Differentiation of pancreatic islet cells

Abstract

Disclosed herein are compositions and methods related to differentiation of stem cells into pancreatic islet cells. In some aspects, the methods provided herein relate to generation of pancreatic β cell, α cell, δ cells, and EC cells in vitro. In some aspects, the disclosure provides pharmaceutical compositions including the cells generated according to the methods disclosed herein, as well as methods of treatment making use thereof.

Description

DIFFERENTIATION OF PANCREATIC ISLET CELLS CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.63 / 643,004, filed May 6, 2024, and U.S. Provisional Application No.63 / 675,641, filed July 25, 2024, which are incorporated by reference in their entireties. SEQUENCE LISTING

[0002] The Sequence Listing is submitted as an XML file in the form of the file named “Sequence.xml” (39,431 bytes), which was created on May 5, 2025, which is incorporated by reference herein. BACKGROUND

[0003] Transplantation of pancreas or pancreatic islets has been used for treating diabetes, such as type I diabetes. Pancreatic islet transplantation does not need major surgery and the function of the islet grafts can be maintained for years in a recipient. However, a shortage of pancreatic islets donors prevents this therapy from being effectively implemented. Artificial pancreas or pancreatic islets provide an alternative source of transplantable islets. Thus, there is a need for methods of in vitro restitution of pancreatic islets whose function and characteristics resemble endogenous pancreatic islets. INCORPORATION BY REFERENCE

[0004] 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. Absent any indication otherwise, publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entireties. SUMMARY

[0005] In some embodiments, the disclosure provides for a composition comprising a plurality of stem cells (e.g., pluripotent stem cells) and an AKT-inhibitor. In some embodiments, the disclosure provides for a composition comprising a plurality of stem cells (e.g., pluripotent stem cells), a bone morphogenic protein (BMP) pathway inhibitor, and a JNK inhibitor. In some embodiments, the disclosure provides for a composition comprising a plurality of stem cells (e.g., pluripotent stem cells) and insulin at a concentration of 21-1000, 400-600, 200-400, 600-1000, 21-500, 21-300, 21-200, 21- 100, 21-50, 50-500, 50-300, 50-250, 50-100, 75-125, 150-500, 150-250, or 175-225 µg / L. In some embodiments, the disclosure provides for a composition comprising a plurality of stem cells (e.g.,pluripotent stem cells) and greater than 8 mM glucose. In some embodiments, the disclosure provides for a composition comprising a plurality of stem cells (e.g., pluripotent stem cells) and a medium, wherein the medium comprises a Wnt pathway activator and does not comprise a member of the transforming growth factor-beta superfamily. In some embodiments, the composition comprises greater than 8 mM glucose. In some embodiments, the composition comprises a bone morphogenic protein (BMP) pathway inhibitor and a JNK inhibitor. In some embodiments, the composition does not comprise a bone morphogenic protein (BMP) pathway inhibitor and a JNK inhibitor. In some embodiments, the composition further comprises insulin. In some embodiments, the composition further comprises insulin at a concentration of 21-1000, 400-600, 200-400, 600-1000, 21-500, 21-300, 21-200, 21-100, 21-50, 50-500, 50-300, 50-250, 50-100, 75-125, 150-500, 150-250, or 175-225 µg / L. In some embodiments, the composition further comprises a Wnt pathway activator. In some embodiments, the Wnt pathway activator is a Wnt protein, CHIR99021, 3F8, A1070722, AR-A 014418, BIO, BlO-acetoxime, FRATide, lOZ-Hymenial disine, 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 embodiments, the Wnt pathway activator is a Wnt protein, and wherein the Wnt protein is a Wnt3a protein or functional fragment or derivative thereof. In some embodiments, the Wnt pathway activator is CHIR99021. In some embodiments, the composition further comprises an AKT inhibitor. In some embodiments, the AKT inhibitor is any one of BEZ235, LY294002, GDC-0941, BYL719, GSK2636771, TGX-221, AS25242, CAL-101, IPI-145, MK-2206, GSK690693, GDC-0068, A-674563, CCT128930, AZD8055, INK128, rapamycin, PF-04691502, everolimus, BI-D1870, H89, PF-4708671, FMK, AT7867, NU7441, PI-103, NU7026, PIK-75, ZSTK474, and PP-121. In some embodiments, the AKT inhibitor comprises the structure of , or apharmaceutically acceptable salt or In some AKT inhibitor is MK-2206. In some embodiments, the composition does not comprise a member of the transforming growth factor-beta superfamily. In some embodiments, the composition does not comprise activin A, GDF8, or GDF11. In some embodiments, at least about 50%, 60%, 65%, 70%, 75%, 80%, or 85% of the cells in the composition are Sox17-positive, Oct4-negative cells. In some embodiments, from 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, the composition comprises 8-15, 8-14, 8-12, 8-11, 8-10,8-9, 9-11, 9-12, or 9.5-10.5 mM glucose. In some embodiments, the stem cells are pluripotent stem cells, and wherein the pluripotent stem cells are embryonic stem cells. In some embodiments, the stem cells are pluripotent stem cells, and wherein the pluripotent stem cells are induced pluripotent stem cells. In some embodiments, the disclosure provides for a composition comprising a plurality of SOX17-positive cells and greater than 8 mM glucose. In some embodiments, the composition comprises 8-15, 8-14, 8-12, 8-11, 8-10, 8-9, 9-11, 9-12, or 9.5-10.5 mM glucose. In some embodiments, the composition further comprises growth factor from the fibroblast growth factor (FGF) family. In some embodiments, the growth factor from the FGF family is any one of KGF, FGF2, FGF8B, FGF10, or FGF21. In some embodiments, the composition comprising a bone morphogenic protein (BMP) pathway inhibitor, a JNK inhibitor, and a plurality of FOXA2-positive, PDX1-negative cells. In some embodiments, 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 fibroblast growth factors (FGF) family, a retinoic acid (RA) signaling pathway activator, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, and a sonic hedgehog (SHH) pathway inhibitor. In some embodiments, the composition further comprises a protein kinase C activator selected from the group consisting of: phorbol 12,13-dibutyrate (PDBU), TPB, phorbol 12-myristate 13-acetate, and bryostatin 1. In some embodiments, the composition further comprises a bone morphogenetic protein signaling pathway inhibitor comprising LDN193189 or DMH-1. In some embodiments, the composition further comprises a growth factor from fibroblast growth factors (FGF) family selected from the group consisting of: keratinocyte growth factor (KGF), FGF2, FGF10, FGF21, and FGF8B. In some embodiments, the composition further comprises a sonic hedgehog pathway inhibitor selected from the group consisting of SANT1, SANT2, SANT4, Cur61414, forskolin, tomatidine, AY9944, triparanol, and cyclopamine. In some embodiments, the composition further comprises 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. In some embodiments, the composition further comprises a ROCK inhibitor selected from the group consisting of Thiazovivin, Y- 27632, Fasudil / HA1077, and 14-1152. In some embodiments, the composition further comprises PDX1-positive cells. In some embodiments, the disclosure provides for an in vitro composition comprising a bone morphogenic protein (BMP) pathway inhibitor, a JNK inhibitor, and a plurality of PDX1-positive and NKX6.1-negative cells. In some embodiments, the disclosure provides for a composition comprising a plurality of PDX1- positive cells comprising any one of the following reagents:ves gents: orprotein (BMP) pathway inhibitor, a JNK inhibitor, and a plurality of PDX1-positive and NKX6.1-negative cells. In some embodiments, the composition comprises a plurality of PDX1-positive, NKX6.1- negative cells. In some embodiments, the composition compriese a plurality of PDX1-positive, NKX6.1-positive cells. In some embodiments, the composition further comprises one or more agents selected from the group consisting of: a growth factor from fibroblast growth factors (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 embodiments, the composition further comprises a growth factor from the transformation growth factor β (TGF-β) superfamily selected from the group consisting of: an Inhibin, an Activin, a Mullerian inhibiting substance (MIS), a bone morphogenic protein (BMP), decapentaplegic (dpp),Vg-1, monoclonal nonspecific suppressor factor (MNSF), growth differentiating factor 8 (GDF8), and growth differentiating factor 11 (GDF11). In some embodiments, the composition further comprises a growth factor from fibroblast growth factors (FGF) family selected from the group consisting of: keratinocyte growth factor (KGF), FGF2, FGF10, FGF21, and FGF8B. In some embodiments, the composition further comprises 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. In some embodiments, the composition further comprises a ROCK inhibitor selected from the group consisting of Thiazovivin, Y- 27632, Fasudil / HA1077, and 14-1152. In some embodiments, the composition further comprises a protein kinase C activator selected from the group consisting of: phorbol 12,13- dibutyrate (PDBU), TPB, phorbol 12-myristate 13-acetate, and bryostatin 1. In some embodiments, the composition further comprises a sonic hedgehog (SHH) pathway inhibitor selected from the group consisting of SANT1, SANT2, SANT4, Cur61414, forskolin, tomatidine, AY9944, triparanol, and cyclopamine. In some embodiments, the composition further comprises a FoxO1 inhibitor, optionally wherein the FoxO1 inhibitor is AS1842856. In some embodiments, the composition further comprises a notch signaling inhibitor, optionally wherein the notch signaling inhibitor is XXI or DAPI. In some embodiments, the BMP inhibitor is noggin, LDN-193189, DMH-1, LDN-212854, ML347 or dorsomorphin. In some embodiments, the JNK inhibitor is a JNK1, JNK2, and / or JNK3 inhibitor. In some embodiments, the JNK inhibitor is a JNK1, JNK2, and JNK3 inhibitor. In some embodiments, the JNK inhibitor is a compound of Formula (I):

[0006] wherein:

[0007] Ring A is a 6 membered monocyclic heteroaryl ring or bicyclic heteroaryl ring; each instance of RAis independently selected from the group consisting of hydrogen, halogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORA1, —N(RA1)2, and —SRA1, wherein each occurrence of RA1is independently hydrogen, acyl, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or two RA1groups are joined to form an optionally substituted heterocyclic ring;

[0008] m is 0, 1, 2, 3, or 4;

[0009] Ring B is a group of the formula: from the group consisting of hydrogen, halogen, optionally substituted acyl,alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORB1a, —N(RB1a)2, and —SRB1a, wherein each occurrence of RB1ais independently hydrogen, acyl, substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or two RB1agroups are joined to form an optionally substituted heterocyclic ring;

[0011] WB is N or CRB2, wherein RB2is selected from the group consisting of hydrogen, halogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORB2a, —N(RB2a)2, and —SRB2a, wherein each occurrence of RB2ais independently hydrogen, acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or two RB2agroups are joined to form an optionally substituted heterocyclic ring;

[0012] optionally wherein RB1and RB2are joined to form an optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, or optionally substituted aryl ring;

[0013] L1is a bond directly attaching Ring A to Ring B;

[0014] represents a single bond;

[0015] X is —NRX—, wherein RXis hydrogen, C1-6 alkyl, or a nitrogen protecting group;

[0016] L2 is —NRL2aC(═O)— or —C(═O)NRL2a—, wherein RL2ais hydrogen, C1-6 alkyl, or a nitrogen protecting group;

[0017] each instance of RCis independently selected from the group consisting of hydrogen, halogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORC1, —N(RC1)2, and —SRC1, wherein each occurrence of RC1is independently hydrogen, acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or two RC1groups are joined to form an optionally substituted heterocyclic ring;

[0018] n is 0, 1, 2, 3, or 4;

[0019] each instance of RDis independently selected from the group consisting of hydrogen, halogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORD1, —N(RD1)2, and —SRD1, wherein each occurrence of RD1is independently hydrogen, acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or two RD1groups are joined to form an optionally substituted heterocyclic ring;

[0020] p is 0, 1, 2, 3, or 4; and

[0021] RE is a group of the formula:

[0022] L3is a bond, —O—, —S—, —NRL3a—, —NRL3aC(═O)—, —C(═O)NRL3a—, — SC(═O)—, —C(═O)S—, —OC(═O)—, —C(═O)O—, —NRL3aC(═S)—, —C(═S)NRL3a—, trans- CRL3b═CRL3b—, cis-CRL3b═CRL3b—, —C≡C—, —OC(RL3b)2—, —C(RL3b)2O—, —NRL3aC(RL3b)2—, —C(RL3b)2NRL3a—, —SC(RL3b)2—, —C(RL3b)2S—, —S(═O)2O—, —OS(═O)2—, —S(═O)2NRL3a—, —NRL3aS(═O)2—, or an optionally substituted C1-4 hydrocarbon chain, optionally wherein one or more carbon units of the hydrocarbon chain is replaced with —O—, —S—, —NRL3a—, — NRL3aC(═O)—, —C(═O)NRL3a—, —SC(═O)—, —C(═O)S—, —OC(═O)—, —C(═O)O—, — NRL3aC(═S)—, —C(═S)NRL3a—, trans-CRL3b═CRL3b—, cis-CRL3b═CRL3b—, —C≡C, —S(═O)2O—, —OS(═O)2—, —S(═O)2NRL3a—, or —NRL3aS(═O)2—, wherein RL3ais hydrogen, C1-6 alkyl, or a nitrogen protecting group, and wherein each occurrence of RL3bis independently selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl, or two RL3bgroups are joined to form an optionally substituted carbocyclic or optionally substituted heterocyclic ring;

[0023] L4 is a bond or an optionally substituted C1-4 hydrocarbon chain;

[0024] RE1is selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, — CH2ORE1a, —CH2N(RE1a)2, —CH2SRE1a, —ORE1a, —N(RE1a)2 and —SRE1a, wherein each occurrence of RE1ais independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl, or two RE1agroups are joined to form an optionally substituted heterocyclic ring;

[0025] RE2is selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, — CH2ORE2a, —CH2N(RE2a)2, —CH2SRE2a, —ORE2a, —N(RE2a)2, and —SRE2a, wherein each occurrence of RE2ais independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl, or two RE2agroups are joined to form an optionally substituted heterocyclic ring; RE3is selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, — CH2ORE3a, —CH2N(RE3a)2, —CH2SRE3a, —ORE3a, —N(RE3a)2, and —SRE3a, wherein each occurrence of RE3ais independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl, or two RE3a groups are joined to form an optionally substituted heterocyclic ring;

[0026] optionally wherein RE1and RE3, or RE2and RE3, or RE1and RE2are joined to form an optionally substituted carbocyclic or optionally substituted heterocyclic ring;

[0027] RE4is a leaving group;

[0028] Y is O, S, or NRE5, wherein RE5is hydrogen, C1-6alkyl, or a nitrogen protecting group;

[0029] a is 1 or 2; and

[0030] z is 0, 1, 2, 3, 4, 5, or 6. In some embodiments, the JNK inhibitor is JNK-IN-8. In some embodiments, the composition does not comprise a bone morphogenic protein (BMP) pathway inhibitor and a JNK inhibitor. In some embodiments, the composition further comprises a water- soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer comprises polyvinyl alcohol, poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), PEG copolymers, poly(N-isopropylacrylamide), or polyacrylamide. In some embodiments, the water-soluble synthetic polymer comprises polyvinyl alcohol. In some embodiments, 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 the composition. In some embodiments, the water-soluble synthetic polymer is present at a concentration of about 0.04% to about 0.06% (w / v) in the composition. In some embodiments, the water-soluble synthetic polymer comprises polyvinyl alcohol that is less than 85% hydrolyzed. In some embodiments, the water- soluble synthetic polymer comprises polyvinyl alcohol that is about 80% hydrolyzed. In someembodiments, the disclosure provides for a composition comprising a plurality of PDX1-positive cells and a thyroid hormone signaling pathway activator at a concentration of 1.5-5, 1.5-3, 1.5-2.5, 1.8-5, 1.8-3, 1.8-2.2, or 1.9-2.1 µM. In some embodiments, the thyroid hormone signaling pathway activator is T3 or GC-1. In some embodiments, the disclosure provides for a composition comprising a plurality of PDX1-positive cells and one or more of an epidermal growth factor, a thyroid hormone, a TGFβ-R1 kinase inhibitor, a notch signaling pathway inhibitor, a sonic hedgehog (SHH) signaling pathway inhibitor, retinoic acid, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, a protein kinase inhibitor, a bone morphogenetic (BMP) signaling pathway inhibitor, a histone methyltransferase EZH2 inhibitor, and a Wnt signaling pathway inhibitor; wherein the composition comprises less than 0.088 mg / ml ascorbic acid (e.g., 0.01-0.087, 0.01-0.05, 0.03-0.087, 0.03-0.08, 0.03-0.07, 0.03-0.06, 0.03-0.05, 0.035-0.06, 0.035-0.045, 0.035-0.045, 0.04-0.06, 0.04- 0.05, or 0.043-0.046 mg / ml ascorbic acid). In some embodiments, the composition comprises less than 0.088 mg / ml ascorbic acid (e.g., 0.01-0.087, 0.01-0.05, 0.03-0.087, 0.03-0.08, 0.03-0.07, 0.03- 0.06, 0.03-0.05, 0.035-0.06, 0.035-0.045, 0.035-0.045, 0.04-0.06, 0.04-0.05, or 0.043-0.046 mg / ml ascorbic acid). In some embodiments, the composition further comprises one or more of an epidermal growth factor, a thyroid hormone, a TGFβ-R1 kinase inhibitor, a notch signaling pathway inhibitor, a sonic hedgehog (SHH) signaling pathway inhibitor, retinoic acid, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, a protein kinase inhibitor, a bone morphogenetic (BMP) signaling pathway inhibitor, a histone methyltransferase EZH2 inhibitor, and a Wnt signaling pathway inhibitor; wherein the composition comprises less than 0.088 mg / ml ascorbic acid (e.g., 0.01-0.087, 0.01-0.05, 0.03-0.087, 0.03-0.08, 0.03-0.07, 0.03-0.06, 0.03-0.05, 0.035-0.06, 0.035-0.045, 0.035- 0.045, 0.04-0.06, 0.04-0.05, or 0.043-0.046 mg / ml ascorbic acid). In some embodiments, the composition further comprises one or more of SANT-1, betacellulin, XXI, Alk5i, GC-1, LDN- 193189, thiazovivin, staurosporine, DZNEP, retinoic acid, or NVP-TNKS656. In some embodiments, the composition comprises one or more agents selected from an acetyl CoA related metabolite, an HDAC inhibitor, a redox homeostasis regulator, and a one carbon metabolism pathway intermediate. In some embodiments, the composition further comprises acetate, β-hydroxybutyrate, taurine, and formate. In some embodiments, the second medium further comprises a vitamin, optimally wherein the vitamin is biotin. In some embodiments, the second medium further comprises glutamine. In some embodiments, the second medium further comprises a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol, poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), PEG copolymers, poly(N-isopropylacrylamide), or polyacrylamide. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol (PVA). In some embodiments, the water-soluble synthetic polymer has a concentration of 0.005% to 0.5% (w / v), 0.01% to 0.2% (w / v), 0.02% to 0.1% (w / v), or 0.03% to 0.08% (w / v) of the medium. In some embodiments, the PVA is at most 90% hydrolyzed, optionally wherein the PVA is about 87%- 89% hydrolyzed. In some embodiments, the composition comprises a plurality of PDX1-positive,NKX6.1-negative cells. In some embodiments, the composition comprises a plurality of PDX1- positive, NKX6.1-positive cells. In some embodiments, the composition comprises a plurality of PDX1-positive, ISL1-positive cells. In some embodiments, the composition is in a sterile container. In some embodiments, the sterile container is a bioreactor.

[0031] In some embodiments, the disclosure provides for a method comprising the step of culturing a plurality of stem cells (e.g., pluripotent stem cells) with a medium comprising greater than 8 mM glucose. In some embodiments, the disclosure provides for a method comprising: a) administering a medium to a container comprising stem cells (e.g., pluripotent stem cells), wherein the medium comprises 4-14 mM glucose; and b) after 4-18 hours of culturing the stem cells (e.g., pluripotent stem cells) in the medium, administering supplemental glucose to the medium, wherein following administration of the supplemental glucose to the medium, the concentration of total glucose in the medium is 4-14 mM. In some embodiments, the disclosure provides for a method comprising the step of culturing a plurality of stem cells (e.g., pluripotent stem cells) with a medium comprising a bone morphogenic protein (BMP) pathway inhibitor and a JNK inhibitor. In some embodiments, the disclosure provides for a method comprising the step of culturing a plurality of stem cells (e.g., pluripotent stem cells) with a medium comprising insulin at a concentration of 21- 1000, 400-600, 200-400, 600-1000, 21-500, 21-300, 21-200, 21-100, 21-50, 50-500, 50-300, 50-250, 50-100, 75-125, 150-500, 150-250, or 175-225 µg / L. In some embodiments, the disclosure provides for a method comprising the step of culturing a plurality of stem cells (e.g., pluripotent stem cells) with a medium comprising an AKT-inhibitor. In some embodiments, the disclosure provides for a method comprising the step of culturing: i) a first population of cells comprising a plurality of stem cells (e.g., pluripotent stem cells) with ii) a first medium comprising a Wnt pathway activator, thereby generating a second population of cells, wherein the first medium does not comprise a member of the transforming growth factor-beta superfamily (e.g., activin A), a bone morphogenic protein (BMP) pathway inhibitor, and / or a JNK inhibitor. In some embodiments, the plurality of stem cells (e.g., pluripotent stem cells) are cultured with the first medium for less than 48 hours or for 12-48 hours, 12-36 hours, 12-30 hours, 12-24 hours, 18-36 hours, 18-30 hours, or 21-27 hours. In some embodiments, further comprising the steps of removing the first medium and culturing the second population of cells in a second medium , thereby generating a third population of cells, wherein the second medium comprises: a) a member of the transforming growth factor-beta superfamily (e.g., activin A) or b) a bone morphogenic protein (BMP) pathway inhibitor and a JNK inhibitor, wherein the second medium lacks a Wnt pathway activator. In some embodiments, the second population of cells are cultured with the second medium for less than 72 hours or for no more than 20-52 hours, 36- 52 hours, 42-42 hours, 20-72 hours, 12-48 hours, 12-36 hours, 12-30 hours, 12-24 hours, 18-36 hours, 18-30 hours, or 21-27 hours. In some embodiments, further comprising the steps of removing the second medium and culturing the third population of cells in a third medium, wherein the third medium does not comprise: a) a Wnt pathway activator, b) a member of the transforming growthfactor-beta superfamily (e.g., activin A), c) a bone morphogenic protein (BMP) pathway inhibitor, and / or d) a JNK inhibitor. In some embodiments, the medium comprises greater than 8 mM glucose. In some embodiments, the medium administered in step a) comprises 4-12, 4-10, 4-8, 6-12, 6-11, 6- 10, 8-10, or 7-9 mM glucose. In some embodiments, after 4-16, 4-12, 4-10, 6-12, 6-16, 6-9, 7-9, 7- 10, 7-12, 8-12, or 10-14 hours of culturing the stem cells (e.g., pluripotent stem cells) in the medium from step a), the supplemental glucose is administered to the medium. In some embodiments, the concentration of total glucose in the medium following administration of the supplemental glucose to the medium in step b) is 4-12, 4-10, 4-8, 6-12, 6-11, 6-10, 8-10, 8-12, 9-12, or 7-9 mM. In some embodiments, the medium comprises a bone morphogenic protein (BMP) pathway inhibitor and a JNK inhibitor. In some embodiments, the medium does not comprise a bone morphogenic protein (BMP) pathway inhibitor or a JNK inhibitor. In some embodiments, the medium further comprises insulin. In some embodiments, the medium further comprises insulin at a concentration of 21-1000, 400-600, 200-400, 600-1000, 21-500, 21-300, 21-200, 21-100, 21-50, 50-500, 50-300, 50-250, 50- 100, 75-125, 150-500, 150-250, or 175-225 µg / L. In some embodiments, the medium further comprises a Wnt pathway activator. In some embodiments, the Wnt pathway activator is a Wnt protein, CHIR99021, 3F8, A1070722, AR-A 014418, BIO, BlO-acetoxime, FRATide, lOZ-Hymenial disine, 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 embodiments, the Wnt pathway activator is a Wnt protein, and wherein the Wnt protein is a Wnt3a protein or functional fragment or derivative thereof. In some embodiments, the Wnt pathway activator is CHIR99021. In some embodiments, the medium further comprises an AKT inhibitor. In some embodiments, the AKT inhibitor is any one of BEZ235, LY294002, GDC-0941, BYL719, GSK2636771, TGX-221, AS25242, CAL-101, IPI-145, MK-2206, GSK690693, GDC-0068, A- 674563, CCT128930, AZD8055, INK128, rapamycin, PF-04691502, everolimus, BI-D1870, H89, PF-4708671, FMK, AT7867, NU7441, PI-103, NU7026, PIK-75, ZSTK474, and PP-121. In some embodiments, the AKT inhibitor comprises the structure of, or a pharmaceutically acceptable salt or derivative thereof. In some embodiments, the AKT inhibitor is MK-2206. In some embodiments, the medium does not comprise a member of the transforming growth factor-beta superfamily. In some embodiments, the medium does not comprise activin A, GDF8, or GDF11. In some embodiments, at least about 50%, 60%, 65%, 70%, 75%, 80%, or 85% of the cells in the medium are Sox17-positive,Oct4-negative cells. In some embodiments, from 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% of the cells are Sox17-positive, Oct4-negative cells. In some embodiments, the medium comprises 8-15, 8-14, 8-12, 8-11, 8-10, 8-9, 9-11, 9-12, or 9.5-10.5 mM glucose. In some embodiments, the disclosure provides for a method comprising culturing a plurality of SOX17-positive cells with greater than 5.5 mM glucose. In some embodiments, the medium comprises 8-15, 8-14, 8-12, 8-11, 8-10, 8-9, 9-11, 9-12, or 9.5-10.5 mM glucose. In some embodiments, the disclosure provides for a method comprising: a) administering a medium to a container SOX17-positive cells, wherein the medium comprises 4-14 mM glucose; b) after 4-18 hours of culturing the SOX17-positive cells in the medium, administering supplemental glucose to the medium, wherein following administration of the supplemental glucose to the medium, the concentration of total glucose in the medium is 4-14 mM. In some embodiments, the medium administered in step a) comprises 4-12, 4-10, 4-8, 6-12, 6-11, 6-10, 8-10, or 7-9 mM glucose. In some embodiments, after 4-16, 4-12, 4-10, 6-12, 6-16, 6-9, 7-9, 7-10, 7-12, 8-12, or 10-14 hours of culturing the stem cells (e.g., pluripotent stem cells) in the medium from step a), the supplemental glucose is administered to the medium. In some embodiments, the concentration of total glucose in the medium following administration of the supplemental glucose to the medium in step b), the concentration of total glucose in the medium is 4-12, 4-10, 4-8, 6-12, 6-11, 6-10, 8-10, 8-12, 9-12, or 7-9 mM. In some embodiments, the medium further comprises growth factor from the fibroblast growth factor (FGF) family. In some embodiments, the growth factor from the FGF family is any one of KGF, FGF2, FGF8B, FGF10, or FGF21. In some embodiments, the disclosure provides for a method comprising culturing a plurality of FOXA2-positive, PDX1-negative cells in a medium comprising a bone morphogenic protein (BMP) pathway inhibitor and a JNK inhibitor. In some embodiments, the medium 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 fibroblast growth factors (FGF) family, a retinoic acid (RA) signaling pathway activator, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, and a sonic hedgehog (SHH) pathway inhibitor. In some embodiments, the medium further comprises a protein kinase C activator selected from the group consisting of: phorbol 12,13-dibutyrate (PDBU), TPB, phorbol 12-myristate 13-acetate, and bryostatin 1. In some embodiments, the medium further comprises a bone morphogenetic protein signaling pathway inhibitor comprising LDN193189 or DMH-1. In some embodiments, the medium further comprises a growth factor from fibroblast growth factors (FGF) family selected from the group consisting of: keratinocyte growth factor (KGF), FGF2, FGF10, FGF21, and FGF8B. In some embodiments, the medium further comprises a sonic hedgehog pathway inhibitor selected from the group consisting of SANT1, SANT2, SANT4, Cur61414, forskolin, tomatidine, AY9944, triparanol, and cyclopamine. In some embodiments, the medium further comprises a retinoic acid signaling pathway activator selected from the group consisting of: retinoicacid, CD1530, AM580, TTHRB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, and CD2314. In some embodiments, the medium further comprises a ROCK inhibitor selected from the group consisting of Thiazovivin, Y-27632, Fasudil / HA1077, and 14-1152. In some embodiments, the medium further comprises PDX1-positive cells. In some embodiments, the disclosure provides for a method comprising culturing a plurality of PDX1-positive and NKX6.1- negative cells in a medium comprising a bone morphogenic protein (BMP) pathway inhibitor and a JNK inhibitor. In some embodiments, the disclosure provides for a method comprising culturing a plurality of PDX1-positive cells in a medium comprising any one of the following reagents:pathway inhibitor, a JNK inhibitor, and a plurality of PDX1-positive and NKX6.1-negative cells. In some embodiments, the medium comprises a plurality of PDX1-positive, NKX6.1-negative cells. Insome embodiments, the medium compriese a plurality of PDX1-positive, NKX6.1-positive cells. In some embodiments, the medium further comprises one or more agents selected from the group consisting of: a growth factor from fibroblast growth factors (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 embodiments, the medium further comprises a growth factor from the transformation growth factor β (TGF-β) superfamily selected from the group consisting of: an Inhibin, an Activin, a Mullerian inhibiting substance (MIS), a bone morphogenic protein (BMP), decapentaplegic (dpp), Vg-1, monoclonal nonspecific suppressor factor (MNSF), growth differentiating factor 8 (GDF8), and growth differentiating factor 11 (GDF11). In some embodiments, the medium further comprises a growth factor from fibroblast growth factors (FGF) family selected from the group consisting of: keratinocyte growth factor (KGF), FGF2, FGF10, FGF21, and FGF8B. In some embodiments, the medium further comprises 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. In some embodiments, the medium further comprises a ROCK inhibitor selected from the group consisting of Thiazovivin, Y- 27632, Fasudil / HA1077, and 14-1152. In some embodiments, the medium further comprises a protein kinase C activator selected from the group consisting of: phorbol 12,13- dibutyrate (PDBU), TPB, phorbol 12-myristate 13-acetate, and bryostatin 1. In some embodiments, the medium further comprises a sonic hedgehog (SHH) pathway inhibitor selected from the group consisting of SANT1, SANT2, SANT4, Cur61414, forskolin, tomatidine, AY9944, triparanol, and cyclopamine. In some embodiments, the medium further comprises a FoxO1 inhibitor, optionally wherein the FoxO1 inhibitor is AS1842856. In some embodiments, the medium further comprises a notch signaling inhibitor, optionally wherein the notch signaling inhibitor is XXI or DAPI. In some embodiments, the BMP inhibitor is noggin, LDN-193189, DMH-1, LDN-212854, ML347 or dorsomorphin. In some embodiments, the JNK inhibitor is a JNK1, JNK2, and / or JNK3 inhibitor. In some embodiments, the JNK inhibitor is a JNK1, JNK2, and JNK3 inhibitor. In some embodiments, the JNK inhibitor is a compound of Formula (I):or a pharmaceutically acceptable salt thereof;

[0032] wherein:

[0033] Ring A is a 6 membered monocyclic heteroaryl ring or bicyclic heteroaryl ring;

[0034] each instance of RAis independently selected from the group consisting of hydrogen, halogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORA1, —N(RA1)2, and —SRA1, wherein each occurrence of RA1is independently hydrogen, acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or two RA1groups are joined to form an optionally substituted heterocyclic ring;

[0035] m is 0, 1, 2, 3, or 4;

[0036] Ring B is a group of the formula:from the group consisting of hydrogen, halogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORB1a, —N(RB1a)2, and —SRB1a, wherein each occurrence of RB1ais independently hydrogen, acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or two RB1agroups are joined to form an optionally substituted heterocyclic ring;

[0038] WBis N or CRB2, wherein RB2is selected from the group consisting of hydrogen, halogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORB2a, —N(RB2a)2, and —SRB2a, wherein each occurrence of RB2ais independently hydrogen, acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substitutedheterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or two RB2agroups are joined to form an optionally substituted heterocyclic ring;

[0039] optionally wherein RB1and RB2are joined to form an optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, or optionally substituted aryl ring;

[0040] L1 is a bond directly attaching Ring A to Ring B;

[0041] represents a single bond;

[0042] X is —NRX—, wherein RXis hydrogen, C1-6alkyl, or a nitrogen protecting group;

[0043] L2is —NRL2aC(═O)— or —C(═O)NRL2a—, wherein RL2ais hydrogen, C1-6alkyl, or a nitrogen protecting group;

[0044] each instance of RCis independently selected from the group consisting of hydrogen, halogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORC1, —N(RC1)2, and —SRC1, wherein each occurrence of RC1is independently hydrogen, acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or two RC1groups are joined to form an optionally substituted heterocyclic ring;

[0045] n is 0, 1, 2, 3, or 4;

[0046] each instance of RDis independently selected from the group consisting of hydrogen, halogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORD1, —N(RD1)2, and —SRD1, wherein each occurrence of RD1is independently hydrogen, acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or two RD1groups are joined to form an optionally substituted heterocyclic ring;

[0047] p is 0, 1, 2, 3, or 4; and

[0048] RE is a group of the formula:

[0050] L3 is a bond, —O—, —S—, —NRL3a—, —NRL3aC(═O)—, —C(═O)NRL3a—, — SC(═O)—, —C(═O)S—, —OC(═O)—, —C(═O)O—, —NRL3aC(═S)—, —C(═S)NRL3a—, trans- CRL3b═CRL3b—, cis-CRL3b═CRL3b—, —C≡C—, —OC(RL3b)2—, —C(RL3b)2O—, —NRL3aC(RL3b)2—, —C(RL3b)2NRL3a—, —SC(RL3b)2—, —C(RL3b)2S—, —S(═O)2O—, —OS(═O)2—, —S(═O)2NRL3a—, —NRL3aS(═O)2—, or an optionally substituted C1-4hydrocarbon chain, optionally wherein one or more carbon units of the hydrocarbon chain is replaced with —O—, —S—, —NRL3a—, — NRL3aC(═O)—, —C(═O)NRL3a—, —SC(═O)—, —C(═O)S—, —OC(═O)—, —C(═O)O—, — NRL3aC(═S)—, —C(═S)NRL3a—, trans-CRL3b═CRL3b—, cis-CRL3b═CRL3b—, —C≡C, —S(═O)2O—, —OS(═O)2—, —S(═O)2NRL3a—, or —NRL3aS(═O)2—, wherein RL3ais hydrogen, C1-6 alkyl, or a nitrogen protecting group, and wherein each occurrence of RL3bis independently selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl, or two RL3bgroups are joined to form an optionally substituted carbocyclic or optionally substituted heterocyclic ring;

[0051] L4 is a bond or an optionally substituted C1-4 hydrocarbon chain;

[0052] RE1is selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, — CH2ORE1a, —CH2N(RE1a)2, —CH2SRE1a, —ORE1a, —N(RE1a)2 and —SRE1a, wherein each occurrence of RE1ais independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl, or two RE1agroups are joined to form an optionally substituted heterocyclic ring;

[0053] RE2is selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, — CH2ORE2a, —CH2N(RE2a)2, —CH2SRE2a, —ORE2a, —N(RE2a)2, and —SRE2a, wherein each occurrence of RE2ais independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl, or two RE2agroups are joined to form an optionally substituted heterocyclic ring;

[0054] RE3is selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, — CH2ORE3a, —CH2N(RE3a)2, —CH2SRE3a, —ORE3a, —N(RE3a)2, and —SRE3a, wherein each occurrence of RE3ais independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl, or two RE3a groups are joined to form an optionally substituted heterocyclic ring;

[0055] optionally wherein RE1and RE3, or RE2and RE3, or RE1and RE2are joined to form an optionally substituted carbocyclic or optionally substituted heterocyclic ring;

[0056] RE4is a leaving group;

[0057] Y is O, S, or NRE5, wherein RE5is hydrogen, C1-6 alkyl, or a nitrogen protecting group;

