.8 μM, about 0.85 μM, about 0.9 μM, about 1 μM, about 1.1 μM, about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM, about 1.8 μM, about 1.9 μM, about 2 μM, about 2.1 μM, about 2.2 μM, about 2.3 μM, about 2.4 μM, about The method includes contacting the RA signaling pathway activator (e.g., retinoic acid) at a concentration of 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. In some examples, the method includes contacting PDX1-positive pancreatic progenitor cells with an RA signaling pathway activator (e.g., retinoic acid) at a concentration of, for example, about 70-130 nM, about 80-120 nM, about 90-110 nM, or about 95-105 nM. In some examples, the method includes contacting PDX1-positive pancreatic progenitor cells with an RA signaling pathway activator (e.g., retinoic acid) at a concentration of about 100 nM.

[0245]

[0254] Any ROCK inhibitor (for example, alone or in combination with at least one growth factor from the FGF family, at least one SHH pathway inhibitor, RA signaling pathway activator, and at least one growth factor from the TGF-β superfamily) that can induce PDX1-positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells can be used. In some embodiments, the ROCK inhibitor comprises thiazovivin, Y-27632, fasudil / HA1077, or H-1152. In some examples, the method includes administering to the PDX1-positive pancreatic progenitor cells 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, The method includes contacting PDX1-positive pancreatic progenitor cells with a ROCK inhibitor (e.g., Y-27632 or thiazovivin) at a concentration of 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. In some examples, the method includes contacting PDX1-positive pancreatic progenitor cells with a ROCK inhibitor (e.g., Y-27632 or thiazovivin) at a concentration of, for example, about 2.2-2.8 μM, about 2.3-2.7 μM, or about 2.4-2.6 μM. In some examples, the method includes contacting the PDX1-positive pancreatic progenitor cells with a ROCK inhibitor (e.g., Y-27632 or thiazovivin) at a concentration of about 2.5 μM.

[0246]

[0255] Any activator from the TGF-β superfamily (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 ROCK inhibitor) can be used to induce PDX1-positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells. In some embodiments, the activator from the TGF-β superfamily comprises activin A or GDF8. In some examples, the methods include administering PDX1-positive pancreatic progenitor cells to a patient at a concentration of, for example, about 0.1 ng / mL, about 0.2 ng / mL, about 0.3 ng / mL, about 0.4 ng / mL, about 0.5 ng / mL, about 0.6 ng / mL, about 0.7 ng / mL, about 0.8 ng / mL, about 1 ng / mL, about 1.2 ng / mL, about 1.4 ng / mL, about 1.6 ng / mL, about 1.8 ng / mL, about 2 ng / mL, about 2.2 ng / mL, about 2.4 ng / mL, about 2.6 ng / mL, about 2.8 ng / mL, about 3 ng / mL, about 3.2 ng / mL, about 3.4 ng / mL, about 3.6 ng / mL, about 3.8 ng / mL, about 4 ng / mL, or about 5 ng / mL. The method includes contacting the cells with a growth factor from the TGF-β superfamily (e.g., activin A) at a concentration of about 4.2ng / mL, about 4.4ng / mL, about 4.6ng / mL, about 4.8ng / mL, about 5ng / mL, about 5.2ng / mL, about 5.4ng / mL, about 5.6ng / mL, about 5.8ng / mL, about 6ng / mL, about 6.2ng / mL, about 6.4ng / mL, about 6.6ng / mL, about 6.8ng / mL, about 7ng / mL, about 8ng / mL, about 9ng / mL, about 10ng / mL, about 20ng / mL, about 30ng / mL, or about 50ng / mL. In some examples, the method includes contacting the PDX1-positive pancreatic progenitor cells with a growth factor from the TGF-β superfamily (e.g., activin A) at a concentration of, for example, about 2-8 ng / ml, about 3-7 ng / ml, about 4-6 ng / ml, or about 4.5-5.5 ng / ml. In some examples, the method includes contacting the PDX1-positive pancreatic progenitor cells with a growth factor from the TGF-β superfamily (e.g., activin A) at a concentration of, for example, about 5 ng / mL.

