Differentiation method for producing immature beta cells

Abstract

Disclosed herein include methods, compositions, and kits suitable for use in cell therapy. In some embodiments, there are provided methods and compositions for differentiating stem cells into pancreatic beta cells capable of producing insulin.

Description

80EM-341790-WO / CT236-PCT1 PATENTDIFFERENTIATION METHOD FOR PRODUCING IMMATURE BETA CELLSRELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application No. 63 / 699,111, filed September 25, 2024; and U.S. Provisional Application No. 63 / 776,284, filed March 24, 2025. The entire contents of these applications are hereby expressly incorporated by reference in their entireties.BACKGROUNDField

[0002] The present disclosure relates generally to the field of methods for producing pancreatic cells (e.g., immature beta cells), from stem cells ex vivo or in vitro.Description of the Related Art

[0003] Beta cells (e.g., stem cell derived beta cells) are cells with many uses, including regulating and maintaining blood glucose level and glycosylated hemoglobin level in subjects. Generation of stem cell derived beta cells can provide a useful step toward the generation of islets and pancreatic organs. One of the rapidly growing diseases that may be treatable by stem cell derived tissues is diabetes. Type 1 diabetes results from autoimmune destruction of beta cells in the pancreatic islet. Type 2 diabetes results from peripheral tissue insulin resistance and beta cell dysfunction. Diabetic patients, particularly those suffering from type 1 diabetes, can potentially be cured through transplantation of new beta cells. Patients transplanted with cadaveric human islets can be made insulin independent for 5 years or longer via this strategy, but this approach is limited because of the scarcity and quality of donor islets. Generation of an unlimited supply of human beta cells from stem cells can extend this therapy to millions of new patients.SUMMARY

[0004] Disclosed herein include methods for generating immature P cells from stem cells, as well as various uses of the immature P cells generated. The immature P cells can, for example, be used for regulating or maintaining blood glucose level (e.g., fasting blood glucose level) and / or glycosylated hemoglobin level, in a subject. The immature P cells can be used, for example, to treat a disease related to abnormal glucose homeostasis, such as hyperglycemia, metabolic syndrome, obesity, hypercholesterolemia, and / or hypertension. In some embodiments, the disease is Type I or Type II diabetes.

[0005] In some embodiments, the method comprises: (a) culturing a population of stem cells, (i) in a first medium comprising Activin A (AA) and a WNT pathway activator to generate a first population of progenitor cells, and (ii) culturing the first population of progenitor cells in a second medium comprising AA and FGF2 for a time sufficient to generate a secondpopulation of progenitor cells comprising definitive endoderm cells; (b) culturing the second population of progenitor cells in a third medium comprising FGF7 for a time sufficient to generate a third population of progenitor cells comprising gut tube cells; (c) culturing the third population of progenitor cells in a fourth medium comprising FGF7, retinoic acid, a Sonic Hedgehog (Shh) pathway inhibitor, and a protein kinase C (PKC) activator for a time sufficient to generate a fourth population of progenitor cells comprising posterior foregut cells; (d) culturing the fourth population of progenitor cells in a fifth medium comprising FGF7, retinoic acid, a Shh pathway inhibitor, and a PKC activator, wherein the fifth medium comprises a lower concentration of FGF7 and a lower concentration of retinoic acid than the fourth medium, for a time sufficient to generate a fifth population of progenitor cells comprising pancreatic endoderm cells, optionally the fifth medium comprises a bone morphogenetic protein (BMP) antagonist; (e) culturing the fifth population of progenitor cells in a sixth medium comprising retinoic acid, a Shh pathway inhibitor, a bone morphogenetic protein (BMP) antagonist, an anaplastic lymphoma kinase 5 (ALK-5) inhibitor, a thyroid hormone, and an anti-coagulant for a time sufficient to generate a sixth population of progenitor cells comprising endocrine precursor cells; and / or (f) culturing the sixth population of progenitor cells in a seventh medium comprising a BMP antagonist, an ALK-5 inhibitor, a thyroid hormone, an anti-coagulant, and a y-secretase inhibitor, for a time sufficient to generate a seventh population of cells comprising immature P cells.

[0006] In some embodiments, the first, second, third, fourth, fifth, sixth, and / or seventh media comprise B27 supplement. In some embodiments, the first, second, third, fourth, fifth, sixth, and / or seventh media comprise about 1% B27 supplement. In some embodiments, the population of stem cells is cultured in the first culture medium for at least 12 hours. In some embodiments, the population of stem cells is cultured in the first culture medium for at least 24 hours.

[0007] In some embodiments, the WNT pathway activator of the first medium is CHIR-99021 (CHIR). In some embodiments, the Shh pathway inhibitor of the fourth, fifth, and / or sixth medium is SANT-1 (SANT). In some embodiments, the PKC activator of the fourth and / or fifth medium is (2E,4E)-N-[(2S,5S)-l,2,3,4,5,6-Hexahydro-5-(hydroxymethyl)-l-methyl-2-(l- methylethyl)-3-oxo-l,4-benzodiazocin-8-yl]-5-[4-(trifluoromethyl)phenyl]-2,4-pentadienamide (TPPB). In some embodiments, the BMP antagonist of the fifth, sixth and / or seventh medium is LDN-193189-HCL (LDN).In some embodiments, the ALK-5 inhibitor of the sixth and / or seventh medium is RepSox. In some embodiments, the thyroid hormone of the sixth and / or seventh medium is 3,3’,5-Triiodo-L-thyronine sodium salt (T3). In some embodiments, the y-secretase inhibitor of the seventh medium is Dibenzazepine (DBZ). In some embodiments, the anticoagulant of the sixth and / or seventh medium is Heparin.

[0008] In some embodiments, the first medium comprises (i) about 100 ng / mL of AA and about 3 pM CHIR or (ii) about 100 ng / mL of AA and about 6 pM CHIR. In some embodiments, the second medium comprises about 20-30 ng / mL FGF2. In some embodiments, the second medium comprises about 25 ng / mL FGF2. In some embodiments, the second medium comprises about 100 ng / mL of AA. In some embodiments, the first medium, the second medium, or both comprise a ROCK inhibitor. The ROCK inhibitor can be Thiazovivin. In some embodiments, the first and / or second media comprise about 2 pM Thiazovivin.

[0009] In some embodiments, the first medium, the second medium, or both, comprise a defined, serum-free serum replacement. In some embodiments, the first medium, the second medium, or both comprise about 0.1% the defined, serum-free serum replacement. In some embodiments, the defined, serum-free serum replacement comprises one or more of insulin, transferrin, ascorbic acid, a plurality of amino acids, a plurality of trace elements, and albumin. In some embodiments, the albumin is AlbuMAX. In some embodiments, the plurality of amino acids comprises two or more of Glycine, L-Histidine, L-Isoleucine, L-Methionine, L- Phenylalanine, L-Proline, L-Hydroxyproline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine, and L-Valine. In some embodiments, the plurality of trace elements comprises two or more of Ag+, Al3+, Ba2+, Cd2+, Co2+, Cr+, Ge4+, Se4+, Br“, L, Mn2+, F“, Si4+, V5+, Mo6+, Ni2+, Rb+, Sn2+and Zr4+. In some embodiments, the defined, serum-free serum replacement is KnockOut serum replacement (KSR).