[0058] a is 1 or 2; and

[0059] z is 0, 1, 2, 3, 4, 5, or 6. In some embodiments, the JNK inhibitor is JNK-IN-8. In some embodiments, the medium does not comprise a bone morphogenic protein (BMP) pathway inhibitor and a JNK inhibitor. In some embodiments, the medium further comprises a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer comprises polyvinyl alcohol, poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), PEG copolymers, poly(N- isopropylacrylamide), or polyacrylamide. In some embodiments, the water-soluble synthetic polymer comprises polyvinyl alcohol. In some embodiments, 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 the medium. In some embodiments, the water-soluble synthetic polymer is present at a concentration of about 0.04% to about 0.06% (w / v) in the medium. In some embodiments, the water-soluble synthetic polymer comprises polyvinyl alcohol that is less than 85% hydrolyzed. In some embodiments, the water-soluble synthetic polymer comprises polyvinyl alcohol that is about 80% hydrolyzed. In some embodiments, the disclosureprovides for a method comprising culturing a plurality of PDX1-positive cells in a medium comprising a thyroid hormone signaling pathway activator at a concentration of 1.5-5, 1.5-3, 1.5-2.5, 1.8-5, 1.8-3, 1.8-2.2, or 1.9-2.1 µM. In some embodiments, the thyroid hormone signaling pathway activator is T3 or GC-1. In some embodiments, the disclosure provides for a method comprising culturing a plurality of PDX1-positive cells in a medium comprising one or more of an epidermal growth factor, a thyroid hormone, a TGFβ-R1 kinase inhibitor, a notch signaling pathway inhibitor, a sonic hedgehog (SHH) signaling pathway inhibitor, retinoic acid, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, a protein kinase inhibitor, a bone morphogenetic (BMP) signaling pathway inhibitor, a histone methyltransferase EZH2 inhibitor, and a Wnt signaling pathway inhibitor; wherein the medium comprises less than 0.088 mg / ml ascorbic acid (e.g., 0.01-0.087, 0.01- 0.05, 0.03-0.087, 0.03-0.08, 0.03-0.07, 0.03-0.06, 0.03-0.05, 0.035-0.06, 0.035-0.045, 0.035-0.045, 0.04-0.06, 0.04-0.05, or 0.043-0.046 mg / ml ascorbic acid). In some embodiments, the medium comprises less than 0.088 mg / ml ascorbic acid (e.g., 0.01-0.087, 0.01-0.05, 0.03-0.087, 0.03-0.08, 0.03-0.07, 0.03-0.06, 0.03-0.05, 0.035-0.06, 0.035-0.045, 0.035-0.045, 0.04-0.06, 0.04-0.05, or 0.043- 0.046 mg / ml ascorbic acid). In some embodiments, the medium further comprises one or more of an epidermal growth factor, a thyroid hormone, a TGFβ-R1 kinase inhibitor, a notch signaling pathway inhibitor, a sonic hedgehog (SHH) signaling pathway inhibitor, retinoic acid, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, a protein kinase inhibitor, a bone morphogenetic (BMP) signaling pathway inhibitor, a histone methyltransferase EZH2 inhibitor, and a Wnt signaling pathway inhibitor; wherein the medium comprises less than 0.088 mg / ml ascorbic acid (e.g., 0.01-0.087, 0.01-0.05, 0.03-0.087, 0.03-0.08, 0.03-0.07, 0.03-0.06, 0.03-0.05, 0.035-0.06, 0.035- 0.045, 0.035-0.045, 0.04-0.06, 0.04-0.05, or 0.043-0.046 mg / ml ascorbic acid). In some embodiments, the medium further comprises one or more of SANT-1, betacellulin, XXI, Alk5i, GC-1, LDN-193189, thiazovivin, staurosporine, DZNEP, retinoic acid, or NVP-TNKS656. In some embodiments, the medium comprises one or more agents selected from an acetyl CoA related metabolite, an HDAC inhibitor, a redox homeostasis regulator, and a one carbon metabolism pathway intermediate. In some embodiments, the medium further comprises acetate, β-hydroxybutyrate, taurine, and formate. In some embodiments, the medium further comprises a vitamin, optimally wherein the vitamin is biotin. In some embodiments, the medium further comprises glutamine. In some embodiments, the medium further comprises a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol, poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), PEG copolymers, poly(N-isopropylacrylamide), or polyacrylamide. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol (PVA). In some embodiments, the water-soluble synthetic polymer has a concentration of 0.005% to 0.5% (w / v), 0.01% to 0.2% (w / v), 0.02% to 0.1% (w / v), or 0.03% to 0.08% (w / v) of the medium. In some embodiments, the PVA is at most 90% hydrolyzed, optionally wherein the PVA is about 87%- 89% hydrolyzed. In some embodiments, the medium comprises a plurality of PDX1-positive,NKX6.1-negative cells. In some embodiments, the medium comprises a plurality of PDX1-positive, NKX6.1-positive cells. In some embodiments, the medium comprises a plurality of PDX1-positive, ISL1-positive cells. In some embodiments, the disclosure provides for a method for culturing a population of cells in vitro, comprising the steps of: a) culturing the population of cells in a medium comprising more than 0.044 mg / ml ascorbic acid (e.g., 0.045-0.15, 0.045-0.1, 0.045-0.09, 0.045-0.07, 0.045-0.05, 0.06-0.1, 0.06-0.09, 0.07-0.1, 0.07-0.09, 0.08-0.09, 0.08-0.1, 0.08-0.12, or 0.085-0.09 mg / ml), and b) culturing the population of cells in a medium comprises less than 0.088 mg / ml ascorbic acid (e.g., 0.01-0.087, 0.01-0.05, 0.03-0.087, 0.03-0.08, 0.03-0.07, 0.03-0.06, 0.03-0.05, 0.035-0.06, 0.035-0.045, 0.035-0.045, 0.04-0.06, 0.04-0.05, or 0.043-0.046 mg / ml ascorbic acid), wherein the population of cells comprises PDX1-positive cells. In some embodiments, step a) comprises culturing the population of cells in a medium comprising more than 0.044 mg / ml ascorbic acid (e.g., 0.045-0.15, 0.045-0.1, 0.045-0.09, 0.045-0.07, 0.045-0.05, 0.06-0.1, 0.06-0.09, 0.07-0.1, 0.07-0.09, 0.08-0.09, 0.08-0.1, 0.08-0.12, or 0.085-0.09 mg / ml) for 12-24 hours, 1-4 days, 1-2 days, 2-3 days, or 3-4 days, or at least 12 hours, 1 day, 2 days, 3 days, or 4 days. In some embodiments, step b) comprises culturing the population of cells in a medium comprising less than 0.088 mg / ml ascorbic acid (e.g., 0.01-0.087, 0.01-0.05, 0.03-0.087, 0.03-0.08, 0.03-0.07, 0.03-0.06, 0.03-0.05, 0.035-0.06, 0.035-0.045, 0.035-0.045, 0.04-0.06, 0.04-0.05, or 0.043-0.046 mg / ml ascorbic acid) for 12-24 hours, 1-4 days, 1-14 days, 1-7 days, 3-5 days, 1-2 days, 2-3 days, 3-4 days, 4-5 days, 6-7 days, or at least 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days. In some embodiments, step a) comprises culturing the population of cells in a medium comprising any one or more of: a growth factor from fibroblast growth factors (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 sonic hedgehog (SHH) pathway inhibitor, a FOXO1 inhibitor, or a gamma secretase inhibitor. In some embodiments, step b) comprises culturing the population of cells in a medium comprising any one or more of: epidermal growth factor, a thyroid hormone, a TGFβ-R1 kinase inhibitor, a notch signaling pathway inhibitor, a sonic hedgehog (SHH) signaling pathway inhibitor, retinoic acid, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, a protein kinase inhibitor, a bone morphogenetic (BMP) signaling pathway inhibitor, a histone methyltransferase EZH2 inhibitor, or a Wnt signaling pathway inhibitor. In some embodiments, the medium in step a) comprises a water-soluble synthetic polymer comprising polyvinyl alcohol, poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), PEG copolymers, poly(N-isopropylacrylamide), or polyacrylamide. In some embodiments, the water-soluble synthetic polymer comprises polyvinyl alcohol that is less than 85% hydrolyzed. In some embodiments, the water-soluble synthetic polymer comprises polyvinyl alcohol that is about 80% hydrolyzed. In some embodiments, the medium in step b) comprises a water-soluble synthetic polymer comprising polyvinyl alcohol, poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), PEG copolymers, poly(N-isopropylacrylamide), or polyacrylamide. In some embodiments, the water-soluble syntheticpolymer comprises polyvinyl alcohol that is at most 90% hydrolyzed, optionally wherein the polyvinyl alcohol is about 87%-89% hydrolyzed. In some embodiments, the medium is in a sterile container. In some embodiments, the sterile container is a bioreactor.

[0060] In some embodiments, the disclosure provides for a method for culturing a population of cells in vitro, comprising the step of culturing the population of cells in a medium comprising a growth factor from the fibroblast growth factor (FGF) family and a retinoic acid signaling pathway activator; wherein the population of cells comprises a plurality of PDX1-positive; NKX6.1-negative cells; and wherein the cells are cultured in the medium for a period of time, wherein the period of time is at least 12 hours but not more than 4 days. In some embodiments, the disclosure provides for a method for culturing a population of cells in vitro, comprising the step of culturing the population of cells in a medium comprising a sonic hedgehog pathway inhibitor and a retinoic acid signaling pathway activator; wherein the population of cells comprises a plurality of PDX1-positive; NKX6.1- negative cells; and wherein the cells are cultured in the medium for a period of time, wherein the period of time is at least 12 hours but not more than 4 days. In some embodiments, the disclosure provides for a method for culturing a population of cells in vitro, comprising the step of culturing the population of cells in a medium comprising a growth factor from the TGF-β superfamily and a retinoic acid signaling pathway activator; wherein the population of cells comprises a plurality of PDX1-positive; NKX6.1-negative cells; and wherein the cells are cultured in the medium for a period of time, wherein the period of time is at least 12 hours but not more than 4 days. In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or between 30-100%, 50-100%, 70- 100%, 85-100%, 80-95%, 85-95%, or 90-95% of the cells in the population are PDX1-positive; NKX6.1-negative cells. In some embodiments, at least 50% of the cells in the population are PDX1- positive; NKX6.1-negative cells. In some embodiments, at least 80% of the cells in the population are PDX1-positive; NKX6.1-negative cells. In some embodiments, between 70-100% of the cells in the population are PDX1-positive; NKX6.1-negative cells. In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or between 30-100%, 50-100%, 70-100%, 85-100%, 80-95%, 85-95%, or 90-95% of the cells in the population are PDX1-positive; NKX6.1-positive cells. In some embodiments, at least 50% of the cells in the population are PDX1-positive; NKX6.1-positive cells. In some embodiments, at least 80% of the cells in the population are PDX1-positive; NKX6.1- positive cells. In some embodiments, between 70-100% of the cells in the population are PDX1- positive; NKX6.1-positive cells. In some embodiments, the cells are cultured in the medium for at least 12 hours but not more than 3 days. In some embodiments, the cells are cultured in the medium for at least 12 hours but not more than 2 days. In some embodiments, the cells are cultured in the medium for at least 12 hours but not more than 1 day. In some embodiments, the cells are cultured in the medium for 1 day, 2 days, 3 days, 4 days, 1-4 days, 1-3 days, 1-2 days, 2-4 days, or 2-3 days. In some embodiments, the medium comprises a sonic hedgehog pathway inhibitor. In some embodiments, the medium comprises a growth factor from the fibroblast growth factor (FGF) family.In some embodiments, the medium comprises a growth factor from the TGF-β superfamily. In some embodiments, the growth factor from the FGF family is any one of KGF, FGF2, FGF8B, FGF10, or FGF21. In some embodiments, the retinoic acid signaling pathway activator is any one of retinoic acid, CD1530, AM580, TTNPB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, and CD2314. In some embodiments, the sonic hedgehog pathway inhibitor selected from the group consisting of SANT1, SANT2, SANT4, Cur61414, forskolin, tomatidine, AY9944, triparanol, and cyclopamine. In some embodiments, the growth factor from the TGF-β superfamily is any one of activin A, GDF8, or GDF11. In some embodiments, the plurality of agents further comprises a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor. In some embodiments, the ROCK inhibitor is any one of Thiazovivin, Y- 27632, Fasudil / HA1077, or 14-1152. In some embodiments, the medium further comprises a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer comprises polyvinyl alcohol, poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), PEG copolymers, poly(N-isopropylacrylamide), or polyacrylamide. In some embodiments, the water-soluble synthetic polymer comprises polyvinyl alcohol. In some embodiments, the water-soluble synthetic polymer is present at a concentration of about 0.04% to about 0.06% (w / v) in the medium. In some embodiments, the water-soluble synthetic polymer comprises polyvinyl alcohol that is about 80% hydrolyzed. In some embodiments, the medium does not comprise a protein kinase C activator. In some embodiments, the medium does not comprise a bone morphogenetic (BMP) signaling pathway inhibitor. In some embodiments, the medium does not comprise a FOXO1 inhibitor. In some embodiments, the medium does not comprise a notch signaling pathway inhibitor. In some embodiments, the medium does not comprise an epidermal growth factor, a thyroid hormone, a TGFβ-R1 kinase inhibitor, a histone methyltransferase EZH2 inhibitor, and / or a Wnt signaling pathway inhibitor. In some embodiments, following the period of time, the cells are cultured in a medium comprising one or more of an epidermal growth factor, a thyroid hormone, a TGFβ-R1 kinase inhibitor, a notch signaling pathway inhibitor, a sonic hedgehog (SHH) signaling pathway inhibitor, a retinoic acid signaling pathway activator, a Rho- associated, coiled-coil containing protein kinase (ROCK) inhibitor, a protein kinase inhibitor, a bone morphogenetic (BMP) signaling pathway inhibitor, a histone methyltransferase EZH2 inhibitor, and / or a Wnt signaling pathway inhibitor. In some embodiments, prior to the period of time, the population of cells were cultured in a medium comprising a protein kinase C activator.

[0061] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a likenumeral. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:

[0063] FIG.1 shows a series of flow plots in which cells following the end of Stage 1 were assessed for SOX17 (x-axis) and OCT4 (y-axis). “Wild type” corresponds to cells generated following stage 1 of Protocol A; “Chir only” corresponds to cells generated following stage 1 of the Protocol A, but where activin A was excluded from stage 1; and “Activin only” corresponds to cells generated following stage 1 of the Protocol A, but where CHIR99021 was excluded from stage 1.

[0064] FIGS.2A and 2B show a series of flow plots in which cells following the end of Stage 1 were assessed for SOX17 (x-axis) and OCT4 (y-axis). In FIG.2A, “Act D1 Only”, “Act D2 Only”, and “Act D3 Only” correspond to cells harvested following stage 1 of experiments where activin A was included in stage 1 only on day 1, day 2, or day 3, respectively. In FIG.2B, “Act-D1”, “Act-D2”, and “Act-D3” correspond to cells harvested following stage 1 of experiments where activin A was excluded in stage 1 on day 1, day 2, or day 3, respectively.

[0065] FIG.3 shows a line graph illustrating the induction of PDX1 following stage 3 of Protocol A (“WT”) or following stage 3 in different conditions of removal / inclusion of activin A in stage 1. “ChiR only” corresponds to cells harvested following stage 3 of Protocol A where activin A was excluded from stage 1. “Activin only” corresponds to cells harvested following stage 3 of Protocol A where CHIR99021 was excluded from stage 1. “Activin D1 only”, “Activin D2 only”, and “Activin D3 only” correspond to cells harvested following stage 3 of experiments where activin A was included in stage 1 only on day 1, day 2, or day 3, respectively. “Activin -D1”, “Activin -D2”, and “Activin -D3” correspond to cells harvested following stage 3 of experiments where activin A was excluded in stage 1 on day 1, day 2, or day 3, respectively. The arrow compares the induction between WT and “Activin D2 only.” “Meso Inh” corresponds to a control condition in which cells were treated with a mesoderm inhibitor.

[0066] FIG.4 is a bar graph showing the yield of cells following stage 5 of Protocol A (“WT”) or in different conditions of removal / inclusion of activin A in stage 1. “CHIR only” corresponds to cells harvested following stage 5 of Protocol A where activin A was excluded from stage 1. “Activin only” corresponds to cells harvested following stage 5 of Protocol A where CHIR99021 was excluded from stage 1. “Act D1 only”, “Act D2 only”, and “Act D3 only” correspond to cells harvested following stage 5 of experiments where activin A was included in stage 1 only on day 1, day 2, or day 3, respectively. “Act-D1”, “Act-D2”, and “Act-D3” correspond to cells harvested following stage 5 of experiments where activin A was excluded in stage 1 on day 1, day 2, or day 3, respectively.

[0067] FIGS.5A and 5B are a series of flow plots of cells following stage 5 of Protocol A (“WT#1” and “WT#2”) or in different conditions of removal / inclusion of activin A in stage 1. ISL1 is plotted on the y-axis, and NKX6.1 is plotted on the x-axis. “CHIR Only” corresponds to cells harvested following stage 5 of Protocol A where activin A was excluded from stage 1. “Act Only”corresponds to cells harvested following stage 5 of Protocol A where CHIR99021 was excluded from stage 1. “Act D1 only”, “Act D2 only”, and “Act D3 only” correspond to cells harvested following stage 5 of experiments where activin A was included in stage 1 only on day 1, day 2, or day 3, respectively. “Act-D1”, “Act-D2”, and “Act-D3” correspond to cells harvested following stage 5 of experiments where activin A was excluded in stage 1 on day 1, day 2, or day 3, respectively.

[0068] FIG.6 shows a bar graph illustrating the glucose stimulation and the human C peptide secretion co-relation at Weeks 2, Week 4, and Week 6 post-transplantation (“post-TX”) of SC-islet cells generated using Protocol A (first two bars in each panel), or a modified version of that protocol. The middle two bars in each panel correspond to mice treated with SC-islets derived from Protocol A, but where activin A was included in Stage 1 only on days 2 and 3. The last two bars in each panel correspond to mice treated with SC-islets derived from Protocol A, but where activin A was included in Stage 1 only on day 2. C-peptide levels were tested using an ELISA assay following an in vivo glucose-stimulated insulin secretion (GSIS) assay. Blood samples were collected following fasting for ~16 hours (“fasted”) or 30 minutes after feeding with glucose (“30 min glucose”). “SI” corresponds to the stimulation index calculated using the GSIS assay comparing “fasted” blood samples with “30 min glucose” blood samples. Error bars show the standard deviation from 4 mice per condition. Each mouse received 5 million cells implanted in the kidney capsule.

[0069] FIGS.7A-7B are a series of flow plots plotting ISL1 on the y-axis and NKX6.1 on the x- axis. Cells were generated either at the end of stage 5 from the protocol of Protocol A (“Protocol A - 100% AA”), or at the end of stage 5 of a modified version of Protocol A. “0% AA” corresponds to post-stage 5 cells from Protocol A but where activin A was not included in stage 1. “DM+JNK+ITS- X” corresponds to post-stage 5 cells from Protocol A, but where activin A was replaced (“0% AA”) in stage 1 with 1 µM dorsomorphin (“DM”) and 1 µM JNK-IN-8 (“JNK”) and supplemented with ITS-X at 20 µg / L (“1X”), 100 µg / L (“5X”), or 200 µg / L (“10X”).

[0070] FIGS.8A-8B are a series of flow plots plotting ISL1 on the y-axis and NKX6.1 on the x- axis. Cells were generated either at the end of stage 5 from Protocol A (“Protocol A - 100% AA”), or at the end of stage 5 of a modified version of Protocol A. “0% AA” corresponds to post-stage 5 cells from Protocol A but where activin A was not included in stage 1. “DM+JNK+ITS-X” corresponds to post-stage 5 cells from Protocol A, but where activin A was replaced (“0% AA”) in stage 1 with 1 µM dorsomorphin (“DM”) and 1 µM JNK-IN-8 (“JNK”) and supplemented with ITS-X at 100 µg / L (“5X”) or 200 µg / L (“10X”) and with or without Akt inhibitor at 0.1 µM.

[0071] FIGS.9A-9B are a series of flow plots plotting ISL1 on the y-axis and NKX6.1 on the x- axis. Cells were generated either at the end of stage 5 from Protocol A (“Protocol A - 100% AA”), or at the end of stage 5 of a modified version of Protocol A. “0% AA” corresponds to post-stage 5 cells from Protocol A but where activin A was not included in stage 1. “DM+JNK+AKTi+ITS-X” corresponds to post-stage 5 cells from Protocol A, but where activin A was replaced (“0% AA”) instage 1 with 1 µM dorsomorphin (“DM”), 0.1 µM Akt inhibitor (“AKTi”), and 1 µM JNK-IN-8 (“JNK”) and supplemented with ITS-X at 100 µg / L (“5X”) or 200 µg / L (“10X”). The protocol used to generate post-stage 5 cells of “DM+JNK+AKTi+ITS-X (5X)” and “DM+JNK+AKTi+ITS-X (10X)” also differed from the cells generated by Protocol 1 in that AS1842856 was replaced in stage 4 with either FOXO1i-compound B or FOXO1i-compound C.

[0072] FIG.10 is a bar graph tabulating the number of ISL1-positive, NKX6.1-positive cells (“Beta”); ISL1-positive, NKX6.1-negative cells (“Non-beta islet”); ISL1-negative, NKX6.1-positive cells (“EC”); and ISL1-negative, NKX6.1-negative cells (“DN”). The first bar for each cell type corresponds to post-stage 5 cells generated in spinner flasks and in Biotts (combined data) using Protocol A (n=4). The second bar for each cell type corresponds to post-stage 5 cells generated in spinner flasks and in Biotts (combined data) using Protocol A, but where activin A is removed from stage 1 (n=2). The third bar for each cell type corresponds to post-stage 5 cells generated using Protocol A, but where activin A is replaced in stage 1 with 1 µM dorsomorphin and 1 µM JNK-IN-8 and supplemented with ITS-X at 200 µg / L (n=2). The fourth bar for each cell type corresponds to post-stage 5 cells generated using Protocol A, but where activin A is replaced in stage 1 with 1 µM dorsomorphin, 0.1 µM Akt inhibitor, and 1 µM JNK-IN-8 and supplemented with ITS-X at 200 µg / L (n=3). n=2 independent experiments.

[0073] FIG.11 is a bar graph tabulating the percentage of ISL1-positive, NKX6.1-positive cells (“Beta”); ISL1-positive, NKX6.1-negative cells (“Non-beta islet”); ISL1-negative, NKX6.1-positive cells (“EC”); and ISL1-negative, NKX6.1-negative cells (“DN”). The first bar for each cell type corresponds to post-stage 5 cells generated in spinner flasks and in Biotts (combined data) using Protocol A (n=4). The second bar for each cell type corresponds to post-stage 5 cells generated in spinner flasks and in Biotts (combined data) using Protocol A, but where activin A is replaced in stage 1 with 1 µM dorsomorphin, 0.1 µM Akt inhibitor, and 1 µM JNK-IN-8 and supplemented with ITS-X at 200 µg / L (n=3).

[0074] FIG.12 is a line graph illustrating the amount of cells yielded at the completion of the first five stages of Protocol A (darker line) or a modified version of Protocol A (lighter line) where activin A is replaced in stage 1 with 1 µM dorsomorphin, 0.1 µM Akt inhibitor, and 1 µM JNK-IN-8 and supplemented with ITS-X at 200 µg / L. “S0C” means Stage 0 complete, “S1C” means Stage 1 complete, etc. n=1 experiment.

[0075] FIG.13 shows several flow plots (top panels) of post-stage 5 cells generated in spinner flasks using Protocol A (“Protocol A - 100% AA”) or a modified version of Protocol A where activin A is replaced in stage 1 with 1 µM dorsomorphin, 0.1 µM Akt inhibitor, and 1 µM JNK-IN-8 and supplemented with ITS-X at 200 µg / L (“0% AA; DM+JNK+AKT+ITS-X (10X)”). The y-axis corresponds to ISL1 and x-axis corresponds to NKX6.1. The bottom panels provide microscopyimages of the cell clusters generated using either protocol; magnification 4x using a Nikon Eclipse microscope.

[0076] FIG.14 is a bar graph tabulating the percentage of ISL1-positive, NKX6.1-positive cells (“Beta”); ISL1-positive, NKX6.1-negative cells (“Non-beta islet”); ISL1-negative, NKX6.1-positive cells (“EC”); ISL1-negative, NKX6.1-negative cells (“DN”). The first bar for each cell type corresponds to stage 6, day 7 cells generated using Protocol A . The second bar for each cell type corresponds to stage 6, day 7 cells generated using Protocol A, but where activin A is replaced in stage 1 with 1 µM dorsomorphin, 0.1 µM Akt inhibitor, and 1 µM JNK-IN-8 and supplemented with ITS-X at 200 µg / L. n=1 experiment.

[0077] FIG.15 is a line graph illustrating the amount of cells yielded at the completion of days 1, 4 and 7 of stage 6 of Protocol A (darker line) or a modified version of Protocol A (lighter line) where activin A is replaced in stage 1 with 1 µM dorsomorphin, 0.1 µM Akt inhibitor, and 1 µM JNK-IN-8 and supplemented with ITS-X at 200 µg / L. n=1 experiment.

[0078] FIG.16 shows several flow plots (top panels) of stage 6 day 7 cells generated in spinner flasks using Protocol A (“Protocol A - 100% AA”) or a modified version of Protocol A where activin A is replaced in stage 1 with 1 µM dorsomorphin, 0.1 µM Akt inhibitor, and 1 µM JNK-IN-8 and supplemented with ITS-X at 200 µg / L (“0% AA; DM+JNK+AKT+ITS-X (10X)”). The y-axis corresponds to ISL1 and x-axis corresponds to NKX6.1. The bottom panels provide microscopy images of the cell clusters generated using either protocol; magnification 4x using a Nikon Eclipse microscope.

[0079] FIG.17 is a bar graph showing the effect of a modified protocol (Protocol A with glucose spiking) relative to Protocol A (“control”) on yield of cells following completion of stage 5 (“St5C”). The graph indicates data from 3L PBS reactors (triangles) and spinners (circles).

[0080] FIG.18 show a series of flow plots of post-stage 5 cells generated using Protocol A (“Protocol A”) or a modified version of the protocol where GC1 was removed from Stage 5 (“-GC1 (Stage 5)”) or where half as much vitamin C (i.e., 0.044 mg / ml) was added to stage 5 (“1 / 2 Vit. C (Stage 5)”). The y-axis corresponds to ISL1 and x-axis corresponds to NKX6.1.

[0081] FIG.19 are bar graphs showing the effect of a modified protocol (i.e., Protocol A, but where two times as much GC1 is added and 1 / 2 as much ascorbic acid is added) on the percentage of cell types (first panel) and total yield of cells (second panel) following stage 5. The modified protocol (second bar in each cell type in the first panel, and second bar in second panel) is compared to Protocol A (“control”- first bar in each cell type in the first panel, and first bar in the second panel). ISL1-positive, NKX6.1-positive cells (“Beta”); ISL1-positive, NKX6.1-negative cells (“Non-beta islet”); ISL1-negative, NKX6.1-positive cells (“SC-EC”); ISL1-negative, NKX6.1-negative cells (“DN”). Cells were generated in spinner flasks.

[0082] FIG.20 is a set of bar graphs showing the effect of a modified protocol (i.e., Protocol A but where two times as much GC1 is added and 1 / 2 as much ascorbic acid is added) on the percentage of cell types at Stage 6, Day 4 (first panel) and total yield of cells (second panel) at Stage 6, Days 4, 7 and 11. The modified protocol (second bar in each cell type in the first panel, and second bar in second panel) is compared to Protocol A (“control”- first bar in each cell type in the first panel, and first bar in the second panel). ISL1-positive, NKX6.1-positive cells (“Beta”); ISL1-positive, NKX6.1-negative cells (“Non-beta islet”); ISL1-negative, NKX6.1-positive cells (“SC-EC”); ISL1- negative, NKX6.1-negative cells (“DN”). Cells were generated in spinner flasks.

[0083] FIG.21 shows a series of microscopy images of cell clusters generated following completion of each of stages 0-5 (“Stage 0C”, “Stage 1C”, etc.) of Protocol A (first two rows from two separate experiments) or of a modified version of Protocol A (“modified protocol”) where a) glucose was spiked in at stages 1 and 2, and b) half as much ascorbic acid (vitamin C) and twice as much GC-1 was added at stage 5.

[0084] FIG.22 shows a bar graph illustrating the effect on cell yield following completion of stage 5 of Protocol A (first bar) or a modified version of Protocol A (second bar) where a) glucose was spiked in at stages 1 and 2, and b) half as much ascorbic acid (vitamin C) and twice as much GC- 1 was added at stage 5.

[0085] FIG.23 shows a series of flow plots of post-stage 5 cells generated using Protocol A (“Protocol A”) from two separate experiments (first two panels) or using a modified version of Protocol A (third panel) where a) glucose was spiked in at stages 1 and 2 and b) half as much ascorbic acid (vitamin C) and twice as much GC-1 was added at stage 5). The y-axis corresponds to ISL1 and x-axis corresponds to NKX6.1.

[0086] FIG.24 shows several graphs illustrating the percentage of different cell types generated following stage 5 of a modified version of Protocol A where a) glucose was spiked in at stages 1 and 2 and b) half as much ascorbic acid (vitamin C) and twice as much GC-1 was added at stage 5 in 3L PBS bioreactors in two separate experiments (first and second panel). ISL1-positive, NKX6.1- positive cells (“Beta”); ISL1-positive, NKX6.1-negative cells (“Non-beta islet”); ISL1-negative, NKX6.1-positive cells (“SC-EC”); ISL1-negative, NKX6.1-negative cells (“DN”).

[0087] FIG.25 shows several graphs illustrating the percentage of different cell types generated at stage 6 of a modified version of Protocol A where a) glucose was spiked in at stages 1 and 2 and b) half as much ascorbic acid (vitamin C) and twice as much GC-1 was added at stage 5. The first panel corresponds to cells starting at the start of stage 6, while the second and third panels correspond to cells generated at day 4 of stage 6. ISL1-positive, NKX6.1-positive cells (“Beta”); ISL1-positive, NKX6.1-negative cells (“Non-beta islet”); ISL1-negative, NKX6.1-positive cells (“SC-EC”); ISL1- negative, NKX6.1-negative cells (“DN”).

[0088] FIG.26 is a simplified schematic showing the transitions (dashed lines) between each of Stages 3-5 of Protocol A (Table 1) and modified versions of Protocol A. Rows 2-7 illustrate modified versions of Stage 4 of Protocol A, where Stage 4 is eliminated completely (second row), or where days are removed from the end of Stage 4 (third through seventh rows).

[0089] FIG.27A is a bar graph showing the percentage of NKX6.1-positive, ISL1-positive cells. The graph shows t test pairwise comparison between cells collected following stage 5 from the Protocol A (“WT”) or from versions of Protocol where Stage 5 (S4) was removed (second bar; corresponding to protocol from second row of FIG.26) or shortened (third through seventh bars; corresponding to protocol from third through seventh rows of FIG.26).

[0090] FIG.27B is a bar chart showing total composition of harvest results, comprising NKX6.1- positive, ISL1-negative; NKX6.1-positive, ISL1-positive; NKX6.1-negative, ISL1-positive; and NKX6.1-negative, ISL1-negative cells. One million living cells at stage 5 complete were fixed with 4% PFA and then washed with PBS. For staining, 200,000 cells from each sample were taken and stained with antibodies at the ratio of 1:40000 for ISL1 and 1:4000 for NKX6-1. ISL1 antibody was primary-conjugated with PE, while NKX6.1 was probed with secondary antibody bearing Alexa 647 fluorophore. The composition shown here is an average of three independent differentiation runs each having a different seed train from the same cell bank. Each run had a technical duplicate. Error bars represent the values from 3 independent experiments in biotts, with each experiment containing 2 technical replicates.

[0091] FIG.28 shows a bar graph showing the average values from three independent harvests with each harvest having 2 replicates per condition. Biotts were harvested, and then resuspended in 10 mls of media. From the resuspension, 20 ul of media was taken and diluted 10 times to take final counts from viacell blue. The counts / ml were then multiplied with total volume to get final yield per biott. P values represent statistics by paired-student T test between conditions at their respective harvest. The first through seventh bars of the graph correspond to cells generated using the Protocols described in rows 1-7 of FIG.26, respectively.

[0092] FIG.29A shows a flow dot plot showing the percentage of cells expressing chromogranin A. Both Protocol A (left) and the S4D1-Short protocol (right; protocol corresponding to Row 3 of FIG.26) show 96% of aggregated cells are endocrine in nature. FIG.29B shows a flow dot plot showing the percentage of NKX6.1-positive, ISL1-positive cells from Protocol A (left) or S4D1-Short (right) after reaggregation and seven days of Stage 6 conditions (see Table 1). For stage 6 thaw, cells were thawed 2-vials each and pooled at S6D7 harvest. One million cells were fixed with 4% PFA and stained accordingly.

[0093] FIG.30A shows a flow dot plot showing the dynamics of cell fate specification in Protocol A (left) and S4D1-Short (right; protocol corresponding to Row 3 of FIG.26) at harvest. FIG.30B shows the yield per spinner (alive cells) in three independent runs of differentiation, witheach run having a separate seed train. One hundred-and-twenty-five million cells per spinner were seeded in either conditions at stage 0, and the cells were then differentiated with Protocol A or the S4D1-Short conditions. At the harvest, 2X spinners from each condition were pooled together and harvested. One million cells per ml viable cells were taken and fixed with 4% paraformaldehyde. The 200,000 cells from the fixed cells were stained with antibodies against ISL1 and NKX6.1 at 1:4000 each. Statistics were performed with unpaired student T test. DETAILED DESCRIPTION

[0094] The following description and examples illustrate embodiments of the present disclosure in detail. It is to be understood that this disclosure is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there are numerous variations and modifications of this disclosure, which are encompassed within its scope.

[0095] All terms are intended to be understood as they would be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

[0096] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0097] Although various features of the present disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarity, the present disclosure can also be implemented in a single embodiment.

[0098] The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated case, e.g., to any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present disclosure, the preferred materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0099] In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. As used herein, the term “media” includes a singular medium or multiple media, unless clearly indicated otherwise.

[0100] In this application, the use of “or” means “and / or” unless stated otherwise. The terms “and / or” and “any combination thereof” and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and / or C” or “A, B, C, or any combination thereof” can mean “A individually; B individually; C individually; A and B; B and C; A and C; andA, B, and C.” The term “or” can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.

[0101] Furthermore, use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting.

[0102] Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures.

[0103] As used in this specification and claim(s), 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 contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

[0104] The term “about” or “approximately” means within an acceptable error range for the 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 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. In another example, the amount “about 10” includes 10 and any amounts from 9 to 11. In yet another example, the term “about” in relation to a reference numerical value can also include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from 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. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0105] The term “diabetes” and its grammatical equivalents as used herein can refer to is a disease characterized by high blood sugar levels over a prolonged period. For example, the term “diabetes” and its grammatical equivalents as used herein can refer to all or any type of diabetes, including, but not limited to, type 1, type 2, cystic fibrosis-related, surgical, gestational diabetes, and mitochondrial diabetes. In some embodiments, diabetes can be a form of hereditary diabetes. In some embodiments, diabetes can be an autoimmune form of diabetes.

[0106] The term “endocrine cell(s),” if not particularly specified, can refer to hormone-producing cells present in the pancreas of an organism, such as “islet”, “islet cells”, “islet equivalent”, “islet-likecells”, “pancreatic islets” and their grammatical equivalents. In an embodiment, the endocrine cells can be differentiated from pancreatic progenitor cells or precursors. Islet cells can comprise different types of cells, including, but not limited to, pancreatic α cells, pancreatic β cells, pancreatic δ cells, pancreatic F cells, and / or pancreatic ε cells. Islet cells can also refer to a group of cells, cell clusters, or the like. In particular embodiments, islet cells are stem cell-derived islet cells, e.g., islet cells that are derived from stem cells in vitro.

[0107] The terms “progenitor” and “precursor” cell are used interchangeably herein and refer to cells that have a cellular phenotype that is more primitive (e.g., is at an earlier step along a developmental pathway or progression than is a fully differentiated cell) relative to a cell which it can give rise to by differentiation. Often, progenitor cells can also have significant or very high proliferative potential. Progenitor cells can give rise to multiple distinct differentiated cell types or to a single differentiated cell type, depending on the developmental pathway and on the environment in which the cells develop and differentiate.

[0108] A “precursor thereof” as the term related to an insulin-positive endocrine cell can refer to any cell that is capable of differentiating into an insulin-positive endocrine cell, including for example, a pluripotent stem cell, a definitive endoderm cell, a primitive gut tube cell, a pancreatic progenitor cell, or endocrine progenitor cell, that if cultured under suitable conditions will differentiate the precursor cell into the insulin-positive endocrine cell.

[0109] The terms “stem cell-derived β cell,” “SC-β cell,” “functional β cell,” “functional pancreatic β cell,” “mature SC-β cell,” “β-like cell” and their grammatical equivalents can refer to cells (e.g., non-native pancreatic β cells) that display at least one marker indicative of a pancreatic β cell (e.g., PDX-1, ISL1 or NKX6.1), expresses insulin. In some embodiments, the SC-β cell displays a glucose stimulated insulin secretion (GSIS) response similar or superior to that of an endogenous mature β cell (e.g., a mature β from a healthy functioning pancreas from a healthy adult non-diabetic patient). For simplicity, SC-β cells may be referred to as simply “β cells” in this disclosure. In some embodiments, the terms “SC-β cell” and “non-native β cell” as used herein are interchangeable. In some embodiments, the “SC-β cell” expresses lower levels of MAFA than a pancreatic β cell from a healthy adult human patient. In some embodiments, the “SC-β cell” expresses higher levels of MAFB than a pancreatic β cell from a healthy adult human patient. In some embodiments, the “SC-β cell” expresses higher levels of SIX2, HOPX, IAPP and / or UCN3 than a pancreatic β cell from a healthy adult human patient. In some embodiments, the “SC-β cell” comprises a mature pancreatic cell. It is to be understood that the SC-β cells need not be derived (e.g., directly) from stem cells, as the methods of the disclosure are capable of deriving SC-β cells from any insulin-positive endocrine cell or precursor thereof using any cell as a starting point (e.g., one can use embryonic stem cells, induced- pluripotent stem cells, progenitor cells such as definitive endoderm cells, partially reprogrammed somatic cells (e.g., a somatic cell which has been partially reprogrammed to an intermediate statebetween an induced pluripotent stem cell and the somatic cell from which it was derived), multipotent cells, totipotent cells, a transdifferentiated version of any of the foregoing cells, etc., as the invention is not intended to be limited in this manner). In some embodiments, the induced pluripotent stem cells are chemically-induced pluripotent stem cells (see, e.g., Wang et al., 2024, Cell, 187, 1–13). In some embodiments, the induced pluripotent stem cells are derived from cells taken from a subject (e.g., a diabetic subject), and the induced pluripotent stem cells are then differentiated using any of the methods disclosed herein in order to make SC-islet cells or precursors thereof that may be administered back to the patient, i.e., the induced pluripotent stem cells are autologous cells to the subject (see, e.g., Wang et al., 2024, Cell, 187, 1–13). In some embodiments, the SC-β cells exhibit a response to multiple glucose challenges (e.g., at least one, at least two, or at least three or more sequential glucose challenges). In some embodiments, the response resembles the response of endogenous islets (e.g., human islets) to multiple glucose challenges. In some embodiments, the morphology of the SC-β cell resembles the morphology of an endogenous β cell. In some embodiments, the SC-β cell exhibits an in vitro GSIS response that resembles the GSIS response of an endogenous β cell. In some embodiments, the SC-β cell exhibits an in vivo GSIS response that resembles the GSIS response of an endogenous β cell. In some embodiments, the SC-β cell exhibits both an in vitro and in vivo GSIS response that resembles the GSIS response of an endogenous β cell. In some embodiments, the GSIS response of the SC-β cell can be observed within two weeks of transplantation of the SC-β cell into a host (e.g., a human or animal). In some embodiments, the GSIS response of the SC-β cell can be observed within three weeks of transplantation of the SC-β cell into a host (e.g., a human or animal). In some embodiments, the GSIS response of the SC-β cell can be observed within four weeks of transplantation of the SC-β cell into a host (e.g., a human or animal). In some embodiments, the GSIS response of the SC-β cell can be observed between one month and three months of transplantation of the SC-β cell into a host (e.g., a human or animal). In some embodiments, the SC-β cells package insulin into secretory granules. In some embodiments, the SC-β cells exhibit encapsulated crystalline insulin granules. In some embodiments, the SC-β cells exhibit a stimulation index of greater than 1. In some embodiments, the SC-β cells exhibit a stimulation index of greater than 1.1. In some embodiments, the SC-β cells exhibit a stimulation index of greater than 2. In some embodiments, the stimulation index of the cell is characterized by the ratio of insulin secreted in response to high glucose concentrations (e.g., 15 mM) compared to low glucose concentrations (e.g., 2.5 mM).