[0247]

[0256] Any FoxO1 inhibitor that can induce PDX1-positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (e.g., alone or in any combination with at least one growth factor from the FGF family, at least one retinoic acid signaling pathway activator, a ROCK inhibitor, at least one growth factor from the TGF-β superfamily, a PKC activator, and a Notch signaling inhibitor) can be used in the methods provided herein. In some embodiments, the FoxO1 inhibitor is AS1842856. In some examples, the methods include administering PDX1-positive pancreatic progenitor cells at a concentration of 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, The method includes contacting the PDX1-positive pancreatic progenitor cells with a FoxO1 inhibitor (e.g., AS1842856) at a concentration of about 0.7-1.3 μM, about 0.8-1.2 μM, about 0.9-1.1 μM, or the like. In some examples, the method includes contacting PDX1-positive pancreatic progenitor cells with a FoxO1 inhibitor (such as AS1842856) at a concentration such as about 1 μM.

[0248]

[0257] Any PKC activator that can induce PDX1-positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (e.g., alone or in any combination with at least one growth factor from the FGF family, at least one retinoic acid signaling pathway activator, a ROCK inhibitor, at least one growth factor from the TGF-β super pathway, a FoxO1 inhibitor, and a Notch signaling inhibitor) can be used in the methods provided herein. In some embodiments, the PKC activator is PDBU. In some examples, the method includes administering to the PDX1-positive pancreatic progenitor cells a concentration of, for example, 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 μM, about 110 μM, about 111 μM, about 112 μM, about 113 μM, about 114 μM, about 115 μM, about 116 μM, about 117 μM, about 118 μM, about 119 μM, about 120 μM, about 121 μM, about 122 μM, about 123 μM, about 124 μM, about 125 μM, about 126 μM, about 127 μM, about 128 μM, about 129 μM, about 130 μM, about 131 μM, about 132 μM, about 133 μM, about 134 μM, about 135 μM, about 136 μ The method includes contacting PDX1-positive pancreatic progenitor cells with a PKC activator (e.g., PDBU) at a concentration of 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. In some examples, the method includes contacting PDX1-positive pancreatic progenitor cells with a PKC activator (e.g., PDBU) at a concentration of, for example, about 0.2-0.8 μM, about 0.3-0.7 μM, or about 0.4-0.6 μM. In some examples, the method includes contacting the PDX1-positive pancreatic progenitor cells with a PKC activator (eg, PDBU) at a concentration of, for example, about 0.5 μM.

[0249]

[0258] Any Notch signaling inhibitor that can induce PDX1-positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (e.g., alone or in any combination with at least one growth factor from the FGF family, at least one retinoic acid signaling pathway activator, a ROCK inhibitor, at least one growth factor from the TGF-β superfamily, a FoxO1 inhibitor, and a PKC activator) can be used in the methods provided herein. In some embodiments, the Notch signaling inhibitor is XXI. In some examples, the method includes administering PDX1-positive pancreatic progenitor cells at a concentration of 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 PDX1-positive pancreatic progenitor cells with a Notch signaling inhibitor (e.g., XXI) at a concentration of about 7 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. In some examples, the method includes contacting PDX1-positive pancreatic progenitor cells with a Notch signaling inhibitor (e.g., XXI) at a concentration of about 1.7-2.3 μM, about 1.8-2.2 μM, or about 1.9-2.1 μM. In some examples, the method includes contacting PDX1-positive pancreatic progenitor cells with a Notch signaling inhibitor (e.g., XXI) at a concentration of about 2 μM.