[0010] In some embodiments, the third medium comprises about 50 ng / mL FGF7. In some embodiments, the fourth medium comprises about 25 ng / mL FGF7, about 1 pM Retinoic Acid, about 0.25 pM SANT, and about 300 nM TPPB. In some embodiments, the fifth medium comprises about 2 ng / mL FGF7 or about 4 ng / mL FGF7, about 50 nM Retinoic Acid or about 100 nM Retinoic Acid, about 0.25 pM SANT, and about 300 nM TPPB. In some embodiments, the fifth medium comprises about 200 nM LDN. In some embodiments, the sixth medium comprises about 50 nM Retinoic Acid, about 0.25 pM SANT, about 100 nM LDN-193189-HCL, about 10 pM RepSox, about IpM T3, and about 10 pg / mL Heparin. In some embodiments, the seventh medium comprises about 100 nM LDN-193189-HCL, about 10 pM RepSox, about 1 pM T3, about 10 pg / mL Heparin, and about 100 nM DBZ.

[0011] In some embodiments, the population of stem cells is generated by culturing a plurality of stem cells in a 3D culture medium comprising a ROCK inhibitor. In some embodiments, the ROCK inhibitor is thiazovivin. In some embodiments, the 3D culture medium comprises 1 pM to 10 pM thiazovivin. In some embodiments, the 3D culture medium comprises about 5 pM thiazovivin. In some embodiments, the starting concentration of the plurality of stem cells added to the 3D culture medium is about 2 x 105cells / mL. In some embodiments, the 3Dculture medium comprises StemScale media. In some embodiments, the plurality of stem cells are cultured in the 3D culture medium for at least 3 days. In some embodiments, at least half of the media is replaced with fresh 3D culture medium at Day 2.

[0012] In some embodiments, the seventh medium further comprises a DNA- damaging agent. In some embodiments, the DNA-damaging agent is selected from the group comprising: radiation, cisplatin, oxaliplatin, carboplatin, nedaplatin, lobaplatin, triplatin, tetranitrate, picoplatin, satraplatin, prolindac, aroplatin, camptothecin, topotecan, irinotecan / sn38, rubitecan, belotecan, idarubicin, amrubicin, pirarubicin, valrubicin, zorubicin, teniposide, aminopterin, methotrexate, pemetrexed, raltitrexed, pentostatin, cladribine, clofarabine, fludarabine, thioguanine, mercaptopurine, fluorouracil, capecitabine, tegafur, carmofur, floxuridine, cytarabine, gemcitabine, azacitidine, hydroxyurea, mechlorethamine, cyclophosphamide, ifosfamide, trofosfamide, chlorambucil, melphalan, prednimustine, bendamustine, uramustine, estramustine, carmustine, lomustine, semustine, fotemustine, nimustine, ranimustine, streptozocin, busulfan, mannosulfan, treosulfan, carboquone, thiotepa, triaziquone, triethylenemelamine, procarbazine, dacarbazine, etopside, temozolomide, altretamine, mitobronitol, actinomycin, bleomycin, mitomycin and plicamycin. In some embodiments, the DNA-damaging agent is bleomycin. In some embodiments, the seventh medium comprises about 50 nM bleomycin. In some embodiments, the sixth population of cells is cultured in the seventh medium comprising the DNA-damaging agent for two days. In some embodiments, the sixth population of cells is cultured in the seventh medium for at least seven days. In some embodiments, the method comprises removing the DNA-damaging agent from the seventh medium following the first two days of culture in the seventh medium comprising the DNA- damaging agent.

[0013] In some embodiments, the time sufficient to generate the first population of progenitor cells of step (a) (i) comprises culturing for at least 12 hours. In some embodiments, the time sufficient to generate the first population of progenitor cells of step (a) (i) comprises culturing for at least 24 hours. In some embodiments, the time sufficient to generate the second population of progenitor cells of step (a) (ii) comprises culturing for at least 12 hours. In some embodiments, the time sufficient to generate the second population of progenitor cells of step (a) (ii) comprises culturing for at least 24 hours. In some embodiments, the time sufficient to generate the third population of progenitor cells of step (b) comprises culturing for at least three days. In some embodiments, the time sufficient to generate the fourth population of progenitor cells of step (c) comprises culturing for at least two days. In some embodiments, the time sufficient to generate the fifth population of progenitor cells of step (d) comprises culturing for at least three days. In some embodiments, the time sufficient to generate the sixth population of progenitor cells of step(e) comprises culturing for at least three days. In some embodiments, the time sufficient to generate the seventh population of cells of step (f) comprises culturing for at least seven days. In some embodiments, steps (a) - (f) occur in 20 days or less. In some embodiments, steps (a) - (f) occur in more than 20 days. In some embodiments, the method is carried out under suspension agitation. In some embodiments, suspension agitation comprises rotation. In some embodiments, the rotation speed is at least about 35 RPM to about 45 RPM. In some embodiments, rotation speed of steps (a)-(e) is at least 45 RPM, and the rotation speed of step (f) is at least 35 RPM.

[0014] In some embodiments, the first, second, third, fourth, fifth, sixth, and / or seventh media comprises MCDB131 medium or S3S6 (BLAR001) medium. In some embodiments, the first, second, third, fourth, fifth, sixth and seventh media comprise GlutaMax, non-human serum, glucose, sodium bicarbonate, or any combination thereof. In some embodiments, the non-human serum is BSA or fatty acid free BSA (FAF BSA). In some embodiments, the concentration of non-human serum is 0.05%-3%, the concentration of glucose is 5 mM to 30 mM, the concentration of sodium bicarbonate is 0.005%-0.02%, and any combination thereof. In some embodiments, ITS-X is added to the first, second, third, fourth, fifth, sixth, and / or seventh media at a v:v ratio of about 1 : 50000 to about 1 :200. In some embodiments, the third medium comprises a plurality of trace elements comprising two or more elements selected from the group consisting of Cupric Sulfate, Ferric Citrate, Sodium Selenite, Zinc Sulfate, Ammonium Molybdate, Ammonium Vanadate, Manganese Sulfate, Nickel Sulfate, Sodium Silicate, Stannous Chloride and Hydrochloric Acid. In some embodiments, the third, fourth, and / or fifth media comprise vitamin C. In some embodiments, the concentration of vitamin C is 0.1 mM to 0.5 mM. In some embodiments, the sixth and seventh media comprise zinc sulfate. In some embodiments, the concentration of zinc sulfate is 5 pM to 15 pM.

[0015] In some embodiments, the first medium comprises about 0.1% FAF -BSA, about 14.4 mM glucose, and / or about 0.01% sodium bicarbonate. In some embodiments, the first medium comprises about 0.5% FAF-BSA, about 10 mM glucose, and / or about 0.015% sodium bicarbonate. In some embodiments, the second medium comprises about 0.1% FAF-BSA, about 14.4 mM glucose, and / or about 0.01% sodium bicarbonate. In some embodiments, the second medium comprises about 0.5% FAF-BSA, about 10 mM glucose, and / or about 0.015% sodium bicarbonate. In some embodiments, the third and / or fourth media comprise about 0.5% FAF-BSA, about 10 mM glucose, about 0.015% sodium bicarbonate, and / or about 0.25 mM vitamin C. In some embodiments, the fifth medium comprises about 0.25% FAF-BSA, about 10 mM glucose, about 0.015% sodium bicarbonate, and / or about 0.25 mM vitamin C. In some embodiments, the sixth and / or seventh media comprise about 2% FAF-BSA, about 20 mM glucose, about 0.015% sodium bicarbonate, and / or about 10 pM zinc sulfate. In some embodiments, any of the first,second, third, fourth, fifth, sixth, and seventh media comprise an antibiotic.