[0110] In some embodiments, the SC-β cells exhibit cytokine-induced apoptosis in response to cytokines. In some embodiments, insulin secretion from the SC-β cells is enhanced in response to known antidiabetic drugs (e.g., secretagogues). In some embodiments, the SC-β cells are monohormonal. In some embodiments, the SC-β cells do not abnormally co-express other hormones, such as glucagon, somatostatin or pancreatic polypeptide. In some embodiments, the SC-β cellsexhibit a low rate of replication. In some embodiments, the SC-β cells increase intracellular Ca2+ in response to glucose.

[0111] The terms “stem cell-derived α cell,” “SC-α cell,” “functional α cell,” “functional pancreatic α cell,” “mature SC-α cell,” “α-like cell” and their grammatical equivalents can refer to cells (e.g., non-native pancreatic α cells) that display at least one marker indicative of a pancreatic α cell (e.g., glucagon, expressing ISL1 but not NKX6.1), expresses glucagon, and is capable of secreting functional glucagon in response to a stimulus that induces an endogenous pancreatic α cell to secrete functional glucagon. In some embodiments, the “SC-α cell” does not express somatostatin. In some embodiments, the “SC-α cell” does not express insulin. In some embodiments, the terms “SC-α cell” and “non-native α cell” as used herein are interchangeable. In some embodiments, the “SC-α cell” comprises a mature pancreatic cell. For short, these cells may be referred to as simply “α cells” in this disclosure.

[0112] The terms “stem cell-derived δ cell,” “SC-δ cell,” “functional δ cell,” “functional pancreatic δ cell,” “mature SC-δ cell,” “δ-like cell” and their grammatical equivalents can refer to cells (e.g., non-native pancreatic δ cells) that display at least one marker indicative of a pancreatic δ cell (e.g., somatostatin), expresses and is capable of secreting somatostatin in response to a stimulus that induces an endogenous pancreatic δ cell to secrete functional glucagon. For simplicity, SC- δ cells may be referred to as simply “δ cells” in this disclosure. In some embodiments, “SC-δ cell” does not express glucagon. In some embodiments, “SC-δ cell” does not express insulin. In some embodiments, the terms “SC-δ cell” and “non-native δ cell” as used herein are interchangeable. In some embodiments, the “SC-δ cell” comprises a mature pancreatic cell.

[0113] The terms “stem cell-derived enterochromaffin (EC) cell,” “SC-EC cell,” and their grammatical equivalents can refer to cells (e.g., non-native pancreatic EC cells) that display at least one marker indicative of a pancreatic EC cell (e.g., VMAT1 (vesicular monoamine transporter 1), expressing NKX6.1 but not ISL1). In some embodiments, the terms “SC-EC cell” and “non-native EC cell” as used herein are interchangeable.

[0114] Similar to SC-β cells, it is to be understood that the SC-α, SC-δ cells, and SC-EC cells need not be derived (e.g., directly) from stem cells, as the methods of the disclosure are capable of deriving SC-α cells from other precursor cells generated during in vitro differentiation of SC-β cells as a starting point (e.g., one can use embryonic stem cells, induced-pluripotent stem cells, progenitor cells, partially reprogrammed somatic cells (e.g., a somatic cell which has been partially reprogrammed to an intermediate state between an induced pluripotent stem cell and the somatic cell from which it was derived), multipotent cells, totipotent cells, a transdifferentiated version of any of the foregoing cells, etc., as the invention is not intended to be limited in this manner).

[0115] As used herein, the term “insulin producing cell” and its grammatical equivalent refer to a cell differentiated from a pancreatic progenitor, or precursor thereof, which secretes insulin. Aninsulin-producing cell can include a pancreatic β cell as that term is described herein, as well as pancreatic β-like cells (e.g., insulin-positive, endocrine cells) that synthesize (e.g., transcribe the insulin gene, translate the proinsulin mRNA, and modify the proinsulin mRNA into the insulin protein), express (e.g., manifest the phenotypic trait carried by the insulin gene), or secrete (release insulin into the extracellular space) insulin in a constitutive or inducible manner. A population of insulin producing cells e.g., produced by differentiating insulin-positive endocrine cells or a precursor thereof 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 least characteristics of an endogenous β cell and exhibit a glucose stimulated insulin secretion (GSIS) response that resembles an endogenous adult β cell). The population of insulin-producing cells, e.g., produced by the methods as disclosed herein can comprise mature pancreatic β cell or SC-β cells, and can also contain non-insulin- producing cells (e.g., cells of cell like phenotype with the exception they do not produce or secrete insulin).

[0116] The terms “insulin-positive β-like cell,” “insulin-positive endocrine cell,” and their grammatical equivalents can refer to cells (e.g., pancreatic endocrine cells) that display at least one marker indicative of a pancreatic β cell and also expresses insulin but, unless specified otherwise, lack a glucose stimulated insulin secretion (GSIS) response characteristic of an endogenous β cell. Exemplary markers of “insulin-positive endocrine cell” include, but are not limited to, NKX6.1 (NK6 homeobox 1), ISL1 (Islet1), and insulin.

[0117] The term “β cell marker” refers to, without limitation, proteins, peptides, nucleic acids, polymorphism of proteins and nucleic acids, splice variants, fragments of proteins or nucleic acids, elements, and other analyte which are expressed or present in pancreatic β cells. Exemplary β cell markers include, but are not limited to, pancreatic and duodenal homeobox 1 (PDX1) polypeptide, insulin, c-peptide, amylin, E-cadherin, Hnf3β, PCI / 3, B2, Nkx2.2, GLUT2, PC2, ZnT-8, ISL1, Pax6, Pax4, NeuroD, 1 Inf1b, Hnf-6, Hnf-3beta, VMAT2, NKX6.1, and MafA, and those described in Zhang et al., Diabetes.50(10):2231-6 (2001). In some embodiments, the β cell marker is a nuclear β- cell marker. In some embodiments, the β cell marker is PDX1 or PH3. In some embodiments, the β cell markers are NKX6.1 and ISL1.

[0118] The term “pancreatic endocrine marker” can refer to without limitation, proteins, peptides, nucleic acids, polymorphism of proteins and nucleic acids, splice variants, fragments of proteins or nucleic acids, elements, and other analytes which are expressed or present in pancreatic endocrine cells. Exemplary pancreatic endocrine cell markers include, but are not limited to, Ngn-3, NeuroD and Islet-1.

[0119] The term “pancreatic progenitor,” “pancreatic endocrine progenitor,” “pancreatic precursor,” “pancreatic endocrine precursor” and their grammatical equivalents are used interchangeably herein and can refer to a stem cell which is capable of becoming a pancreatichormone expressing cell capable of forming pancreatic endocrine cells, pancreatic exocrine cells or pancreatic duct cells. These cells are committed to differentiating towards at least one type of pancreatic cell, e.g. β cells that produce insulin; α cells that produce glucagon; δ cells (or D cells) that produce somatostatin; and / or F cells that produce pancreatic polypeptide. In some embodiments, such cells can express at least one of the following markers: NGN3, NKX2.2, NeuroD, ISL-1, Pax4, Pax6, or ARX.

[0120] The term “PDX1-positive pancreatic progenitor” as used herein can refer to a cell which is a pancreatic endoderm (PE) cell which has the capacity to differentiate into SC-β cells, such as pancreatic β cells. A PDX1-positive pancreatic progenitor expresses the marker PDX1. Other markers include, but are not limited to Cdcp1, or Ptf1a, or HNF6 or NRx2.2. The expression of PDX1 may be assessed by any method known by the skilled person such as immunochemistry using an anti-PDX1 antibody or quantitative RT-PCR. In some embodiments, a PDX1-positive pancreatic progenitor cell lacks expression of NKX6.1. In some embodiments, a PDX1-positive pancreatic progenitor cell can also be referred to as PDX1-positive, NKX6.1-negative pancreatic progenitor cell due to its lack of expression of NKX6.1. In some embodiments, the PDX1-positive pancreatic progenitor cells can also be termed as “pancreatic foregut endoderm cells.”

[0121] The term “pancreatic foregut precursor cells” means a pancreatic endoderm (PE) cell which has the capacity to differentiate into insulin-producing cells, such as pancreatic β cells. Pancreatic foregut precursor cells express PDX1 and markers such as NKX6.1, Cdcp1, or Ptf1a, or HNF6 or NRx2.2. In particular embodiments, pancreatic foregut precursor cells are PDX1-positive, NKX6.1-positive cells. “PDX1-positive, NKX6.1-positive pancreatic progenitor,” and “NKX6.1- positive pancreatic progenitor” are used interchangeably herein. The expression of NKX6-1 may be assessed by any method known by the skilled person such as immunochemistry using an anti-NKX6-1 antibody or quantitative RT-PCR. As used herein, the terms “NKX6.1” and “NKX6-1” are equivalent and interchangeable.

[0122] The terms “NeuroD” and “NeuroD1” are used interchangeably and identify a protein expressed in pancreatic endocrine progenitor cells and the gene encoding it.

[0123] The term “differentiated cell” or its grammatical equivalents means any primary cell that is not, in its native form, pluripotent as that term is defined herein. Stated another way, 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) in a cellular differentiation process. Without wishing to be limited to theory, a pluripotent stem cell in the course of normal ontogeny can differentiate first to an endoderm cell that is capable of forming pancreas cells and other endoderm cell types. Further differentiation of an endoderm cell may lead to the pancreatic pathway, where ~98% of the cells become exocrine, ductular, or matrix cells, and ~2% become endocrine cells. Early endocrine cells are islet progenitors, which can then differentiatefurther into insulin-producing cells (e.g. functional endocrine cells) which secrete insulin, glucagon, somatostatin, or pancreatic polypeptide. Endoderm cells can also be differentiated into other cells of endodermal origin, e.g. lung, liver, intestine, thymus etc.

[0124] As used herein, the term “somatic cell” can refer to any cells forming the body of an organism, as opposed to germline cells. In mammals, germline cells (also known as “gametes”) are the spermatozoa and ova which fuse during fertilization to produce a cell called a zygote, from which the entire mammalian embryo develops. Every other cell type in the mammalian body – apart from the sperm and ova, the cells from which they are made (gametocytes) and undifferentiated stem cells – is a somatic cell: internal organs, skin, bones, blood, and connective tissue are all made up of somatic cells. In some embodiments the somatic cell is a “non-embryonic somatic cell”, by which is meant a somatic cell that is not present in or obtained from an embryo and does not result from proliferation of such a cell in vitro. In some embodiments the somatic cell is an “adult somatic cell”, by which is meant a cell that is present in or obtained from an organism other than an embryo or a fetus or results from proliferation of such a cell in vitro. Unless otherwise indicated the methods for converting at least one insulin-positive endocrine cell or precursor thereof to an insulin-producing, glucose responsive cell can be performed both in vivo and in vitro (where in vivo is practiced when at least one insulin-positive endocrine cell or precursor thereof are present within a subject, and where in vitro is practiced using an isolated at least one insulin-positive endocrine cell or precursor thereof maintained in culture).

[0125] As used herein, the term “adult cell” can refer to a cell found throughout the body after embryonic development.

[0126] The term “endoderm cell” as used herein can refer to a cell which is from one of the three primary germ cell layers in the very early embryo (the other two germ cell layers are the mesoderm and ectoderm). The endoderm is the innermost of the three layers. An endoderm cell differentiates to give rise first to the embryonic gut and then to the linings of the respiratory and digestive tracts (e.g., the intestine), the liver and the pancreas.

[0127] The term “a cell of endoderm origin” as used herein can refer to any cell which has developed or differentiated from an endoderm cell. For example, a cell of endoderm origin includes cells of the liver, lung, pancreas, thymus, intestine, stomach and thyroid. Without wishing to be bound by theory, liver and pancreas progenitors (also referred to as pancreatic progenitors) are developed from endoderm cells in the embryonic foregut. Shortly after their specification, liver and pancreas progenitors rapidly acquire markedly different cellular functions and regenerative capacities. These changes are elicited by inductive signals and genetic regulatory factors that are highly conserved among vertebrates. Interest in the development and regeneration of the organs has been fueled by the intense need for hepatocytes and pancreatic β cells in the therapeutic treatment of liver failure and type I diabetes. Studies in diverse model organisms and humans have revealedevolutionarily conserved inductive signals and transcription factor networks that elicit the differentiation of liver and pancreatic cells and provide guidance for how to promote hepatocyte and β cell differentiation from diverse stem and progenitor cell types.

[0128] The term “definitive endoderm” as used herein can refer to a cell differentiated from an endoderm cell and which can be differentiated into a SC-β cell (e.g., a pancreatic β cell). A definitive endoderm cell expresses the marker Sox17. Other markers characteristic of definitive endoderm cells may 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, and do not express significant levels of GATA4, SPARC, APF or DAB. Definitive endoderm cells are not positive for the marker PDX1 (e.g. they are PDX1-negative). Definitive endoderm cells have the capacity to differentiate into cells including those of the liver, lung, pancreas, thymus, intestine, stomach and thyroid. The expression of Sox17 and other markers of definitive endoderm may be assessed by any method known by the skilled person such as immunochemistry, e.g., using an anti- Sox17 antibody, or quantitative RT-PCR.

[0129] The term “pancreatic endoderm” can refer to a cell of endoderm origin which is capable of differentiating into multiple pancreatic lineages, including pancreatic β cells, but no longer has the capacity to differentiate into non-pancreatic lineages.

[0130] The term “pancreatic islet cells” refers to a population of cells that include different types of pancreatic endocrine cells (β-cells, α-cells, δ-cells, ε-cells) and enterochromaffin (EC) cells, e.g., as described in Xavier et al. (J Clin Med.2018 Mar; 7(3): 54), incorporated herein by reference.

[0131] The term “primitive gut tube cell” or “gut tube cell” as used herein can refer to a cell differentiated from an endoderm cell and which can be differentiated into a SC-β cell (e.g., a pancreatic β cell). A primitive gut tube cell expresses at least one of the following markers: HNP1-β, HNF3-β or HNF4-α. In some embodiments, a primitive gut tube cell is FOXA2-positive and SOX2- positive, i.e., expresses both FOXA2 (also known as HNF3-β) and SOX2. In some embodiments, a primitive gut tube cell is FOXA2-positive and PDX1-negative, i.e., expresses FOXA2 but not PDX1. Primitive gut tube cells have the capacity to differentiate into cells including those of the lung, liver, pancreas, stomach, and intestine. The expression of HNF1-β and other markers of primitive gut tube may be assessed by any method known by the skilled person such as immunochemistry, e.g., using an anti-HNF1-β antibody.

[0132] The term “phenotype” can refer to one or a number of total biological characteristics that define the cell or organism under a particular set of environmental conditions and factors, regardless of the actual genotype.

[0133] The terms “patient,” “subject,” and “individual” may be used interchangeably and refer to either a human or a non-human animal. The “non-human animals” and “non-human mammals” asused interchangeably herein, includes 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. However, advantageously, the subject is a mammal such as a human, or other mammals such as a domesticated mammal, e.g., dog, cat, horse, and the like, or production mammal, e.g. cow, sheep, pig, and the like. “Patient in need thereof” or “subject in need thereof” is referred to herein as a patient diagnosed with or suspected of having a disease or disorder, for instance, but not restricted to diabetes.

[0134] “Administering” as used herein can refer to providing one or more compositions described herein to a patient or a subject. By way of example and not limitation, composition administration, e.g., injection, can be performed by intravenous (i.v.) injection, sub-cutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, or intramuscular (i.m.) injection. One or more such routes can be employed. Parenteral administration can be, for example, by bolus injection or by gradual perfusion over time. Alternatively, or concurrently, administration can be by the oral route. Additionally, administration can also be by surgical deposition of a bolus or pellet of cells, or positioning of a medical device. In an embodiment, a composition of the present disclosure can comprise engineered cells or host cells expressing nucleic acid sequences described herein, or a vector comprising at least one nucleic acid sequence described herein, in an amount that is effective to treat or prevent proliferative disorders. A pharmaceutical composition can comprise the cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions can comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0135] The ranges disclosed throughout are sometimes referred to as, for example, “X is administered on or on about day 1 to 2; or 2 to 3 [or any numerical range].” This range includes the numbers themselves (e.g., the endpoints of the range) and any individual numbers present in this range.

[0136] All these different combinations are contemplated by the ranges disclosed throughout. All disclosed ranges should be interpreted in this manner, whether it refers to an administration of a therapeutic agent or referring to days, months, years, weight, dosage amounts, etc., unless otherwise specifically indicated to the contrary. Differentiation stages

[0137] Pancreatic differentiation as disclosed herein may be carried out in a step-wise manner. In an exemplary embodiment of the step-wise progression, “Stage 1” or “S1” or “St1” refers to the first step in the differentiation process, the differentiation of pluripotent stem cells into cells expressingmarkers characteristic of definitive endoderm cells (“DE”, “Stage 1 cells” or “St1 cells” or “S1 cells”). In some embodiments, “Stage 2” refers to the second step, the differentiation of cells expressing markers characteristic of definitive endoderm cells into cells expressing markers characteristic of gut tube cells (“GT”, “Stage 2 cells” “St2 cells” or “S2 cells”). In some embodiments, “Stage 3” refers to the third step, the differentiation of cells expressing markers characteristic of gut tube cells into cells expressing markers characteristic of pancreatic progenitor 1 cells (“PP1”, “Stage 3 cells” or “St3 cells” or “S3 cells”). In some embodiments, “Stage 4” refers to the fourth step, the 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 “St4 cells” or “S4 cells”). In some embodiments, “Stage 5” refers to the fifth step, the 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 “St5 cells” or “S5 cells”). In some embodiments, “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 β cells (“SC-β cells”) or pancreatic endocrine α cells (“SC-α cells”). It should be appreciated, however, that not all cells in a particular population progress through these stages at the same rate, i.e., some cells may have progressed less, or more, down the differentiation pathway than the majority of cells present in theparticular population. For example, in some embodiments, SC-β cells can be identified during stage 5, at the conclusion of stage 5, at the beginning of stage 6, etc. Examples of methods of making cells of any one of stages 1-6 are provided in, for example, US Patent 10,030,229; US Patent 10,443,042; US Patent No. US 11,466,256; published application US 20200332262; and published application US 20210198632, published application US 20220090020, published application US 2022-0233646; published application US 2022-0090020; published application US 20230218676; and published application WO2022147056, each of which is incorporated by reference in its entirety. Compositions and methods for producing pancreatic islet cells

[0138] In some aspects, the present disclosure provides compositions and methods of differentiating pancreatic islet cells (e.g., differentiating from stem cells such as human embryonic stem cells or human pluripotent stem cells). The compositions and methods provided herein can, in some embodiments, offer pancreatic SC-islet cells, cell populations, or cell clusters containing pancreatic SC-β cells and pancreatic SC-α cells. In some embodiments, such pancreatic SC-islet cells, cell populations or cell clusters exhibit, high insulin content, superior glucose-dependent insulin secretion response, as well as a percentage of pancreatic SC-α, SC-β, and SC-δ cells and enterochromaffin (EC) cells, which can resemble native pancreatic islets both structurally and functionally. In some embodiments, a population of pancreatic islet cells (e.g., stem cell derivedpancreatic islet cells) produced using the compositions and methods described herein comprises at least 50% pancreatic SC-β cells, up to 30% pancreatic SC-α cells, 3-10% pancreatic SC-δ cells, and / or less than SC-20% EC cells. In some embodiments, a population of pancreatic islet cells (e.g., stem cell derived pancreatic islet cells) produced using the compositions and methods described herein has improved glucose-stimulated insulin secretion (GSIS) response as compared to cell compositions generated according to conventional methods. In some embodiments, a population of pancreatic islet cells (e.g., stem cell derived pancreatic islet cells) produced using the compositions and methods described herein has dynamic GSIS response similar to native pancreatic islets (e.g., pancreatic islets from a healthy functioning pancreas from a healthy adult non-diabetic subject).

[0139] It should be noted that the disclosure provides for compositions prepared according to any of the methods disclosed herein, and methods of administering any of the reagents to the compositions disclosed herein.

[0140] In some embodiments, any of the methods or compositions disclosed herein comprises a medium, which may be a basal media. Such basal media comprises essential nutrients for survival of cells. Such basal media allows survival of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of cells (e.g., 30-100%, 50-100%, 70-100%, 90-100%, 30-80%, 30-60%, 50-80%, 50-70%, 70- 90%, 80-95% or 90-98% of cells) in a culture for at least 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days or 5 days in culture (e.g., 6-12 hours, 12-24 hours, 1-5 days, 1-4 days, 1-3 days, 1-2 days). In particular embodiments, basal media may be replaced in a culture after a period of time (e.g., 12-24 hours, 1-5 days, 1-4 days, 1-3 days, 1-2 days). In some embodiments, basal media comprises any one or more of glucose, nucleotides, amino acids, calcium, ascorbic acid, niacin, potassium, iron, manganous sulfate, cuprous sulfate, zinc, nickel, vitamin B-12, Folinic acid, riboflavin, biotin, ethanolamine, transferrin and / or insulin. In some embodiments, the basal media comprises any one of or any combination of StemScale, NutriStem, TeSR-E8, StemFit, StemPro, MCDB-131, ITS-X, GlutaMax, RPMI-1640, MEM, DMEM (e.g., DMEM-F12), NS-GFs media, MCDB131 medium, and / or CMRL medium.

[0141] The disclosure may refer to a composition that does not comprise, or that lacks, a particular reagent. Such compositions lack exogenous instances of such reagents. For example, if a composition comprising cells does not comprise a growth factor of the TGF-beta superfamily, the composition does not comprise exogenously added growth factor of the TGF-beta superfamily but may include endogenous levels (e.g., low levels) of growth factor secreted by the cells. Methods of producing pancreatic islet cells

[0142] In aspects, the present disclosure relates to compositions and methods of generating endocrine cells from pancreatic progenitor cells or precursors. Certain exemplary detailed protocols of generating endocrine cells to provide at least one SC-β cell are described in US Patent 10,030,229; US Patent 10,443,042; US Patent No. US 11,466,256; published application US 20200332262; andpublished application US 20210198632, published application US 20220090020, published application US 2022-0233646; published application US 2022-0090020; published application US 20230218676; and published application WO2022147056, each of which is herein incorporated by reference in its entirety.

[0143] In some embodiments, a method of generating a population of endocrine cells leads to increased percentage of pancreatic α and / or δ cells and decreased percentage of pancreatic EC cells when generating pancreatic β cells. In some embodiments, a method described herein may be used to obtain an enriched population of α cells. In some embodiments, a method described herein may be used to obtain an enriched population of β cells. In some embodiments, a method described herein may be used to obtain an enriched population of α cells and β cells. In some embodiments, a method described herein may be used to obtain an increased yield of pancreatic endocrine cells.

[0144] The differentiation of hPSC cells to hormone-expressing pancreatic endocrine cells may be conducted by transitioning hPSC cells through major stages of embryonic development; differentiation to mesendoderm and definitive endoderm, establishment of the primitive gut endoderm, patterning of the posterior foregut, and specification and maturation of pancreatic endoderm and endocrine precursors. Through these stages, hPSC cells can obtain pancreatic endocrine phenotype and ability of glucose responsive insulin secretion in vitro.

[0145] Generally, the at least one pancreatic SC-α, SC-β and / or SC-δ cell or precursor thereof, e.g., pancreatic progenitors produced according to the methods disclosed herein can comprise a mixture or combination of different cells, e.g., for example a mixture of cells such as a PDX1-positive pancreatic progenitors, pancreatic progenitors co-expressing PDX1 and NKX6.1, a Ngn3-positive endocrine progenitor cell, an insulin-positive endocrine cell (e.g., NKX6.1-positive, ISL1-positive cells, or β-like cells), and / or other pluripotent or stem cells.

[0146] The at least one pancreatic α, β and / or δ cell or precursor thereof can be produced according to any suitable culturing protocol to differentiate a stem cell or pluripotent cell to a desired stage of differentiation. In some embodiments, the at least one pancreatic α, β and / or δ cell or the precursor thereof are produced by culturing at least one pluripotent cell for a period of time and under conditions suitable for the at least one pluripotent cell to differentiate into the at least one pancreatic α, β and / or δ cell or the precursor thereof.

[0147] In some embodiments, the at least one pancreatic α, β and / or δ cell or precursor thereof is a substantially pure population of pancreatic α, β and / or δ cells or precursors thereof. In some embodiments, a population of pancreatic α, β and / or δ cells or precursors thereof comprises a mixture of pluripotent cells or differentiated cells. In some embodiments, a population pancreatic α, β and / or δ cells or precursors thereof are substantially free or devoid of embryonic stem cells or pluripotent cells or iPS cells. In some embodiments, a method described herein produces a population of cells comprising pancreatic α, β and / or δ cells at a ratio that resembles that of a natural pancreatic islet.

[0148] In some embodiments, in a method described herein, the first medium and / or the second medium further comprises a TGF-β ligand (e.g., activin A). In some embodiments, the first medium further comprises a Wnt signaling pathway activator (e.g., a glycogen synthase kinase 3 (GSK3) inhibitor such as CHIR99021). In some embodiments, the first medium and / or the second medium further comprises an inhibitor of PI3K / Akt / mTOR signaling (e.g., GSK-690693). In some embodiments, the first medium and / or the second medium further comprises a water-soluble synthetic polymer (e.g., PVA). In some embodiments, the first medium is supplemented with additional metabolites such as amino acids (e.g., aspartate, glycine, and / or serine) and further comprises a TGF-β ligand (e.g., activin A) and a Wnt signaling pathway activator (e.g., a glycogen synthase kinase 3 (GSK3) inhibitor such as CHIR99021), and optionally further comprises an inhibitor of PI3K / Akt / mTOR signaling (e.g., GSK-690693) and / or a water-soluble synthetic polymer (e.g., PVA). In some embodiments, the second medium is supplemented with additional amino acids (e.g., aspartate, glycine, and / or serine) and further comprises a TGF-β ligand (e.g., activin A), and optionally further comprises an inhibitor of PI3K / Akt / mTOR signaling (e.g., GSK-690693) and / or a water-soluble synthetic polymer (e.g., PVA), and does not comprise a Wnt signaling pathway activator.

[0149] In some embodiments, in a method described herein, the first medium and / or second medium further comprises a TGF-β ligand (e.g., activin A). In some embodiments, the TGF-β ligand (e.g., activin A) has a concentration of about 1-200 ng / ml (e.g., about 1-200, 1-150, 1-125, 1-110, 1- 100, 1-90, 1-75, 1-50, 1-25, 1-15, 1-12, 1-10, 1-8, 1-5, 5-200, 5-150, 5-125, 5-110, 5-100, 5-90, 5-75, 5-50, 5-25, 5-15, 5-12, 5-10, 5-8, 8-200, 8-150, 8-125, 8-110, 8-100, 8-90, 8-75, 8-50, 8-25, 8-15, 8- 12, 8-10, 10-200, 10-150, 10-125, 10-110, 10-100, 10-90, 10-75, 10-50, 10-25, 10-15, 10-12, 12-200, 12-150, 12-125, 12-110, 12-100, 12-90, 12-75, 12-50, 12-25, 12-15, 15-200, 15-150, 15-125, 15-110, 15-100, 15-90, 15-75, 15-50, 15-25, 25-200, 25-150, 25-125, 25-110, 25-100, 25-90, 25-75, 25-50, 50-200, 50-150, 50-125, 50-110, 50-100, 50-90, 50-75, 75-200, 75-150, 75-125, 75-110, 75-100, 75- 90, 90-200, 90-150, 90-125, 90-110, 90-100, 100-200, 100-150, 100-125, 100-110, 110-200, 110-250, 110-125, 125-200, 125-150, or 150-200 ng / ml). In some embodiments, the TGF-β ligand (e.g., activin A) has a concentration of about 1-50, 1-25, 5-50, 5-25, 5-15, 8-12, 10-1000, 10-500, 10-250, 10-125, 75-1000, 75-500, 75-250, 75-125, or 90-110 ng / ml. In some embodiments, the TGF-β ligand (e.g., activin A) has a concentration of about 90-110 ng / ml (e.g., 90, 95, 100, 105, or 110 ng / ml). In some embodiments, the TGF-β ligand (e.g., activin A) has a concentration of about 8-12 ng / ml (e.g., 8, 9, 10, 11, or 12 ng / ml).

[0150] In some embodiments, in a method described herein, the first medium and / or second medium further comprises an inhibitor of PI3K / Akt / mTOR signaling. In some embodiments, the inhibitor of PI3K / Akt / mTOR signaling may be selected from, but is not limited to, 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 embodiments, the inhibitor of PI3K / Akt / mTOR signaling is GSK-690693 or an analog or derivative thereof. In some embodiments, the inhibitor of PI3K / Akt / mTOR signaling (e.g., GSK- 690693, or an analog or a derivative thereof) has a concentration of about 0.01-1 μM (e.g., about 0.01- 1, 0.01-0.8, 0.01-0.6, 0.01-0.4, 0.01-0.2, 0.01-0.1, 0.05-1, 0.05-0.8, 0.05-0.6, 0.05-0.4, 0.05-0.2, 0.05- 0.1, 0.1-1, 0.1-0.8, 0.1-0.6, 0.1-0.4, 0.1-0.2, 0.2-1, 0.2-0.8, 0.2-0.5, 0.2-0.4, 0.4-1, 0.4-0.8, 0.4-0.6, 0.6-1, 0.6-0.8, or 0.8-1 μM). In some embodiments, the inhibitor of PI3K / Akt / mTOR signaling (e.g., GSK-690693, or an analog or a derivative thereof) has a concentration of about 0.01-1 μM, 0.02-0.8 μM, 0.05-0.5 μM, 0.06-0.2 μM, 0.07-0.15 μM, or 0.08-0.12 μM. In some embodiments, the inhibitor of PI3K / Akt / mTOR signaling (e.g., GSK-690693, or an analog or a derivative thereof) has a concentration of about 0.1 μM.

[0151] In some embodiments, in a method described herein, the first medium further comprises a Wnt signaling pathway activator. In some embodiments, the Wnt signaling pathway activator may be a glycogen synthase kinase 3 (GSK3) inhibitor. In some embodiments, the glycogen synthase kinase 3 (GSK3) inhibitor is CHIR99021. In some embodiments, the Wnt signaling pathway activator (e.g., CHIR99021) has a concentration of about 0.1-50 μM (e.g., about 0.1-50, 0.1-25, 0.1-10, 0.1-5, 0.1-4, 0.1-3, 0.1-2, 0.1-1, 0.1-0.5, 0.5-50, 0.5-25, 0.5-10, 0.5-5, 0.5-4, 0.5-3, 0.5-2, 0.5-1, 1-50, 1-25, 1-10, 1-5, 1-4, 1-3, 1-2, 2-50, 2-25, 2-10, 2-5, 2-4, 2-3, 3-50, 3-25, 3-10, 3-5, 3-4, 4-50, 4-25, 4-10, 4-5, 5- 50, 5-25, 5-10, 10-50, 10-25, 25-50 μM). In some embodiments, the Wnt signaling pathway activator (e.g., CHIR99021) has a concentration of 2-4 μM (e.g., 2, 3, or 4 μM).

[0152] In some embodiments, in a method described herein, the first medium and / or second medium further comprises a water-soluble synthetic polymer. In some embodiments, the water- soluble synthetic polymer is polyvinyl alcohol (PVA), poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), PEG copolymers, poly(N-isopropylacrylamide), or polyacrylamide, optionally wherein the water-soluble synthetic polymer is polyvinyl alcohol. In some embodiments, the water water- soluble synthetic polymer is polyvinyl alcohol (PVA). In some embodiments, the water-soluble synthetic polymer has a concentration of about 0.005% to 0.5% (w / v), 0.01% to 0.2% (w / v), 0.02% to 0.1% (w / v), or 0.03% to 0.08% (w / v) of the culture medium. In some embodiments, the water-soluble synthetic polymer has a concentration of about 0.005% (w / v), 0.01% (w / v), 0.05% (w / v), 0.1% (w / v), 0.15% (w / v), 0.2% (w / v), 0.25% (w / v), 0.3% (w / v), 0.35% (w / v), to 0.4% (w / v), 0.45% (w / v), or 0.5% (w / v) of the medium. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol (PVA), and the PVA is at most 85% (e.g., 75%-80%) hydrolyzed. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol (PVA), and the PVA is about 80% hydrolyzed.

[0153] In some embodiments, in a method described herein, the first population of cells is cultured in the first medium for a period of about 18-48 hours (e.g., about 18-48 hours, 18-42 hours, 18-36 hours, 18-30 hours, 18-24 hours, 24-48 hours, 24-42 hours, 24-36 hours, 24-30 hours, 30-48hours, 30-42 hours, 30-36 hours, 36-48 hours, 36-42 hours, or 42-48 hours). In some embodiments, the first population of cells is cultured in the first medium for a period of about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours. In some embodiments, the first population of cells is cultured in the first medium for a period of about 24 hours. In some embodiments, culturing the first population of cells in the first media for a contacting period described herein (e.g., 24 hours) results in a second population of cells.

[0154] In some embodiments, a method described herein further comprises culturing the second population of cells with the second medium for a period of 36-72 hours (e.g., 36-72 hours, 36-66 hours, 36-60 hours, 36-54 hours, 36-48 hours, 36-42 hours, 42-72 hours, 42-66 hours, 42-60 hours, 42-54 hours, 42-48 hours, 48-72 hours, 48-66 hours, 48-60 hours, 48-54 hours, 54-72 hours, 54-66 hours, 54-60 hours, 60-72 hours, 60-66 hours, or 66-72 hours). In some embodiments, the second population of cells is cultured in the second medium for a period of about 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours. In some embodiments, the second population of cells is cultured in the second medium for a period of about 48 hours. In some embodiments, culturing the second population of cells in the second media for a contacting period described herein (e.g., 24 hours) results in a third population of cells. In some embodiments, the third population of cells comprise definitive endoderm cells. In some embodiments, the third population of cells further comprise pluripotent stem cells and / or cells that are at a differentiation stage of between pluripotent stem cells and definitive endoderm cells.

[0155] In some embodiments, the pluripotent stem cells used in a method described herein are embryonic stem cells. In some embodiments, the pluripotent stem cells used in a method described herein are induced pluripotent stem cells. In some embodiments, the pluripotent stem cells used in a method described herein are human pluripotent stem cells. In some embodiments, the pluripotent stem cells are ABO blood group type O. In some embodiments, the pluripotent stem cells are genetically modified such that the cell is ABO blood group type O. In some embodiments, the pluripotent stem cells have reduced expression or activity of one or more of beta-2 microglobulin, CIITA, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, renalase, tissue factor, and HLA-DR, relative to cells that are not genetically modified. In some embodiments, the pluripotent stem cells have increased expression or activity of CD47, PDL1, HLA-G, CD46, CD55, CD59 and CTLA, relative to cells that are not genetically modified.

[0156] In some embodiments, a method described herein further comprises differentiating (e.g., using any methods described herein or known in the art) the definitive endoderm cells to pancreatic endocrine cells (e.g., β cells, α cells, and δ cells).Cell types during pancreatic differentiation

[0157] Aspects of the present disclosure provide cell types of the pancreatic lineage obtained during differentiation of stem cells to generate pancreatic islet cells. Such cells include any cell that is capable of differentiating into a pancreatic islet cell, including for example, a pluripotent stem cell, a definitive endoderm cell, a primitive gut tube cell, a pancreatic progenitor cell, or endocrine progenitor cell, when cultured under conditions suitable for differentiating the precursor cell into the pancreatic islet cell. The present disclosure refers to culturing cells in a media (e.g., a media with one or more reagents) for a period of time (e.g., 1 day, 2 days, 3 days etc.). It should be understood that such culturing encompasses replacement of media or reagents with fresh media or reagents. For example, if a method refers to culturing or incubating a population of cells with a specific reagent for five days, the method encompasses replacement of the media and the reagent with fresh versions of the media and the reagent during that five day period.

[0158] The present disclosure also refers to culturing or incubating specific cells in a media comprising one or more reagents. It should be understood that the specific cells in the media may differentiate into other cells over time, but that at least one time (e.g., at initiation of a period of time that the media and reagents being introduced to the cells, or at the completion of a recited period of time) the media comprised the recited cells. For example, the disclosure may provide for methods of culturing a plurality of NKX6.1-negative; PDX1-positive cells in a media comprising multiple reagents for a period of three days. In such an instance, the multiple reagents may facilitate alone or in part the differentiation of the cells, and, as a result, by the end of the three day period the NKX6.1- negative; PDX1-positive cells may have been replaced with other cells. Alternatively, the one or more reagents may promote selective proliferation of one cell type, or selective cell death of another cell type, such that recited percentages of cell types may change over a period of time. By way of another example, the disclosure may provide for methods of culturing a population of cells in a media comprising multiple reagents for a period of three days; wherein at least 60% of the cells in the population of cells NKX6.1-negative; PDX1-positive cells. In such an instance, the multiple reagents may facilitate alone or in part the differentiation of the cells, and, as a result, by the end of the three days the population may comprise a lower percentage of NKX6.1-negative; PDX1-positive cells (e.g., less than 20%). As such, the specific cells or percentage of specific cells recited in connection with a disclosed method step may reflect the cells or percentage of cells present at a given moment in time for the method. In some embodiments, the given moment in time is at the start of the disclosed method step. In other embodiments, the given moment in time is at the end of the disclosed method step. Stem Cells

[0159] “Stem cell” refers to a cell (e.g., plant stem cell, vertebrate stem cell) that has the ability both to self-renew and to generate a differentiated cell type (Morrison et al. (1997) Cell 88:287-298).In the context of cell ontogeny, the adjective “differentiated,” or “differentiating” is a relative term. A “differentiated cell” is a cell that has progressed further down the developmental pathway than the cell it is being compared with. Thus, pluripotent stem cells can differentiate into lineage-restricted progenitor cells (e.g., mesodermal stem cells), which in turn can differentiate into cells that are further restricted (e.g., neuron progenitors), which can differentiate into end-stage cells (i.e., terminally differentiated cells, e.g., neurons, cardiomyocytes, etc.), which play a characteristic role in a certain tissue type, and can or cannot retain the capacity to proliferate further. Stem cells can be characterized by both the presence of specific markers (e.g., proteins, RNAs, etc.) and the absence of specific markers. Stem cells can also be identified by functional assays both in vitro and in vivo, particularly assays relating to the ability of stem cells to give rise to multiple differentiated progeny. In an embodiment, the host cell is an adult stem cell, a somatic stem cell, a non- embryonic stem cell, an embryonic stem cell, hematopoietic stem cell, an include pluripotent stem cells, and a trophoblast stem cell.