[0250]

[0259] In some embodiments, the cells are further contacted with a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol. In some examples, the polyvinyl alcohol is at least 78% hydrolyzed, e.g., 79-81% hydrolyzed, 87-89% hydrolyzed, 87-90% hydrolyzed, or 99% hydrolyzed. In some embodiments, the polyvinyl alcohol (PVA) 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 PVA is 80% hydrolyzed.

[0251]

[0260] In some embodiments, PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive pancreatic progenitor cells with KGF, Sant1, and RA for 5 or 6 days under conditions that promote cell clustering. In some embodiments, PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive pancreatic progenitor cells with KGF, Sant1, RA, thiazovivin, and activin A for 5 or 6 days under conditions that promote cell clustering. In some embodiments, PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive pancreatic progenitor cells with KGF for 5 days under conditions that promote cell clustering. In some embodiments, PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive pancreatic progenitor cells with KGF for 6 days under conditions that promote cell clustering. In some embodiments, PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by a) contacting PDX1-positive pancreatic progenitor cells with KGF, Sant1, RA, thiazovivin, and activin A for 3, 4, or 5 days (e.g., 4 days), followed by b) contacting the cells of a) with PDBU, XXI, KGF, Sant1, RA, thiazovivin, and activin A, and optionally, AS1842856 for 1, 2, or 3 days (e.g., 2 days).

[0252] Insulin-positive endocrine cells

[0261] Embodiments of the present disclosure involve insulin-positive endocrine cells (e.g., NKX6.1-positive, ISL1-positive, or β-like cells), and additional methods for producing insulin-positive endocrine cells.The insulin-positive endocrine cells used herein can be derived from any source or produced according to any suitable protocol.In some embodiments, 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.

[0253]

[0262] In some aspects, a method for generating insulin-positive endocrine cells from NKX6.1-positive pancreatic progenitor cells includes contacting a cell population containing NKX6-1-positive pancreatic progenitor cells (e.g., under conditions that promote cell clustering) with: a) a TGF-β signaling pathway inhibitor; b) a thyroid hormone signaling pathway activator; c) a BMP pathway inhibitor; and / or d) a protein kinase inhibitor to induce differentiation of at least one NKX6.1-positive pancreatic progenitor cell in the population into an insulin-positive endocrine cell, wherein the insulin-positive endocrine cell expresses insulin. In some embodiments, the insulin-positive endocrine cell expresses PDX1, NKX6.1, ISL1, NKX2.2, Mafb, glis3, Sur1, Kir6.2, Znt8, SLC2A1, SLC2A3, and / or insulin.

[0254]

[0263] Any TGF-β signaling pathway inhibitor that can induce differentiation of NKX6.1-positive pancreatic progenitor cells into insulin-positive endocrine cells (e.g., alone or in combination with other β cell differentiation factors, such as thyroid hormone signaling pathway activators) can be used. In some embodiments, the TGF-β signaling pathway comprises TGF-β type I receptor kinase signaling. In some embodiments, the TGF-β signaling pathway inhibitor comprises Alk5 inhibitor II. In some examples, the method includes administering NKX6.1 positive pancreatic progenitor cells at about 0.1 μM, about 0.5 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 4.5 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 10.5 μM, about 11 μM, about 11.5 μM, about 12 μM, about 12.5 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 31 μM, about 32 μM, about 33 μM, about 34 μM, about 35 μM, about 36 μM, about 37 μM, about 38 μM, about 39 μM, about 40 μM, about 41 μM, about 42 μM, about 43 μM, about 44 μM, about 45 μM, about 46 μM, about 47 μM, about 48 μM, about 49 μM, about 50 μM, about 51 μM, about 52 μM, about 53 μM, about 54 μM, about In some examples, the method includes contacting the NKX6.1-positive pancreatic progenitor cells with a TGF-β signaling pathway inhibitor (e.g., an Alk5 inhibitor such as Alk5 inhibitor II) at a concentration of about 7-13 μM, about 8-12 μM, or about 9-11 μM, such as about 13.5 μM, about 14 μM, about 14.5 μM, about 15 μM, about 15.5 μM, about 16 μM, about 16.5 μM, about 17 μM, about 17.5 μM, about 18 μM, about 18.5 μM, about 19 μM, about 19.5 μM, about 20 μM, about 25 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM, or about 50 μM. In some examples, the method includes contacting the NKX6.1-positive pancreatic progenitor cells with a TGF-β signaling pathway inhibitor (e.g., an Alk5 inhibitor such as Alk5 inhibitor II) at a concentration of about 7-13 μM, about 8-12 μM, or about 9-11 μM. In some examples, the method includes contacting the NKX6.1-positive pancreatic progenitor cells with a TGF-β signaling pathway inhibitor (e.g., an Alk5 inhibitor such as Alk5 inhibitor II) at a concentration of about 10 μM, or the like.