[0016] In some embodiments, the first medium comprises MCDB 131 medium or S3 S6 (BLAR001) medium, GlutaMax, 0.1% FAF-BSA, 14.4 mM glucose, 0.01% sodium bicarbonate, 100 ng / mL of AA, and 3 pM CHIR. In some embodiments, the first medium comprises MCDB 131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.015% sodium bicarbonate, 100 ng / mL of AA, 3 pM CHIR, and 2 pM Thiazovivin. In some embodiments, the first medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.015% sodium bicarbonate, 0.1% KSR, 100 ng / mL of AA, 3 pM CHIR, and 2 pM Thiazovivin. In some embodiments, the first medium comprises MCDB 131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.01% sodium bicarbonate, 1% B27, 100 ng / mL of AA, 6 pM CHIR, and 2 pM Thiazovivin.

[0017] In some embodiments, the second medium comprises MCDB 131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.1% FAF-BSA, 14.4 mM glucose, 0.01% sodium bicarbonate, 100 ng / mL AA and 25 ng / mL FGF2. In some embodiments, the second medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.015% sodium bicarbonate, 100 ng / mL AA, 2 pM Thiazovivin, and 25 ng / mL FGF2. In some embodiments, the second medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.015% sodium bicarbonate, 0.1% KSR, 100 ng / mL AA, 2 pM Thiazovivin, and 25 ng / mL FGF2. In some embodiments, the second medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.01% sodium bicarbonate, 1% B27, 100 ng / mL AA, 2 pM Thiazovivin, and 25 ng / mL FGF2.

[0018] In some embodiments, the third medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.015% sodium bicarbonate, 0.25 mM Vitamin C, and 50 ng / mL FGF7. In some embodiments, the third medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.015% sodium bicarbonate, 0.25 mM Vitamin C, trace elements, and 50 ng / mL FGF7. In some embodiments, the third medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.015% sodium bicarbonate, 0.25 mM Vitamin C, trace elements, 1% B27, and 50 ng / mL FGF7.

[0019] In some embodiments, the fourth medium comprises MCDB 131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :200 (v:v) ITS-X, 0.015% sodium bicarbonate, 0.25 mM Vitamin C, 25 ng / mL FGF7, 1 pM retinoic acid, 0.25 pMSANT, and 300 nM TPPB. In some embodiments, the fourth medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :200 (v:v) ITS-X, 0.015% sodium bicarbonate, 0.25 mM Vitamin C, 1% B27, 25 ng / mL FGF7, 1 pM retinoic acid, 0.25 pM SANT, and 300 nM TPPB.

[0020] In some embodiments, the fifth medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.25% FAF-BSA, 10 mM glucose, 1 :200 (v:v) ITS-X, 0.015% sodium bicarbonate, 0.25 mM Vitamin C, 2 ng / mL FGF7, 50 nM retinoic acid, 0.25 pM SANT, and 300 nM TPPB. In some embodiments, the fifth medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.25% FAF-BSA, 10 mM glucose, 1 :200 (v:v) ITS-X, 0.015% sodium bicarbonate, 0.25 mM Vitamin C, 1% B27, 4 ng / mL FGF7, 100 nM retinoic acid, 0.25 pM SANT, 300 nM TPPB, and 200 nM LDN.

[0021] In some embodiments, the sixth medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 2% FAF-BSA, 20 mM glucose, 1 :200 (v:v) ITS-X, 0.015% sodium bicarbonate, 10 pM Zinc Sulfate, 50 nM retinoic acid, 0.25 pM SANT, 100 nM LDN, 10 pM RepSOX, 1 pM T3, and 10 pg / mL Heparin. In some embodiments, the sixth medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 2% FAF-BSA, 20 mM glucose, 1 :200 (v:v) ITS-X, 0.015% sodium bicarbonate, 10 pM Zinc Sulfate, 1% B27, 50 nM retinoic acid, 0.25 pM SANT, 100 nM LDN, 10 pM RepSOX, 1 pM T3, and 10 pg / mL Heparin.

[0022] In some embodiments, the seventh medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 2% FAF-BSA, 20 mM glucose, 1 :200 (v:v) ITS-X, 0.015% sodium bicarbonate, 10 pM Zinc Sulfate, 100 nM LDN, 10 pM RepSOX, 1 pM T3, 10 pg / mL Heparin, and 100 nM DBZ. In some embodiments, the seventh medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 2% FAF-BSA, 20 mM glucose, 1 :200 (v:v) ITS-X, 0.015% sodium bicarbonate, 10 pMZinc Sulfate, 1% B27, 100 nMLDN, 10 pM RepSOX, 1 pM T3, 10 pg / mL Heparin, and 100 nM DBZ.

[0023] In some embodiments, at least 25% of the seventh population of cells express PDX1 and NKX6.1, at least 20% of the seventh population of cells express ISL1 and NKX6.1, at least 30% of the seventh population of cells express NKX6.1 and INS, less than 1% of the seventh population of cells express GCG and INS, or any combination thereof. In some embodiments, the proportion of cells expressing FOXA2, PDX1, NKX6.1, INS, ISL1, or any combination thereof, is increased by at least 0.5-fold in the seventh population of cells relative to a population of stem cells. In some embodiments, at least a portion of the fifth population of progenitor cells and / or the seventh population of cells express one or more markers of differentiation. In some embodiments, each of the one or more markers comprise CHGA, FOXA2, PDX1, NKX6.1, or NGN3. In some embodiments, at least 25% of the fifth population of progenitor cells express NKX6.1 and PDX1.In some embodiments, at least 0.5-fold more cells of the fifth population of progenitor cells and / or the seventh population of cells express one or more markers of differentiation each selected from the group consisting of CHGA, F0XA2, PDX1, NKX6.1 and NGN3; as compared to a fifth population of progenitor cells and / or a seventh population of cells generated without culturing in a medium comprising FGF2 in step (a). In some embodiments, the average C-peptide levels capable of being secreted from the sixth population of progenitor cells or seventh population of cells is increased by at least 5-fold as compared to a sixth population of progenitor cells or seventh population of cells generated without culturing in a medium comprising FGF2 in step (a). In some embodiments, at least 75% of the second population of progenitor cells express F0XA2 and SOX17.