[0160] In some embodiments, the stem cell is a multipotent and is not a pluripotent stem cell. In some embodiments, the stem cell is a stem cell reprogrammed from a primary pancreatic islet cell. In some embodiments, the multipotent stem cell is the SR1423 cell line described in Ratiu et al., 2023, bioRxiv, https: / / doi.org / 10.1101 / 2023.10.20.563345.

[0161] In some embodiments, cells of interest include pluripotent stem cells (PSCs). The term “pluripotent stem cell” or “PSC” is used herein to mean a stem cell capable of producing all cell types of the organism. Therefore, a PSC can give rise to cells of all germ layers of the organism (e.g., the endoderm, mesoderm, and ectoderm of a vertebrate). Pluripotent cells are capable of forming teratomas and of contributing to ectoderm, mesoderm, or endoderm tissues in a living organism. Pluripotent stem cells of plants are capable of giving rise to all cell types of the plant (e.g., cells of the root, stem, leaves, etc.).

[0162] PSCs of animals can be derived in a number of 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) whereas induced pluripotent stem cells (iPSCs) are derived from somatic cells (Takahashi et. al, Cell.2007 Nov.30; 13 l(5):86l-72; Takahashi et. al, Nat Protoc.2007; 2(l2):308l-9; Yu et. al, Science.2007 Dec.21; 318(5858): 1917-20. Epub 2007 Nov.20). Because the term PSC refers to pluripotent stem cells regardless of their derivation, the term PSC encompasses the terms ESC and iPSC, as well as the term embryonic germ stem cells (EGSC), which are another example of a PSC. PSCs can be in the form of an established cell line, they can be obtained directly from primary embryonic tissue, or they can be derived from a somatic cell.

[0163] By “embryonic stem cell” (ESC) is meant a PSC that is isolated from an embryo, typically from the inner cell mass of the blastocyst. ESC lines are listed in the NIH Human Embryonic Stem Cell Registry, e.g. hESBGN-Ol, hESBGN-02, hESBGN-03, hESBGN-04 (BresaGen, Inc); HES-l,HES-2, HES-3, HES-4, HES-5, HES-6 (ES Cell International); Miz- hESl (MizMedi Hospital-Seoul National University); HSF-l, HSF-6 (University of California at San Francisco); and Hl, H7, H9, H13, H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute)). Stem cells of interest also include embryonic stem cells from other primates, such as Rhesus stem cells and marmoset stem cells. The stem cells can be obtained from any mammalian species, e.g., human, equine, bovine, porcine, canine, feline, rodent, e.g. mice, rats, hamster, primate, etc. (Thomson et al. (1998) Science 282: 1145; Thomson et al. (1995) Proc. Natl. Acad. Sci USA 92:7844; Thomson et al. (1996) Biol. Reprod. 55:254; Shamblott et al., Proc. Natl. Acad. Sci. USA 95: 13726, 1998). In culture, ESCs typically grow as flat colonies with large nucleo-cytoplasmic ratios, defined borders and prominent nucleoli. In addition, ESCs express SSEA-3, SSEA-4, TRA-l-60, TRA-1-81, and Alkaline Phosphatase, but not SSEA-l. Examples of methods of generating and characterizing ESCs may be found in, for example, U.S. Pat. No.7,029,913, U.S. Pat. No.5,843,780, and U.S. Pat. No.6,200,806, each of which is incorporated herein by its entirety. Methods for proliferating hESCs in the undifferentiated form are described in WO 99 / 20741, WO 01 / 51616, and WO 03 / 020920, each of which is incorporated herein by its entirety.

[0164] By “embryonic germ stem cell” (EGSC) or “embryonic germ cell” or “EG cell,” it is meant a PSC that is derived from germ cells and / or germ cell progenitors, e.g., primordial germ cells, i.e. those that can become sperm and eggs. Embryonic germ cells (EG cells) are thought to have properties similar to embryonic stem cells as described above. Examples of methods of generating and characterizing EG cells may be found in, for example, U.S. Pat. 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 are incorporated herein by its entirety. By “induced pluripotent stem cell” or “iPSC,” it is meant a PSC that is derived from a cell that is not a PSC (i.e., from a cell this is differentiated relative to a PSC). iPSCs can be derived from multiple different cell types, including terminally differentiated cells. iPSCs have an ES cell-like morphology, growing as flat colonies with large nucleo-cytoplasmic ratios, defined borders and prominent nuclei. In addition, iPSCs express one or more key pluripotency markers known by one of ordinary skill in the art, including but not limited to Alkaline Phosphatase, SSEA3, SSEA4, Sox2, Oct3 / 4, Nanog, TRA160, TRA181, TDGF 1, Dnmt3b, FoxD3, GDF3, Cyp26al, TERT, and zfp42. Examples of methods of generating and characterizing iPSCs can be found in, for example, Patent Publication Nos. US20090047263, US20090068742, US20090191159, US20090227032, US20090246875, and US20090304646, each of which are incorporated herein by its entirety. Generally, to generate iPSCs, somatic cells are provided with reprogramming factors (e.g., Oct4, SOX2, KLF4, MYC, Nanog, Lin28, etc.) known in the art to reprogram the somatic cells to become pluripotent stem cells. In some embodiments, the induced pluripotent stem cells are generated from mesenchymal stromal cells. In some embodiments, the induced pluripotent stem cells are generated from adipose-derived mesenchymal stromal cells(ADSCs) isolated. In some embodiments, the induced pluripotent stem cells are chemically-induced pluripotent stem cells (see, e.g., Guan et al., 2022, Nature, 605:325-331; Wang et al., 2024, Cell, 187, 1–13). In some embodiments, the induced pluripotent stem cells are derived from cells taken from a subject (e.g., a diabetic subject), and the induced pluripotent stem cells are then differentiated using any of the methods disclosed herein in order to make SC-islet cells or precursors thereof that may be administered back to the patient, i.e., the induced pluripotent stem cells are autologous cells to the subject (see, e.g., Wang et al., 2024, Cell, 187, 1–13).

[0165] By “somatic cell,” it is meant any cell in an organism that, in the absence of experimental manipulation, does not ordinarily give rise to all types of cells in an organism. In other words, somatic cells are cells that have differentiated sufficiently that they do not naturally generate cells of all three germ layers of the body, i.e., ectoderm, mesoderm and endoderm. For example, somatic cells can include both neurons and neural progenitors, the latter of which is able to naturally give rise to all or some cell types of the central nervous system but cannot give rise to cells of the mesoderm or endoderm lineages.

[0166] In certain examples, the stem cells can be undifferentiated (e.g., a cell not committed to a specific lineage) prior to exposure to at least one cell maturation factor according to the methods as disclosed herein, whereas in other examples it may be desirable to differentiate the stem cells to one or more intermediate cell types prior to exposure of the at least one cell maturation factor (s) described herein. For example, the stems cells may display morphological, biological or physical characteristics of undifferentiated cells that can be used to distinguish them from differentiated cells of embryo or adult origin. In some examples, undifferentiated cells may appear in the two dimensions of a microscopic view in colonies of cells with high nuclear / cytoplasmic ratios and prominent nucleoli. The stem cells may be themselves (for example, without substantially any undifferentiated cells being present) or may be used in the presence of differentiated cells. In certain examples, the stem cells may be cultured in the presence of suitable nutrients and optionally other cells such that the stem cells can grow and optionally differentiate. For example, embryonic fibroblasts or fibroblast-like cells may be present in the culture to assist in the growth of the stem cells. The fibroblast may be present during one stage of stem cell growth but not necessarily at all stages. For example, the fibroblast may be added to stem cell cultures in a first culturing stage and not added to the stem cell cultures in one or more subsequent culturing stages.

[0167] Stem cells used in all aspects of the present invention can be any cells derived from any kind of tissue (for example embryonic tissue such as fetal or pre-fetal tissue, or adult tissue), which stem cells have the characteristic of being capable under appropriate conditions of producing progeny of different cell types, e.g., derivatives of all of at least one of the 3 germinal layers (endoderm, mesoderm, and ectoderm). These cell types may be provided in the form of an established cell line, or they may be obtained directly from primary embryonic tissue and used immediately fordifferentiation. Included are cells listed in the NIH Human Embryonic Stem Cell Registry, e.g. hESBGN-Ol, hESBGN-02, hESBGN-03, hESBGN-04 (BresaGen, Inc.); HES-l, HES-2, HES-3, HES- 4, HES-5, HES-6 (ES Cell International); Miz-hESl (MizMedi Hospital-Seoul National University); HSF-l, FISF-6 (University of California at San Francisco); and Hl, H7, H9, H13, 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 destroying a human embryo.

[0168] In another embodiment, the stem cells can be isolated from tissue including solid tissue. In some embodiments, the tissue is skin, fat tissue (e.g., adipose tissue), muscle tissue, heart or cardiac tissue. In other embodiments, the tissue is for example but not limited to, umbilical cord blood, placenta, bone marrow, or chondral.

[0169] Stem cells of interest also include embryonic cells of various types, exemplified by human embryonic stem (hES) cells, described by Thomson et al, (1998) Science 282: 1145; embryonic stem cells from other primates, such as Rhesus 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 of interest are lineage committed stem cells, such as mesodermal stem cells and other early cardiogenic cells (see Reyes et al, (2001) Blood 98:2615-2625; Eisenberg & Bader (1996) Circ Res.78(2):205-l6; etc.). The stem cells may be obtained from any mammalian species, e.g., human, equine, bovine, porcine, canine, feline, rodent, e.g., mice, rats, hamster, primate, etc. In some embodiments, a human embryo was not destroyed for the source of pluripotent cell used on the methods and compositions as disclosed herein.

[0170] A mixture of cells from a suitable source of endothelial, muscle, and / or neural stem cells can be harvested from a mammalian donor by methods known in the art. A suitable source is the hematopoietic microenvironment. For example, circulating peripheral blood, preferably mobilized (i.e., recruited), may be removed from a subject. In an embodiment, the stem cells can be reprogrammed stem cells, such as stem cells derived from somatic or differentiated cells. In such an embodiment, the de-differentiated stem cells can be for example, but not limited to, neoplastic cells, tumor cells and cancer cells or alternatively induced reprogrammed cells such as induced pluripotent stem cells or iPS cells.

[0171] In some embodiments, the stem cells are embryonic stem cells. In some embodiments, the stem cells are induced pluripotent stem cells. In some embodiments, the stem cells used in a method described herein are human stem cells. In some embodiments, the stem cells are ABO blood group type O. In some embodiments, the stem cells are genetically modified such that the cell is ABO blood group type O. In some embodiments, the stem cells have reduced expression of one or more of beta-2 microglobulin, CIITA, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, renalase, CXCL10,or tissue factor, relative to cells that are not genetically modified. In some embodiments, the stem cells have increased expression or activity of one or more of CD47, PDL1, HLA-G, CD46, CD55, CD59 and CTLA, relative to cells that are not genetically modified. Definitive Endoderm Cells

[0172] The definitive endoderm can be generated in vivo from the inner cell mass by the process of gastrulation of embryogenesis, in which epiblast cells are instructed to form the three germ layers. Definitive endoderm can give rise to diverse cells and tissues that contribute to vital organs as the pancreatic β cells, liver hepatocytes, lung alveolar cells, thyroid, thymus, and the epithelial lining of the alimentary and respiratory tract. It is different from the primitive endoderm of extraembryonic tissues, which can give rise to the visceral and parietal endoderm. The definitive endoderm derived from ES cells is theoretically capable of becoming any endoderm derivatives.

[0173] Precise patterning of anterior-posterior axis of the definitive endoderm can eventually form the primitive gut tube. The definitive endoderm-derived primitive gut tube induces the pharynx, esophagus, stomach, duodenum, small and large intestine along the anterior-posterior axis as well as associated organs, including pancreas, lung, thyroid, thymus, parathyroid, and liver. The anterior portion of the foregut of the primitive gut tube becomes lung, thyroid, esophagus, and stomach. The pancreas, liver, and duodenum originate from the posterior portion of the foregut. The midgut and hindgut of primitive gut tube gives rise to the small and large intestine. The anterior foregut expresses developmental markers, NK2 homeobox 1 (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), one cut homeobox 1 (ONECUT1, known as HNF6), and hepatocyte nuclear factor 4 alpha (HNF4A); and the midgut / hindgut expresses caudal type homeobox 1 (CDX1), caudal type homeobox 2 (CDX2), and motor neuron and pancreas homeobox 1 (MNX1) (3, 19, 20).

[0174] As described herein definitive endoderm cells of use herein can be derived from any source or generated in accordance with any suitable protocol. In some aspects, stem cells, e.g., iPSCs or hESCs, are differentiated to endoderm cells. In some aspects, the endoderm cells (stage 1) are further differentiated, e.g., to primitive gut tube cells (stage 2), PDX1-positive pancreatic progenitor cells (stage 3), NKX6.1-positive pancreatic progenitor cells (stage 4), or Ngn3-positive endocrine progenitor cells or insulin-positive endocrine cells (stage 5), followed by induction or maturation to SC-β cells (stage 6). In some embodiments, definitive endoderm cells can be obtained by differentiating at least some stem cells (e.g., pluripotent stem cells) in a population into definitive endoderm cells, e.g., by contacting a population of stem cells with i) at least one growth factor from the TGF-β superfamily, and ii) a WNT signaling pathway activator, to induce the differentiation of atleast some of the pluripotent cells into definitive endoderm cells, wherein the definitive endoderm cells express at least one marker characteristic of definitive endoderm.

[0175] In some embodiments, the disclosure provides for a composition comprising a plurality of stem cells (e.g., pluripotent stem cells), and any of the bone morphogenic protein (BMP) pathway inhibitors disclosed herein. In some embodiments, the BMP pathway inhibitor is dorsomorphin. In some embodiments, the concentration of the BMP pathway inhibitor is 0.1-100 µM, 0.1-50 µM, 0.1- 25 µM, 0.1-10 µM, 0.1-1 µM, 0.5-25 µM, 0.5-10 µM, 0.5-5 µM, 0.5-2 µM, or 0.8-1.2 µM. In particular embodiments, the concentration of the BMP pathway inhibitor is 0.5-2 µM.

[0176] In some embodiments, the disclosure provides for a composition comprising a plurality of stem cells (e.g., pluripotent stem cells), and any of the JNK inhibitors disclosed herein. In some embodiments, the JNK inhibitor is a JNK1, JNK2, and / or JNK3 inhibitor. In some embodiments, the JNK inhibitor is a JNK1, JNK2, and JNK3 inhibitor. In some embodiments, the JNK inhibitor is JNK-IN-8. In some embodiments, the concentration of the JNK inhibitor is 0.1-100 µM, 0.1-50 µM, 0.1-25 µM, 0.1-10 µM, 0.1-1 µM, 0.5-25 µM, 0.5-10 µM, 0.5-5 µM, 0.5-2 µM, or 0.8-1.2 µM. In particular embodiments, the concentration of the JNK inhibitor is 0.5-2 µM.

[0177] In some embodiments, the disclosure provides for a composition comprising a plurality of stem cells (e.g., pluripotent stem cells) and any of the BMP pathway inhibitors disclosed herein and any of the JNK inhibitors disclosed herein. In some embodiments, the composition does not comprise an exogenous growth factor from the TGF-β superfamily (e.g., activin A, GDF8, or GDF11).

[0178] In some embodiments, the disclosure provides for a composition comprising a plurality of stem cells (e.g., pluripotent stem cells) and an AKT-inhibitor. In some embodiments, the AKT inhibitor is any one of BEZ235, LY294002, GDC-0941, BYL719, GSK2636771, TGX-221, AS25242, CAL-101, IPI-145, MK-2206, GSK690693, GDC-0068, A-674563, CCT128930, AZD8055, INK128, rapamycin, PF-04691502, everolimus, BI-D1870, H89, PF-4708671, FMK, AT7867, NU7441, PI-103, NU7026, PIK-75, ZSTK474, and PP-121. In some embodiments, the AKT inhibitor comprises the structure , or apharmaceutically acceptable salt or derivative thereof. In some embodiments, the AKT inhibitor is MK-2206.

[0179] In some embodiments, the disclosure provides for a composition comprising a plurality of stem cells (e.g., pluripotent stem cells), any of the AKT inhibitors disclosed herein and any of theBMP pathway inhibitors disclosed herein. In some embodiments, the disclosure provides for a composition comprising a plurality of stem cells (e.g., pluripotent stem cells), any of the AKT inhibitors disclosed herein and any of the JNK inhibitors disclosed herein. In some embodiments, the disclosure provides for a composition comprising a plurality of stem cells (e.g., pluripotent stem cells), any of the AKT inhibitors disclosed herein, any of the BMP pathway inhibitors disclosed herein, and any of the JNK inhibitors disclosed herein. In some embodiments, the composition does not comprise an exogenous growth factor from the TGF-β superfamily (e.g., activin A, GDF8, or GDF11).

[0180] In some embodiments, the disclosure provides for a method comprising the step of culturing a plurality of stem cells (e.g., pluripotent stem cells) with a media comprising a bone morphogenic protein (BMP) pathway inhibitor. In some embodiments, the disclosure provides for a method comprising the step of culturing a plurality of stem cells (e.g., pluripotent stem cells) with a media comprising a JNK inhibitor. In some embodiments, the disclosure provides for a method comprising the step of culturing a plurality of stem cells (e.g., pluripotent stem cells) with a media comprising an AKT inhibitor. In some embodiments, the disclosure provides for a method comprising the step of culturing a plurality of stem cells (e.g., pluripotent stem cells) with a media comprising a BMP pathway inhibitor and a JNK inhibitor or an AKT inhibitor. In some embodiments, the disclosure provides for a method comprising the step of culturing a plurality of stem cells (e.g., pluripotent stem cells) with a media comprising a JNK inhibitor and a BMP pathway inhibitor or an AKT inhibitor. In some embodiments, the disclosure provides for a method comprising the step of culturing a plurality of stem cells (e.g., pluripotent stem cells) with a media comprising a JNK inhibitor, and a BMP pathway inhibitor, an AKT inhibitor. In some embodiments, the method does not comprise a step of administering a growth factor from the TGF-β superfamily (e.g., activin A, GDF8, or GDF11) to the media.

[0181] In some embodiments, the disclosure provides for a composition comprising a plurality of stem cells (e.g., pluripotent stem cells) and insulin. In some embodiments, the insulin is present at a concentration of 21-1000, 400-600, 200-400, 600-1000, 21-500, 21-300, 21-200, 21-100, 21-50, 50- 500, 50-300, 50-250, 50-100, 75-125, 150-500, 150-250, or 175-225 µg / L. In some embodiments, the insulin is a component of ITS-X (insulin-transferrin-selenium-ethanolamine) in the composition. In some embodiments, the ITS-X is present in the composition at a concentration of 21-1000, 400-600, 200-400, 600-1000, 21-500, 21-300, 21-200, 21-100, 21-50, 50-500, 50-300, 50-250, 50-100, 75-125, 150-500, 150-250, or 175-225 µg / L. In some embodiments, the disclosure provides for a method comprising the step of culturing a plurality of pluripotent stem cells with a media comprising insulin at a concentration of 21-1000, 400-600, 200-400, 600-1000, 21-500, 21-300, 21-200, 21-100, 21-50, 50-500, 50-300, 50-250, 50-100, 75-125, 150-500, 150-250, or 175-225 µg / L.

[0182] In some embodiments, the disclosure provides for a composition comprising a plurality of stem cells (e.g., pluripotent stem cells) and greater than 8 mM glucose. In some embodiments, the composition comprises 8-15, 8-14, 8-12, 8-11, 8-10, 8-9, 9-11, 9-12, or 9.5-10.5 mM glucose. In some embodiments, the composition does not comprise less than 4 mM glucose. In some embodiments, the disclosure provides for a method comprising the step of culturing a plurality of stem cells (e.g., pluripotent stem cells) with a media comprising greater than 8 mM glucose. In some embodiments, the disclosure provides for a method comprising: a) administering a media to a container comprising stem cells (e.g., pluripotent stem cells), wherein the media comprises 4-14 mM glucose; and b) after 4-18 hours of culturing the pluripotent stem cells in the media, administering supplemental glucose to the media, wherein following administration of the supplemental glucose to the media, the concentration of total glucose in the media is 4-14 mM. In some embodiments, the media administered in step a) comprises 4-12, 4-10, 4-8, 6-12, 6-11, 6-10, 8-10, or 7-9 mM glucose. In some embodiments, 2-10, 2-8, 2-6, 2-4, 3-9, 3-7, 3-5 mM glucose is added in step b). In some embodiments, after 4-16, 4-12, 4-10, 6-12, 6-16, 6-9, 7-9, 7-10, 7-12, 8-12, or 10-14 hours of culturing the pluripotent stem cells in the media from step a), the supplemental glucose is administered to the media. In some embodiments, the concentration of total glucose in the media following administration of the supplemental glucose to the media in step b) is 4-12, 4-10, 4-8, 6-12, 6-11, 6-10, 8-10, 8-12, 9-12, or 7-9 mM. In some embodiments, steps a) and b) are repeated over about 2-5 days, about 2-4 days, or about 2-3 days. In some embodiments, the disclosure provides for a method of administering media to a container comprising stem cells (e.g., pluripotent stem cells), wherein the media comprises 4-14 mM glucose and, before the glucose in the media is below 4 mM, administering supplemental glucose to the media such that the concentration of total glucose in the media is maintained at 4-14 mM. In some embodiments, the disclosure provides for a method comprising culturing a population of stem cells (e.g., pluripotent stem cells) in a media comprising at least 4 mM glucose, wherein the concentration of glucose in the media is maintained such that it does not fall below 1-4 mM (e.g., does not fall below 4 mM). In some embodiments, the concentration of glucose in the media is maintained such that it does not fall below 1-4 mM by supplementing the media with glucose (e.g., 4-10 mM glucose). In some embodiments, the concentration of glucose in the media following supplementation with glucose is 4-12, 4-10, 4-8, 6-12, 6-11, 6-10, 8-10, 8-12, 9- 12, or 7-9 mM. In some embodiments, 2-10, 2-8, 2-6, 2-4, 3-9, 3-7, 3-5 mM glucose is supplemented to the media. In some embodiments, 10-100%, 10-80%, 10-60%, 10-40%, 10-20%, 30-100%, 30- 80%, 30-60%, 50-100%, 50-80%, 70-100%, or 90-100% of the cells in the composition are stem cells (e.g., pluripotent stem cells). In some embodiments, 10-100%, 10-80%, 10-60%, 10-40%, 10-20%, 30-100%, 30-80%, 30-60%, 50-100%, 50-80%, 70-100%, or 90-100% of the cells in the composition express OCT4. In some embodiments, 10-100%, 10-80%, 10-60%, 10-40%, 10-20%, 30-100%, 30- 80%, 30-60%, 50-100%, 50-80%, 70-100%, or 90-100% of the cells in the composition are definitive endoderm cells. In some embodiments, 10-100%, 10-80%, 10-60%, 10-40%, 10-20%, 30-100%, 30-80%, 30-60%, 50-100%, 50-80%, 70-100%, or 90-100% of the cells in the composition express SOX17.

[0183] In some embodiments, at least about 50%, 60%, 65%, 70%, 75%, 80%, or 85% of the cells in any of the definitive endoderm cell compositions disclosed herein are Sox17-positive, Oct4- negative cells. In some embodiments, 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% of the cells in any of the definitive endoderm cell compositions disclosed herein are Sox17-positive, Oct4-negative cells.

[0184] In some embodiments, the disclosure provides for a method comprising the step of culturing: i) a first population of cells comprising a plurality of stem cells (e.g., pluripotent stem cells) with ii) a first media comprising a Wnt pathway activator, thereby generating a second population of cells, wherein the first media does not comprise a member of the transforming growth factor-beta superfamily (e.g., activin A), a bone morphogenic protein (BMP) pathway inhibitor, an AKT inhibitor, and / or a JNK inhibitor. In some embodiments, the plurality of pluripotent stem cells are cultured with the first media for less than 48 hours or for 12-48 hours, 12-36 hours, 12-30 hours, 12- 24 hours, 18-36 hours, 18-30 hours, or 21-27 hours. In some embodiments, the method further comprises the steps of removing the first media and culturing the second population of cells in a second media, thereby generating a third population of cells, wherein the second media comprises: a) a member of the transforming growth factor-beta superfamily (e.g., activin A) or b) a bone morphogenic protein (BMP) pathway inhibitor, AKT inhibitor and / or a JNK inhibitor, wherein the second media lacks a Wnt pathway activator. In some embodiments, the second population of cells are cultured with the second media for less than 72 hours or for no more than 20-52 hours, 36-52 hours, 42-42 hours, 20-72 hours, 12-48 hours, 12-36 hours, 12-30 hours, 12-24 hours, 18-36 hours, 18-30 hours, or 21-27 hours. In some embodiments, the method further comprises the steps of removing the second media and culturing the third population of cells in a third media, wherein the third media does not comprise: a) a Wnt pathway activator, b) a member of the transforming growth factor-beta superfamily (e.g., activin A), c) a bone morphogenic protein (BMP) pathway inhibitor, and / or d) a JNK inhibitor.In some embodiments, the disclosure provides for a composition comprising stem cells (e.g., pluripotent stem cells) and ascorbic acid. In some embodiments, the composition comprises 0.01-1 mg / ml ascorbic acid. In some embodiments, the composition comprises more than 0.044 mg / ml ascorbic acid (e.g., 0.045-0.15, 0.045-0.1, 0.045-0.09, 0.045-0.07, 0.045-0.05, 0.06-0.1, 0.06-0.09, 0.07-0.1, 0.07-0.09, 0.08-0.09, 0.08-0.1, 0.08-0.12, or 0.085-0.09 mg / ml. In some embodiments, the disclosure provides for a method of administering ascorbic acid to a composition comprising stem cells (e.g., pluripotent stem cells).

[0185] Any growth factor from the TGF-β superfamily capable of inducing the pluripotent stem cells to differentiate into definitive endoderm cells (e.g., alone, or in combination with a WNTsignaling pathway activator) can be used in the method 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 differentiating factor 8 (GDF8). Any WNT signaling pathway activator capable of inducing the pluripotent stem cells to differentiate into definitive endoderm cells (e.g., alone, or in combination with a growth factor from the TGF-β superfamily) can be used in the method provided herein. In some embodiments, the WNT signaling pathway activator comprises CHIR99021. In some embodiments, the WNT signaling pathway activator comprises Wnt3a recombinant protein.

[0186] In some embodiments, differentiating at least some stem cells (e.g., pluripotent stem cells) in a population into definitive endoderm cells is achieved by a process of contacting a population of pluripotent cells with i) Activin A, and ii) CHIR99021 for a suitable period of time, e.g., about 2 days, about 3 days, about 4 days, or about 5 days to induce the differentiation of at least some of the pluripotent cells in the population into definitive endoderm cells, wherein the definitive endoderm cells express at least one marker characteristic of definitive endoderm. In some embodiments, the process comprises contacting a population of stem cells (e.g., pluripotent stem cells) with activin A and CHIR99021 for 1 day, and then with activin A (in the absence of CHIR99021) for a further 1 or 2 days. In some embodiments, on each of the days, the cells are further in contact with an inhibitor of PI3K / Akt / mTOR signaling.

[0187] In some examples, the method comprises differentiating stem cells (e.g., pluripotent stem cells) into definitive endoderm cells by contacting a population of stem cells (e.g., pluripotent stem cells) with a suitable concentration of the growth factor from the TGF-β superfamily (e.g., Activin A, GDF 8, or GDF11), such as, about 10 ng / mL, about 20 ng / mL, about 50 ng / mL, about 75 ng / mL, about 80 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 110 ng / mL, about 120 ng / mL, about 130 ng / mL, about 140 ng / mL, about 150 ng / mL, about 175 ng / mL, about 180 ng / mL, about 200 ng / mL, about 250 ng / mL, or about 300 ng / mL. In some embodiments, the method comprises use of about 70-130 ng.ml, 80-120 ng / ml, or 90-110 ng / ml Activin A for differentiation of stem cells (e.g., pluripotent stem cells) into definitive endoderm cells. In some embodiments, the method comprises use of about 100 ng / mL Activin A for differentiation of stem cells (e.g., pluripotent stem cells) into definitive endoderm cells. In some embodiments, the method comprises use of about 200 ng / mL Activin A for differentiation of stem cells (e.g., pluripotent stem cells) into definitive endoderm cells.

[0188] In some examples, the method comprises differentiating stem cells (e.g., pluripotent stem cells) into definitive endoderm cells by contacting a population of stem cells (e.g., pluripotent stem cells) with a suitable concentration of the WNT signaling pathway activator (e.g., CHIR99021), such as, about 0.01 µM, about 0.05 µM, about 0.1 µM, about 0.2 µM, about 0.5 µM, about 0.8 µM, about 1 µM, about 1.5 µM, about 2 µM, about 2.5 µM, about 3 µM, about 3.5 µM, about 4 µM, about 5 µM,about 8 µM, about 10 µM, about 12 µM, about 15 µM, about 20 µM, about 30 µM, about 50 µM, about 100 µM, or about 200 µM. In some embodiments, the method comprises use of about 1-5 µM or 2-4 µM CHIR99021 for differentiation of stem cells (e.g., pluripotent stem cells) into definitive endoderm cells. In some embodiments, the method comprises use of about 2 µM CHIR99021 for differentiation of stem cells (e.g., pluripotent stem cells) into definitive endoderm cells. In some embodiments, the method comprises use of about 3 µM CHIR99021 for differentiation of stem cells (e.g., pluripotent stem cells) into definitive endoderm cells. In some embodiments, the method comprises use of about 5 µM CHIR99021 for differentiation of stem cells (e.g., pluripotent stem cells) into definitive endoderm cells.

[0189] In some embodiments, the method comprises use of about 1-5 µM or 2-4 µM CHIR99021 for differentiation of stem cells (e.g., pluripotent stem cells) into definitive endoderm cells. In some embodiments, the method comprises use of about 2 µM CHIR99021 for differentiation of stem cells (e.g., pluripotent stem cells) into definitive endoderm cells. In some embodiments, the method comprises use of about 3 µM CHIR99021 for differentiation of stem cells (e.g., pluripotent stem cells) into definitive endoderm cells. In some embodiments, the method comprises use of about 5 µM CHIR99021 for differentiation of stem cells (e.g., pluripotent stem cells) into definitive endoderm cells.

[0190] In some examples, the method comprises differentiating stem cells (e.g., pluripotent stem cells) into definitive endoderm cells by contacting a population of stem cells (e.g., pluripotent stem cells) with a suitable concentration of the an inhibitor of PI3K / Akt / mTOR signaling (e.g., an AKT inhibitor such as MK-2206), such as, about 0.01-1 μM (e.g., 0.01-1, 0.01-0.8, 0.01-0.6, 0.01-0.4, 0.01- 0.2, 0.01-0.1, 0.05-1, 0.05-0.8, 0.05-0.6, 0.05-0.4, 0.05-0.2, 0.05-0.1, 0.1-1, 0.1-0.8, 0.1-0.6, 0.1-0.4, 0.1-0.2, 0.2-1, 0.2-0.8, 0.2-0.5, 0.2-0.4, 0.4-1, 0.4-0.8, 0.4-0.6, 0.6-1, 0.6-0.8, or 0.8-1 μM). In some embodiments, the method comprises use of about 0.01-1 μM, 0.02-0.8 μM, 0.05-0.5 μM, 0.06-0.2 μM, 0.07-0.15 μM, or 0.08-0.12 μM of the inhibitor of PI3K / Akt / mTOR signaling (e.g., GSK-690693 or MK-2206, or an analog or a derivative thereof) for differentiation of pluripotent cells into definitive endoderm cells. In some embodiments, the method comprises use of about 0.1 μM of the inhibitor of PI3K / Akt / mTOR signaling (e.g., GSK-690693 or MK-2206, or an analog or a derivative thereof) for differentiation of pluripotent cells into definitive endoderm cells.

[0191] 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.

[0192] In some embodiments, a definitive endoderm cell produced by the methods as disclosed herein expresses 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, wherein the expression of at least one marker is upregulated to by a statistically significant amount in the definitive endoderm cell relative to the pluripotent stem cell from which it was derived. In some embodiments, a definitive endoderm cell produced by the methods as disclosed herein does not express by a statistically significant amount at least one marker selected the group consisting of: Gata4, SPARC, AFP and Dab2 relative to the pluripotent stem cell from which it was derived. In some embodiments, a definitive endoderm cell produced by the methods as disclosed herein does not express by a statistically significant amount at least one marker selected the group consisting of: Zic1, Pax6, Flk1 and CD31 relative to the pluripotent stem cell from which it was derived. In some embodiments, a definitive endoderm cell produced by the methods as disclosed herein has a higher level of phosphorylation of Smad2 by a statistically significant amount relative to the pluripotent stem cell from which it was derived. In some embodiments, a definitive endoderm cell produced by the methods as disclosed herein has the capacity to form gut tube in vivo. In some embodiments, a definitive endoderm cell produced by the methods as disclosed herein can differentiate into a cell with morphology characteristic of a gut cell, and wherein a cell with morphology characteristic of a gut cell expresses FoxA2 and / or Claudin6. In some embodiments, a definitive endoderm cell produced by the methods as disclosed herein can be further differentiated into a cell of endoderm origin.

[0193] In some embodiments, a population of stem cells (e.g., pluripotent stem cells) are cultured in the presence of at least one β cell differentiation factor prior to any differentiation or during the first stage of differentiation. One can use any stem cell, such as a human pluripotent stem cell, or a human iPS cell or any of pluripotent stem cell as discussed herein or other suitable pluripotent stem cells. In some embodiments, a β cell differentiation factor as described herein can be present in the culture medium of a population of stem cells (e.g., pluripotent stem cells) or may be added in bolus or periodically during growth (e.g. replication or propagation) of the population of pluripotent stem cells. In certain examples, a population of stem cells (e.g., pluripotent stem cells) can be exposed to at least one β cell differentiation factor prior to any differentiation. In other examples, a population of stem cells (e.g., pluripotent stem cells) may be exposed to at least one β cell differentiation factor during the first stage of differentiation. Primitive Gut Tube Cells

[0194] Aspects of the disclosure involve primitive gut tube cells. Primitive gut tube cells of use herein can be derived from any source or generated in accordance with any suitable protocol. In some aspects, definitive endoderm cells are differentiated to primitive gut tube cells. In some aspects, the primitive gut tube cells are further differentiated, e.g., to PDX1-positive pancreatic progenitor cells,NKX6.1-positive pancreatic progenitor cells, Ngn3-positive endocrine progenitor cells, insulin- positive endocrine cells, followed by induction or maturation to SC-β cells.

[0195] In some embodiments, primitive gut tube cells can be obtained by differentiating at least some definitive endoderm cells in a population into primitive gut tube cells, e.g., by contacting definitive endoderm cells with at least one growth factor from the fibroblast growth factor (FGF) family, to induce the differentiation of at least some of the definitive endoderm cells into primitive gut tube cells, wherein the primitive gut tube cells express at least one marker characteristic of primitive gut tube cells.

[0196] Any growth factor from the FGF family capable of inducing definitive endoderm cells to differentiate into primitive gut tube cells (e.g., alone, or in combination with other factors) can be used in the method 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 comprises FGF2. In some embodiments, the at least one growth factor from the FGF family comprises FGF8B. In some embodiments, the at least one growth factor from the FGF family comprises FGF10. In some embodiments, the at least one growth factor from the FGF family comprises FGF21.

[0197] In some embodiments, primitive gut tube cells can be obtained by differentiating at least some definitive endoderm cells in a population into primitive gut tube cells, e.g., by contacting 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, to induce the differentiation of at least some of the definitive endoderm cells into primitive gut tube cells.

[0198] In some embodiments, the method comprises differentiating definitive endoderm cells into primitive gut tube cells by contacting definitive endoderm cells with a suitable concentration of the growth factor from the FGF family (e.g., KGF), such as, about 10 ng / mL, about 20 ng / mL, about 50 ng / mL, about 75 ng / mL, about 80 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 110 ng / mL, about 120 ng / mL, about 130 ng / mL, about 140 ng / mL, about 150 ng / mL, about 175 ng / mL, about 180 ng / mL, about 200 ng / mL, about 250 ng / mL, or about 300 ng / mL. In some embodiments, the method comprises use of about 20-80 ng / ml, 30-70 ng / ml, or 40-60 ng / mL KGF for differentiation of definitive endoderm cells into primitive gut tube cells. In some embodiments, the method comprises use of about 50 ng / mL KGF for differentiation of definitive endoderm cells into primitive gut tube cells. In some embodiments, the method comprises use of about 100 ng / mL KGF for differentiation of definitive endoderm cells into primitive gut tube cells.