[0255]

[0264] Any thyroid hormone signaling pathway activator that can induce differentiation of NKX6.1-positive pancreatic progenitor cells into insulin-positive endocrine cells (e.g., alone or in combination with other beta cell differentiation factors, such as TGF-β signaling pathway inhibitors) can be used. In some embodiments, the thyroid hormone signaling pathway activator comprises triiodothyronine (T3). In some embodiments, the thyroid hormone signaling pathway activator comprises GC-1. In some examples, the method comprises inducing NKX6.1-positive pancreatic progenitor cells with a thyroid hormone signaling pathway activator at 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, or about 0.30 μM. The method includes contacting the NKX6.1-positive pancreatic progenitor cells with a thyroid hormone signaling pathway activator (e.g., GC-1) at a concentration of 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, etc. In some examples, the method includes contacting the NKX6.1-positive pancreatic progenitor cells with a thyroid hormone signaling pathway activator (e.g., GC-1) at a concentration of about 0.7-1.3 μM, about 0.8-1.2 μM, or about 0.9-1.1 μM, etc. In some examples, the methods include contacting the NKX6.1-positive pancreatic progenitor cells with a thyroid hormone signaling pathway activator (eg, GC-1) at a concentration such as about 1 μM.

[0256]

[0265] In some embodiments, the method comprises contacting a cell population (e.g., NKX6.1-positive pancreatic progenitor cells) with at least one additional factor. In some embodiments, the method comprises contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells with at least one of the following: i) an SHH pathway inhibitor, ii) a gamma-secretase inhibitor, iii) at least one growth factor from the epidermal growth factor (EGF) family, iv) a TGF-β signaling pathway inhibitor, or vii) a thyroid hormone signaling pathway activator. In some embodiments, the method comprises contacting a cell population (e.g., NKX6.1-positive pancreatic progenitor cells) with at least one additional factor. In some embodiments, the method comprises contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells with 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, viiii) a wnt signaling pathway inhibitor, or ix) a PKC activator.

[0257]

[0266] In some embodiments, 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, or ix) a ROCK inhibitor.

[0258]

[0267] In some embodiments, 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. In some embodiments, the method includes contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells in culture 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 inhibitor, viii) an epigenetic modifying compound, ix) a protein kinase inhibitor, x) a ROCK inhibitor, xi) a PKC activator, and xii) a Wnt signaling pathway inhibitor for 1 day, 2 days, or 3 days (e.g., 1-2 days, 1-3 days, or 2-3 days), and then contacting the cells in culture with i) a γ-secretase inhibitor, v) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, vi) a TGF-β signaling pathway inhibitor, vii) a thyroid hormone signaling pathway inhibitor, viii) an epigenetic modifying compound, ix) a protein kinase inhibitor, x) a ROCK inhibitor, xi) a PKC activator, and xii) a Wnt signaling pathway inhibitor for 1 day, 2 days, or 3 days (e.g., 1-2 days, 1-3 days, or 2-3 days). the method comprises contacting the cells with a secretase inhibitor, ii) at least one growth factor from the epidermal growth factor (EGF) family, iii) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, iv) a TGF-β signaling pathway inhibitor, v) a thyroid hormone signaling pathway activator, vi) an epigenetic modifying compound, vii) a protein kinase inhibitor, and viii) a ROCK inhibitor for 1, 2, 3, 4, 5, 6, or 7 days (e.g., 1 to 7 days, 1 to 5 days, 1 to 3 days, 3 to 7 days, 3 to 5 days, 5 to 7 days, or 4 to 6 days) in the absence of an SHH pathway inhibitor, an RA signaling pathway activator, a Wnt signaling pathway inhibitor, a PKC activator, and / or an epidermal growth factor (EGF) family growth factor.