[0024] In some embodiments, the yield of the seventh population of cells is increased by at least 10% when culturing in a medium comprising the defined, serum-free serum replacement as compared to culturing in a medium that does not comprise the defined, serum-free serum replacement in step (a). In some embodiments, the yield of the seventh population of cells is increased by at least 2-fold when culturing in: a first medium comprising B27 and 6 pM CHIR, a second medium comprising B27, a third medium comprising B27, a fourth medium comprising B27, a fifth medium comprising B27, 4 ng / mL FGF7, 100 nM retinoic acid, and 200 nM LDN, a sixth medium comprising B27, and a seventh medium comprising B27; e.g., as compared to culturing in a first medium that does not comprise B27 and 6 pM CHIR, a second medium that does not comprise B27, a third medium that does not comprise B27, a fourth medium that does not comprise B27, a fifth medium that does not comprise B27, 4 ng / mL FGF7, 100 nM retinoic acid, and 200 nM LDN, a sixth medium that does not comprise B27, and a seventh medium that does not comprise B27.

[0025] In some embodiments, the immature P cells are capable of differentiating into mature P cells capable of secreting insulin in response to glucose. In some embodiments, about 60 x io6to about 80 * 106immature P cells are produced from a population of stem cells comprising about 20 * 106cells.

[0026] In some embodiments, the population of stem cells is a population of genetically modified cells. In some embodiments, the stem cells are genetically modified by an RNA-guided endonuclease system. The RNA-guided endonuclease system can be, e.g., a CRISPR system comprising a CRISPR nuclease and a guide RNA. In some embodiments, the stem cells are induced pluripotent stem cells (iPSC), pluripotent stem cells (PSC), embryonic stem cells (ESC), or adult stem cells (ASC). In some embodiments, the stem cells are mammalian cells, e.g., human cells. The method can comprise differentiating the immature P cells into mature P cells capable of secreting insulin in response to glucose.

[0027] Also provided herein are populations of immature P cells differentiated by or obtainable by any of the methods of the disclosure. Disclosed herein include populations of cells comprising at least 10% or 20% pancreatic progenitor cells, wherein the pancreatic progenitor cells express one or more markers each selected from the group consisting of ISL1, NKX6.1, PDX1, INS, CHGA, FOXA2, PDX1, and NGN3. In some embodiments, at least 10% of the population of cells express ISL1 and NKX6.1. In some embodiments, at least 25% of the population of cells express PDX1 and NKX6.1. In some embodiments, at least 30% of the population of cells express NKX6.1 and INS. In some embodiments, the pancreatic progenitor cells comprise Stage 6 immature P cells. In some embodiments, the population of cells are capable of differentiating into mature P cells capable of secreting insulin in response to glucose, following administration to a subject. In some embodiments, at least 800 pmol / L of C-peptide is detected in the blood of the subject following glucose stimulation, wherein the subject is administered about 3.5 million cells, optionally C-peptide is measured 8 weeks following the administration, further optionally C-peptide is measured 12 weeks following the administration.

[0028] Disclosed herein include compositions comprising populations of immature P cells differentiated by or obtainable by any of the methods of the disclosure, for use as a medicament, optionally wherein the composition is a pharmaceutical composition. Disclosed herein include populations of immature P cells differentiated by or obtainable by any of the methods of the disclosure and compositions comprising populations of immature P cells differentiated by or obtainable by any of the methods of the disclosure for use in treating a subject in need thereof. Disclosed herein include populations of immature P cells differentiated by or obtainable by any of the methods of the disclosure and compositions comprising populations of immature P cells differentiated by or obtainable by any of the methods of the disclosure for use in treating diabetes. Disclosed herein include compositions comprising populations of cells comprising pancreatic progenitor cells (e.g. , immature P cells), for use as a medicament, optionally wherein the composition is a pharmaceutical composition. Disclosed herein include pancreatic progenitor cells for use in treating a subject in need thereof. Disclosed herein include pancreatic progenitor cells for use in treating a disease related to abnormal glucose homeostasis. In some embodiments, the disease is hyperglycemia, metabolic syndrome, obesity, hypercholesterolemia, and / or hypertension. In some embodiments, the disease is Type I or Type II diabetes.

[0029] Disclosed herein include methods for treating a subject in need thereof. In some embodiments, the method comprises administering to the subject a population of immature P cells or a population of cells comprising pancreatic progenitor cells of the disclosure. In some embodiments, the population of immature P cells or the population of cells comprising pancreatic progenitor cells are administered as one or more pharmaceutical compositions. In someembodiments, glucose-stimulated insulin secretion in the subject increases by at least 1.5-fold after about 20 weeks following the administration; e.g., as compared to insulin secretion after about 8 weeks following administration. In some embodiments, body weight of the subject increases after about two weeks following administration. In some embodiments, fasting blood glucose levels of the subject are decreased by at least 25% after about one month following administration. In some embodiments, fasting blood glucose levels of the subject are decreased by at least 50% after about two months following administration. In some embodiments, at least 3.0 x io6cells are administered to the subject. In some embodiments, about 7.0 x io6cells are administered to the subject. In some embodiments, the subject is a mouse.

[0030] In some embodiments, the subject has, is suspected of having, or is at risk for a disease related to abnormal glucose homeostasis. In some embodiments, the subject has, is suspected of having, or is at risk for hyperglycemia, metabolic syndrome, obesity, hypercholesterolemia, and / or hypertension. In some embodiments, the subject has, is suspected of having, or is at risk for diabetes. In some embodiments, the diabetes is type I or type II diabetes. In some embodiments, the subject is a mammal, e.g., a human.BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1 depicts exemplary images of different stages of pancreatic progenitor cell differentiation from stem cells to Stage 6 immature beta cells.

[0032] FIG. 2 displays exemplary data related to the differentiation yield as measured by aggregate pellet volume (APV) of a differentiation protocol of the disclosure.

[0033] FIG. 3 displays exemplary data related to FACS profiling of populations of cells for expression ISL1 and PDX1 or PDX1 and NKX6.1.

[0034] FIG. 4 displays exemplary gene expression data for populations of cells differentiated from stem cells to mature islet cells.

[0035] FIG. 5 displays exemplary data showing that the differentiated cells are capable of producing and secreting insulin as shown by levels of C-peptide.

[0036] FIG. 6A-FIG. 6H display non-limiting exemplary gene expression data for differentiation markers at S4 (FIG. 6A-FIG. 6D) and S6 stage (FIG. 6E-FIG. 6H) from experiments where FGF2 was added to Stage 1, Day 2 media (e.g., second medium) or where FGF2 was not added. Adding FGF2 at stage 1 improved the iPSC S4 and S6 differentiation as indicated by the increased expression of differentiation markers at each stage.

[0037] FIG. 7 displays a schematic of differentiation of stem cells to Stage 6 cells. Also shown is a rotation culture device used for culture of cells according to some embodiments.

[0038] FIG. 8 displays data showing improvement in performance (indicated by C- peptide levels) of the differentiation protocol with FGF2 and Thiazovivin (e.g , Protocol 3 in Table32B below) of the present disclosure (right panel) as compared to protocols without addition of FGF2 and Thiazovivin (left panel, Protocol 1 in Table 32A below), e.g., during SI. The data in the left panel is also shown in FIG. 5.

[0039] FIG. 9 displays exemplary data related to the yield of a differentiation protocol in which KSR is added to Stage 1, Day 1 media (e.g., Protocol B).

[0040] FIG. 10 displays data of C-peptide levels detected in blood after implantation with the indicated numbers of S6 cells generated by Protocol 3 according to Table 32B below.