[0199] In some embodiments, the disclosure provides for a composition comprising a plurality of SOX17-positive cells and greater than 8 mM glucose. In some embodiments, the composition comprises 8-15, 8-14, 8-12, 8-11, 8-10, 8-9, 9-11, 9-12, or 9.5-10.5 mM glucose. In some embodiments, the composition does not comprise less than 4 mM glucose. In some embodiments, thedisclosure provides for a method comprising the step of culturing a plurality of SOX17-positive cells with a media comprising greater than 8 mM glucose. In some embodiments, the disclosure provides for a method comprising: a) administering a media to a container comprising SOX17-positive cells, wherein the media comprises 4-14 mM glucose; and b) after 4-18 hours of culturing the SOX17- positive cells in the media, administering supplemental glucose to the media, wherein following administration of the supplemental glucose to the media, the concentration of total glucose in the media is 4-14 mM. In some embodiments, the media administered in step a) comprises 4-12, 4-10, 4- 8, 6-12, 6-11, 6-10, 8-10, or 7-9 mM glucose. In some embodiments, 2-10, 2-8, 2-6, 2-4, 3-9, 3-7, 3-5 mM glucose is added in step b). In some embodiments, after 4-16, 4-12, 4-10, 6-12, 6-16, 6-9, 7-9, 7- 10, 7-12, 8-12, or 10-14 hours of culturing the SOX17-positive cells in the media from step a), the supplemental glucose is administered to the media. In some embodiments, the concentration of total glucose in the media following administration of the supplemental glucose to the media in step b) is 4- 12, 4-10, 4-8, 6-12, 6-11, 6-10, 8-10, 8-12, 9-12, or 7-9 mM. In some embodiments, steps a) and b) are repeated over about 2-5 days, about 2-4 days, or about 2-3 days. In some embodiments, the disclosure provides for a method of administering media to a container comprising SOX17-positive cells, wherein the media comprises 4-14 mM glucose and, before the glucose in the media is below 4 mM, administering supplemental glucose to the media such that the concentration of total glucose in the media is maintained at 4-14 mM. In some embodiments, the disclosure provides for a method comprising culturing a population of SOX17-positive cells in a media comprising at least 4 mM glucose, wherein the concentration of glucose in the media is maintained such that it does not fall below 1-4 mM (e.g., does not fall below 4 mM). In some embodiments, the concentration of glucose in the media is maintained such that it does not fall below 1-4 mM by supplementing the media with glucose (e.g., 4-10 mM glucose). In some embodiments, the concentration of glucose in the media following supplementation with glucose is 4-12, 4-10, 4-8, 6-12, 6-11, 6-10, 8-10, 8-12, 9-12, or 7-9 mM. In some embodiments, 2-10, 2-8, 2-6, 2-4, 3-9, 3-7, 3-5 mM glucose is supplemented to the media. In some embodiments, 10-100%, 10-80%, 10-60%, 10-40%, 10-20%, 30-100%, 30-80%, 30- 60%, 50-100%, 50-80%, 70-100%, or 90-100% of the cells in the composition are SOX17-positive cells. In some embodiments, 10-100%, 10-80%, 10-60%, 10-40%, 10-20%, 30-100%, 30-80%, 30- 60%, 50-100%, 50-80%, 70-100%, or 90-100% of the cells in the composition are primitive gut cells. In some embodiments, 10-100%, 10-80%, 10-60%, 10-40%, 10-20%, 30-100%, 30-80%, 30-60%, 50- 100%, 50-80%, 70-100%, or 90-100% of the cells in the composition express SOX17.

[0200] In some embodiments, the disclosure provides for a composition comprising SOX-17- positive cells and ascorbic acid. In some embodiments, the composition comprises 0.01-1 mg / ml ascorbic acid. In some embodiments, the composition comprises more than 0.044 mg / ml ascorbic acid (e.g., 0.045-0.15, 0.045-0.1, 0.045-0.09, 0.045-0.07, 0.045-0.05, 0.06-0.1, 0.06-0.09, 0.07-0.1, 0.07-0.09, 0.08-0.09, 0.08-0.1, 0.08-0.12, or 0.085-0.09 mg / ml. In some embodiments, the disclosureprovides for a method of administering ascorbic acid to a composition comprising SOX17-positive cells.

[0201] 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. PDX1-positive Pancreatic Progenitor Cells

[0202] Aspects of the disclosure involve PDX1-positive pancreatic progenitor cells. PDX1- positive pancreatic progenitor cells of use herein can be derived from any source or generated in accordance with any suitable protocol. In some aspects, primitive gut tube cells are differentiated to PDX1 -positive pancreatic progenitor cells. In some aspects, the PDX1-positive pancreatic progenitor cells are NKX6.1 negative, and can be further differentiated to, e.g., NKX6.1-positive pancreatic progenitor cells, Ngn3-positive endocrine progenitor cells, insulin-positive endocrine cells, followed by induction or maturation to SC-β cells.

[0203] In some aspects, PDX1-positive pancreatic progenitor cells can be obtained by differentiating at least some primitive gut tube cells in a population into PDX1-positive pancreatic progenitor cells, e.g., by contacting primitive gut tube cells with one or more of i) at least one BMP signaling pathway inhibitor, ii) a growth factor from 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 the differentiation of at least some of the primitive gut tube cells into PDX1-positive pancreatic progenitor cells, wherein the PDX1-positive pancreatic progenitor cells express PDX1.

[0204] In some aspects, PDX1-positive pancreatic progenitor cells can be obtained by differentiating at least some primitive gut tube cells in a population into PDX1-positive pancreatic progenitor cells, e.g., by contacting primitive gut tube cells with one or more of i) at least one BMP signaling pathway inhibitor, ii) a growth factor from 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 the differentiation of at least some of the primitive gut tube cells into PDX1-positive pancreatic progenitor cells, wherein the PDX1-positive pancreatic progenitor cells express PDX1.

[0205] In some embodiments, PDX1-positive pancreatic progenitor cells can be obtained by differentiating at least some primitive gut tube cells in a population into PDX1-positive pancreatic progenitor cells, e.g., by contacting primitive gut tube cells with one or more of i) at least one BMP signaling pathway inhibitor, ii) at least one growth factor from the FGF family, iii) at least one SHHpathway inhibitor, iv) at least one retinoic acid (RA) signaling pathway activator; and v) at least one protein kinase C activator, to induce the differentiation of at least some of the primitive gut tube cells into PDX1-positive pancreatic progenitor cells, wherein the PDX1-positive pancreatic progenitor cells express PDX1.

[0206] In some embodiments, PDX1-positive pancreatic progenitor cells can be obtained by differentiating at least some primitive gut tube cells in a population into PDX1-positive pancreatic progenitor cells, e.g., by contacting primitive gut tube 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.

[0207] In some embodiments, PDX1-positive pancreatic progenitor cells can be obtained by differentiating at least some primitive gut tube cells in a population into PDX1-positive pancreatic progenitor cells, e.g., by contacting primitive gut tube 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 the differentiation of at least some of the primitive gut tube cells into PDX1-positive pancreatic progenitor cells, wherein the PDX1 -positive pancreatic progenitor cells express PDX1.

[0208] Any BMP signaling pathway inhibitor capable of inducing primitive gut tube cells to differentiate into PDX1-positive pancreatic progenitor cells (e.g., alone, or with any combination of a growth factor from 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 ROCK inhibitor) can be used in the method provided herein. In some embodiments, the BMP signaling pathway inhibitor comprises LDN193189 or DMH-1. In some examples, the method comprises contacting primitive gut tube cells with a concentration of BMP signaling pathway inhibitor (e.g., LDN1931189), such as, 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 1µM. In some examples, the method comprises contacting primitive gut tube cells with a concentration of BMP signaling pathway inhibitor (e.g., DMH-1), such as, about 0.01 µM, about 0.02µM, about 0.05µM, about 0.1µM, about 0.2µM, about 0.5 µM, about 0.8 µM, about 1 µM, about 1.2 µM, about 1.5µM, about 1.75µM, about 2 µM, about 2.2 µM, about 2.5µM, about 2.75µM, 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 comprises contacting primitive gut tube cells with a concentration of BMP signaling pathway inhibitor (e.g., DMH-1), such as, about 220-280 nM, about 230-270 nM, about 240-260 nM, or about245-255 nM. In some examples, the method comprises contacting primitive gut tube cells with a concentration of BMP signaling pathway inhibitor (e.g., DMH-1) about 250 nM.

[0209] Any growth factor from the TGF-β superfamily capable of inducing primitive gut tube cells to differentiate into PDX1-positive pancreatic progenitor cells (e.g., alone, or with any combination of 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 ROCK inhibitor) can be used. In some embodiments, the growth factor from TGF-β family comprises Activin A. In some embodiments, the growth factor from TGF-β family comprises GDF8. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a growth factor from TGF-β superfamily (e.g., Activin A), such as, 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 comprises contacting primitive gut tube cells with a concentration of a growth factor from TGF-β superfamily (e.g., Activin A), such as, about 17-23 ng / ml, about 18-22 ng / ml, or about 19-21 ng / ml. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a growth factor from TGF-β superfamily (e.g., Activin A) of about 20 ng / ml. In some embodiments, the method does not comprise contacting primitive gut tube cells with any TGF-beta superfamily growth factor.

[0210] Any growth factor from the FGF family capable of inducing primitive gut tube cells to differentiate into PDX1-positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one BMP signaling pathway inhibitor, a growth factor from 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 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 comprises contacting primitive gut tube cells with a concentration of a growth factor from FGF family (e.g., KGF), such as, about 10 ng / mL, about 20 ng / mL, about 50 ng / mL, about 75 ng / mL, about 80 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 110 ng / mL, about 120 ng / mL, about 130 ng / mL, about 140 ng / mL, about 150 ng / mL, about 175 ng / mL, about 180 ng / mL, about 200 ng / mL, about 250 ng / mL, or about 300 ng / mL. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a growth factor from FGF family (e.g., KGF), such as, 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 comprisescontacting primitive gut tube cells with a concentration of a growth factor from FGF family (e.g., KGF) of about 50 ng / ml.

[0211] Any SHH pathway inhibitor capable of inducing primitive gut tube cells to differentiate into PDX1-positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one BMP signaling pathway inhibitor, at least one growth factor from the FGF family, a growth factor from TGF-β superfamily, at least one retinoic acid signaling pathway activator, at least one protein kinase C activator, and ROCK inhibitor) can be used. In some embodiments, the SHH pathway inhibitor comprises Sant1. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a SHH pathway inhibitor (e.g., Sant1), such as, about 0.001 µM, about 0.002 µM, about 0.005 µM, about 0.01 µM, about 0.02 µM, about 0.03µM, about 0.05µM, about 0.08 µM, about 0.1µM, about 0.12 µM, about 0.13 µM, about 0.14 µM, about 0.15 µM, about 0.16 µM, about 0.17 µM, about 0.18 µM, about 0.19 µM, about 0.2 µM, about 0.21 µM, about 0.22 µM, about 0.23 µM, about 0.24 µM, about 0.25 µM, about 0.26 µM, about 0.27 µM, about 0.28 µM, about 0.29 µM, about 0.3 µM, about 0.31 µM, about 0.32 µM, about 0.33 µM, about 0.34 µM, about 0.35 µM, about 0.4 µM, about 0.45 µM, about 0.5 µM, about 0.6 µM, about 0.8 µM, about 1 µM, about 2 µM, or about 5 µM. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a SHH pathway inhibitor (e.g., Sant1), such as, about 220-280 nM, about 230-270 nM, about 240-260 nM, or about 245-255 nM. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a SHH pathway inhibitor (e.g., Sant1) of about 250 nM.

[0212] Any RA signaling pathway activator capable of inducing primitive gut tube cells to differentiate into PDX1-positive pancreatic progenitor cells (e.g., alone, or with any combination of 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 ROCK inhibitor) can be used. In some embodiments, the RA signaling pathway activator comprises retinoic acid. In some examples, the method comprises contacting primitive gut tube cells with a concentration of an RA signaling pathway activator (e.g., retinoic acid), such as, about 0.02 µM, about 0.1µM, about 0.2 µM, about 0.25 µM, about 0.3 µM, about 0.4 µM, about 0.45 µM, about 0.5 µM, about 0.55 µM, about 0.6 µM, about 0.65 µM, about 0.7 µM, about 0.75 µM, about 0.8 µM, about 0.85 µM, about 0.9 µM, about 1 µM, about 1.1 µM, about 1.2 µM, about 1.3 µM, about 1.4 µM, about 1.5 µM, about 1.6 µM, about 1.7 µM, about 1.8 µM, about 1.9 µM, about 2 µM, about 2.1 µM, about 2.2 µM, about 2.3 µM, about 2.4 µM, about 2.5 µM, about 2.6 µM, about 2.7 µM, about 2.8 µM, about 3 µM, about 3.2 µM, about 3.4 µM, about 3.6 µM, about 3.8 µM, about 4 µM, about 4.2 µM, about 4.4 µM, about 4.6 µM, about 4.8 µM, about 5 µM, about 5.5 µM, about 6 µM, about 6.5 µM, about 7 µM, about 7.5 µM, about 8 µM, about 8.5 µM, about 9 µM, about 9.5 µM, about 10 µM, about 12 µM, about 14 µM, about 15 µM, about 16 µM, about 18 µM, about 20 µM, about 50 µM, or about 100 µM. In some examples, the method comprises contacting primitive gut tube cells with a concentration of an RAsignaling pathway activator (e.g., retinoic acid), such as, about 1.7-2.3 µM, about 1.8-2.2 µM, or about 1.9-2.1 µM. In some examples, the method comprises contacting primitive gut tube cells with a concentration of an RA signaling pathway activator (e.g., retinoic acid) of about 2 µM.

[0213] Any PKC activator capable of inducing primitive gut tube cells to differentiate into PDX1- positive pancreatic progenitor cells (e.g., alone, or with any combination of 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 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 contacting primitive gut tube cells with a concentration of a PKC activator (e.g., PdBU or TPPB), such as, about 10 nM, 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM, 1 µM, 10 µM, about 20 µM, about 50 µM, about 75 µM, about 80 µM, about 100 µM, about 120 µM, about 140 µM, about 150 µM, about 175 µM, about 180 µM, about 200 µM, about 210 µM, about 220 µM, about 240 µM, about 250 µM, about 260 µM, about 280 µM, about 300 µM, about 320 µM, about 340 µM, about 360 µM, about 380 µM, about 400 µM, about 420 µM, about 440 µM, about 460 µM, about 480 µM, about 500 µM, about 520 µM, about 540 µM, about 560 µM, about 580 µM, about 600 µM, about 620 µM, about 640 µM, about 660 µM, about 680 µM, about 700 µM, about 750 µM, about 800 µM, about 850 µM, about 900 µM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, or about 5 mM. In some embodiments, the method comprises contacting primitive gut tube cells with a concentration of a PKC activator (e.g., PdBU or TPPB) of 10 nM-1 mM, 10 nM-500 µM, 10 nM-1 µM, 10-800 nM, 100-900 nM, 300-800 nM, 300-600 nM, 400-600 nM, 450-550 nM, or about 500 nM. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a PKC activator (e.g., PdBU or TPPB), such as, about 450-550 mM, about 475-525 nM, about 490-510 nM, or about 495-505 nM. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a PKC activator (e.g., PdBU or TPPB) of about 500 nM. In some embodiments, primitive gut tube cells are not treated with a PKC activator (e.g., PDBU).

[0214] Any ROCK inhibitor capable of inducing primitive gut tube cells to differentiate into PDX1-positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one BMP signaling pathway inhibitor, at least one growth factor from the FGF family, at least one SHH pathway inhibitor, PKC activator, and at least one RA 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 contacting primitive gut tube cells with a concentration of a ROCK inhibitor (e.g., Y-27632 or Thiazovivin), such as, about 0.2 µM, about 0.5 µM, about 0.75 µM, about 1 µM, about 2 µM, about 3 µM, about 4 µM, about 5 µM, about 6 µM, about 7 µM, about 7.5 µM, about 8 µM, about 9 µM, about 10 µM,about 11 µM, about 12 µM, about 13 µM, about 14 µM, about 15 µM, about 16 µM, about 17 µM, about 18 µM, about 19 µM, about 20 µM, about 21 µM, about 22 µM, about 23 µM, about 24 µM, about 25 µM, about 26 µM, about 27 µM, about 28 µM, about 29 µM, about 30 µM, about 35 µM, about 40 µM, about 50 µM, or about 100 µM. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a ROCK inhibitor (e.g., Y-27632 or Thiazovivin), such as, about 2.2-2.8 µM, about 2.3-2.7 µM, or about 2.4-2.6 µM. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a ROCK inhibitor (e.g., Y-27632 or Thiazovivin) of about 2.5 µM.

[0215] In some embodiments, the disclosure provides for a composition comprising a plurality of primitive gut tube cells (e.g., FOXA2-positive, PDX1-negative cells), and any of the bone morphogenic protein (BMP) pathway inhibitors disclosed herein. In some embodiments, the BMP pathway inhibitor is dorsomorphin. In some embodiments, the concentration of the BMP pathway inhibitor is 0.1-100 µM, 0.1-50 µM, 0.1-25 µM, 0.1-10 µM, 0.1-1 µM, 0.5-25 µM, 0.5-10 µM, 0.5-5 µM, 0.5-2 µM, or 0.8-1.2 µM. In particular embodiments, the concentration of the BMP pathway inhibitor is 0.5-2 µM.

[0216] In some embodiments, the disclosure provides for a composition comprising a plurality of primitive gut tube cells (e.g., FOXA2-positive, PDX1-negative cells), and any of the JNK inhibitors disclosed herein. In some embodiments, the JNK inhibitor is a JNK1, JNK2, and / or JNK3 inhibitor. In some embodiments, the JNK inhibitor is a JNK1, JNK2, and JNK3 inhibitor. In some embodiments, the JNK inhibitor is JNK-IN-8. In some embodiments, the concentration of the JNK inhibitor is 0.1-100 µM, 0.1-50 µM, 0.1-25 µM, 0.1-10 µM, 0.1-1 µM, 0.5-25 µM, 0.5-10 µM, 0.5-5 µM, 0.5-2 µM, or 0.8-1.2 µM. In particular embodiments, the concentration of the JNK inhibitor is 0.5-2 µM.

[0217] In some embodiments, the disclosure provides for a composition comprising a plurality of primitive gut tube cells (e.g., FOXA2-positive, PDX1-negative cells) and any of the BMP pathway inhibitors disclosed herein and any of the JNK inhibitors disclosed herein. In some embodiments, the composition does not comprise an exogenous growth factor from the TGF-β superfamily (e.g., activin A, GDF8, or GDF11).

[0218] In some embodiments, the disclosure provides for a composition comprising a plurality of primitive gut tube cells (e.g., FOXA2-positive, PDX1-negative cells) and an AKT-inhibitor. In some embodiments, the AKT inhibitor is any one of BEZ235, LY294002, GDC-0941, BYL719, GSK2636771, TGX-221, AS25242, CAL-101, IPI-145, MK-2206, GSK690693, GDC-0068, A- 674563, CCT128930, AZD8055, INK128, rapamycin, PF-04691502, everolimus, BI-D1870, H89, PF-4708671, FMK, AT7867, NU7441, PI-103, NU7026, PIK-75, ZSTK474, and PP-121. In some embodiments, the AKT inhibitor comprises the structure of, or a pharmaceutically acceptable salt or AKT inhibitor is MK-2206.

[0219] In some embodiments, the disclosure provides for a composition comprising a plurality of primitive gut tube cells (e.g., FOXA2-positive, PDX1-negative cells), any of the AKT inhibitors disclosed herein and any of the BMP pathway inhibitors disclosed herein. In some embodiments, the disclosure provides for a composition comprising a plurality of primitive gut tube cells (e.g., FOXA2- positive, PDX1-negative cells), any of the AKT inhibitors disclosed herein and any of the JNK inhibitors disclosed herein. In some embodiments, the disclosure provides for a composition comprising a plurality of primitive gut tube cells (e.g., FOXA2-positive, PDX1-negative cells), any of the AKT inhibitors disclosed herein, any of the BMP pathway inhibitors disclosed herein, and any of the JNK inhibitors disclosed herein. In some embodiments, the composition does not comprise an exogenous growth factor from the TGF-β superfamily (e.g., activin A, GDF8, or GDF11).

[0220] In some embodiments, the disclosure provides for a method comprising the step of culturing a plurality of primitive gut tube cells (e.g., FOXA2-positive, PDX1-negative cells) with a media comprising a bone morphogenic protein (BMP) pathway inhibitor. In some embodiments, the disclosure provides for a method comprising the step of culturing a plurality of primitive gut tube cells (e.g., FOXA2-positive, PDX1-negative cells) with a media comprising a JNK inhibitor. In some embodiments, the disclosure provides for a method comprising the step of culturing a plurality of primitive gut tube cells (e.g., FOXA2-positive, PDX1-negative cells) with a media comprising an AKT inhibitor. In some embodiments, the disclosure provides for a method comprising the step of culturing a plurality of primitive gut tube cells (e.g., FOXA2-positive, PDX1-negative cells) with a media comprising a BMP pathway inhibitor and a JNK inhibitor or an AKT inhibitor. In some embodiments, the disclosure provides for a method comprising the step of culturing a plurality of primitive gut tube cells (e.g., FOXA2-positive, PDX1-negative cells) with a media comprising a JNK inhibitor and a BMP pathway inhibitor or an AKT inhibitor. In some embodiments, the disclosure provides for a method comprising the step of culturing a plurality of primitive gut tube cells (e.g., FOXA2-positive, PDX1-negative cells) with a media comprising a JNK inhibitor, a BMP pathway inhibitor, and an AKT inhibitor. In some embodiments, the method does not comprise a step of administering a growth factor from the TGF-β superfamily (e.g., activin A, GDF8, or GDF11) to the media.

[0221] In some embodiments, the disclosure provides for a composition comprising primitive gut tube cells (e.g., FOXA2-positive, PDX1-negative cells) and ascorbic acid. In some embodiments, the composition comprises 0.01-1 mg / ml ascorbic acid. In some embodiments, the composition comprises more than 0.044 mg / ml ascorbic acid (e.g., 0.045-0.15, 0.045-0.1, 0.045-0.09, 0.045-0.07, 0.045-0.05, 0.06-0.1, 0.06-0.09, 0.07-0.1, 0.07-0.09, 0.08-0.09, 0.08-0.1, 0.08-0.12, or 0.085-0.09 mg / ml. In some embodiments, the disclosure provides for a method of administering ascorbic acid to a composition comprising primitive gut tube cells (e.g., FOXA2-positive, PDX1-negative cells).

[0222] 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.

[0223] In some embodiments, PDX1-positive pancreatic progenitor cells can be obtained by differentiating at least some primitive gut tube cells in a population into PDX1-positive pancreatic progenitor cells, e.g., by contacting primitive gut tube 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 primitive gut tube cells in a population into PDX1-positive pancreatic progenitor cells, e.g., by contacting primitive gut tube 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 some primitive gut tube cells in a population into PDX1-positive pancreatic progenitor cells, e.g., by contacting primitive gut tube cells with retinoic acid, KGF, Sant1, DMH-1, PdBU, thiazovivin, and Activin A for 1 day, followed by contacting the cells with retinoic acid, KGF, Sant1, PdBU, thiazovivin, and Activin A for 1 day (in the absence of DMH-1). NKX6.1-positive Pancreatic Progenitor Cells

[0224] Aspects of the disclosure involve NKX6.1-positive pancreatic progenitor cells. NKX6.1- positive pancreatic progenitor cells of use herein can be derived from any source or generated in accordance with any suitable protocol. In some aspects, PDX1-positive, NKX6.1-negative pancreatic progenitor cells are differentiated to PDX1-positive, NKX6.1-positive pancreatic progenitor cells. In some aspects, the NKX6.1-positive pancreatic progenitor cells are further differentiated, e.g., to Ngn3-positive endocrine progenitor cells, or insulin-positive endocrine cells, followed by induction or maturation to SC-β cells.

[0225] In some aspects, a method of producing a NKX6.1-positive pancreatic progenitor cell from a PDX1-positive pancreatic progenitor cell comprises contacting a population of cells (e.g., underconditions that promote cell clustering and / or promoting cell survival) comprising PDX1-positive pancreatic progenitor cells with at least two β cell-differentiation factors comprising a) at least one growth factor from the fibroblast growth factor (FGF) family, b) a sonic hedgehog pathway inhibitor, and optionally c) a low concentration of a retinoic acid (RA) signaling pathway activator, to induce the differentiation of at least one PDX1-positive pancreatic progenitor cell in the population into NKX6.1-positive pancreatic progenitor cells, wherein the NKX6.1-positive pancreatic progenitor cells expresses NKX6.1.

[0226] 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) a RA signaling pathway activator, to induce the differentiation of at least some of the PDX1-positive pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells, wherein the PDX1- positive, NKX6.1- positive pancreatic progenitor cells express PDX1 and NKX6.1.

[0227] 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) a RA signaling pathway activator, iv) ROCK inhibitor, and v) at least one growth factor from the TGF-β superfamily, to induce the differentiation of at least some of the PDX1-positive pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells. In some embodiments, following 3, 4, or 5 days of contacting the PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) a RA signaling pathway activator, iv) ROCK inhibitor, and v) at least one growth factor from the TGF-β superfamily; the cells are then contacted with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) a RA signaling pathway activator, iv) ROCK inhibitor, and v) at least one growth factor from the TGF-β superfamily, and vi) a PKC activator and optionally vii) a gamma-secretase inhibitor. In some embodiments, the PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive pancreatic progenitor cells under conditions that promote cell clustering with at least one growth factor from the FGF family. In some embodiments, the growth factor from the FGF family is KGF.

[0228] In some embodiments, the disclosure provides for a method in which a first population of cells comprising PDX1-positive, NKX6.1-negative cells is cultured in a media comprising any one or combination of: i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, iii) a RA signaling pathway activator, iv) a ROCK inhibitor, and v) a growth factor from the TGF-β superfamily for a period of about 1, 2, 3, 4 or 5 days (e.g., 2-4, 3-4, or 4-5 days); thereby generating a second population of cells. In some embodiments, the second population of cells is thenincubated 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) a 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 for about 1, 2, or 3 days (e.g., 1-2, 1-3, or 2-3 days).

[0229] In some embodiments, in the media 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, 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, 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.

[0230] In some embodiments, in the media 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, 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, the growth factor from the TGF-β superfamily is present at a concentration of 2 about -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, and 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.

[0231] In some embodiments, the PDX1-positive pancreatic progenitor cells are produced from a population of pluripotent cells. In some embodiments, the PDX1-positive pancreatic progenitor cells are produced from a population of iPS cells. In some embodiments, the PDX1-positive pancreatic progenitor cells are produced from a population of ESC cells. In some embodiments, the PDX1- positive pancreatic progenitor cells are produced from a population of definitive endoderm cells. In some embodiments, the PDX1-positive pancreatic progenitor cells are produced from a population of primitive gut tube cells.

[0232] Any growth factor from the FGF family capable of inducing PDX1-positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one SHH pathway inhibitor, a ROCK inhibitor, a growth factor from the TGF-β superfamily, and at least one retinoic acid signaling pathway activator) can be used in themethod 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, FGF 10, and FGF21. In some examples, the method comprises contacting PDX1-positive pancreatic progenitor cells with a concentration of a growth factor from FGF family (e.g., KGF), such as, about 10 ng / mL, about 20 ng / mL, about 50 ng / mL, about 75 ng / mL, about 80 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 110 ng / mL, about 120 ng / mL, about 130 ng / mL, about 140 ng / mL, about 150 ng / mL, about 175 ng / mL, about 180 ng / mL, about 200 ng / mL, about 250 ng / mL, or about 300 ng / mL. In some examples, the method comprises contacting PDX1-positive pancreatic progenitor cells with a concentration of a growth factor from FGF family (e.g., KGF), such as, 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 comprises contacting PDX1-positive pancreatic progenitor cells with a concentration of a growth factor from FGF family (e.g., KGF) of about 50 ng / ml.

[0233] Any SHH pathway inhibitor capable of inducing PDX1-positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one growth factor from the FGF family, a retinoic acid signaling pathway activator, ROCK inhibitor, and at least one growth factor from the TGF-β superfamily) can be used in the method provided herein. In some embodiments, the SHH pathway inhibitor comprises Sant1. In some examples, the method comprises contacting PDX1-positive pancreatic progenitor cells with a concentration of a SHH pathway inhibitor (e.g., Sant1), such as, about 0.001 µM, about 0.002 µM, about 0.005 µM, about 0.01 µM, about 0.02 µM, about 0.03µM, about 0.05µM, about 0.08 µM, about 0.1µM, 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 0.21µM, about 0.22µM, about 0.23µM, 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 comprises contacting PDX1-positive pancreatic progenitor cells with a concentration of a SHH pathway inhibitor (e.g., Sant1), such as, about 220-280 nM, about 230- 270 nM, about 240-260 nM, or about 245-255 nM. In some examples, the method comprises contacting PDX1-positive pancreatic progenitor cells with a concentration of a SHH pathway inhibitor (e.g., Sant1) of about 250 nM.

[0234] Any RA signaling pathway activator capable of inducing PDX1-positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one growth factor from the FGF family, at least one SHH pathway inhibitor, ROCK inhibitor, and at least one growth factor from the TGF-β superfamily) can be used. In some embodiments, the RA signaling pathway activator comprises retinoic acid. In some examples, the method comprises contacting PDX1-positive pancreatic progenitor cells with a concentration of anRA signaling pathway activator (e.g., retinoic acid), such as, about 0.02 µM, about 0.1µM, about 0.2 µM, about 0.25 µM, about 0.3 µM, about 0.4 µM, about 0.45 µM, about 0.5 µM, about 0.55 µM, about 0.6 µM, about 0.65 µM, about 0.7 µM, about 0.75 µM, about 0.8 µM, about 0.85 µM, about 0.9 µM, about 1 µM, about 1.1 µM, about 1.2 µM, about 1.3 µM, about 1.4 µM, about 1.5 µM, about 1.6 µM, about 1.7 µM, about 1.8 µM, about 1.9 µM, about 2 µM, about 2.1 µM, about 2.2 µM, about 2.3 µM, about 2.4 µM, about 2.5 µM, about 2.6 µM, about 2.7 µM, about 2.8 µM, about 3 µM, about 3.2 µM, about 3.4 µM, about 3.6 µM, about 3.8 µM, about 4 µM, about 4.2 µM, about 4.4 µM, about 4.6 µM, about 4.8 µM, about 5 µM, about 5.5 µM, about 6 µM, about 6.5 µM, about 7 µM, about 7.5 µM, about 8 µM, about 8.5 µM, about 9 µM, about 9.5 µM, about 10 µM, about 12 µM, about 14 µM, about 15 µM, about 16 µM, about 18 µM, about 20 µM, about 50 µM, or about 100 µM. In some examples, the method comprises contacting PDX1-positive pancreatic progenitor cells with a concentration of an RA signaling pathway activator (e.g., retinoic acid), such as, about 70-130 nM, about 80-120 nM, about 90-110 nM, or about 95-105 nM. In some examples, the method comprises contacting PDX1-positive pancreatic progenitor cells with a concentration of an RA signaling pathway activator (e.g., retinoic acid) of about 100 nM.

[0235] Any ROCK inhibitor capable of inducing PDX1-positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one growth factor from the FGF family, at least one SHH pathway inhibitor, a RA signaling pathway activator, and at least one growth factor from the TGF-β superfamily) can be used. In some embodiments, the ROCK inhibitor comprises Thiazovivin, Y-27632, Fasudil / HA1077, or 14-1152. In some examples, the method comprises contacting PDX1-positive pancreatic progenitor cells with a concentration of a ROCK inhibitor (e.g., Y-27632 or Thiazovivin), such as, about 0.2 µM, about 0.5 µM, about 0.75 µM, about 1 µM, about 2 µM, about 3 µM, about 4 µM, about 5 µM, about 6 µM, about 7 µM, about 7.5 µM, about 8 µM, about 9 µM, about 10 µM, about 11 µM, about 12 µM, about 13 µM, about 14 µM, about 15 µM, about 16 µM, about 17 µM, about 18 µM, about 19 µM, about 20 µM, about 21 µM, about 22 µM, about 23 µM, about 24 µM, about 25 µM, about 26 µM, about 27 µM, about 28 µM, about 29 µM, about 30 µM, about 35 µM, about 40 µM, about 50 µM, or about 100 µM. In some examples, the method comprises contacting PDX1-positive pancreatic progenitor cells with a concentration of a ROCK inhibitor (e.g., Y-27632 or Thiazovivin), such as, about 2.2-2.8 µM, about 2.3-2.7 µM, or about 2.4-2.6 µM. In some examples, the method comprises contacting PDX1-positive pancreatic progenitor cells with a concentration of a ROCK inhibitor (e.g., Y-27632 or Thiazovivin) of about 2.5 µM.

[0236] Any activator from the TGF-β superfamily capable of inducing PDX1-positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one growth factor from the FGF family, at least one SHH pathway inhibitor, a RA signaling pathway activator, and ROCK inhibitor) can be used. In some embodiments, the activator from the TGF-β superfamily comprises Activin A or GDF8. In some examples, themethod comprises contacting PDX1-positive pancreatic progenitor cells with a concentration of a growth factor from TGF-β superfamily (e.g., Activin A), such as, about 0.1 ng / mL, about 0.2 ng / mL, about 0.3 ng / mL, about 0.4 ng / mL, about 0.5 ng / mL, about 0.6 ng / mL, about 0.7 ng / mL, about 0.8 ng / mL, about 1 ng / mL, about 1.2 ng / mL, about 1.4 ng / mL, about 1.6 ng / mL, about 1.8 ng / mL, about 2 ng / mL, about 2.2 ng / mL, about 2.4 ng / mL, about 2.6 ng / mL, about 2.8 ng / mL, about 3 ng / mL, about 3.2 ng / mL, about 3.4 ng / mL, about 3.6 ng / mL, about 3.8 ng / mL, about 4 ng / mL, about 4.2 ng / mL, about 4.4 ng / mL, about 4.6 ng / mL, about 4.8 ng / mL, about 5 ng / mL, about 5.2 ng / mL, about 5.4 ng / mL, about 5.6 ng / mL, about 5.8 ng / mL, about 6 ng / mL, about 6.2 ng / mL, about 6.4 ng / mL, about 6.6 ng / mL, about 6.8 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, or about 50 ng / mL. In some examples, the method comprises contacting PDX1-positive pancreatic progenitor cells with a concentration of a growth factor from TGF-β superfamily (e.g., Activin A), such as, 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 comprises contacting PDX1-positive pancreatic progenitor cells with a concentration of a growth factor from TGF-β superfamily (e.g., Activin A), such as, about 5 ng / mL.

[0237] Any FoxO1 inhibitor capable of inducing PDX1-positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one growth factor from the FGF family, at least one retinoic acid signaling pathway activator, ROCK inhibitor, at least one growth factor from the TGF-β superfamily, PKC activator, and Notch signaling inhibitor) can be used in the method provided herein. In some embodiments, the FoxO1 inhibitor is AS1842856. In particular embodiments, the FOXO1 inhibitor iscells with a concentration of a FoxO1 inhibitor, such as, about 0.1µM, 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 0.21µM, about 0.22µM, about 0.23µM, 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 comprises contacting PDX1-positive pancreatic progenitor cells with a concentration of a FoxO1 inhibitor, such as, about 0.7-1.3 µM, about 0.8-1.2 µM, about or 0.9-1.1 µM. In some examples, the method comprises contacting PDX1-positive pancreatic progenitor cells with a concentration of a FoxO1 inhibitor, such as, about 1 µM.

[0238] Any PKC activator capable of inducing PDX1-positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one growth factor from the FGF family, at least one retinoic acid signaling pathway activator, ROCK inhibitor, at least one growth factor from the TGF-β superfamily, FoxO1 inhibitor, and Notch signaling inhibitor) can be used in the method provided herein. In some embodiments, the PKC activator is PDBU. In some examples, the method comprises contacting PDX1-positive pancreatic progenitor cells with a concentration of a PKC activator (e.g., PDBU), such as, about 0.1µM, 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 0.21µM, about 0.22µM, about 0.23µM, 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 comprises contacting PDX1-positive pancreatic progenitor cells with a concentration of a PKC activator (e.g., PDBU), such as, about 0.2-0.8 µM, about 0.3-0.7 µM, about 0.4-0.6 µM. In some examples, the method comprises contacting PDX1-positive pancreatic progenitor cells with a concentration of a PKC activator (e.g., PDBU), such as, about 0.5 µM.

[0239] Any Notch signaling inhibitor capable of inducing PDX1-positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one growth factor from the FGF family, at least one retinoic acid signaling pathway activator, ROCK inhibitor, at least one growth factor from the TGF-β superfamily, FoxO1 inhibitor, and PKC activator) can be used in the method provided herein. In some embodiments, the Notch signaling inhibitor is XXI. In some examples, the method comprises contacting PDX1-positive pancreatic progenitor cells with a concentration of a Notch signaling inhibitor (e.g., XXI), such as, about 0.1µM, about 0.12 µM, about 0.13 µM, about 0.14 µM, about 0.15 µM, about 0.16 µM, about 0.17 µM, about 0.18 µM, about 0.19 µM, about 0.2 µM, about 0.21 µM, about 0.22 µM, about 0.23 µM, about 0.24 µM, about 0.25 µM, about 0.26 µM, about 0.27 µM, about 0.28 µM, about 0.29 µM, about 0.3 µM, about 0.31 µM, about 0.32 µM, about 0.33 µM, about 0.34 µM, about 0.35 µM, about 0.4 µM, about 0.45 µM, about 0.5 µM, about 0.6 µM, about 0.8 µM, about 1 µM, about 2 µM, or about 5 µM. In some examples, the method comprises contacting PDX1-positive pancreaticprogenitor cells with a concentration of a Notch signaling inhibitor (e.g., XXI), such as, about 1.7-2.3 µM, about 1.8-2.2 µM, or about 1.9- 2.1 µM. In some examples, the method comprises contacting PDX1-positive pancreatic progenitor cells with a concentration of a Notch signaling inhibitor (e.g., XXI), such as, about 2 µM.

[0240] In some embodiments, the disclosure provides for a composition comprising a plurality of PDX1-positive cells, and any of the bone morphogenic protein (BMP) pathway inhibitors disclosed herein. In some embodiments, the BMP pathway inhibitor is dorsomorphin. In some embodiments, the concentration of the BMP pathway inhibitor is 0.1-100 µM, 0.1-50 µM, 0.1-25 µM, 0.1-10 µM, 0.1-1 µM, 0.5-25 µM, 0.5-10 µM, 0.5-5 µM, 0.5-2 µM, or 0.8-1.2 µM. In particular embodiments, the concentration of the BMP pathway inhibitor is 0.5-2 µM.