[0259]

[0268] In some embodiments, in the method of 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 embodiments, some of the differentiation factors, such as an SHH pathway inhibitor, an RA signaling pathway activator, a PKC activator, and at least one growth factor from the EGF family, are removed from the medium after the first 1, 2, or 3 days of culture.

[0260]

[0269] Any gamma-secretase inhibitor that can induce the differentiation of NKX6.1-positive pancreatic progenitor cells in the population into insulin-positive endocrine cells (e.g., alone or in combination with a TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator) can be used. In some embodiments, the gamma-secretase inhibitor comprises XXI. In some embodiments, the gamma-secretase inhibitor comprises DAPT. In some examples, the method comprises administering to the patient a dose of NKX6.1-positive pancreatic progenitor cells at a concentration of, for example, about 0.01 μM, about 0.02 μM, about 0.05 μM, about 0.075 μM, about 0.1 μM, about 0.2 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1 μM, about 1.1 μM, about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM, about 1.8 μM, about 1.9 μM, about 2 μM, about 2.1 μM, about 2.2 μM, about 2.3 μM, about 2.4 μM, about 2.5 μM, about 2 ... The method includes contacting the antibody with a gamma secretase inhibitor (e.g., XXI) at a concentration of about 0.6 μM, about 2.7 μM, about 2.8 μM, about 2.9 μM, about 3 μM, about 3.2 μM, about 3.4 μM, about 3.6 μM, about 3.8 μM, about 4 μM, about 4.2 μM, about 4.4 μM, about 4.6 μM, about 4.8 μM, about 5 μM, about 5.2 μM, about 5.4 μM, about 5.6 μM, about 5.8 μM, about 6 μM, about 6.2 μM, about 6.4 μM, about 6.6 μM, about 6.8 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 20 μM, about 30 μM, or about 50 μM. In some examples, the method includes contacting the NKX6.1-positive pancreatic progenitor cells with a gamma-secretase inhibitor (e.g., XXI) at a concentration of about 1.7-2.3 μM, about 1.8-2.2 μM, or about 1.9-2.1 μM, etc. In some examples, the method includes contacting the NKX6.1-positive pancreatic progenitor cells with a gamma-secretase inhibitor (e.g., XXI) at a concentration of about 2 μM, etc.

[0261]

[0270] Any growth factor from the EGF family that can induce differentiation of NKX6.1-positive pancreatic progenitor cells in the population into insulin-positive endocrine cells (e.g., alone or in combination with any TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway activator) can be used. In some embodiments, at least one growth factor from the EGF family comprises betacellulin. In some embodiments, at least one growth factor from the EGF family comprises EGF. In some examples, the method includes contacting the NKX6.1-positive pancreatic progenitor cells with a growth factor from the EGF family (e.g., betacellulin) at a concentration of, for example, about 1 ng / mL, about 2 ng / mL, about 4 ng / mL, about 6 ng / mL, about 8 ng / mL, about 10 ng / mL, about 12 ng / mL, about 14 ng / mL, about 16 ng / mL, about 18 ng / mL, about 20 ng / mL, about 22 ng / mL, about 24 ng / mL, about 26 ng / mL, about 28 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 75 ng / mL, about 80 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 150 ng / mL, about 200 ng / mL, about 250 ng / mL, or about 300 ng / mL. In some examples, the method includes contacting the NKX6.1-positive pancreatic progenitor cells with an EGF family growth factor (e.g., betacellulin) at a concentration of about 17-23 ng / ml, about 18-22 ng / ml, or about 19-21 ng / ml, etc. In some examples, the method includes contacting the NKX6.1-positive pancreatic progenitor cells with an EGF family growth factor (e.g., betacellulin) at a concentration of about 20 ng / ml, etc.