[0041] FIG. 11 displays data of C-peptide levels detected in blood after implantation with S6 cells generated by Protocol 3 according to Table 32B below.

[0042] FIG. 12 displays data of C-peptide levels detected in blood after implantation of indicated model with S6 cells generated by a method disclosed herein.

[0043] FIG. 13 displays a graph of yield of S6 cells from stem cells, using the compositions and methods of the disclosure.

[0044] FIG. 14A-FIG. 14B display data related to characterization of S6 cells differentiated according to the compositions and methods of the disclosure. FIG. 14A displays exemplary images of S4 and S6 cells differentiated using Protocol C. FIG. 14B displays characterization of cell markers at the indicated stage by FACS.

[0045] FIG. 15 displays data related to in vivo maturation of the S6 cells. 3.5 * 106S6 cells differentiated using protocol A were implanted (kidney capsule transplantation) into NSG mice. Shown are C-peptide levels detected from mice transplanted with the indicated S6 clone.

[0046] FIG. 16A-FIG. 16C display exemplary in vivo data showing that implanted S6 cells (kidney capsule transplantation) obtained using protocol A (derived from hypoimmunogenic iPSCs) were able to reverse STZ-induced type 1 diabetes model to normal glycemia at week 7 post-implantation. Blood glucose data is shown in FIG. 16A. Body weight of mice is shown in FIG. 16B. C-peptide levels of mice at week 8 is shown in FIG. 16C.

[0047] FIG. 17 displays a schematic related to safety concerns for S6 products and method of mitigating said concerns.

[0048] FIG. 18A-FIG. 18B display data showing in vitro differentiation with bleomycin reduced proliferated cells and iPSC residues.

[0049] FIG. 19A-FIG. 19C display data showing in vitro differentiation with bleomycin reduced off-target differentiation gene expression.

[0050] FIG. 20A-FIG. 20B display data showing in vitro differentiation with bleomycin can reduce cyst formation in vivo.DETAILED DESCRIPTION

[0051] In the following detailed description, reference is made to the accompanyingdrawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein and made part of the disclosure herein.

[0052] All patents, published patent applications, other publications, and sequences from GenBank, and other databases referred to herein are incorporated by reference in their entirety with respect to the related technology.

[0053] Disclosed herein include methods for generating immature P cells from stem cells. In some embodiments, the method comprises: (a) culturing a population of stem cells, (i) in a first medium comprising AA and a WNT pathway activator to generate a first population of progenitor cells, and (ii) culturing the first population of progenitor cells in a second medium comprising AA and FGF2 for a time sufficient to generate a second population of progenitor cells comprising definitive endoderm cells; (b) culturing the second population of progenitor cells in a third medium comprising FGF7 for a time sufficient to generate a third population of progenitor cells comprising gut tube cells; (c) culturing the third population of progenitor cells in a fourth medium comprising FGF7, retinoic acid, a Sonic Hedgehog (Shh) pathway inhibitor, and a protein kinase C (PKC) activator for a time sufficient to generate a fourth population of progenitor cells comprising posterior foregut cells; (d) culturing the fourth population of progenitor cells in a fifth medium comprising FGF7, retinoic acid, a Shh pathway inhibitor, and a PKC activator, wherein the fifth medium comprises a lower concentration of FGF7 and a lower concentration of retinoic acid than the fourth medium, for a time sufficient to generate a fifth population of progenitor cells comprising pancreatic endoderm cells, optionally the fifth medium comprises a bone morphogenetic protein (BMP) antagonist; (e) culturing the fifth population of progenitor cells in a sixth medium comprising retinoic acid, a Shh pathway inhibitor, a bone morphogenetic protein (BMP) antagonist, an anaplastic lymphoma kinase 5 (ALK-5) inhibitor, a thyroid hormone, and an anti-coagulant for a time sufficient to generate a sixth population of progenitor cells comprising endocrine precursor cells; and / or (f) culturing the sixth population of progenitor cells in a seventh medium comprising a BMP antagonist, an ALK-5 inhibitor, a thyroid hormone, an anti-coagulant, and a y-secretase inhibitor, for a time sufficient to generate a seventh population of cells comprising immature P cells.

[0054] Also provided herein are populations of immature P cells differentiated by orobtainable by any of the methods of the disclosure. Disclosed herein include compositions comprising populations of immature P cells of differentiated by or obtainable by any of the methods of the disclosure, for use as a medicament, optionally wherein the composition is a pharmaceutical composition.

[0055] Disclosed herein include populations of immature P cells differentiated by or obtainable by any of the methods of the disclosure, and compositions comprising populations of immature P cells differentiated by or obtainable by any of the methods of the disclosure for use in treating a subject in need thereof. Disclosed herein include using populations of immature P cells differentiated by or obtainable by any of the methods of the disclosure and compositions comprising populations of Immature P cells differentiated by or obtainable by any of the methods of the disclosure to maintaining blood glucose level in a subject, and to treat diseases related to abnormal glucose homeostasis (e.g., diabetes).

[0056] Disclosed herein include methods for treating a subject in need thereof. In some embodiments, the method comprises administering to the subject a population of immature P cells of the disclosure. In some embodiments, the population of immature P cells are administered as one or more pharmaceutical compositions.Definitions

[0057] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. See, e.g. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For purposes of the present disclosure, the following terms are defined below.

[0058] As used herein, the term “about” can mean plus or minus 5% of the provided value.

[0059] “Markers”, as used herein, can refer to nucleic acid or polypeptide molecules that are differentially expressed in a cell of interest. In this context, differential expression means an increased level for a positive marker and a decreased level for a negative marker as compared to an undifferentiated cell or a cell at another stage of differentiation. The detectable level of the marker nucleic acid or polypeptide is sufficiently higher or lower in the cells of interest compared to other cells, such that the cell of interest can be identified and distinguished from other cells using any of a variety of methods known in the art.

[0060] As used herein, a cell is “positive for” a specific marker, “positive”, or “+” when the specific marker is sufficiently detected in the cell. Similarly, the cell is “negative for”, “negative” or for a specific marker when the specific marker is not sufficiently detected in thecell. For example, positive by fluorescence activated cell sorting cytometry (“FACS”) is usually greater than about 2%, whereas the negative threshold by FACS is usually less than about 1%. Positive by polymerase chain reaction cytometry (“PCR”) is usually less than or equal to about 30 cycles (Cts); whereas negative by PCR is usually more than about 31 cycles.

[0061] As used herein, the term “deletion” which may be used interchangeably with the terms “genetic deletion”, “knock-out”, or “KO”, generally refers to a genetic modification wherein a site or region of genomic DNA is removed by any molecular biology method, e.g., methods described herein, e.g., by delivering to a site of genomic DNA an endonuclease and at least one gRNA. Any number of nucleotides can be deleted. In some embodiments, a deletion involves the removal of at least one, at least two, at least three, at least four, at least five, at least ten, at least fifteen, at least twenty, or at least 25 nucleotides. In some embodiments, a deletion involves the removal of 10-50, 25-75, 50-100, 50-200, or more than 100 nucleotides. In some embodiments, a deletion involves the removal of an entire target gene. In some embodiments, a deletion involves the removal of part of a target gene. In some embodiments, a deletion involves the removal of a transcriptional regulator, e.g., a promoter region, of a target gene. In some embodiments, a deletion involves the removal of all or part of a coding region such that the product normally expressed by the coding region is no longer expressed, is expressed as a truncated form, or expressed at a reduced level. In some embodiments, a deletion leads to a decrease in expression of a gene relative to an unmodified cell.