[0241] In some embodiments, the disclosure provides for a composition comprising a plurality of PDX1-positive cells, and any of the JNK inhibitors disclosed herein. In some embodiments, the JNK inhibitor is a JNK1, JNK2, and / or JNK3 inhibitor. In some embodiments, the JNK inhibitor is a JNK1, JNK2, and JNK3 inhibitor. In some embodiments, the JNK inhibitor is JNK-IN-8. In some embodiments, the concentration of the JNK inhibitor is 0.1-100 µM, 0.1-50 µM, 0.1-25 µM, 0.1-10 µM, 0.1-1 µM, 0.5-25 µM, 0.5-10 µM, 0.5-5 µM, 0.5-2 µM, or 0.8-1.2 µM. In particular embodiments, the concentration of the JNK inhibitor is 0.5-2 µM.

[0242] In some embodiments, the disclosure provides for a composition comprising a plurality of PDX1-positive cells and any of the BMP pathway inhibitors disclosed herein and any of the JNK inhibitors disclosed herein. In some embodiments, the composition does not comprise an exogenous growth factor from the TGF-β superfamily (e.g., activin A, GDF8, or GDF11).

[0243] In some embodiments, the disclosure provides for a composition comprising a plurality of PDX1-positive cells and an AKT-inhibitor. In some embodiments, the AKT inhibitor is any one of BEZ235, LY294002, GDC-0941, BYL719, GSK2636771, TGX-221, AS25242, CAL-101, IPI-145, MK-2206, GSK690693, GDC-0068, A-674563, CCT128930, AZD8055, INK128, rapamycin, PF- 04691502, everolimus, BI-D1870, H89, PF-4708671, FMK, AT7867, NU7441, PI-103, NU7026, PIK-75, ZSTK474, and PP-121. In some embodiments, the AKT inhibitor comprises the structure of, or a pharmaceutically acceptable salt or derivative thereof. In some embodiments, the AKT inhibitor is MK-2206.

[0244] In some embodiments, the disclosure provides for a composition comprising a plurality of PDX1-positive cells, any of the AKT inhibitors disclosed herein and any of the BMP pathway inhibitors disclosed herein. In some embodiments, the disclosure provides for a composition comprising a plurality of PDX1-positive cells, any of the AKT inhibitors disclosed herein and any of the JNK inhibitors disclosed herein. In some embodiments, the disclosure provides for a composition comprising a plurality of PDX1-positive cells, any of the AKT inhibitors disclosed herein, any of the BMP pathway inhibitors disclosed herein, and any of the JNK inhibitors disclosed herein. In some embodiments, the composition does not comprise an exogenous growth factor from the TGF-β superfamily (e.g., activin A, GDF8, or GDF11).

[0245] In some embodiments, the disclosure provides for a method comprising the step of culturing a plurality of PDX1-positive cells with a media comprising a bone morphogenic protein (BMP) pathway inhibitor. In some embodiments, the disclosure provides for a method comprising the step of culturing a plurality of PDX1-positive cells with a media comprising a JNK inhibitor. In some embodiments, the disclosure provides for a method comprising the step of culturing a plurality of PDX1-positive cells with a media comprising an AKT inhibitor. In some embodiments, the disclosure provides for a method comprising the step of culturing a plurality of PDX1-positive cells with a media comprising a BMP pathway inhibitor and a JNK inhibitor or an AKT inhibitor. In some embodiments, the disclosure provides for a method comprising the step of culturing a plurality of PDX1-positive cells with a media comprising a JNK inhibitor and a BMP pathway inhibitor or an AKT inhibitor. In some embodiments, the disclosure provides for a method comprising the step of culturing a plurality of PDX1-positive cells with a media comprising a JNK inhibitor, a BMP pathway inhibitor, and an AKT inhibitor. In some embodiments, the method does not comprise a step of administering a growth factor from the TGF-β superfamily (e.g., activin A, GDF8, or GDF11) to the media.

[0246] In some embodiments, the disclosure provides for a composition comprising PDX1- positive cells and ascorbic acid. In some embodiments, the composition comprises 0.01-1 mg / ml ascorbic acid. In some embodiments, the composition comprises more than 0.044 mg / ml ascorbic acid (e.g., 0.045-0.15, 0.045-0.1, 0.045-0.09, 0.045-0.07, 0.045-0.05, 0.06-0.1, 0.06-0.09, 0.07-0.1, 0.07-0.09, 0.08-0.09, 0.08-0.1, 0.08-0.12, or 0.085-0.09 mg / ml. In some embodiments, the disclosure provides for a method of administering ascorbic acid to a composition comprising PDX1-positive cells.

[0247] 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.

[0248] In some embodiments, the PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive pancreatic progenitor cells under conditions that promote cell clustering with KGF, Sant1, and RA, for a period of 5 days or 6 days. In some embodiments, the PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1- positive pancreatic progenitor cells under conditions that promote cell clustering with KGF, Sant1, RA, thiazovivin, and Activin A, for a period of 5 or 6 days. In some embodiments, the PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive pancreatic progenitor cells under conditions that promote cell clustering with KGF for a period of 5 days. In some embodiments, the PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive pancreatic progenitor cells under conditions that promote cell clustering with KGF for a period of 6 days. In some embodiments, the 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 a period of 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 orInsulin-positive Endocrine Cells

[0249] Aspects of the disclosure involve insulin-positive endocrine cells (e.g., β-like cells) and additional methods of generating insulin-positive endocrine cells. Insulin-positive endocrine cells of use herein can be derived from any source or generated in accordance with any suitable protocol. In some aspects, pancreatic foregut precursor cells are differentiated to insulin-positive endocrine cells(e.g., β-like cells), In some aspects, the insulin-positive endocrine cells are further differentiated, e.g., by induction or maturation to SC-β cells.

[0250] In some aspects, a method of producing an insulin-positive endocrine cell from a pancreatic foregut precursor cell comprises contacting a population of cells (e.g., under conditions that promote cell clustering) comprising pancreatic foregut precursor cells 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 the differentiation of at least one pancreatic foregut precursor cell in the population into an insulin-positive endocrine cell, wherein the insulin-positive endocrine ceil expresses insulin. In some embodiments, insulin-positive endocrine cells express PDX1, NKX6.1, ISL1, NKX2.2, Mafb, glis3, Sur1, Kir6.2, Znt8, SLC2A1, SLC2A3 and / or insulin.

[0251] Any TGF-β signaling pathway inhibitor capable of inducing the differentiation of pancreatic foregut precursor cells to differentiate into insulin-positive endocrine cells (e.g., alone, or in combination with other β cell-differentiation factors, e.g., a thyroid hormone signaling pathway activator) can be used. In some embodiments, the TGF-β signaling pathway comprises TGF-β receptor type I kinase signaling. In some embodiments, the TGF-β signaling pathway inhibitor comprises Alk5 inhibitor II. In some examples, the method comprises contacting pancreatic foregut precursor cells with a concentration of a TGF-β signaling pathway inhibitor (e.g., Alk5 inhibitor such as Alk5 inhibitor II), such as, 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 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 18.5µM, 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 comprises contacting pancreatic foregut precursor cells with a concentration of a TGF-β signaling pathway inhibitor (e.g., Alk5 inhibitor such as Alk5 inhibitor II), such as, about 7-13 µM, about 8-12 µM, about 9-11 µM. In some examples, the method comprises contacting pancreatic foregut precursor cells with a concentration of a TGF-β signaling pathway inhibitor (e.g., Alk5 inhibitor such as Alk5 inhibitor II), such as, about 10 µM.

[0252] Any thyroid hormone signaling pathway activator capable of inducing the differentiation of pancreatic foregut precursor cells to differentiate into insulin-positive endocrine cells (e.g., alone, or in combination with other β cell-differentiation factors, e.g., a TGF-β signaling pathway inhibitor) 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 contacting pancreatic foregut precursor cells with a concentration of thyroid hormone signaling pathway activator (e.g., GC-1), such as, about0.1µM, 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 0.21µM, about 0.22µM, about 0.23µM, 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 comprises contacting pancreatic foregut precursor cells with a concentration of thyroid hormone signaling pathway activator (e.g., GC-1), such as, about 0.7-1.3 µM, about 0.8-1.2 µM, or about 0.9-1.1 µM. In some examples, the method comprises contacting pancreatic foregut precursor cells with a concentration of thyroid hormone signaling pathway activator (e.g., GC-1), such as, about 1 µM. In some embodiments, the disclosure provides for a composition comprising a plurality of PDX1-positive cells and a thyroid hormone signaling pathway activator at a concentration of 1.5-5, 1.5-3, 1.5-2.5, 1.8-5, 1.8-3, 1.8-2.2, or 1.9-2.1 µM. In some embodiments, the disclosure provides for a method comprising culturing a plurality of PDX1-positive cells in a media comprising a thyroid receptor activator at a concentration of 1.5-5, 1.5-3, 1.5-2.5, 1.8- 5, 1.8-3, 1.8-2.2, or 1.9-2.1 µM.

[0253] In some embodiments, the method comprises contacting the population of cells (e.g., pancreatic foregut precursor cells) with at least one additional factor. In some embodiments, the method comprises contacting the pancreatic foregut precursor cells with at least one of i) a SHH pathway inhibitor, ii) a γ-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 the population of cells (e.g., pancreatic foregut precursor cells) with at least one additional factor. In some embodiments, the method comprises contacting the pancreatic foregut precursor cells with at least one of i) a SHH pathway inhibitor, ii) a RA signaling pathway activator, iii) a γ-secretase inhibitor, iv) at least one growth factor from the epidermal growth factor (EGF) family, v) a protein kinase inhibitor, vi) a TGF-β signaling pathway inhibitor, vii) a thyroid hormone signaling pathway activator, viii) a wnt signaling pathway inhibitor, or ix) a PKC activator.

[0254] In some embodiments, the method comprises contacting the pancreatic foregut precursor cells with at least one of i) a SHH pathway inhibitor, ii) a RA signaling pathway activator, iii) a γ- secretase inhibitor, iv) at least one growth factor from the epidermal growth factor (EGF) family, v) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, vi) a TGF-β signaling pathway inhibitor, vii) a thyroid hormone signaling pathway activator, viii) a protein kinase inhibitor, or ix) a ROCK inhibitor.

[0255] In some embodiments, the method comprises contacting the pancreatic foregut precursor cells with at least one of i) a SHH pathway inhibitor, ii) a RA signaling pathway activator, iii) a γ- secretase inhibitor, iv) at least one growth factor from the epidermal growth factor (EGF) family, v) atleast 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 comprises contacting the pancreatic foregut precursor cells in a culture with a i) a SHH pathway inhibitor, ii) a RA signaling pathway activator, iii) a γ-secretase inhibitor, iv) at least one growth factor from the epidermal growth factor (EGF) family, v) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, vi) a TGF-β signaling pathway inhibitor, vii) a thyroid hormone signaling pathway activator, viii) an epigenetic modifying compound, ix) a protein kinase inhibitor, x) a ROCK inhibitor, xi) a PKC activator and xii) a Wnt signaling pathway inhibitor for 1, 2, or 3 days (e.g., 1-2, 1-3, or 2-3 days), and then contacting the cells in the culture with i) a γ-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 a period of 1, 2, 3, 4, 5, 6, or 7 days (e.g., 1-7, 1-5, 1-3, 3-7, 3-5, 5-7, or 4-6 days) in the absence of a SHH pathway inhibitor, a RA signaling pathway activator, a Wnt signaling pathway inhibitor, PKC activator, and / or growth factor from the epidermal growth factor (EGF) family.

[0256] In some embodiments, in the method of generating the insulin-positive endocrine cells from the pancreatic foregut precursor 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 the SHH pathway inhibitor, the RA signaling pathway activator, the PKC activator, and the at least one growth factor from the EGF family are removed from the culture medium after the first 1, 2, or 3 days of incubation.

[0257] Any γ-secretase inhibitor that is capable of inducing the differentiation of pancreatic foregut precursor cells in a population into insulin-positive endocrine cells (e.g., alone, or in combination with any of a TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator) can be used. In some embodiments, the γ-secretase inhibitor comprises XXI. In some embodiments, the γ-secretase inhibitor comprises DAPT. In some examples, the method comprises contacting pancreatic foregut precursor cells with a concentration of a γ-secretase inhibitor (e.g., XXI), such as, about 0.01 µM, about 0.02 µM, about 0.05 µM, about 0.075 µM, about 0.1 µM, about 0.2 µM, about 0.3 µM, about 0.4 µM, about 0.5 µM, about 0.6 µM, about 0.7 µM, about 0.8 µM, about 0.9 µM, about 1 µM, about 1.1 µM, about 1.2 µM, about 1.3 µM, about 1.4 µM, about 1.5 µM, about 1.6 µM, about 1.7 µM, about 1.8 µM, about 1.9 µM, about 2 µM, about 2.1 µM, about 2.2 µM, about 2.3 µM, about 2.4 µM, about 2.5 µM, about 2.6 µM, about 2.7 µM, about 2.8 µM, about 2.9 µM, about 3 µM, about 3.2 µM, about 3.4 µM, about 3.6 µM, about 3.8 µM, about 4 µM, about 4.2 µM, about 4.4 µM, about 4.6 µM, about 4.8 µM, about 5 µM, about 5.2 µM, about 5.4 µM, about 5.6 µM, about 5.8 µM, about 6 µM, about 6.2 µM, about 6.4 µM, about 6.6 µM, about 6.8 µM, about7 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 comprises contacting pancreatic foregut precursor cells with a concentration of a γ-secretase inhibitor (e.g., XXI), such as, about 1.7-2.3 µM, about 1.8-2.2 µM, or about 1.9-2.1 µM. In some examples, the method comprises contacting pancreatic foregut precursor cells with a concentration of a γ-secretase inhibitor (e.g., XXI), such as about 2 µM.

[0258] Any growth factor from the EGF family capable of inducing the differentiation of pancreatic foregut precursor cells in a population into insulin-positive endocrine cells (e.g., alone, or in combination with any of a TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator) can be used. In some embodiments, the 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 comprises contacting pancreatic foregut precursor cells with a concentration of a growth factor from EGF family (e.g., betacellulin), such as, 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 comprises contacting pancreatic foregut precursor cells with a concentration of a growth factor from EGF family (e.g., betacellulin), such as, about 17-23 ng / ml, about 18-22 ng / ml, or about 19-21 ng / ml. In some examples, the method comprises contacting NKX6.1-positive pancreatic progenitor cells with a concentration of a growth factor from EGF family (e.g., betacellulin), such as, about 20 ng / ml.

[0259] Any RA signaling pathway activator capable of inducing the differentiation of pancreatic foregut precursor cells to differentiate into insulin-positive endocrine cells (e.g., alone, or in combination with any of a TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator) can be used. In some embodiments, the RA signaling pathway activator comprises RA. In some examples, the method comprises contacting pancreatic foregut precursor cells with a concentration of an RA signaling pathway activator (e.g., retinoic acid), such as, about 0.02 µM, about 0.05 µM, about 0.1µM, about 0.2 µM, about 0.25 µM, about 0.3 µM, about 0.4 µM, about 0.45 µM, about 0.5 µM, about 0.55 µM, about 0.6 µM, about 0.65 µM, about 0.7 µM, about 0.75 µM, about 0.8 µM, about 0.85 µM, about 0.9 µM, about 1 µM, about 1.1 µM, about 1.2 µM, about 1.3 µM, about 1.4 µM, about 1.5 µM, about 1.6 µM, about 1.7 µM, about 1.8 µM, about 1.9 µM, about 2 µM, about 2.1 µM, about 2.2 µM, about 2.3 µM, about 2.4 µM, about 2.5 µM, about 2.6 µM, about 2.7 µM, about 2.8 µM, about 3 µM, about 3.2 µM, about 3.4 µM, about 3.6 µM, about 3.8 µM, about 4 µM, about 4.2 µM, about 4.4 µM, about 4.6 µM, about 4.8 µM, about 5 µM, about 5.5 µM, about 6 µM, about 6.5 µM, about 7 µM, about 7.5 µM, about 8 µM, about 8.5 µM, about 9 µM, about 9.5 µM, about 10 µM, about 12 µM, about 14 µM, about 15 µM, about 16 µM, about 18 µM, about 20 µM, about 50 µM, or about 100 µM. In some examples, the method comprises contacting pancreaticforegut precursor cells with a concentration of an RA signaling pathway activator (e.g., retinoic acid), such as, about 20-80 nM, about 30-70 nM, or about 40-60 nM. In some examples, the method comprises contacting pancreatic foregut precursor cells with a concentration of an RA signaling pathway activator (e.g., retinoic acid), such as, about 50 nM.

[0260] Any SHH pathway inhibitor capable of inducing the differentiation of pancreatic foregut precursor cells to differentiate into insulin-positive endocrine cells (e.g., alone, or in combination with any of a TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator) can be used in the method provided herein. In some embodiments, the SHH pathway inhibitor comprises Sant1. In some examples, the method comprises contacting pancreatic foregut precursor cells with a concentration of a SHH pathway inhibitor (e.g., Sant1), such as, about 0.001 µM, about 0.002 µM, about 0.005 µM, about 0.01 µM, about 0.02 µM, about 0.03µM, about 0.05µM, about 0.08 µM, about 0.1µM, 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 0.21µM, about 0.22µM, about 0.23µM, 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 comprises contacting pancreatic foregut precursor cells with a concentration of a SHH pathway inhibitor (e.g., Sant1), such as, about 220-280 nM, about 230-270 nM, about 240-260 nM, or about 245-255 nM. In some examples, the method comprises contacting pancreatic foregut precursor cells with a concentration of a SHH pathway inhibitor (e.g., Sant1), such as, about 250 nM.

[0261] Any BMP signaling pathway inhibitor capable of inducing the differentiation of pancreatic foregut precursor cells to differentiate into insulin-positive endocrine cells (e.g., alone, or in combination with any of a TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator) can be used. In some embodiments, the BMP signaling pathway inhibitor comprises LDN193189 or DMH-1. In some examples, the method comprises contacting pancreatic foregut precursor cells with a concentration of BMP signaling pathway inhibitor (e.g., LDN1931189), such as, 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 1µM. In some examples, the method comprises contacting pancreatic foregut precursor cells with a concentration of BMP signaling pathway inhibitor (e.g., LDN1931189), such as, about 70-130 nM, about 80-120 nM, about 90-110 nM. In some examples, the method comprises contacting pancreatic foregut precursor cells with a concentration of BMP signaling pathway inhibitor (e.g., LDN1931189), such as, about 100 nM.

[0262] Any ROCK inhibitor that is capable of inducing the differentiation of pancreatic foregut precursor cells in a population into insulin-positive endocrine cells (e.g., alone, or in combination with any of a TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator) can be used. In some 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 contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a concentration of a ROCK inhibitor (e.g., Y-27632 or Thiazovivin), such as, about 0.2 µM, about 0.5 µM, about 0.75 µM, about 1 µM, about 2 µM, about 3 µM, about 4 µM, about 5 µM, about 6 µM, about 7 µM, about 7.5 µM, about 8 µM, about 9 µM, about 10 µM, about 11 µM, about 12 µM, about 13 µM, about 14 µM, about 15 µM, about 16 µM, about 17 µM, about 18 µM, about 19 µM, about 20 µM, about 21 µM, about 22 µM, about 23 µM, about 24 µM, about 25 µM, about 26 µM, about 27 µM, about 28 µM, about 29 µM, about 30 µM, about 35 µM, about 40 µM, about 50 µM, or about 100 µM. In some embodiments, the ROCK inhibitor comprises Thiazovivin. In some examples, the method comprises contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a concentration of a ROCK inhibitor (e.g., Y-27632 or Thiazovivin), such as, about 2.2-2.8 µM, about 2.3-2.7 µM, or about 2.4-2.6 µM. In some embodiments, the ROCK inhibitor comprises Thiazovivin. In some examples, the method comprises contacting pancreatic foregut precursor cells with a concentration of a ROCK inhibitor (e.g., Y-27632 or Thiazovivin), such as, about 2.5 µM.

[0263] Any epigenetic modifying compound that is capable of inducing the differentiation of pancreatic foregut precursor cells in a population into insulin-positive endocrine cells (e.g., alone, or in combination with any of a TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator) can be used. In some embodiments, the epigenetic modifying compound comprises a histone methyltransferase inhibitor or a HDAC inhibitor. In some embodiments, the epigenetic modifying compound comprises a histone methyltransferase inhibitor, e.g., DZNep. In some embodiments, the epigenetic modifying compound comprises a HDAC inhibitor, e.g., KD5170. In some examples, the method comprises contacting pancreatic foregut precursor cells with a concentration of an epigenetic modifying compound (e.g., DZNep or KD5170), such as, about 0.01 µM, about 0.025 µM, about 0.05 µM, about 0.075 µM, about 0.1 µM, about 0.15 µM, about 0.2 µM, about 0.5 µM, about 0.75 µM, about 1 µM, about 2 µM, about 3 µM, about 4 µM, about 5 µM, about 6 µM, about 7 µM, about 7.5 µM, about 8 µM, about 9 µM, about 10 µM, about 15 µM, about 20 µM, about 25 µM, about 30 µM, about 35 µM, about 40 µM, about 50 µM, or about 100 µM. In some examples, the method comprises contacting pancreatic foregut precursor cells with a concentration of an epigenetic modifying compound (e.g., DZNep or KD5170), such as, about 70-130 nM, about 80- 120 nM, or about 90-110 nM. In some examples, the method comprises contacting pancreatic foregut precursor cells with a concentration of an epigenetic modifying compound (e.g., DZNep or KD5170), such as, about 100 nM.

[0264] Any Wnt signaling pathway inhibitor that is capable of inducing the differentiation of pancreatic foregut precursor cells in a population into insulin-positive endocrine cells (e.g., alone, or in combination with any of a TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator) can be used. In some embodiments, the Wnt signaling pathway inhibitor comprises a tankyrase inhibitor. In some embodiments, the tankyrase inhibitor is NVP-TNKS656. In some examples, the method comprises contacting pancreatic foregut precursor cells with a concentration of a Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor such as NVP- TNKS656), such as, about 0.1µM, about 0.15 µM, about 0.2 µM, about 0.25 µM, about 0.3 µM, about 0.35 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 0.95 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, or about 5 µM. In some examples, the method comprises contacting pancreatic foregut precursor cells with a concentration of a Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor such as NVP-TNKS656), such as, about 1.7-2.3 µM, about 1.8-2.2 µM, or about 1.9-2.1 µM. In some examples, the method comprises contacting pancreatic foregut precursor cells with a concentration of a Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor such as NVP-TNKS656), such as, about 2 µM.

[0265] Any PKC activator that is capable of inducing the differentiation of pancreatic foregut precursor cells in a population into insulin-positive endocrine cells (e.g., alone, or in combination with any of a TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator) can be used. In some embodiments, the PKC activator is TPB or PDBU. In some examples, the method comprises contacting pancreatic foregut precursor cells with a concentration of a PKC activator (TPB or PDBU), such as, about 0.01 µM, about 0.025 µM, about 0.05 µM, about 0.075 µM, about 0.1 µM, about 0.15 µM, about 0.2 µM, about 0.25 µM, about 0.3 µM, about 0.35 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 0.95 µM, about 1 µM, about 2 µM, about 3 µM, about 4 µM, about 5 µM, about 6 µM, about 7 µM, about 7.5 µM, about 8 µM, about 9 µM, about 10 µM, about 15 µM, or about 20 µM. In some examples, the method comprises contacting pancreatic foregut precursor cells with a concentration of a PKC activator (TPB or PDBU), such as, about 450-550 mM, about 475-525 nM, about 490-510 nM, or about 495-505 nM. In some examples, the method comprises contacting pancreatic foregut precursor cells with a concentration of a PKC activator (TPB or PDBU), such as, about 500 nM.

[0266] In some embodiments, the population of cells is optionally contacted with a protein kinase inhibitor. In some embodiments, the population of cells is not contacted with the protein kinase inhibitor. In some embodiments, the population of cells is contacted with the protein kinase inhibitor. Any protein kinase inhibitor that is capable of inducing the differentiation of pancreatic foregutprecursor cells in a population into insulin-positive endocrine cells (e.g., alone, or in combination with any of a TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator). In some embodiments, the protein kinase inhibitor comprises staurosporine. In some examples, the method comprises contacting pancreatic foregut precursor cells with a concentration of a protein kinase inhibitor (e.g., staurosporine), such as, about 0.1 nM, about 0.2 nM, about 0.3 nM, about 0.4 nM, about 0.5 nM, about 0.6 nM, about 0.7 nM, about 0.8 nM, about 0.9 nM, about 1 nM, about 1.1 nM, about 1.2 nM, about 1.3 nM, about 1.4 nM, about 1.5 nM, about 1.6 nM, about 1.7 nM, about 1.8 nM, about 1.9 nM, about 2.0 nM, about 2.1 nM, about 2.2 nM, about 2.3 nM, about 2.4 nM, about 2.5 nM, about 2.6 nM, about 2.7 nM, about 2.8 µM, about 2.9 nM, about 3 nM, about 3.1 nM, about 3.2 nM, about 3.3 nM, about 3.4 nM, about 3.5 nM, about 3.6 nM, about 3.7 nM, about 3.8 nM, about 3.9 nM, about 4.0 nM, about 4.1 nM, about 4.2 nM, about 4.3 nM, about 4.4 nM, about 4.5 nM, about 4.6 nM, about 4.7 nM, about 4.8 µM, about 4.9 nM, or about 5 nM. In some examples, the method comprises contacting pancreatic foregut precursor cells with a concentration of a protein kinase inhibitor (e.g., staurosporine), such as, about 1-5 nM, about 2-4 nM, or about 2.5-3.5 nM. In some examples, the method comprises contacting pancreatic foregut precursor cells with a concentration of a protein kinase inhibitor (e.g., staurosporine), such as, about 3 nM.

[0267] 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 89% hydrolyzed.

[0268] In some embodiments, the method comprises contacting the population of cells (e.g., pancreatic foregut precursor cells) with XXI, Alk5i, T3 or GC-1, RA, Sant1, and betacellulin, PDBU, and NVP-TNKS656 for a period of 7 days, to induce the 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 method comprises contacting the population of cells (e.g., pancreatic foregut precursor cells) with XXI, Alk5i, T3 or GC-1, RA, Sant1, betacellulin, and LDN193189 for a period of 7 days, to induce the differentiation of at least one pancreatic foregut precursor cell in the population into an insulin-positive endocrine cell, wherein the insulin-positive endocrine cell expresses insulin. In some embodiments, one or more differentiation factors are added in a portion of the Stage 5, for instance, only the first 1, 2, 3, 4, 5, or 6 days of the period of time for Stage 5, or the last 1, 2, 3, 4, 5, or 6 days of the period of time for Stage 5. In one example, the cells are contacted with SHH signaling pathway inhibitor the PKC activator, the retinoic acid, and / or the wnt signaling pathway inhibitor for only the first 2, 3, 4, or 5 days during Stage 5, after which the SHH signaling pathway inhibitor, the PKC activator, the retinoic acid, and / or the wnt signaling pathway inhibitor are not included in or removed from the culturemedium. In another example, the cells are contacted with BMP signaling pathway inhibitor for only the first 1, 2, or 3 days during Stage 5, after which the BMP signaling pathway inhibitor is removed from the culture medium.

[0269] In some embodiments, the method comprises contacting the population of cells (e.g., pancreatic foregut precursor cells) with one or more metabolites. In some embodiments, the method comprises contacting the population of cells (e.g., pancreatic foregut precursor cells) with one or more of an acetyl CoA-related metabolite, a vitamin, histone deacetylase inhibitor (HDACi), a redox homeostasis regulator, a one

[0270] carbon metabolism pathway intermediate, and / or glutamine. Examples of metabolites include glutamine, taurine, acetate, beta-hydroxybutyrate, biotin, and formate.

[0271] In some embodiments, a composition (e.g., medium) of the disclosure comprises an acetyl CoA-related metabolite. Exemplary acetyl CoA-related metabolites include, but are not limited to acetate, pyruvate, ketogenic amino acids, valine, leucine, isoleucine, phenylalanine, tyrosine, lysine, tryptophan, fatty acids, CoA, Isovaleryl-CoA, and β-hydroxybutyrate. In some embodiments, the acetyl CoA-related metabolite is acetate. In some embodiments, the acetyl CoA-related metabolite is present in or is added to a composition of the disclosure at a concentration of about 10 nM, about 50 nM, about 80 nM, about 100 nM, about 120 nM, about 140 nM, about 150 nM, about 200 nM, about 300 nM, about 500 nM, about 800 nM, about 1 μM, about 10 μM, about 100 μM, about 500 μM, about 800 μM, about 900 μM, about 1 mM, about 2 mM, about 3 mM, about 5 mM, or about 10 mM. In some embodiments, the acetyl CoA-related metabolite is present in or is added to a composition of the disclosure at a concentration of about 0.01-50 mM, 0.1-50 mM, 0.5-50 mM, 0.01-20 mM, 0.1-20 mM, 0.5-20 mM, 0.01-10 mM, 0.1-10 mM, 0.5-10 mM, 0.8-25 mM, 0.8-10 mM, 0.8-5 mM, 0.8-2 mM, 0.8-1.5 mM, 0.8-1.2 mM, 0.9-1.1 mM, or 0.95-1.05 mM. In some embodiments, the acetyl CoA- related metabolite is acetate present at a concentration of about 1 mM. In some embodiments, the acetyl CoA-related metabolite is acetate present at a concentration of about 50-1000 nM, 50-800 nM, 50-500 nM, 50-300 nM, 50-250 nM, 100-200 nM, or 125-175 nM. In some embodiments, the acetyl CoA-related metabolite is acetate present at a concentration of about 160 nM.

[0272] In some embodiments, a composition (e.g., medium) of the disclosure comprises one or more vitamins. Exemplary vitamins include, but are not limited to vitamin C, biotin, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B6 (pyridoxine) and vitamin B12 (cyanocobalamin). In some embodiments the vitamin modulates fatty acid synthesis. In some embodiments the vitamin modulates branched-chain amino acid metabolism. In some embodiments the vitamin modulates or participates as a co-factor in the TCA cycle, e.g., as a cofactor for pyruvate carboxylase. In some embodiments, the vitamin is biotin. In some embodiments, the vitamin is present in or is added to a composition of the disclosure at a concentration of about 100 nM, about 300 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM, about 1 μM,about 1.5 μM, about 3 μM, about 5 μM, about 10 μM, or about 100 μM. In some embodiments, the vitamin is biotin present at a concentration of about 800 nM. In some embodiments, the vitamin is present in or is added to a composition of the disclosure at a concentration of about 1 nM to 500 μM, 1 nM to 100 μM, 1 nM to 10 μM, 1 nM to 1 μM, 1 nM to 800 nM, 1 nM to 600 nM, 1 nM to 400 nM, 1 nM to 300 nM, 1 nM to 200 nM, 25 nM to 500 μM, 25 nM to 100 μM, 25 nM to 10 μM, 25 nM to 1 μM, 25 nM to 800 nM, 25

[0273] nM to 600 nM, 25 nM to 400 nM, 25 nM to 300 nM, 25 nM to 200 nM, 50 nM to 500 μM, 50 nM to 100 μM, 50 nM to 10 μM, 50 nM to 1 μM, 50 nM to 800 nM, 50 nM to 600 nM, 50 nM to 400 nM, 50 nM to 300 nM, 50 nM to 200 nM, 100 nM to 500 μM, 100 nM to 100 μM, 100 nM to 10 μM, 100 nM to 1 μM, 100 nM to 800 nM, 100 nM to 600 nM, 100 nM to 400 nM, 100 nM to 300 nM, or 100 nM to 200 nM.

[0274] In some embodiments, the disclosure provides for a composition comprising a plurality of pancreatic foregut precursor cells and ascorbic acid. In some embodiments, the composition comprises less than 0.088 mg / ml ascorbic acid. In some embodiments, the composition comprises 0.01-0.087, 0.01-0.05, 0.03-0.087, 0.03-0.08, 0.03-0.07, 0.03-0.06, 0.03-0.05, 0.035-0.06, 0.035- 0.045, 0.035-0.045, 0.04-0.06, 0.04-0.05, or 0.043-0.046 mg / ml ascorbic acid. In some embodiments, the disclosure provides for a method for culturing a population of cells (e.g., PDX-1 positive cells) in vitro, comprising the steps of: a) culturing the population of cells in a media comprising more than 0.044 mg / ml ascorbic acid (e.g., 0.045-0.15, 0.045-0.1, 0.045-0.09, 0.045-0.07, 0.045-0.05, 0.06-0.1, 0.06-0.09, 0.07-0.1, 0.07-0.09, 0.08-0.09, 0.08-0.1, 0.08-0.12, or 0.085-0.09 mg / ml), and

[0275] b) culturing the population of cells in a media comprises less than 0.088 mg / ml ascorbic acid (e.g., 0.01-0.087, 0.01-0.05, 0.03-0.087, 0.03-0.08, 0.03-0.07, 0.03-0.06, 0.03-0.05, 0.035-0.06, 0.035-0.045, 0.035-0.045, 0.04-0.06, 0.04-0.05, or 0.043-0.046 mg / ml ascorbic acid), wherein the population of cells comprises PDX1-positive cells. In some embodiments, the disclosure provides for a method for culturing a population of cells (e.g., PDX-1 positive cells) in vitro, comprising the steps of: a) culturing the population of cells in a media comprising 0.08-0.12 mg / ml ascorbic acid, and b) culturing the population of cells in a media comprising 0.03-0.06 mg / ml ascorbic acid. In some embodiments, step a) comprises culturing the population of cells in a media comprising more than 0.044 mg / ml ascorbic acid (e.g., 0.045-0.15, 0.045-0.1, 0.045-0.09, 0.045-0.07, 0.045-0.05, 0.06-0.1, 0.06-0.09, 0.07-0.1, 0.07-0.09, 0.08-0.09, 0.08-0.1, 0.08-0.12, or 0.085-0.09 mg / ml) for 12-24 hours, 1-4 days, 1-2 days, 2-3 days, or 3-4 days, or at least 12 hours, 1 day, 2 days, 3 days, or 4 days. In some embodiments, step b) comprises culturing the population of cells in a media comprising less than 0.088 mg / ml ascorbic acid (e.g., 0.01-0.087, 0.01-0.05, 0.03-0.087, 0.03-0.08, 0.03-0.07, 0.03- 0.06, 0.03-0.05, 0.035-0.06, 0.035-0.045, 0.035-0.045, 0.04-0.06, 0.04-0.05, or 0.043-0.046 mg / ml ascorbic acid) for 12-24 hours, 1-4 days, 1-14 days, 1-7 days, 3-5 days, 1-2 days, 2-3 days, 3-4 days,4-5 days, 6-7 days, or at least 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days.

[0276] In some embodiments, a composition (e.g., medium) of the disclosure comprises a histone deacetylase inhibitor (HDACi). Exemplary histone deacetylase inhibitors (HDACi) include, but are not limited to β-Hydroxybutyrate, butyric acid, class I HDACi, class IIA HDACi, class IIB HDACi, class III HDACi, class IV HDACi, HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-6, HDAC-7, HDAC-8, HDAC-9, HDAC-10, HDAC-11, sirtuins, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, Vorinostat (suberoylanilide hydroxamic acid, SAHA, MK0683), Entinostat (MS-275, SNDX-275), Panobinostat (LBH589, NVP-LBH589), Trichostatin A (TSA), Mocetinostat (MGCD0103, MG0103), GSK3117391 (GSK3117391A, HDAC-IN-3), BRD3308, BRD3308, Tubastatin A TFA (Tubastatin A trifluoroacetate salt), Tubastatin A, SIS17, NKL 22, BML-210 (CAY10433), TC-H 106, SR-4370, Belinostat (PXD101, NSC726630, PX-105684), Romidepsin (FK228, Depsipeptide, FR 901228, NSC 630176), MC1568, Givinostat (ITF2357), Dacinostat (LAQ824, NVP-LAQ824), CUDC-101, Quisinostat (JNJ-26481585), Pracinostat (SB939), PCI- 34051, Droxinostat (NS 41080), Abexinostat (PCI- 24781), Abexinostat (PCI-24781, CRA-024781), RGFP966, AR-42 (HDAC-42), Ricolinostat (ACY-1215, Rocilinostat), Valproic acid sodium salt (Sodium valproate), Tacedinaline (CI994, PD-123654, GOE-5549, Acetyldinaline), Fimepinostat (CUDC-907), Sodium butyrate (NaB), Curcumin, Diferuloylmethane, M344, Tubacin, RG2833 (RGFP109), RG2833 (RGFP109), Resminostat (RAS2410), Divalproex Sodium, Scriptaid (GCK 1026), Sodium Phenylbutyrate, Sinapinic acid (Sinapic acid), TMP269, Santacruzamate A (CAY10683), TMP195 (TFMO 2), Valproic acid (VPA), UF010, Tasquinimod (ABR-215050), SKLB-23bb, Isoguanosine, Sulforaphane, BRD73954, Citarinostat (ACY-241, HDAC-IN-2), Suberohydroxamic acid, Splitomicin, HPOB, LMK-235, Biphenyl-4-sulfonyl chloride (p- Phenylbenzenesulfonyl, 4- Phenylbenzenesulfonyl, p-Biphenylsulfonyl), Nexturastat A, TH34, Tucidinostat (Chidamide, HBI-8000, CS-055), (-)-Parthenolide, WT161, CAY10603, CAY10603, ACY-738, Raddeanin A, Tinostamustine(EDO-S101), Domatinostat (4SC-202), and BG45. In some embodiments, the HDACi is β-Hydroxybutyrate. In some embodiments, the HDACi is present in or is added to a composition of the disclosure at a concentration of about 100 nM, about 300 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM, about 1 μM, about 1.5 μM, about 3 μM, about 5 μM, about 10 μM, or about 100 μM. In some embodiments, the HDACi is β- Hydroxybutyrate present at a concentration of about 200 nM. In some embodiments, the HDACi is present in or is added to a composition of the disclosure at a concentration of about 1 nM to 500 μM, 1 nM to 100 μM, 1 nM to 10 μM, 1 nM to 1 μM, 1 nM to 800 nM, 1 nM to 600 nM, 1 nM to 400 nM, 1 nM to 300 nM, 1 nM to 200 nM, 5 nM to 500 μM, 25 nM to 100 μM, 25 nM to 10 μM, 25 nM to 1 μM, 25 nM to 800 nM, 25 nM to 600 nM, 25 nM to 400 nM, 25 nM to 300 nM, 25 nM to 200 nM, 50 nM to 500 μM, 50 nM to 100 μM, 50 nM to 10 μM, 50 nM to 1 μM, 50 nM to 800 nM, 50 nM to 600 nM, 50 nM to 400 nM, 50 nM to 300 nM, 50 nM to 200 nM, 100 nM to 500 μM, 100 nM to 100 μM,100 nM to 10 μM, 100 nM to 1 μM, 100 nM to 800 nM, 100 nM to 600 nM, 100 nM to 400 nM, 100 nM to 300 nM, or 100 nM to 200 nM.