[0262]

[0271] Any RA signaling pathway activator that can induce the differentiation of NKX6.1-positive pancreatic progenitor cells into insulin-positive endocrine cells (e.g., alone or in combination with any TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway activator) can be used. In some embodiments, the RA signaling pathway activator comprises RA. In some examples, the methods include administering NKX6.1 positive pancreatic progenitor cells to a patient at a concentration of, for example, about 0.02 μM, about 0.05 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. In some examples, the method includes contacting the NKX6.1-positive pancreatic progenitor cells with an RA signaling pathway activator (e.g., retinoic acid) at a concentration of about 20-80 nM, about 30-70 nM, or about 40-60 nM, etc. In some examples, the method includes contacting the NKX6.1-positive pancreatic progenitor cells with an RA signaling pathway activator (e.g., retinoic acid) at a concentration of about 50 nM, etc.

[0263]

[0272] Any SHH pathway inhibitor that can induce differentiation of NKX6.1-positive pancreatic progenitor cells into insulin-positive endocrine cells (e.g., alone or in combination with any TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway activator) can be used in the methods provided herein. In some embodiments, the SHH pathway inhibitor comprises Sant1. In some examples, the method comprises administering to the patient NKX6.1-positive pancreatic progenitor cells 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, 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. In some examples, the method includes contacting NKX6.1-positive pancreatic progenitor cells with an SHH pathway inhibitor (e.g., Sant1) at a concentration of about 220-280 nM, about 230-270 nM, about 240-260 nM, or about 245-255 nM, etc. In some examples, the method includes contacting NKX6.1-positive pancreatic progenitor cells with an SHH pathway inhibitor (e.g., Sant1) at a concentration of about 250 nM, etc.

[0264]

[0273] Any BMP signaling pathway inhibitor that can induce the differentiation of NKX6.1-positive pancreatic progenitor cells into insulin-positive endocrine cells (e.g., alone or in combination with any TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway activator) can be used. In some embodiments, the BMP signaling pathway inhibitor comprises LDN193189 or DMH-1. In some examples, the method includes contacting NKX6.1-positive pancreatic progenitor cells with a BMP signaling pathway inhibitor (e.g., LDN1931189) at a concentration of, for example, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 110 nM, about 120 nM, about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about 200 nM, about 210 nM, about 220 nM, about 230 nM, about 240 nM, about 250 nM, about 280 nM, about 300 nM, about 400 nM, about 500 nM, or about 1 μM. In some examples, the method includes contacting the NKX6.1-positive pancreatic progenitor cells with a BMP signaling pathway inhibitor (e.g., LDN1931189) at a concentration of about 70-130 nM, about 80-120 nM, about 90-110 nM, etc. In some examples, the method includes contacting the NKX6.1-positive pancreatic progenitor cells with a BMP signaling pathway inhibitor (e.g., LDN1931189) at a concentration of about 100 nM, etc.

[0265]

[0274] Any ROCK inhibitor that can induce the differentiation of NKX6.1-positive pancreatic progenitor cells in the population into insulin-positive endocrine cells (e.g., alone or in combination with any TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway activator) can be used. In some embodiments, the ROCK inhibitor comprises thiazovivin, Y-27632, Fasudil / HA1077, or H-1152. In some embodiments, the ROCK inhibitor comprises Y-27632. In some embodiments, 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, a...