[0062] As used herein, the term “insertion” which may be used interchangeably with the terms “genetic insertion” or “knock-in”, generally refers to a genetic modification wherein a polynucleotide is introduced or added into a site or region of genomic DNA by any molecular biological method, e.g., methods described herein, e.g., by delivering to a site of genomic DNA an endonuclease and at least one gRNA. In some embodiments, an insertion may occur within or near a site of genomic DNA that has been the site of a prior genetic modification, e.g., a deletion or insertion-deletion mutation. In some embodiments, an insertion occurs at a site of genomic DNA that partially overlaps, completely overlaps, or is contained within a site of a prior genetic modification, e.g., a deletion or insertion-deletion mutation. In some embodiments, an insertion involves the introduction of a polynucleotide that encodes a protein of interest. In some embodiments, an insertion involves the introduction of an exogenous promoter, e.g., a constitutive promoter, e.g., a CAG promoter. In some embodiments, an insertion involves the introduction of a polynucleotide that encodes a noncoding gene. In general, a polynucleotide to be inserted is flanked by sequences (e.g., homology arms) having substantial sequence homology with genomic DNA at or near the site of insertion.

[0063] The term “cell culture medium” as used herein refers to a solution containingnutrients needed for culturing a cell. The term “cell culture medium” as used herein may be used interchangeably with the term “culture medium,” “medium,” or “growth medium.” The medium includes a commercialized or prepared medium used in culturing the cell.

[0064] As used herein, the term “subject” refers to a mammal. In some embodiments, a subject is non-human primate or rodent. In some embodiments, a subject is a human. In some embodiments, a subject has, is suspected of having, or is at risk for, a disease or disorder. In some embodiments, a subject has one or more symptoms of a disease or disorder.

[0065] As used herein, “cell density” and “seeding density” are used interchangeably herein and refer to the number of cells seeded per unit area of a solid or semisolid planar or curved substrate.

[0066] The term “suspension culture” or “suspension agitation”, as used herein, refers to the culture of cells, dispersed in a liquid nutrient medium. Due to this culture technique, the cells do not adhere to the solid support or the culture vessel. In some embodiments, in suspension culture the culture vessel is constantly moving or agitated. It is readily understood that suspension cultures can comprise cells in various stages of aggregation. A range of aggregate sizes are encountered in the suspensions with sizes ranging from tens of microns in diameter (single cells or couple of hundred aggregated cells) to aggregates hundreds of microns in diameter, consisting of many thousands of cells.

[0067] As used herein the term “time sufficient to generate” a specific cell type (e.g., immature beta cells) may be understood as a time frame sufficient to observe at least one differentiated cell (e.g., an immature beta cell) among the cell population. In some embodiments, the “time sufficient to generate immature beta cells” includes time to achieve a plurality of immature beta cells in the cell population (e.g., more than one immature beta cell). In some embodiments, the “time sufficient to generate immature beta cells” includes time to achieve a maj ority of immature beta cells in the cell population (e.g. , time to achieve a cell population having more than 50%, more than 60%, more than 70%, more than 80% or more than 90% of immature beta cells).

[0068] Disclosed herein include methods for generating immature P cells from stem cells. In some embodiments, the method comprises: (a) culturing a population of stem cells, (i) in a first medium comprising AA and a WNT pathway activator to generate a first population of progenitor cells (e.g., Stage 1, Day 1 cells), and (ii) culturing the first population of progenitor cells in a second medium comprising AA and FGF2 for a time sufficient to generate a second population of progenitor cells (e.g., Stage 1, Day 2 cells) comprising definitive endoderm cells; (b) culturing the second population of progenitor cells in a third medium comprising FGF7 for a time sufficient to generate a third population of progenitor cells (e.g., Stage 2 cells) comprisinggut tube cells; (c) culturing the third population of progenitor cells in a fourth medium comprising FGF7, retinoic acid, a Sonic Hedgehog (Shh) pathway inhibitor, and a protein kinase C (PKC) activator for a time sufficient to generate a fourth population of progenitor cells (e.g., Stage 3 cells) comprising posterior foregut cells; (d) culturing the fourth population of progenitor cells in a fifth medium comprising FGF7, retinoic acid, a Shh pathway inhibitor, and a PKC activator, wherein the fifth medium comprises a lower concentration of FGF7 and a lower concentration of retinoic acid than the fourth medium, for a time sufficient to generate a fifth population of progenitor cells (e.g., Stage 4 cells) comprising pancreatic endoderm cells, optionally the fifth medium comprises a bone morphogenetic protein (BMP) antagonist; (e) culturing the fifth population of progenitor cells in a sixth medium comprising retinoic acid, a Shh pathway inhibitor, a bone morphogenetic protein (BMP) antagonist, an anaplastic lymphoma kinase 5 (ALK-5) inhibitor, a thyroid hormone, and an anti-coagulant for a time sufficient to generate a sixth population of progenitor cells (e.g., Stage 5 cells) comprising endocrine precursor cells; and / o (f) culturing the sixth population of progenitor cells in a seventh medium comprising a BMP antagonist, an ALK-5 inhibitor, a thyroid hormone, an anti-coagulant, and a y-secretase inhibitor, for a time sufficient to generate a seventh population of cells (e.g., Stage 6 cells) comprising immature P cells. In some embodiments, the first and / or second medium comprise a ROCK inhibitor. In some embodiments, the first and / or second medium comprise a defined, serum-free serum replacement.

[0069] For differentiation of pluripotent stem cells into, e.g., immature beta cells, in vitro cell cultures, the differentiation process can be viewed as progressing through a number of consecutive stages. In particular, the differentiation process is commonly viewed as progressing through multiple stages. In this step-wise differentiation, “Stage 1” refers to the first step in the differentiation process, the differentiation of stem cells (e.g., pluripotent stem cells) into cells expressing markers characteristic of the definitive endoderm (“Stage 1 cells”). “Stage 2” refers to the second step, the differentiation of cells expressing markers characteristic of the definitive endoderm cells into cells expressing markers characteristic of gut tube cells (e.g., primitive gut tube cells) (“Stage 2 cells”). “Stage 3” refers to the third step, differentiation of cells expressing markers characteristic of primitive gut tube cells into cells expressing markers characteristic of foregut cells (e.g., posterior foregut cells, also referred to as foregut endoderm cells) (“Stage 3 cells”). “Stage 4” refers to the fourth step, the differentiation of cells expressing markers characteristic of foregut endoderm cells (e.g., posterior foregut cells) into cells expressing markers characteristic of pancreatic endoderm cells (“Stage 4 cells”). “Stage 5” refers to the fifth step, the differentiation of cells expressing markers characteristic of pancreatic endoderm cells into cells expressing markers characteristic of one or both of pancreatic endoderm cells and pancreaticendocrine precursor cells (collectively referred to as “Stage 5 cells” or, alternatively, “pancreatic endoderm / endocrine precursor cells”). Stage 6 refers to the sixth step, the differentiation of cells expressing markers characteristic of pancreatic endocrine precursor cells into cells expressing markers characteristic of immature beta-cells (“Stage 6 cells”). Stage 6 cells are capable of differentiating into mature or functional beta cells (“Stage 7 cells”) either by in vitro methods known in the art, or following transplantation into a subject. By “functional beta-cells that have a more matured phenotype” or “Stage 7 cells” is meant a pancreatic endocrine cell that, when compared to a Stage 6 cell, is not only single hormonal insulin +, MAFA+, NKX6.1+, UCN3+, SLC2A1+ and PDX1+, but also expresses MAFA at a higher level than a less mature pancreatic endocrine cell, in particular an immature beta cell.