[0277] In some embodiments, a composition (e.g., medium) of the disclosure comprises a redox homeostasis regulator. Exemplary redox homeostasis regulators include, but are not limited to taurine, respiratory chain regulators, free radical scavengers, regulators of mitochondrial protein synthesis, allium sulphur compounds, anthocyanins, beta-carotene, catechins, copper, cryptoxanthins, flavonoids, indoles, isoflavonoids, lignans, lutein, lycopene, alpha lipoic acid, ellagic acid, manganese, polyphenols, selenium, glutathione, vitamin A, vitamin C, vitamin E, zinc, superoxide disutases, GSHPx, Prx-I, catalase, and co-enzyme Q10. In some embodiments, the redox homeostasis regulator is taurine. In some embodiments, the redox homeostasis regulator is present in or is added to a composition of the disclosure at a concentration of about 100 nM, about 500 nM, 1 μM, about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, about 100 μM, about 110 μM, about 110 μM, about 150 μM, or about 200 μM. In some embodiments, the redox homeostasis regulator is taurine. In some embodiments, the redox homeostasis regulator is taurine present at a concentration of about 90 μM. In some embodiments, the redox homeostasis regulator intermediate is present or is added at a concentration of about 100 nM to 1 mM, 500 nM to 1 mM, 1 μM to 1 mM, 10 μM to 1 mM, 20 μM to 1 mM, 30 μM to 1 mM, 30 μM to 1 mM, 40 μM to 1 mM, 50 μM to 1 mM, 60 μM to 1 mM, 70 μM to 1 mM, 80 μM to 1 mM, 100 nM to 250 μM, 500 nM to 250 μM, 1 μM to 250 μM, 10 μM to 250 μM, 20 μM to 250 μM, 30 μM to 250 μM, 30 μM to 250 μM, 40 μM to 250 μM, 50 μM to 250 μM, 60 μM to 250 μM, 70 μM to 250 μM, 100 nM to 100 μM, 500 nM to 100 μM, 1 μM to 100 μM, 10 μM to 100 μM, 20 μM to 100 μM, 30 μM to 100 μM, 40 μM to 100 μM, 50 μM to 100 μM, 60 μM to 100 μM, 70 μM to 100 μM, or 80 μM to 100 μM.

[0278] In some embodiments, a composition (e.g., medium) of the disclosure comprises a one carbon metabolism pathway intermediate. Exemplary one carbon metabolism pathway intermediates include, but are not limited to formate, tetrahydrofolate (THF), 10-formylTHF; 5,10-meTHF; 5,10- meTHF; and 10-formylTHF. In some embodiments, the one carbon metabolism pathway intermediate is formate present at a concentration of about 50 μM. In some embodiments, the one carbon metabolism pathway intermediate is present or is added at a concentration of about 100 nM to 1 mM, 500 nM to 1 mM, 1 μM to 1 mM, 10 μM to 1 mM, 20 μM to 1 mM, 30 μM to 1 mM, 100 nM to 250 μM, 500 nM to 250 μM, 1 μM to 250 μM, 10 μM to 250 μM, 20 μM to 250 μM, 30 μM to 250 μM, 100 nM to 100 μM, 500 nM to 100 μM, 1 μM to 100 μM, 10 μM to 100 μM, 20 μM to 100 μM, 30 μM to 100 μM, 100 nM to 60 μM, 500 nM to 60 μM, 1 μM to 60 μM, 10 μM to 60 μM, 20 μM to 60 μM, 30 μM to 60 μM, 40 μM to 60 μM, or 45 μM to 55 μM.

[0279] In some embodiments, a composition (e.g., medium) of the disclosure comprises

[0280] glutamine. Thus in some embodiments, compositions and methods of the disclosure utilize glutamine in a form with increased bioavailability, such as a free glutamine form, such as a non-dipeptide form, a non-alanine-glutamine dipeptide form (e.g., a non-alanyl-l-glutamine form), a non-glycine-glutamine dipeptide form (e.g., a non-glycyl-l-glutamine form), a form that in which glutamine is not conjugated to another amino acid or stabilizing moiety, a monomeric form, a free form, or a combination thereof. In some embodiments, glutamine is provided as a protein hydrolysate. In some embodiments, glutamine is present or is added to a composition of the disclosure at a concentration of from 0.5-20 mM, 0.5-10 mM, 0.5-5 mM, 1-5 mM, 2-5 mM, or 1 mM to 10 mM. In some embodiments, glutamine is present or is added to a composition of the disclosure at a concentration of 3.8-4.2 mM. In some embodiments, glutamine is present or is added to a composition of the disclosure at a concentration of 1-10, 1-7, 1-8, 1-6, 1-5, 1-4, 2-10, 2-7, 2-8, 2-6, 2-5, 2-4, 3-10, 3-7, 3-8, 3-6, 3-5, 3-4, 3.5-4.5, 3.8-4.2, or 3.9-4.1 mM. In some

[0281] embodiments, glutamine is present or is added to a composition of the disclosure at a concentration of about 4 mM. In some embodiments, at least 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM of the glutamine is not in a dipeptide form. In some embodiments, at least 500 μM, at least 750 μM, at least 1 mM, at least 1.5 mM, at least 2 mM, at least 2.5 mM, at least 2.6 mM, at least 2.7 mM, at least 2.8 mM, at least 2.9 mM, at least 3 mM, at least 3.1 mM, at least 3.2 mM, at least 3.3 mM, at least 3.4 mM, at least 3.5 mM, at least 3.6 mM, at least 3.7 mM, at least 3.8 mM, at least 3.9 mM, at least 4 mM, at least 5 mM, at least 5.5 mM, at least 6 mM, at least 6.5 mM, at least 7 mM, at least 7.5 mM, at least 8 mM, at least 8.5 mM, at least 9 mM, at least 9.5 mM, or at least 10 mM of the glutamine is in a free form.

[0282] In some embodiments, the method comprises culturing the population of cells (e.g., pancreatic foregut precursor cells) in a medium, to induce the differentiation of at least one pancreatic foregut precursor cell in the population into an insulin-positive endocrine cell, wherein the insulin- positive endocrine cell expresses insulin.

[0283] Aspects of the disclosure involve treatment of cell population comprising pancreatic foregut precursor cells with PKC activator and / or wnt signaling pathway inhibitor, which can lead to increase in percentage of pancreatic α cells, increase in percentage of pancreatic δ cells, increase in percentage of pancreatic β cells, reduction in percentage of EC cells, or any combination thereof, in the cell population of pancreatic endocrine cells generated according to the method disclosed herein.

[0284] In some embodiments, the method comprises contacting a population of cells comprising pancreatic foregut precursor cells with a first composition comprising a FOXO1 inhibitor, notch signaling inhibitor, a PKC activator, a ROCK inhibitor, a growth factor from TGFβ superfamily, a growth factor from FGF family, a RA signaling pathway activator, and a SHH pathway inhibitor, for one to two days, thereby obtaining a first transformation cell population comprising pancreatic foregutprecursor cells; and contacting the first transformation cell population comprising pancreatic foregut precursor cells with a second composition comprising the PKC activator, notch signaling inhibitor, a TGF-β signaling pathway inhibitor, a TH signaling pathway activator, BMP pathway inhibitor, ROCK inhibitor, retinoic acid, and EGF-family growth factor, wnt signaling pathway inhibitor, and / or an epigenetic modifying compound, for one to two days, thereby obtaining a second transformation cell population comprising pancreatic foregut precursor cells.

[0285] In some embodiments, the disclosure provides for a method for culturing a population of cells in vitro, comprising the step of culturing the population of cells in a media comprising a growth factor from the fibroblast growth factor (FGF) family and a retinoic acid signaling pathway activator; wherein the population of cells comprises a plurality of PDX1-positive; NKX6.1-negative cells; and wherein the cells are cultured in the media for a period of time, wherein the period of time is at least 12 hours but not more than 4 days. In some embodiments, the disclosure provides for a method for culturing a population of cells in vitro, comprising the step of culturing the population of cells in a media comprising a sonic hedgehog pathway inhibitor and a retinoic acid signaling pathway activator; wherein the population of cells comprises a plurality of PDX1-positive; NKX6.1-negative cells; and wherein the cells are cultured in the media for a period of time, wherein the period of time is at least 12 hours but not more than 4 days. In some embodiments, the disclosure provides for a method for culturing a population of cells in vitro, comprising the step of culturing the population of cells in a media comprising a growth factor from the TGF-β superfamily and a retinoic acid signaling pathway activator; wherein the population of cells comprises a plurality of PDX1-positive; NKX6.1-negative cells; and wherein the cells are cultured in the media for a period of time, wherein the period of time is at least 12 hours but not more than 4 days. In some embodiments, the period of time is 12 hours to 1 day, 12 hours to 2 days, 12 hours to 3 days, 12 hours to 4 days, 1-4 days, 1-3 days, 1-2 days, 2-4 days or 3-4 days. In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or between 30-100%, 50-100%, 70-100%, 85-100%, 80-95%, 85-95%, or 90-95% of the cells in the population are PDX1-positive; NKX6.1-negative cells. In some embodiments, at least 50% of the cells in the population are PDX1-positive; NKX6.1-negative cells. In some embodiments, at least 80% of the cells in the population are PDX1-positive; NKX6.1-negative cells. In some embodiments, between 70-100% of the cells in the population are PDX1-positive; NKX6.1-negative cells. In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or between 30-100%, 50-100%, 70- 100%, 85-100%, 80-95%, 85-95%, or 90-95% of the cells in the population are PDX1-positive; NKX6.1-positive cells. In some embodiments, at least 50% of the cells in the population are PDX1- positive; NKX6.1-positive cells. In some embodiments, at least 80% of the cells in the population are PDX1-positive; NKX6.1-positive cells. In some embodiments, between 70-100% of the cells in the population are PDX1-positive; NKX6.1-positive cells. In some embodiments, the cells are cultured in the media for at least 12 hours but not more than 3 days. In some embodiments, the cells are cultured in the media for at least 12 hours but not more than 2 days. In some embodiments, the cells arecultured in the media for at least 12 hours but not more than 1 day. In some embodiments, the cells are cultured in the media for 1 day, 2 days, 3 days, 4 days, 1-4 days, 1-3 days, 1-2 days, 2-4 days, or 2-3 days. In some embodiments, the media comprises a sonic hedgehog pathway inhibitor. In some embodiments, the media comprises a growth factor from the fibroblast growth factor (FGF) family. In some embodiments, the media comprises a growth factor from the TGF-β superfamily. In some embodiments, the growth factor from the FGF family is any one of KGF, FGF2, FGF8B, FGF10, or FGF21. In some embodiments, the retinoic acid signaling pathway activator is any one of retinoic acid, CD1530, AM580, TTNPB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, and CD2314. In some embodiments, the sonic hedgehog pathway inhibitor selected from the group consisting of SANT1, SANT2, SANT4, Cur61414, forskolin, tomatidine, AY9944, triparanol, and cyclopamine. In some embodiments, the growth factor from the TGF-β superfamily is any one of activin A, GDF8, or GDF11. In some embodiments, the plurality of agents further comprises a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor. In some embodiments, the ROCK inhibitor is any one of Thiazovivin, Y- 27632, Fasudil / HA1077, or 14-1152. In some embodiments, the media further comprises a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer comprises polyvinyl alcohol, poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), PEG copolymers, poly(N-isopropylacrylamide), or polyacrylamide. In some embodiments, the water-soluble synthetic polymer comprises polyvinyl alcohol. In some embodiments, the water-soluble synthetic polymer is present at a concentration of about 0.04% to about 0.06% (w / v) in the media. In some embodiments, the water-soluble synthetic polymer comprises polyvinyl alcohol that is about 80% hydrolyzed. In some embodiments, the media does not comprise a protein kinase C activator. In some embodiments, the media does not comprise a bone morphogenetic (BMP) signaling pathway inhibitor. In some embodiments, the media does not comprise a FOXO1 inhibitor. In some embodiments, the media does not comprise a notch signaling pathway inhibitor. In some embodiments, the media does not comprise an epidermal growth factor. In some embodiments, the media does not comprise a thyroid hormone. In some embodiments, the media does not comprise a TGFβ-R1 kinase inhibitor. In some embodiments, the media does not comprise a histone methyltransferase EZH2 inhibitor. In some embodiments, the media does not comprise a Wnt signaling pathway inhibitor. In some embodiments, following the period of time, the cells are cultured in a media comprising one or more of an epidermal growth factor, a thyroid hormone, a TGFβ-R1 kinase inhibitor, a notch signaling pathway inhibitor, a sonic hedgehog (SHH) signaling pathway inhibitor, a retinoic acid signaling pathway activator, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, a protein kinase inhibitor, a bone morphogenetic (BMP) signaling pathway inhibitor, a histone methyltransferase EZH2 inhibitor, and / or a Wnt signaling pathway inhibitor. In some embodiments, prior to the period of time, the population of cells were cultured in a media comprising a protein kinase C activator. In some embodiments, the media is in a sterile container. In some embodiments, the sterile container is a bioreactor.Pancreatic β Cells

[0286] Aspects of the disclosure involve generating pancreatic β cells (e.g., non-native pancreatic β cells / SC-β cells) and additional methods of generating them. Non-native pancreatic β cells. In some embodiments, resemble endogenous mature β cells in form and function, but nevertheless are distinct from native β cells.

[0287] In some embodiments, the insulin-positive pancreatic endocrine cells generated using the method provided herein can form a cell cluster, alone or together with other types of cells, e.g., precursors thereof, e.g., stem cell, definitive endoderm cells, primitive gut tube cell, PDX1-positive pancreatic progenitor cells, or pancreatic foregut precursor cells.

[0288] In some embodiments, any of the cells or populations of cells disclosed herein are in a cell cluster. In some aspects, provided herein are cell clusters that resemble the functions and characteristics of endogenous pancreatic islets. Such cell clusters can mimic the function of endogenous pancreatic islets in regulating metabolism, e.g., glucose metabolism in a subject.

[0289] In some embodiments, a composition or cell population of the present disclosure comprises SC-β cells cells that express lower levels of MAFA than SC-beta cells from the pancreas of a healthy control adult subject. In some embodiments, the composition or cell population comprises SC-β cells cells that express higher levels of MAFB than SC-β cells cells from the pancreas of a healthy control adult subject. In some embodiments, the composition or cell population comprises SC-β cells cells that express higher levels of SIX2, HOPX, IAPP and / or UCN3 than SC-β cells cells from the pancreas of a healthy control adult subject.

[0290] In some embodiments, a composition or cell population of the present disclosure comprises SC-β cells that do not express MAFA. In some embodiments, the composition or cell population comprises NKX6.1-positive, ISL-positive cells that express MAFB.

[0291] In some embodiments, the cell population comprising the insulin-positive endocrine cells can be directly induced to mature into SC-β cells without addition of any exogenous differentiation factors (such as inhibitor of TGF-β signaling pathway, thyroid hormone signaling pathway activator, PKC activator, growth factors from TGF-β superfamily, FGF family, or EGF family, SHH signaling pathway inhibitor, γ-secretase inhibitor, ROCK inhibitor, or BMP signaling pathway inhibitor). In some embodiments, the method provided herein comprises contacting a cell population comprising NKX6.1-positive, ISL1-positive endocrine cells with a serum albumin protein, a TGF-β signaling pathway inhibitor, a SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and / or an epigenetic modifying compound. In some embodiments, the method provided herein comprises contacting a cell population comprising NKX6.1-positive, ISL1-positive endocrine cells with human serum albumin protein. In some embodiments, the method provided herein comprises contacting a cell population comprising NKX6.1-positive, ISL1-positive endocrine cells with a PKC activator.

[0292] In some embodiments, the cell population comprising the insulin-positive endocrine cells can be induced to mature into SC-β cells by contacting the insulin-positive endocrine cells with differentiation factors. The differentiation factors can comprise at least one inhibitor of TGF-β signaling pathway and thyroid hormone signaling pathway activator as described herein. In some embodiments, SC-β cells can be obtained by contacting a population of cells comprising insulin- positive endocrine cells with Alk5i and T3 or GC-1.

[0293] In some embodiments, the method provided herein comprises contacting a cell population comprising NKX6.1-positive, ISL1-positive endocrine cells with (i) a TGF-β signaling pathway inhibitor, (ii) a thyroid hormone signaling pathway activator, (iii) an epigenetic modifying compound, (iv) a BMP signaling pathway inhibitor, (v) a ROCK inhibitor, and / or (vi) a protein kinase inhibitor (e.g., staurosporine).

[0294] In some embodiments, the method provided herein comprises contacting a cell population comprising NKX6.1-positive, ISL1-positive endocrine cells with (i) a growth factor from the FGF family, (ii) a TGF-β signaling pathway inhibitor, (iii) a thyroid hormone signaling pathway activator, (iv) an epigenetic modifying compound, (v) a protein kinase inhibitor, (vi) a ROCK inhibitor, (vii) a BMP signaling pathway inhibitor, and (viii) a lipase inhibitor for about one two five days. In some embodiments, the contacting is for about three days.

[0295] Any TGF-β signaling pathway inhibitor capable of inducing the differentiation of insulin- positive endocrine cells to mature into SC-β cells (e.g., alone, or in combination with other β cell- differentiation factors, e.g., a thyroid hormone signaling pathway activator) can be used. In some embodiments, the TGF-β signaling pathway comprises TGF-β receptor type I kinase signaling. In some embodiments, the TGF-β signaling pathway inhibitor comprises Alk5 inhibitor II. In some examples, the method comprises contacting insulin-positive endocrine cells with a concentration of a TGF-β signaling pathway inhibitor (e.g., Alk5 inhibitor such as Alk5 inhibitor II), such as, 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 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 18.5µM, 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 comprises contacting insulin-positive endocrine cells with a concentration of a TGF-β signaling pathway inhibitor (e.g., Alk5 inhibitor such as Alk5 inhibitor II), such as, about 7-13 µM, about 8-12 µM, or about 9-11 µM. In some examples, the method comprises contacting insulin- positive endocrine cells with a concentration of a TGF-β signaling pathway inhibitor (e.g., Alk5 inhibitor such as Alk5 inhibitor II), such as, about 10 µM.

[0296] Any thyroid hormone signaling pathway activator capable of inducing the differentiation of insulin-positive endocrine cells to mature into SC-β cells (e.g., alone, or in combination with other β cell-differentiation factors, e.g., a TGF-β signaling pathway inhibitor) 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 contacting insulin-positive endocrine cells with a concentration of thyroid hormone signaling pathway activator (e.g., GC-1), such as, about 0.1µM, 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 0.21µM, about 0.22µM, about 0.23µM, 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 comprises contacting insulin-positive endocrine cells with a concentration of thyroid hormone signaling pathway activator (e.g., GC-1), such as, about 0.7-1.3 µM, about 0.8-1.2 µM, or about 0.9-1.1 µM. In some examples, the method comprises contacting insulin-positive endocrine cells with a concentration of thyroid hormone signaling pathway activator (e.g., GC-1), such as, about 1 µM. In some embodiments, the disclosure provides for a composition comprising insulin-positive endocrine cells a thyroid receptor activator at a concentration of 1.5-5, 1.5-3, 1.5-2.5, 1.8-5, 1.8-3, 1.8-2.2, or 1.9-2.1 µM.

[0297] Any BMP signaling pathway inhibitor capable of inducing the differentiation of insulin- positive endocrine cells to mature into SC-β cells (e.g., alone, or in combination with any of a TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator) can be used. In some embodiments, the BMP signaling pathway inhibitor comprises LDN193189 or DMH-1. In some examples, the method comprises contacting insulin-positive endocrine cells with a concentration of BMP signaling pathway inhibitor (e.g., LDN1931189), such as, 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 1µM. In some examples, the method comprises contacting insulin-positive endocrine cells with a concentration of BMP signaling pathway inhibitor (e.g., LDN1931189), such as, about 70-130 nM, about 80-120 nM, about 90-110 nM. In some examples, the method comprises contacting NKX6.1-positive pancreatic progenitor cells with a concentration of BMP signaling pathway inhibitor (e.g., LDN1931189), such as, about 100 nM.

[0298] Any ROCK inhibitor that is capable of inducing the differentiation of insulin-positive endocrine cells to mature into SC-β cells (e.g., alone, or in combination with any of a TGF-β signalingpathway inhibitor and / or a 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 contacting insulin-positive endocrine cells with a concentration of a ROCK inhibitor (e.g., Y-27632 or Thiazovivin), such as, about 0.2 µM, about 0.5 µM, about 0.75 µM, about 1 µM, about 2 µM, about 3 µM, about 4 µM, about 5 µM, about 6 µM, about 7 µM, about 7.5 µM, about 8 µM, about 9 µM, about 10 µM, about 11 µM, about 12 µM, about 13 µM, about 14 µM, about 15 µM, about 16 µM, about 17 µM, about 18 µM, about 19 µM, about 20 µM, about 21 µM, about 22 µM, about 23 µM, about 24 µM, about 25 µM, about 26 µM, about 27 µM, about 28 µM, about 29 µM, about 30 µM, about 35 µM, about 40 µM, about 50 µM, or about 100 µM. In some embodiments, the ROCK inhibitor comprises Thiazovivin. In some examples, the method comprises contacting insulin-positive endocrine cells with a concentration of a ROCK inhibitor (e.g., Y-27632 or Thiazovivin), such as, about 2.2-2.8 µM, about 2.3-2.7 µM, or about 2.4-2.6 µM. In some embodiments, the ROCK inhibitor comprises Thiazovivin. In some examples, the method comprises contacting insulin-positive endocrine cells with a concentration of a ROCK inhibitor (e.g., Y-27632 or Thiazovivin), such as, about 2.5 µM.

[0299] Any epigenetic modifying compound that is capable of inducing the differentiation of insulin-positive endocrine cells to mature into SC-β cells (e.g., alone, or in combination with any of a TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator) can be used. In some embodiments, the epigenetic modifying compound comprises a histone methyltransferase inhibitor or a HDAC inhibitor. In some embodiments, the epigenetic modifying compound comprises a histone methyltransferase inhibitor, e.g., DZNep. In some embodiments, the epigenetic modifying compound comprises a HDAC inhibitor, e.g., KD5170. In some examples, the method comprises contacting insulin-positive endocrine...

Claims

CLAIMS What is claimed is:

1. A composition comprising a plurality of stem cells (e.g., pluripotent stem cells) and an AKT- inhibitor.

2. A composition comprising a plurality of stem cells (e.g., pluripotent stem cells), a bone morphogenic protein (BMP) pathway inhibitor, and a c-Jun N-terminal kinase (JNK) inhibitor.

3. A composition comprising a plurality of stem cells (e.g., pluripotent stem cells) and insulin at a concentration of about 21-1000, 400-600, 200-400, 600-1000, 21-500, 21-300, 21-200, 21-100, 21-50, 50-500, 50-300, 50-250, 50-100, 75-125, 150-500, 150-250, or 175-225 µg / L.

4. A composition comprising a plurality of stem cells (e.g., pluripotent stem cells) and greater than 8 mM glucose.

5. A composition comprising a plurality of stem cells (e.g., pluripotent stem cells) and a medium, wherein the medium comprises a Wnt pathway activator and does not comprise a member of the transforming growth factor-beta superfamily.

6. The composition of any one of claims 1-3 and 5, wherein the composition comprises greater than 8 mM glucose.

7. The composition of any one of claims 1 or 3-6, wherein the composition comprises a BMP pathway inhibitor and a JNK inhibitor.

8. The composition of claim 5, wherein the composition does not comprise a BMP pathway inhibitor and a JNK inhibitor.

9. The composition of any one of claims 1-2 or 4-8, wherein the composition further comprises insulin.

10. The composition of claims 1-2 and 4-9, wherein the composition further comprises insulin at a concentration of about 21-1000, 400-600, 200-400, 600-1000, 21-500, 21-300, 21-200, 21-100, 21-50, 50-500, 50-300, 50-250, 50-100, 75-125, 150-500, 150-250, or 175-225 µg / L.

11. The composition of any one of claims 1-4 and 6-10, wherein the composition further comprises a Wnt pathway activator.

12. The composition of 5 or 11, wherein the Wnt pathway activator is a Wnt protein, CHIR99021, 3F8, A1070722, AR-A 014418, BIO, BlO-acetoxime, FRATide, lOZ-Hymenial disine, 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.

13. The composition of claim 12, wherein the Wnt pathway activator is a Wnt protein, and wherein the Wnt protein is a Wnt3a protein or functional fragment or derivative thereof.

14. The composition of claim 12, wherein the Wnt pathway activator is CHIR99021.

15. The composition of any one of claims 2-14, wherein the composition further comprises an AKT inhibitor.

16. The composition of claim 1 or 15, wherein the AKT inhibitor is any one of BEZ235, LY294002, GDC-0941, BYL719, GSK2636771, TGX-221, AS25242, CAL-101, IPI-145, MK-2206, GSK690693, GDC-0068, A-674563, CCT128930, AZD8055, INK128, rapamycin, PF-04691502, everolimus, BI-D1870, H89, PF-4708671, FMK, AT7867, NU7441, PI-103, NU7026, PIK-75, ZSTK474, and PP-121.

17. The composition of claim 1 or 15, wherein the AKT inhibitor comprises the structure of , or a pharmaceutically acceptable salt or18. The composition of claim 1 or 16, wherein the AKT inhibitor is MK-2206.

19. The composition of any one of claims 1-4 and 6-18, wherein the composition does not comprise a member of the transforming growth factor-beta superfamily.

20. The composition of claim 5 or 19, wherein the composition does not comprise activin A, GDF8, or GDF11.

21. The composition of any one of claims 1-20, wherein at least about 50%, 60%, 65%, 70%, 75%, 80%, or 85% of the cells in the composition are Sox17-positive, Oct4-negative cells.

22. The composition of claim 20, wherein from 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% of cells in the composition are Sox17-positive, Oct4-negative cells.

23. The composition of any one of claims 4 or 6-22, wherein the composition comprises about 8-15, 8-14, 8-12, 8-11, 8-10, 8-9, 9-11, 9-12, or 9.5-10.5 mM glucose.

24. The composition of any one of claims 1-23, wherein the stem cells are pluripotent stem cells, and wherein the pluripotent stem cells are embryonic stem cells.

25. The composition of any one of claims 1-24, wherein the stem cells are pluripotent stem cells, and wherein the pluripotent stem cells are induced pluripotent stem cells.

26. A composition comprising a plurality of SOX17-positive cells and greater than 8 mM glucose.

27. The composition of claim 26, wherein the composition comprises about 8-15, 8-14, 8-12, 8-11, 8- 10, 8-9, 9-11, 9-12, or 9.5-10.5 mM glucose.

28. The composition of claim 26 or 27, wherein the composition further comprises a growth factor from the fibroblast growth factor (FGF) family.

29. The composition of claim 28, wherein the growth factor from the FGF family is any one of KGF, FGF2, FGF8B, FGF10, and FGF21.

30. A composition comprising a BMP pathway inhibitor, a JNK inhibitor, and a plurality of FOXA2- positive, PDX1-negative cells.

31. The composition of claim 30, wherein 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 fibroblast growth factors (FGF) family, a retinoicacid (RA) signaling pathway activator, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, and a sonic hedgehog (SHH) pathway inhibitor.

32. The composition of claim 30, wherein the composition further comprises a protein kinase C activator selected from the group consisting of: phorbol 12,13-dibutyrate (PDBU), TPB, phorbol 12- myristate 13-acetate, and bryostatin 1.

33. The composition of any one of claims 30-32, wherein the BMP inhibitor comprisesing LDN193189 or DMH-1.

34. The composition of any one of claims 30-33, wherein the composition further comprises a growth factor from FGF family selected from the group consisting of: keratinocyte growth factor (KGF), FGF2, FGF10, FGF21, and FGF8B.

35. The composition of any one of claims 30-34, wherein the composition further comprises a SHHpathway inhibitor selected from the group consisting of SANT1, SANT2, SANT4, Cur61414, forskolin, tomatidine, AY9944, triparanol, and cyclopamine.

36. The composition of any one of claims 30-35, wherein the composition further comprises a RA signaling pathway activator selected from the group consisting of: RA, CD1530, AM580, TTHRB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, and CD2314.

37. The composition of any one of claims 30-36, wherein the composition further comprises a ROCK inhibitor selected from the group consisting of Thiazovivin, Y- 27632, Fasudil / HA1077, and 14-1152.

38. The composition of any one of claims 30-37, wherein the composition further comprises PDX1- positive cells.

39. An in vitro composition comprising a BMP pathway inhibitor, a JNK inhibitor, and a plurality of PDX1-positive and NKX6.1-negative cells.

40. A composition comprising a plurality of PDX1-positive cells and any one of the following reagents:ves 41. The composition of claim 39, wherein the composition further comprises any one of the following reagents:

42. The composition of claim 40 or 41, wherein the composition further comprises a BMP pathway inhibitor, a JNK inhibitor, and a plurality of PDX1-positive and NKX6.1-negative cells.

43. The composition of any one of claims 39-42, wherein the composition comprises a plurality of PDX1-positive, NKX6.1-negative cells.

44. The composition of any one of claims 39-43, wherein the composition compriese a plurality of PDX1-positive, NKX6.1-positive cells.

45. The composition of any one of claims 39-44, wherein the composition further comprises one or more agents selected from the group consisting of: a growth factor from FGF family, a RA signaling pathway activator, a Rho-associated, ROCK inhibitor, a protein kinase C activator, and a SHH pathway inhibitor.

46. The composition of any one of claims 39-45, wherein the composition further comprises a growth factor from the transformation growth factor β (TGF-β) superfamily selected from the group consisting of: an Inhibin, an Activin, a Mullerian inhibiting substance (MIS), a BMP, decapentaplegic (dpp), Vg-1, monoclonal nonspecific suppressor factor (MNSF), growth differentiating factor 8 (GDF8), and growth differentiating factor 11 (GDF11).

47. The composition of any one of claims 39-46, wherein the composition further comprises a growth factor from FGF family selected from the group consisting of: keratinocyte growth factor (KGF), FGF2, FGF10, FGF21, and FGF8B.

48. The composition of any one of claims 39-47, wherein the composition further comprises a 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.

49. The composition of any one of claims 39-48, wherein the composition further comprises a ROCK inhibitor selected from the group consisting of Thiazovivin, Y- 27632, Fasudil / HA1077, and 14-1152.

50. The composition of any one of claims 39-49, wherein the composition further comprises a protein kinase C activator selected from the group consisting of: phorbol 12,13- dibutyrate (PDBU), TPB, phorbol 12-myristate 13-acetate, and bryostatin 1.

51. The composition of any one of claims 39-50, wherein the composition further comprises a SHH pathway inhibitor selected from the group consisting of SANT1, SANT2, SANT4, Cur61414, forskolin, tomatidine, AY9944, triparanol, and cyclopamine.

52. The composition of any one of claims 39-51, wherein the composition further comprises a FoxO1 inhibitor, optionally wherein the FoxO1 inhibitor is AS1842856.

53. The composition of any one of claims 39-52, wherein the composition further comprises a notch signaling inhibitor, optionally wherein the notch signaling inhibitor is XXI or DAPI.

54. The composition of any of claims 2, 7-30, or 32-53, wherein the BMP inhibitor is noggin, LDN- 193189, DMH-1, LDN-212854, ML347 or dorsomorphin.

55. The composition of any one of claims 2, 7-30, or 32-54, wherein the JNK inhibitor is a JNK1, JNK2, and / or JNK3 inhibitor.

56. The composition of claim 55, wherein the JNK inhibitor is a JNK1, JNK2, and JNK3 inhibitor.

57. The composition of claim 55, wherein the JNK inhibitor is a compound of Formula (I):wherein: Ring A is a 6 membered monocyclic heteroaryl ring or bicyclic heteroaryl ring; each instance of RAis independently selected from the group consisting of hydrogen, halogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORA1, —N(RA1)2, and —SRA1, wherein each occurrence of RA1is independently hydrogen, acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or two RA1groups are joined to form an optionally substituted heterocyclic ring; m is 0, 1, 2, 3, or 4; Ring B is a group of the formula:he group consisting of hydrogen, halogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORB1a, —N(RB1a)2, and —SRB1a, wherein each occurrence of RB1ais independently hydrogen, acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or two RB1agroups are joined to form an optionally substituted heterocyclic ring; WB is N or CRB2, wherein RB2is selected from the group consisting of hydrogen, halogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORB2a, —N(RB2a)2, and —SRB2a, wherein each occurrence of RB2ais independently hydrogen, acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or two RB2agroups are joined to form an optionally substituted heterocyclic ring; optionally wherein RB1and RB2are joined to form an optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, or optionally substituted aryl ring; L1 is a bond directly attaching Ring A to Ring B; represents a single bond; X is —NRX—, wherein RXis hydrogen, C1-6alkyl, or a nitrogen protecting group; L2is —NRL2aC(═O)— or —C(═O)NRL2a—, wherein RL2ais hydrogen, C1-6alkyl, or a nitrogen protecting group; each instance of RCis independently selected from the group consisting of hydrogen, halogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORC1, —N(RC1)2, and —SRC1, wherein eachoccurrence of RC1is independently hydrogen, acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or two RC1groups are joined to form an optionally substituted heterocyclic ring; n is 0, 1, 2, 3, or 4; each instance of RDis independently selected from the group consisting of hydrogen, halogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORD1, —N(RD1)2, and —SRD1, wherein each occurrence of RD1is independently hydrogen, acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or two RD1groups are joined to form an optionally substituted heterocyclic ring; p is 0, 1, 2, 3, or 4; and RE is a group of the formula:w ere n: L3 is a bond, —O—, —S—, —NRL3a—, —NRL3aC(═O)—, —C(═O)NRL3a—, —SC(═O)—, — C(═O)S—, —OC(═O)—, —C(═O)O—, —NRL3aC(═S)—, —C(═S)NRL3a—, trans-CRL3b═CRL3b—, cis-CRL3b═CRL3b—, —C≡C—, —OC(RL3b)2—, —C(RL3b)2O—, —NRL3aC(RL3b)2—, — C(RL3b)2NRL3a—, —SC(RL3b)2—, —C(RL3b)2S—, —S(═O)2O—, —OS(═O)2—, —S(═O)2NRL3a—, —NRL3aS(═O)2—, or an optionally substituted C1-4 hydrocarbon chain, optionally wherein one or more carbon units of the hydrocarbon chain is replaced with —O—, —S—, —NRL3a—, — NRL3aC(═O)—, —C(═O)NRL3a—, —SC(═O)—, —C(═O)S—, —OC(═O)—, —C(═O)O—, — NRL3aC(═S)—, —C(═S)NRL3a—, trans-CRL3b═CRL3b—, cis-CRL3b═CRL3b—, —C≡C, —S(═O)2O—, —OS(═O)2—, —S(═O)2NRL3a—, or —NRL3aS(═O)2—, wherein RL3ais hydrogen, C1-6alkyl, or anitrogen protecting group, and wherein each occurrence of RL3bis independently selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl, or two RL3bgroups are joined to form an optionally substituted carbocyclic or optionally substituted heterocyclic ring; L4is a bond or an optionally substituted C1-4hydrocarbon chain; RE1is selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, — CH2ORE1a, —CH2N(RE1a)2, —CH2SRE1a, —ORE1a, —N(RE1a)2 and —SRE1a, wherein each occurrence of RE1ais independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl, or two RE1agroups are joined to form an optionally substituted heterocyclic ring; RE2is selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, — CH2ORE2a, —CH2N(RE2a)2, —CH2SRE2a, —ORE2a, —N(RE2a)2, and —SRE2a, wherein each occurrence of RE2ais independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl, or two RE2agroups are joined to form an optionally substituted heterocyclic ring; RE3is selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, — CH2ORE3a, —CH2N(RE3a)2, —CH2SRE3a, —ORE3a, —N(RE3a)2, and —SRE3a, wherein each occurrence of RE3ais independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl, or two RE3a groups are joined to form an optionally substituted heterocyclic ring; optionally wherein RE1and RE3, or RE2and RE3, or RE1and RE2are joined to form an optionally substituted carbocyclic or optionally substituted heterocyclic ring; RE4is a leaving group; Y is O, S, or NRE5, wherein RE5is hydrogen, C1-6 alkyl, or a nitrogen protecting group; a is 1 or 2; and z is 0, 1, 2, 3, 4, 5, or 6.

58. The composition of claim 55, wherein the JNK inhibitor is JNK-IN-8.

59. The composition of claim 5, wherein the composition does not comprise a BMP pathway inhibitor and a JNK inhibitor.

60. The composition of any one of claims 1-59, wherein the composition further comprises a water- soluble synthetic polymer.

61. The composition of claim 60, wherein the water-soluble synthetic polymer comprises polyvinyl alcohol, poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), PEG copolymers, poly(N- isopropylacrylamide), or polyacrylamide.

62. The composition of claim 60, wherein the water-soluble synthetic polymer comprises polyvinyl alcohol.

63. The composition of any one of claims 60-62, wherein 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 the composition.

64. The composition of any one of claims 60-62, wherein the water-soluble synthetic polymer is present at a concentration of about 0.04% to about 0.06% (w / v) in the composition.