Claims

1. A method comprising contacting a plurality of stem cells in vitro with an inhibitor of PI3K / Akt / mTOR signaling.

2. 2. The method of claim 1, wherein the inhibitor of PI3K / Akt / mTOR signaling comprises an inhibitor of PI3K protein, an inhibitor of Akt protein, an inhibitor of mTOR, or any combination thereof.

3. 2. The method of claim 1, wherein the inhibitor of PI3K / Akt / mTOR signaling comprises one or more of GSK-690693, IPI-3063, AZD8055, omipalisib, GNE-477, VS-5584, BYL319, YM201636, PI4KIIIbeta-IN-10, nemiralisib, BYL719, FT113, apitolisib, or any analogue or derivative thereof.

4. The method of claim 1 , wherein the inhibitor of PI3K / Akt / mTOR signaling comprises an inhibitor of PI3K protein and an inhibitor of Akt protein.

5. The method of claim 1, wherein the inhibitor of PI3K / Akt / mTOR signaling comprises GSK-690693, an analog or a derivative thereof.

6. 2. The method of claim 1, wherein the inhibitor of PI3K / Akt / mTOR signaling comprises BYL719, an analog or a derivative thereof.

7. 6. The method of claim 5, wherein the contacting step comprises contacting the plurality of stem cells with about 0.01 μM to about 1 μM, about 0.02 μM to about 0.8 μM, about 0.05 μM to about 0.5 μM, about 0.06 μM to about 0.2 μM, about 0.07 μM to about 0.15 μM, or about 0.08 μM to about 0.12 μM of GSK-690693.

8. 6. The method of claim 5, wherein the contacting step comprises contacting the plurality of stem cells with about 0.01 μM, 0.02 μM, 0.04 μM, 0.06 μM, 0.08 μM, 0.1 μM, 0.12 μM, 0.15 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.8 μM, or 1 μM of GSK-690693.

9. 7. The method of claim 6, wherein the contacting step comprises contacting the plurality of stem cells with about 1 nM to about 500 nM, about 5 nM to about 250 nM, about 10 nM to about 200 nM, about 15 nM to about 150 nM, about 20 nM to about 100 nM, about 30 nM to about 80 nM, about 30 nM to about 60 nM, or about 35 nM to about 50 nM of BYL719.

10. The method of any one of claims 1 to 9, comprising contacting a plurality of stem cells with an inhibitor of PI3K / Akt / mTOR signaling and a growth factor from the TGF-β superfamily.

11. 11. The method of claim 10, wherein the growth factors from the TGF-β superfamily include activin A, GDF8, or both.

12. 11. The method of claim 10, comprising contacting the plurality of stem cells with about 0.5 ng / mL to about 500 ng / mL, about 1 ng / mL to about 250 ng / mL, about 10 ng / mL to about 200 ng / mL, about 20 ng / mL to about 150 ng / mL, about 50 ng / mL to about 120 ng / mL, about 1 ng / mL to about 50 ng / mL, about 2 ng / mL to about 25 ng / mL, or about 5 ng / mL to about 20 ng / mL of activin A.

13. 10. The method of any one of claims 1 to 9, comprising contacting a plurality of stem cells with an inhibitor of PI3K / Akt / mTOR signaling for about 24 hours to about 96 hours, about 36 hours to about 84 hours, about 48 hours to about 84 hours, about 60 hours to about 84 hours, or about 3 days.

14. The method of any one of claims 1 to 9, further comprising contacting the plurality of stem cells with an activator of the WNT signaling pathway.

15. 15. The method of claim 14, wherein the activator of the WNT signaling pathway comprises one or more of Wnt3a, CHIR99021, 3F8, A 1070722, AR-A 014418, BIO, BIO-acetoxime, FRATide, 10Z-hymenialdisine, indirubin-3'oxime, kenpaullone, L803, L803-mts, lithium carbonate, NSC693868, SB 216763, SB 415286, TC-G 24, TCS 2002, TCS 21311, TWS 119, and any analogs or derivatives thereof.

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