[0070] It is to be noted that not all cells in a particular population progress through these stages at the same rate or at the same stage (or developed to the same stage). Consequently, it is not uncommon in in vitro cell cultures to detect the presence of cells that have progressed less, or more, down the differentiation pathway than the majority of cells present in the population, particularly at the later differentiation stages. For purposes of illustrating the present invention, characteristics of the various cell types associated with the above-identified stages are described herein.

[0071] “Definitive endoderm” as used herein, refers to cells which bear the characteristics of cells arising from the epiblast during gastrulation and which form the gastrointestinal tract and its derivatives. Definitive endoderm cells express at least one of the following markers: FOXA2 (also known as hepatocyte nuclear factor 3-P (“HNF3-P”)), GATA4, SOX17, CXCR4, Brachyury, Cerberus, OTX2, goosecoid, C-Kit, CD99, and MIXL1. Markers characteristic of the definitive endoderm cells are CXCR4, FOXA2, and SOX17. Thus, definitive endoderm cells may be characterized by their expression of CXCR4, FOXA2, and SOX17. In addition, depending on the length of time cells are allowed to remain in Stage 1, an increase in HNF4a may be observed.

[0072] “Primitive gut tube cells” or “gut tube cells”, as used herein, refers to cells derived from definitive endoderm and that can give rise to all endodermal organs, such as lungs, liver, pancreas, stomach, and intestine. Gut tube cells may be characterized by their substantially increased expression of HNF4a over that expressed by definitive endoderm cells. For example, a ten- to forty -fold increase in mRNA expression of HNF4a may be observed during Stage 2.

[0073] “Foregut endoderm cells”, as used herein, refers to cells that give rise to the esophagus, lungs, stomach, liver, pancreas, gall bladder, and a portion of the duodenum. The posterior foregut cells give rise to the liver and pancreas. Foregut endoderm cells express at least one of the following markers: PDX1, FOXA2, CDX2, SOX2, and HNF4[. Foregut endoderm cellsmay be characterized by an increase in expression of PDX1 compared to gut tube cells. For example, greater than fifty percent of the cells in Stage 3 cultures typically express PDX1.

[0074] “Pancreatic endoderm cells”, as used herein, refers to cells that express at least one of the following markers: PDX1, NKX6.1, HNFip, PTFla, HNF6, HNF4a, SOX9, NGN3, gastrin; HB9, or PROXI. Pancreatic endoderm cells may be characterized by their lack of substantial expression of CDX2 or SOX2.

[0075] “Pancreatic endocrine precursor cells” or “endocrine precursor cells”, as used herein, refers to pancreatic endoderm cells capable of becoming a pancreatic hormone expressing cell. Pancreatic endocrine precursor cells express at least one of the following markers: NGN3; NKX2.2; NeuroDl; ISL1; PAX4; PAX6; or ARX. Pancreatic endocrine precursor cells may be characterized by their expression of NKX2.2 and NeuroDl.

[0076] “Pancreatic endocrine cells”, as used herein, refer to cells capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin, ghrelin, and pancreatic polypeptide. In addition to these hormones, markers characteristic of pancreatic endocrine cells include one or more of NeuroDl, ISL1, PDX1, NKX6.1, PAX4, ARX, NKX2.2, HB9 and PAX6.

[0077] “Beta cells” (“P cells”) are pancreatic endocrine cells capable of expressing insulin, but not glucagon, somatostatin, ghrelin, and pancreatic polypeptide. Pancreatic endocrine cells expressing markers characteristic of beta cells can be characterized by their expression of insulin and at least one of the following transcription factors: PDX1, NKX2.2, NKX6.1, NeuroDl, ISL1, HNF3P, HB9, MAFA and PAX6. “Immature beta cells” (e.g., generated by the methods disclosed herein) express and are capable of producing insulin, but typically do not secrete insulin in response to glucose or exhibit other characteristics of mature beta cells. “Immature beta cells” are pancreatic endocrine cells that do not display glucose-dependent mitochondrial respiration / activity, and biphasic GSIS. Immature beta cells expressing markers characteristic of beta cells can be characterized by their expression of insulin and at least one of the following transcription factors: PDX1, NKX2.2, NKX6.1, NeuroDl, ISL1, HNF3P, HB9, MAFA, and PAX6.

[0078] “Functional beta-cells” or “mature beta cells” are pancreatic endocrine cells that display the well-established processes that ensure rapid and regulated glucose-stimulated insulin secretion (“GSIS”), specifically an increase in mitochondrial respiration / activity followed by the first phase and second phase of insulin secretion (“biphasic GSIS”). In detail, functional beta-cells exhibit at least one of the following characteristics of biphasic GSIS: (i) coupling of mitochondrial respiration / activity with insulin secretion; (ii) rapid insulin secretion response to heightened demand (here defined as high glucose concentration); (iii) ability to rapidly turn offinsulin secretion after demand has subsided; (iv) ability for multiple rounds of “on-off’ switching of insulin secretion; (v) ability to secrete the correct amount of insulin as dictated by demand; and (vi) ability to respond to multiple insulin secretagogues (for example, Exendin-4, or amino acids- L-Glutamine and L-Arginine). Mature beta-cells can be characterized by their expression of insulin and at least one of the following transcription factors: PDX1, NKX2.2, NKX6.1, NeuroDl, ISL1, HNF3P, HB9, PAX6, MAFA, SLC2A1, UCN3, and GLP1R. In some embodiments, the immature beta cells of the disclosure of capable of developing into mature beta cells. In some embodiments, immature beta cells develop into mature beta cells in vitro, ex vivo, or in vivo (e.g., following implantation in a subject). The method can comprise differentiating the immature P cells into mature P cells capable of producing insulin.Cell Compositions

[0079] Some embodiments disclosed herein provide cell compositions that include immature beta (P) (e.g., Stage 6 immature beta (P) cells).