65. The composition of any one of claims 60-64, wherein the water-soluble synthetic polymer comprises polyvinyl alcohol that is less than 85% hydrolyzed.

66. The composition of any one of claims 65, wherein the water-soluble synthetic polymer comprises polyvinyl alcohol that is about 80% hydrolyzed.

67. A composition comprising a plurality of PDX1-positive cells and a thyroid hormone signaling pathway activator at a concentration of about 1.5-5, 1.5-3, 1.5-2.5, 1.8-5, 1.8-3, 1.8-2.2, or 1.9-2.1 µM.

68. The composition of claim 67, wherein the thyroid hormone signaling pathway activator is T3 or GC-1.

69. A composition comprising a plurality of PDX1-positive cells and one or more of an epidermal growth factor, a thyroid hormone, a TGFβ-R1 kinase inhibitor, a notch signaling pathway inhibitor, aSHH signaling pathway inhibitor, retinoic acid, a ROCK inhibitor, a protein kinase inhibitor, a BMP signaling pathway inhibitor, a histone methyltransferase EZH2 inhibitor, and a Wnt signaling pathway inhibitor; wherein the composition comprises less than about 0.088 mg / ml ascorbic acid (e.g., about 0.01-0.087, 0.01-0.05, 0.03-0.087, 0.03-0.08, 0.03-0.07, 0.03-0.06, 0.03-0.05, 0.035-0.06, 0.035- 0.045, 0.035-0.045, 0.04-0.06, 0.04-0.05, or 0.043-0.046 mg / ml ascorbic acid).

70. The composition of claim 67 or 68, wherein the composition comprises less than about 0.088 mg / ml ascorbic acid (e.g., about 0.01-0.087, 0.01-0.05, 0.03-0.087, 0.03-0.08, 0.03-0.07, 0.03-0.06, 0.03-0.05, 0.035-0.06, 0.035-0.045, 0.035-0.045, 0.04-0.06, 0.04-0.05, or 0.043-0.046 mg / ml ascorbic acid).

71. The composition of claim 67 or 68, wherein the composition further comprises one or more of an epidermal growth factor, a thyroid hormone, a TGFβ-R1 kinase inhibitor, a notch signaling pathway inhibitor, a SHH signaling pathway inhibitor, retinoic acid, a ROCK inhibitor, a protein kinase inhibitor, a BMP signaling pathway inhibitor, a histone methyltransferase EZH2 inhibitor, and a Wnt signaling pathway inhibitor; wherein the composition comprises less than about 0.088 mg / ml ascorbic acid (e.g., about 0.01-0.087, 0.01-0.05, 0.03-0.087, 0.03-0.08, 0.03-0.07, 0.03-0.06, 0.03-0.05, 0.035- 0.06, 0.035-0.045, 0.035-0.045, 0.04-0.06, 0.04-0.05, or 0.043-0.046 mg / ml ascorbic acid).

72. The composition of any one of claims 67, 69 or 71, wherein the composition further comprises one or more of SANT-1, betacellulin, XXI, Alk5i, GC-1, LDN-193189, thiazovivin, staurosporine, DZNEP, retinoic acid, andNVP-TNKS656.

73. The composition of any one of claims 67-72, wherein the composition comprises one or more agents selected from the group consisting of an acetyl CoA related metabolite, an HDAC inhibitor, a redox homeostasis regulator, and a one carbon metabolism pathway intermediate.

74. The composition of any one of claims 67-73, wherein the composition further comprises acetate, β-hydroxybutyrate, taurine, and formate.

75. The composition of any one of claims 67-74, wherein the composition further comprises a vitamin, optimally wherein the vitamin is biotin.

76. The composition of any one of claims 67-75, wherein the composition further comprises glutamine.

77. The composition of any one of claims 67-76, wherein the composition further comprises a water-soluble synthetic polymer.

78. The composition of 77, wherein the water-soluble synthetic polymer is polyvinyl alcohol, poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), PEG copolymers, poly(N- isopropylacrylamide), or polyacrylamide.

79. The composition of 77, wherein the water-soluble synthetic polymer is polyvinyl alcohol (PVA).

80. The composition of any one of claims 77-79, wherein the water-soluble synthetic polymer has a concentration of about 0.005% to 0.5% (w / v), 0.01% to 0.2% (w / v), 0.02% to 0.1% (w / v), or 0.03% to 0.08% (w / v) of the composition .

81. The composition of claim 79 or 80, wherein the PVA is at most 90% hydrolyzed, optionally wherein the PVA is about 87%-89% hydrolyzed.

82. The composition of any one of claims 67-81, wherein the composition comprises a plurality of PDX1-positive, NKX6.1-negative cells.

83. The composition of any one of claims 67-82, wherein the composition comprises a plurality of PDX1-positive, NKX6.1-positive cells.

84. The composition of any one of claims 67-83, wherein the composition comprises a plurality of PDX1-positive, ISL1-positive cells.

85. The composition of any one of claims 1-84, wherein the composition is in a sterile container.

86. The composition of claim 1-85, wherein the sterile container is a bioreactor.

87. A method comprising the step of culturing a plurality of stem cells (e.g., pluripotent stem cells) with a medium comprising greater than 8 mM glucose.

88. A method comprising: a) administering a medium to a container comprising stem cells (e.g., pluripotent stem cells), wherein the media comprises 4-14 mM glucose; andb) after 4-18 hours of culturing the stem cells (e.g., pluripotent stem cells) in the medium, administering supplemental glucose to the medium, wherein following administration of the supplemental glucose to the medium, the concentration of total glucose in the medium is 4-14 mM.

89. A method comprising the step of culturing a plurality of stem cells (e.g., pluripotent stem cells) with a medium comprising a BMP pathway inhibitor and a JNK inhibitor.

90. A method comprising the step of culturing a plurality of stem cells (e.g., pluripotent stem cells) with a medium comprising insulin at a concentration of about 21-1000, 400-600, 200-400, 600-1000, 21-500, 21-300, 21-200, 21-100, 21-50, 50-500, 50-300, 50-250, 50-100, 75-125, 150-500, 150-250, or 175-225 µg / L.

91. A method comprising the step of culturing a plurality of stem cells (e.g., pluripotent stem cells) with a medium comprising an AKT-inhibitor.

92. A method comprising the step of culturing: i) a first population of cells comprising a plurality of stem cells (e.g., pluripotent stem cells) with ii) a first medium comprising a Wnt pathway activator, thereby generating a second population of cells, wherein the first medium does not comprise a member of the transforming growth factor-beta superfamily (e.g., activin A), a BMP pathway inhibitor, and / or a JNK inhibitor.

93. The method of claim 92, wherein the plurality of stem cells (e.g., pluripotent stem cells) are cultured with the first medium for less than 48 hours or for 12-48 hours, 12-36 hours, 12-30 hours, 12- 24 hours, 18-36 hours, 18-30 hours, or 21-27 hours.

94. The method of claim 92 or 93, further comprising the steps of removing the first medium and culturing the second population of cells in a second medium, thereby generating a third population of cells, wherein the second medium comprises: a) a member of the transforming growth factor-beta superfamily (e.g., activin A) or b) a BMP pathway inhibitor and a JNK inhibitor, wherein the second medium lacks a Wnt pathway activator.

95. The method of claim 94, wherein the second population of cells are cultured with the second medium for a) less than 72 hours; or b) no more than 20-52 hours, 36-52 hours, 42-42 hours, 20-72 hours, 12-48 hours, 12-36 hours, 12-30 hours, 12-24 hours, 18-36 hours, 18-30 hours, or 21-27 hours.

96. The method of claim 94 or 95, further comprising the steps of removing the second medium and culturing the third population of cells in a third medium, wherein the third medium does not comprise:a) a Wnt pathway activator, b) a member of the transforming growth factor-beta superfamily (e.g., activin A), c) a BMP pathway inhibitor, and / or d) a JNK inhibitor.

97. The method of any one of claims 89-96, wherein the first medium comprises greater than 8 mM glucose.

98. The method of claim 88, wherein the first medium comprises about 4-12, 4-10, 4-8, 6-12, 6-11, 6- 10, 8-10, or 7-9 mM glucose.

99. The method of claim 88 or 98, wherein after 4-16, 4-12, 4-10, 6-12, 6-16, 6-9, 7-9, 7-10, 7-12, 8- 12, or 10-14 hours of culturing the stem cells (e.g., pluripotent stem cells) in the first medium, the supplemental glucose is administered to the medium.

100. The method of any one of claims 88, 98 or 99, wherein the concentration of total glucose in the first medium following administration of the supplemental glucose to the first mediumis 4-12, 4-10, 4-8, 6-12, 6-11, 6-10, 8-10, 8-12, 9-12, or 7-9 mM.

101. The method of any one of claims 87-88 or 90-100, wherein the first medium comprises a BMP pathway inhibitor and a JNK inhibitor.

102. The method of claim 92, wherein the first medium does not comprise a BMP pathway inhibitor or a JNK inhibitor.

103. The method of any one of claims 87-89 or 91-102, wherein the first medium further comprises insulin.

104. The method of claims 87-89 or 91-102, wherein the first medium further comprises insulin at a concentration of about 21-1000, 400-600, 200-400, 600-1000, 21-500, 21-300, 21-200, 21-100, 21-50, 50-500, 50-300, 50-250, 50-100, 75-125, 150-500, 150-250, or 175-225 µg / L.

105. The method of any one of claims 87-91 or 93-104, wherein the first medium further comprises a Wnt pathway activator.

106. The method of 92 or 105, wherein the Wnt pathway activator is a Wnt protein, CHIR99021, 3F8, A1070722, AR-A 014418, BIO, BlO-acetoxime, FRATide, lOZ-Hymenial disine, Indirubin- 3'oxime, kenpaullone, L803, L803-mts, lithium carbonate, NSC693868, SB 216763, SB 415286, TC- G 24, TCS 2002, TCS 21311, TWS 119, analogs or derivatives thereof.

107. The method of claim 106, wherein the Wnt pathway activator is a Wnt protein, and wherein the Wnt protein is a Wnt3a protein, afunctional fragment or a derivative thereof.

108. The method of claim 106, wherein the Wnt pathway activator is CHIR99021.

109. The method of any one of claims 87-90 or 92-108, wherein the first medium further comprises an AKT inhibitor.

110. The method of claim 91 or 109, wherein the AKT inhibitor is any one of BEZ235, LY294002, GDC-0941, BYL719, GSK2636771, TGX-221, AS25242, CAL-101, IPI-145, MK-2206, GSK690693, GDC-0068, A-674563, CCT128930, AZD8055, INK128, rapamycin, PF-04691502, everolimus, BI-D1870, H89, PF-4708671, FMK, AT7867, NU7441, PI-103, NU7026, PIK-75, ZSTK474, and PP-121.

111. The method of claim 91 or 109, wherein the AKT inhibitor comprises the structure of , or a pharmaceutically acceptable salt or112. The method of claim 91 or 110, wherein the AKT inhibitor is MK-2206.

113. The method of any one of claims 87-91 or 93-112, wherein the first medium does not comprise a member of the transforming growth factor-beta superfamily.

114. The method of claim 92 or 113, wherein the first medium does not comprise activin A, GDF8, or GDF11.

115. The method of any one of claims 87-114, wherein at least about 50%, 60%, 65%, 70%, 75%, 80%, or 85% of the cells in the first medium are Sox17-positive, Oct4-negative cells.

116. The method of claim 114, wherein from 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% of the cells are Sox17-positive, Oct4-negative cells.

117. The method of any one of claims 89-116, wherein the first medium comprises about 8-15, 8-14, 8-12, 8-11, 8-10, 8-9, 9-11, 9-12, or 9.5-10.5 mM glucose.

118. A method comprising culturing a plurality of SOX17-positive cells with greater than 5.5 mM glucose.

119. The method of claim 118, wherein the media comprises about 8-15, 8-14, 8-12, 8-11, 8-10, 8-9, 9-11, 9-12, or 9.5-10.5 mM glucose.

120. A method comprising: a) administering a medium to a container comprising SOX17-positive cells, wherein the medium comprises about 4-14 mM glucose; b) after 4-18 hours of culturing the SOX17-positive cells in the medium, administering supplemental glucose to the medium, wherein following administration of the supplemental glucose to the medium, the concentration of total glucose in the media is about 4-14 mM.

121. The method of claim 120, wherein the media administered in step a) comprises about 4-12, 4-10, 4-8, 6-12, 6-11, 6-10, 8-10, or 7-9 mM glucose.

122. The method of claim 120 or 121, wherein after 4-16, 4-12, 4-10, 6-12, 6-16, 6-9, 7-9, 7-10, 7-12, 8-12, or 10-14 hours of culturing the stem cells (e.g., pluripotent stem cells) in the media from step a), the supplemental glucose is administered to the media.

123. The method of any one of claims 120-122, wherein the concentration of total glucose in the medium following administration of the supplemental glucose to the media in step b), the concentration of total glucose in the medium is about 4-12, 4-10, 4-8, 6-12, 6-11, 6-10, 8-10, 8-12, 9- 12, or 7-9 mM.

124. The method of any one of claims 116-123, wherein the medium further comprises a growth factor from the FGF family.

125. The method of claim 124, wherein the growth factor from the FGF family is any one of KGF, FGF2, FGF8B, FGF10, or FGF21.

126. A method comprising culturing a plurality of FOXA2-positive, PDX1-negative cells in a medium comprising a BMP pathway inhibitor and a JNK inhibitor.

127. The method of claim 126, wherein the medium 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 FGF family, a RA signaling pathway activator, a Rho- associated, ROCK inhibitor, and a SHH pathway inhibitor.

128. The method of claim 126 or 127, wherein the medium further comprises a protein kinase C activator selected from the group consisting of: phorbol 12,13-dibutyrate (PDBU), TPB, phorbol 12- myristate 13-acetate, and bryostatin 1.

129. The method of any one of claims 126-128, wherein the medium further comprises a BMP signaling pathway inhibitor comprising LDN193189 or DMH-1.

130. The method of any one of claims 126-129, wherein the medium further comprises a growth factor from the FGF family selected from the group consisting of: KGF, FGF2, FGF10, FGF21, and FGF8B.

131. The method of any one of claims 126-130, wherein the medium further comprises a SHHpathway inhibitor selected from the group consisting of SANT1, SANT2, SANT4, Cur61414, forskolin, tomatidine, AY9944, triparanol, and cyclopamine.

132. The method of any one of claims 126-131, wherein the medium further comprises a 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.

133. The method of any one of claims 126-132, wherein the medium further comprises a ROCK inhibitor selected from the group consisting of Thiazovivin, Y-27632, Fasudil / HA1077, and 14-1152.

134. The method of any one of claims 126-133, wherein the medium further comprises PDX1- positive cells.

135. A method comprising culturing a plurality of PDX1-positive and NKX6.1-negative cells in a medium comprising a BMP pathway inhibitor and a JNK inhibitor.

136. A method comprising culturing a plurality of PDX1-positive cells in a medium comprising any one of the following reagents:

137. The method of claim 135, wherein the medium further comprises any one of the following reagents:

138. The method of claim 136, wherein the medium further comprises a BMP pathway inhibitor, a JNK inhibitor, and a plurality of PDX1-positive and NKX6.1-negative cells.

139. The method of any one of claims 135-137, wherein the medium comprises a plurality of PDX1- positive, NKX6.1-negative cells.

140. The method of any one of claims 135-137 and 139, wherein the medium comprieses a plurality of PDX1-positive, NKX6.1-positive cells.

141. The method of any one of claims 135-140, wherein the medium further comprises one or more agents selected from the group consisting of: a growth factor from the FGF family, a RA signaling pathway activator, a ROCK inhibitor, a protein kinase C activator, and a SHH pathway inhibitor.

142. The method of any one of claims 135-141, wherein the medium further comprises a growth factor from the transformation growth factor β (TGF-β) superfamily selected from the group consisting of: an Inhibin, an Activin, a Mullerian inhibiting substance (MIS), a BMP, decapentaplegic (dpp), Vg-1, monoclonal nonspecific suppressor factor (MNSF), growth differentiating factor 8 (GDF8), and growth differentiating factor 11 (GDF11).

143. The method of any one of claims 135-142, wherein the medium further comprises a growth factor from the FGF family selected from the group consisting of: KGF, FGF2, FGF10, FGF21, and FGF8B.

144. The method of any one of claims 135-143, wherein the medium further comprises a 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.

145. The method of any one of claims 135-144, wherein the mdium further comprises a ROCK inhibitor selected from the group consisting of Thiazovivin, Y- 27632, Fasudil / HA1077, and 14-1152.

146. The method of any one of claims 135-145, wherein the medium further comprises a protein kinase C activator selected from the group consisting of: phorbol 12,13- dibutyrate (PDBU), TPB, phorbol 12-myristate 13-acetate, and bryostatin 1.

147. The method of any one of claims 135-146, wherein the medium further comprises a SHH pathway inhibitor selected from the group consisting of SANT1, SANT2, SANT4, Cur61414, forskolin, tomatidine, AY9944, triparanol, and cyclopamine.

148. The method of any one of claims 135-147, wherein the medium further comprises a FoxO1 inhibitor, optionally wherein the FoxO1 inhibitor is AS1842856.

149. The method of any one of claims 135-148, wherein the medium further comprises a notch signaling inhibitor, optionally wherein the notch signaling inhibitor is XXI or DAPI.

150. The method of any of claims 89, 94-101, or 103-149, wherein the BMP inhibitor is noggin, LDN-193189, DMH-1, LDN-212854, ML347 or dorsomorphin.

151. The method of any one of claims 89, 94-101, or 103-150, wherein the JNK inhibitor is a JNK1, JNK2, and / or JNK3 inhibitor.

152. The method of claim 150, wherein the JNK inhibitor is a JNK1, JNK2, and JNK3 inhibitor.

153. The method of claim 151, wherein the JNK inhibitor is a compound of Formula (I):wherein: Ring A is a 6 membered monocyclic heteroaryl ring or bicyclic heteroaryl ring; each instance of RAis independently selected from the group consisting of hydrogen, halogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORA1, —N(RA1)2, and —SRA1, wherein each occurrence of RA1is independently hydrogen, acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or two RA1groups are joined to form an optionally substituted heterocyclic ring;m is 0, 1, 2, 3, or 4; Ring B is a group of the formula: group consisting of hydrogen, halogen, optionally substituted acyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORB1a, —N(RB1a)2, and —SRB1a, wherein each occurrence of RB1ais independently hydrogen, acyl, substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionallycarbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or two RB1agroups are joined to form an optionally substituted heterocyclic ring; WBis N or CRB2, wherein RB2is selected from the group consisting of hydrogen, halogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORB2a, —N(RB2a)2, and —SRB2a, wherein each occurrence of RB2ais independently hydrogen, acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or two RB2agroups are joined to form an optionally substituted heterocyclic ring; optionally wherein RB1and RB2are joined to form an optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, or optionally substituted aryl ring; L1is a bond directly attaching Ring A to Ring B; represents a single bond; X is —NRX—, wherein RXis hydrogen, C1-6 alkyl, or a nitrogen protecting group; L2 is —NRL2aC(═O)— or —C(═O)NRL2a—, wherein RL2ais hydrogen, C1-6 alkyl, or a nitrogen protecting group; each instance of RCis independently selected from the group consisting of hydrogen, halogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORC1, —N(RC1)2, and —SRC1, wherein each occurrence of RC1is independently hydrogen, acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or two RC1groups are joined to form an optionally substituted heterocyclic ring; n is 0, 1, 2, 3, or 4; each instance of RDis independently selected from the group consisting of hydrogen, halogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORD1, —N(RD1)2, and —SRD1, wherein each occurrence of RD1is independently hydrogen, acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or two RD1groups are joined to form an optionally substituted heterocyclic ring; p is 0, 1, 2, 3, or 4; and RE is a group of the formula:w ere n: L3 is a bond, —O—, —S—, —NRL3a—, —NRL3aC(═O)—, —C(═O)NRL3a—, —SC(═O)—, — C(═O)S—, —OC(═O)—, —C(═O)O—, —NRL3aC(═S)—, —C(═S)NRL3a—, trans-CRL3b═CRL3b—, cis-CRL3b═CRL3b—, —C≡C—, —OC(RL3b)2—, —C(RL3b)2O—, —NRL3aC(RL3b)2—, — C(RL3b)2NRL3a—, —SC(RL3b)2—, —C(RL3b)2S—, —S(═O)2O—, —OS(═O)2—, —S(═O)2NRL3a—, —NRL3aS(═O)2—, or an optionally substituted C1-4 hydrocarbon chain, optionally wherein one or more carbon units of the hydrocarbon chain is replaced with —O—, —S—, —NRL3a—, — NRL3aC(═O)—, —C(═O)NRL3a—, —SC(═O)—, —C(═O)S—, —OC(═O)—, —C(═O)O—, — NRL3aC(═S)—, —C(═S)NRL3a—, trans-CRL3b═CRL3b—, cis-CRL3b═CRL3b—, —C≡C, —S(═O)2O—, —OS(═O)2—, —S(═O)2NRL3a—, or —NRL3aS(═O)2—, wherein RL3ais hydrogen, C1-6alkyl, or anitrogen protecting group, and wherein each occurrence of RL3bis independently selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl, or two RL3bgroups are joined to form an optionally substituted carbocyclic or optionally substituted heterocyclic ring; L4is a bond or an optionally substituted C1-4hydrocarbon chain; RE1is selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, — CH2ORE1a, —CH2N(RE1a)2, —CH2SRE1a, —ORE1a, —N(RE1a)2 and —SRE1a, wherein each occurrence of RE1ais independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl, or two RE1agroups are joined to form an optionally substituted heterocyclic ring; RE2is selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, — CH2ORE2a, —CH2N(RE2a)2, —CH2SRE2a, —ORE2a, —N(RE2a)2, and —SRE2a, wherein each occurrence of RE2ais independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl, or two RE2agroups are joined to form an optionally substituted heterocyclic ring; RE3is selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, — CH2ORE3a, —CH2N(RE3a)2, —CH2SRE3a, —ORE3a, —N(RE3a)2, and —SRE3a, wherein each occurrence of RE3ais independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl, or two RE3a groups are joined to form an optionally substituted heterocyclic ring; optionally wherein RE1and RE3, or RE2and RE3, or RE1and RE2are joined to form an optionally substituted carbocyclic or optionally substituted heterocyclic ring; RE4is a leaving group; Y is O, S, or NRE5, wherein RE5is hydrogen, C1-6 alkyl, or a nitrogen protecting group; a is 1 or 2; and z is 0, 1, 2, 3, 4, 5, or 6.

154. The method of claim 151, wherein the JNK inhibitor is JNK-IN-8.

155. The method of claim 92, wherein the medium does not comprise a BMP pathway inhibitor and a JNK inhibitor.

156. The method of any one of claims 87-155, wherein the medium further comprises a water-soluble synthetic polymer.

157. The method of claim 156, wherein the water-soluble synthetic polymer comprises polyvinyl alcohol, poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), PEG copolymers, poly(N- isopropylacrylamide), or polyacrylamide.

158. The method of claim 156, wherein the water-soluble synthetic polymer comprises polyvinyl alcohol.

159. The method of any one of claims 156-158, wherein 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 the medium.

160. The method of any one of claims 156-158, wherein the water-soluble synthetic polymer is present at a concentration of about 0.04% to about 0.06% (w / v) in the medium.

161. The method of any one of claims 156-160, wherein the water-soluble synthetic polymer comprises polyvinyl alcohol that is less than 85% hydrolyzed.

162. The method of any one of claims 161, wherein the water-soluble synthetic polymer comprises polyvinyl alcohol that is about 80% hydrolyzed.

163. A method comprising culturing a plurality of PDX1-positive cells in a medium comprising a thyroid hormone signaling pathway activator at a concentration of about 1.5-5, 1.5-3, 1.5-2.5, 1.8-5, 1.8-3, 1.8-2.2, or 1.9-2.1 µM.

164. The method of claim 163, wherein the thyroid hormone signaling pathway activator is T3 or GC- 1.

165. A method comprising culturing a plurality of PDX1-positive cells in a medium comprising one or more of an epidermal growth factor, a thyroid hormone, a TGFβ-R1 kinase inhibitor, a notchsignaling pathway inhibitor, a SHH signaling pathway inhibitor, retinoic acid, a ROCK inhibitor, a protein kinase inhibitor, a BMP signaling pathway inhibitor, a histone methyltransferase EZH2 inhibitor, and a Wnt signaling pathway inhibitor; wherein the media comprises less than about 0.088 mg / ml ascorbic acid (e.g., about 0.01-0.087, 0.01-0.05, 0.03-0.087, 0.03-0.08, 0.03-0.07, 0.03-0.06, 0.03-0.05, 0.035-0.06, 0.035-0.045, 0.035-0.045, 0.04-0.06, 0.04-0.05, or 0.043-0.046 mg / ml ascorbic acid).

166. The method of claim 165, wherein the medium comprises less than about 0.088 mg / ml ascorbic acid (e.g., 0.01-0.087, 0.01-0.05, 0.03-0.087, 0.03-0.08, 0.03-0.07, 0.03-0.06, 0.03-0.05, 0.035-0.06, 0.035-0.045, 0.035-0.045, 0.04-0.06, 0.04-0.05, or 0.043-0.046 mg / ml ascorbic acid).

167. The method of claim 165, wherein the medium further comprises one or more of an epidermal growth factor, a thyroid hormone, a TGFβ-R1 kinase inhibitor, a notch signaling pathway inhibitor, a SHH signaling pathway inhibitor, retinoic acid, a ROCK inhibitor, a protein kinase inhibitor, a bone morphogenetic (BMP) signaling pathway inhibitor, a histone methyltransferase EZH2 inhibitor, and a Wnt signaling pathway inhibitor; wherein the media comprises less than about 0.088 mg / ml ascorbic acid (e.g., about 0.01-0.087, 0.01-0.05, 0.03-0.087, 0.03-0.08, 0.03-0.07, 0.03-0.06, 0.03-0.05, 0.035- 0.06, 0.035-0.045, 0.035-0.045, 0.04-0.06, 0.04-0.05, or 0.043-0.046 mg / ml ascorbic acid).

168. The method of claim 165 or 167, wherein the medium further comprises one or more of SANT-1, betacellulin, XXI, Alk5i, GC-1, LDN-193189, thiazovivin, staurosporine, DZNEP, retinoic acid, or NVP-TNKS656.

169. The method of any one of claims 163-168, wherein the medium comprises one or more agents selected from an acetyl CoA related metabolite, an HDAC inhibitor, a redox homeostasis regulator, and a one carbon metabolism pathway intermediate.

170. The method of any one of claims 163-169, wherein the medium further comprises acetate, β- hydroxybutyrate, taurine, and formate.

171. The method of any one of claims 163-170, wherein the medium further comprises a vitamin, optimally wherein the vitamin is biotin.

172. The method of any one of claims 163-171, wherein the medium further comprises glutamine.

173. The method of any one of claims 163-172, wherein the medium further comprises a water- soluble synthetic polymer.

174. The method of 173, wherein the water-soluble synthetic polymer is polyvinyl alcohol, poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), PEG copolymers, poly(N- isopropylacrylamide), or polyacrylamide.

175. The method of 173, wherein the water-soluble synthetic polymer is polyvinyl alcohol (PVA).

176. The method of any one of claims 173-175, wherein the water-soluble synthetic polymer has a concentration of 0.005% to 0.5% (w / v), 0.01% to 0.2% (w / v), 0.02% to 0.1% (w / v), or 0.03% to 0.08% (w / v) of the medium.

177. The method of claim 175 or 176, wherein the PVA is at most 90% hydrolyzed, optionally wherein the PVA is about 87%-89% hydrolyzed.

178. The method of any one of claims 163-177, wherein the medium comprises a plurality of PDX1- positive, NKX6.1-negative cells.

179. The method of any one of claims 163-178, wherein the medium comprises a plurality of PDX1-positive, NKX6.1-positive cells.

180. The method of any one of claims 163-179, wherein the medium comprises a plurality of PDX1-positive, ISL1-positive cells.

181. A method for culturing a population of cells in vitro, comprising the steps of: a) culturing the population of cells in a medium comprising more than about 0.044 mg / ml ascorbic acid (e.g., about 0.045-0.15, 0.045-0.1, 0.045-0.09, 0.045-0.07, 0.045-0.05, 0.06-0.1, 0.06-0.09, 0.07- 0.1, 0.07-0.09, 0.08-0.09, 0.08-0.1, 0.08-0.12, or 0.085-0.09 mg / ml), and b) culturing the population of cells in a medium comprises less than 0.088 mg / ml ascorbic acid (e.g., about 0.01-0.087, 0.01-0.05, 0.03-0.087, 0.03-0.08, 0.03-0.07, 0.03-0.06, 0.03-0.05, 0.035-0.06, 0.035-0.045, 0.035-0.045, 0.04-0.06, 0.04-0.05, or 0.043-0.046 mg / ml ascorbic acid), wherein the population of cells comprises PDX1-positive cells.

182. The method of claim 181, wherein step a) comprises culturing the population of cells in a medium comprising more than 0.044 mg / ml ascorbic acid (e.g., about 0.045-0.15, 0.045-0.1, 0.045- 0.09, 0.045-0.07, 0.045-0.05, 0.06-0.1, 0.06-0.09, 0.07-0.1, 0.07-0.09, 0.08-0.09, 0.08-0.1, 0.08-0.12, or 0.085-0.09 mg / ml) for 12-24 hours, 1-4 days, 1-2 days, 2-3 days, or 3-4 days, or at least 12 hours, 1 day, 2 days, 3 days, or 4 days.

183. The method of claim 181, wherein step b) comprises culturing the population of cells in a medium comprising less than 0.088 mg / ml ascorbic acid (e.g., about 0.01-0.087, 0.01-0.05, 0.03- 0.087, 0.03-0.08, 0.03-0.07, 0.03-0.06, 0.03-0.05, 0.035-0.06, 0.035-0.045, 0.035-0.045, 0.04-0.06, 0.04-0.05, or 0.043-0.046 mg / ml ascorbic acid) for 12-24 hours, 1-4 days, 1-14 days, 1-7 days, 3-5 days, 1-2 days, 2-3 days, 3-4 days, 4-5 days, 6-7 days, or at least 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days.

184. The method of any one of claims 181-183, wherein step a) comprises culturing the population of cells in a medium comprising any one or more of: a growth factor from the FGF family, a RA signaling pathway activator, a ROCK inhibitor, a protein kinase C activator, a SHH pathway inhibitor, a FOXO1 inhibitor, and a gamma secretase inhibitor.

185. The method of any one of claims 181-184, wherein step b) comprises culturing the population of cells in a medium comprising any one or more of: epidermal growth factor, a thyroid hormone, a TGFβ-R1 kinase inhibitor, a notch signaling pathway inhibitor, a sonic hedgehog (SHH) signaling pathway inhibitor, retinoic acid, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, a protein kinase inhibitor, a bone morphogenetic (BMP) signaling pathway inhibitor, a histone methyltransferase EZH2 inhibitor, and a Wnt signaling pathway inhibitor.

186. The method of any one of claims 181-185, wherein the medium in step a) comprises a water- soluble synthetic polymer comprising polyvinyl alcohol, poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), PEG copolymers, poly(N-isopropylacrylamide), or polyacrylamide.

187. The method of claim 186, wherein the water-soluble synthetic polymer comprises polyvinyl alcohol that is less than 85% hydrolyzed.

188. The method of claim 186 or 187, wherein the water-soluble synthetic polymer comprises polyvinyl alcohol that is about 80% hydrolyzed.

189. The method of any one of claims 181-188, wherein the medium in step b) comprises a water- soluble synthetic polymer comprising polyvinyl alcohol, poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), PEG copolymers, poly(N-isopropylacrylamide), or polyacrylamide.

190. The method of claim 186, wherein the water-soluble synthetic polymer comprises polyvinyl alcohol that is at most 90% hydrolyzed, optionally wherein the polyvinyl alcohol is about 87%-89% hydrolyzed.

191. The method of any one of claims 87-190, wherein the medium is in a sterile container.

192. The method of claim 191, wherein the sterile container is a bioreactor.

193. A method for culturing a population of cells in vitro, comprising the step of culturing the population of cells in a medium comprising a growth factor from the FGF family and a RA signaling pathway activator; wherein the population of cells comprises a plurality of PDX1-positive; NKX6.1- negative cells; and wherein the cells are cultured in the medium for a period of time, wherein the period of time is at least 12 hours but not more than 4 days.

194. A method for culturing a population of cells in vitro, comprising the step of culturing the population of cells in a medium comprising a SHH pathway inhibitor and a RA signaling pathway activator; wherein the population of cells comprises a plurality of PDX1-positive; NKX6.1-negative cells; and wherein the cells are cultured in the medium for a period of time, wherein the period of time is at least 12 hours but not more than 4 days.

195. A method for culturing a population of cells in vitro, comprising the step of culturing the population of cells in a medium comprising a growth factor from the TGF-β superfamily and a RA signaling pathway activator; wherein the population of cells comprises a plurality of PDX1-positive; NKX6.1-negative cells; and wherein the cells are cultured in the medium for a period of time, wherein the period of time is at least 12 hours but not more than 4 days.

196. The method of any one of claims 193-195, wherein: a) at least 30%, 40%, 50%, 60%, 70%, 80%, or 90%; or b) about 30-100%, 50-100%, 70-100%, 85-100%, 80-95%, 85-95%, or 90-95%, of the cells in the population are PDX1-positive; NKX6.1-negative cells.

197. The method of claim 196, wherein at least 50% of the cells in the population are PDX1- positive; NKX6.1-negative cells.

198. The method of claim 196, wherein at least 80% of the cells in the population are PDX1- positive; NKX6.1-negative cells.

199. The method of claim 196, wherein about 70-100% of the cells in the population are PDX1- positive; NKX6.1-negative cells.

200. The method of any one of claims 193-195, wherein: a) at least 30%, 40%, 50%, 60%, 70%, 80%, or 90%; or b) about 30-100%, 50-100%, 70-100%, 85-100%, 80-95%, 85-95%, or 90-95%, of the cells in the population are PDX1-positive; NKX6.1-positive cells.

201. The method of claim 196, wherein at least 50% of the cells in the population are PDX1- positive; NKX6.1-positive cells.

202. The method of claim 196, wherein at least 80% of the cells in the population are PDX1- positive; NKX6.1-positive cells.

203. The method of claim 196, wherein about 70-100% of the cells in the population are PDX1- positive; NKX6.1-positive cells.

204. The method of any one of claims 193-203, wherein the period of time is at least 12 hours but not more than 3 days.

205. The method of any one of claims 193-204, wherein the period of time is at least 12 hours but not more than 2 days.

206. The method of any one of claims 193-205, wherein the period of time is at least 12 hours but not more than 1 day.

207. The method of any one of claims 193-206, wherein the period of time is 1 day, 2 days, 3 days, 4 days, 1-4 days, 1-3 days, 1-2 days, 2-4 days, or 2-3 days.

208. The method of claim 193 or 195, wherein the medium comprises a sonic hedgehog pathway inhibitor.

209. The method of claim 194 or 195, wherein the medium comprises a growth factor from the fibroblast growth factor (FGF) family.

210. The method of claim 193 or 194, wherein the medium comprises a growth factor from the TGF-β superfamily.

211. The method of claim 193 or 196-210, wherein the growth factor from the FGF family is any one of KGF, FGF2, FGF8B, FGF10, or FGF21.

212. The method of any one of claims 193-211, wherein the RA signaling pathway activator is any one of retinoic acid, CD1530, AM580, TTNPB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, and CD2314.

213. The method of any one of claims 194 or 196-212, wherein the SHH pathway inhibitor selected from the group consisting of SANT1, SANT2, SANT4, Cur61414, forskolin, tomatidine, AY9944, triparanol, and cyclopamine.

214. The method of any one of claims 195-212, wherein the growth factor from the TGF-β superfamily is any one of activin A, GDF8, and GDF11.

215. The method of any one of claims 193-214, wherein the medium further comprises a ROCK inhibitor.

216. The method of claim 215, wherein the ROCK inhibitor is any one of Thiazovivin, Y- 27632, Fasudil / HA1077, or 14-1152.

217. The method of any one of claims 193-216, wherein the medium further comprises a water- soluble synthetic polymer.

218. The method of claim 217, wherein the water-soluble synthetic polymer comprises polyvinyl alcohol, poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), PEG copolymers, poly(N- isopropylacrylamide), or polyacrylamide.

219. The method of claim 218, wherein the water-soluble synthetic polymer comprises polyvinyl alcohol.

220. The method of any one of claims 217-219, wherein the water-soluble synthetic polymer is present at a concentration of about 0.04% to about 0.06% (w / v) in the medium.

221. The method of any one of claims 217-220, wherein the water-soluble synthetic polymer comprises polyvinyl alcohol that is about 80% hydrolyzed.

222. The method of any one of claims 193-221, wherein the medium does not comprise a protein kinase C activator.

223. The method of any one of claims 193-222, wherein the medium does not comprise a BMP signaling pathway inhibitor.

224. The method of any one of claims 193-223, wherein the medium does not comprise a FOXO1 inhibitor.

225. The method of any one of claims 193-224, wherein the medium does not comprise a notch signaling pathway inhibitor.

226. The method of any one of claims 193-225, wherein the medium does not comprise an epidermal growth factor, a thyroid hormone, a TGFβ-R1 kinase inhibitor, a histone methyltransferase EZH2 inhibitor, and / or a Wnt signaling pathway inhibitor.

227. The method of any one of claims 193-226, wherein following the period of time, the cells are cultured in a medium comprising one or more of an epidermal growth factor, a thyroid hormone, a TGFβ-R1 kinase inhibitor, a notch signaling pathway inhibitor, a SHH signaling pathway inhibitor, a retinoic acid signaling pathway activator, a ROCK inhibitor, a protein kinase inhibitor, a BMP signaling pathway inhibitor, a histone methyltransferase EZH2 inhibitor, and / or a Wnt signaling pathway inhibitor.

228. The method of any one of claims 193-227, wherein prior to the period of time, the population of cells were cultured in a medium comprising a protein kinase C activator.

229. The method of any one of claims 193-228, wherein prior to the period of time, the population of cells were cultured in a medium comprising a BMP inhibitor.

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