[0080] In some embodiments, the immature beta cells are capable of differentiating into Stage 7 beta cells, which are functionally similar to pancreatic islet cells found in vivo. Such Stage 7 cells of share many characteristic features of beta cells which are important for normal beta cell function. In some embodiments, the Stage 7 cell exhibits a glucose stimulated insulin secretion (GSIS) response in vitro. In some embodiments, the Stage 7 cell exhibits a GSIS response in vivo. In some embodiments, the Stage 7 cell exhibits in vitro and in vivo GSIS responses. In some embodiments, the GSIS responses resemble the GSIS responses of an endogenous mature pancreatic beta cell. In some embodiments, the Stage 7 cell exhibits a GSIS response to at least one glucose challenge. In some embodiments, the Stage 7 cell exhibits a GSIS response to at least two sequential glucose challenges. In some embodiments, the Stage 7 cell exhibits a GSIS response to at least three sequential glucose challenges. In some embodiments, the GSIS responses resemble the GSIS response of endogenous human islets to multiple glucose challenges. In some embodiments, the GSIS response is observed immediately upon transplanting the cell into a human or animal. In some embodiments, the GSIS response is observed within approximately 24 hours of transplanting the cell into a human or animal. In some embodiments, the GSIS response is observed within approximately one week of transplanting the cell into a human or animal. In some embodiments, the GSIS response is observed within approximately two weeks of transplanting the cell into a human or animal. In some embodiments, the stimulation index of the cell as characterized by the ratio of insulin secreted in response to high glucose concentrations compared to low glucose concentrations is similar to the stimulation index of an endogenous mature pancreatic beta cell. In some embodiments, the Stage 7 cell exhibits astimulation index of greater than 1. In some embodiments, the Stage 7 cell exhibits a stimulation index of greater than or equal to 1. In some embodiments, the Stage 7 cell exhibits a stimulation index of greater than 1.1. In some embodiments, the beta cell exhibits a stimulation index of greater than or equal to 1.1. In some embodiments, the Stage 7 cell exhibits a stimulation index of greater than 2. In some embodiments, the Stage 7 cell exhibits a stimulation index of greater than or equal to 1. In some embodiments, the Stage 7 cell exhibits a stimulation index of at least 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 or greater.

[0081] In some embodiments, there are provided populations of cells comprising immature beta cells. In some embodiments, there are provided compositions comprising a population of immature beta cells. In some embodiments, at least 25% of the cells express PDX1 and NKX6.1, at least 10% of the cells express ISL1 and NKX6.1, at least 30% of the cells express NKX6.1 and INS, less than 1% of the seventh population of cells express GCG and INS, or any combination thereof. The proportion of cells expressing FOXA2, PDX1, NKX6.1, INS, ISL1, or any combination thereof, can be increased by at least 0.5-fold in the population of cells relative to a population of stem cells. In some embodiments, at least a portion of cells express one or more markers of differentiation. Each of the one or more markers can comprise CHGA, FOXA2, PDX1, or NGN3. In some embodiments, at least 0.5-fold more cells of the population of cells express one or more markers of differentiation each selected from the group consisting of CHGA, FOXA2, PDX1, and NGN3; as compared to a population of immature beta cells generated by previous methods.

[0082] The immature P cells can be capable of differentiating into mature P cells capable of secreting insulin in response to glucose. The method can comprise differentiating the immature P cells into mature P cells capable of secreting insulin in response to glucose. In some embodiments, the mature P cells capable of secreting insulin in response to glucose differentiated from immature P cells of the disclosure are more potent (e.g., have a greater response to glucose) than cells obtained using other known methods.

[0083] In some cases, any of the cell populations disclosed herein are unsorted, e.g., isolated cell populations that have not been through cell sorting process. In some embodiments, the cell populations disclosed herein can refer to a cell cluster formed by self-aggregation of cells cultured in a given environment, for instance, in a 3D suspension culture. In some embodiments, cell clusters disclosed herein are intermediate cell clusters formed during the differentiation process as described herein. In some cases, the intermediate cell clusters, e.g., cell clusters comprising PDX1 -positive, NKX6.1 -negative pancreatic progenitor cells (e.g., Stage 3 cell clusters) or cell clusters comprising PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells(e.g., Stage 4 cell clusters), are not subjected to cell sorting. In some case, cell populations going through cell sorting may not be able to form the intermediate cell clusters disclosed herein. For instance, PDX1 -positive pancreatic progenitor cells, after going through cell sorting, may not be able to form a cell cluster as disclosed herein.

[0084] Cell sorting as described herein can refer to a process of isolating a group of cells from a plurality of cells by relying on differences in cell size, shape (morphology), surface protein expression, endogenous signal protein expression, or any combination thereof. In some cases, cell sorting comprises subjecting the cells to flow cytometry. Flow cytometry can be a laser- or impedance-based, biophysical technology. During flow cytometry, one can suspend cells in a stream of fluid and pass them through an electronic detection apparatus. In one type of flow cytometry, fluorescent-activated cell sorting (FACS), based on one or more parameters of the cells’ optical properties (e.g., emission wavelength upon laser excitation), one can physically separate and thereby purify cells of interest using flow cytometry. As described herein, an unsorted cell cluster can be cell cluster that formed by a plurality of cells that have not been subject to an active cell sorting process, e.g, flow cytometry. In some cases, flow cytometry as discussed herein can be based on one or more signal peptides expressed in the cells. For example, a cell cluster can comprise cells that express a signal peptide (e.g., a fluorescent protein, e.g., green fluorescent protein (GFP) or tdTomato). In some cases, the signal peptide is expressed as an indicator of insulin expression in the cells. For instance, a cell cluster can comprise cell harboring an exogenous nucleic acid sequence coding for GFP under the control of an insulin promoter. The insulin promoter can be an endogenous or exogenous promoter. In some cases, the expression of GFP in these cells can be indicative of insulin expression in the cells. The GFP signal can thus be a marker of a pancreatic beta cell. In some cases, cell sorting as described herein can comprise magnetic-activated flow cytometry, where magnetic antibody or other ligand is used to label cells of different types, and the differences in magnetic properties can be used for cell sorting.

[0085] The percentage of cells expressing one or more particular markers, like PDX1, NKX6.1, insulin, NGN3, or CHGA, described herein can be the percentage value detected using techniques like flow cytometry assay. In some cases, during a flow cytometry assay, cell populations discussed herein are dispersed into single-cell suspension by incubation in digesting enzyme like trypsin or TrypLE™ Express. Dispersed cells can be washed in suitable buffer like PBS, centrifuged and then re-suspended in fixation buffer like 4%PFA. Incubation with primary antibodies against the cell markers of interest can then be conducted, which can be followed by incubation with the secondary antibodies. After antibody incubation, the cells can be washed and the subject to segregation by flow cytometry. Techniques other than flow cytometry can also be used to characterize the cells described herein, e.g., determine the cell percentages. Non-limitingexamples of cell characterization methods include gene sequencing, microscopic techniques (fluorescence microscopy, atomic force microscopy), karyotyping, isoenzyme analysis, DNA properties, and viral susceptibility.Stem Cells and Reprogramming

[0086] Differentiation is the process by which an unspecialized (“uncommitted”) or less specialized cell acquires the features of a specialized cell, for example a nerve cell or a muscle cell. A differentiated cell is one that has taken on a more specialized (“committed”) position within the lineage of a cell. The term “committed”, when applied to the process of differentiation, refers to a cell that has proceeded in the differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type. “De-differentiation” refers to the process by which a cell reverts to a less specialized (or committed) position within the lineage of a cell. As used herein, the lineage of a cell defines the heredity of the cell, i.e., which cells it came from and to what cells it can give rise. The lineage of a cell places the cell within a hereditary scheme of development and differentiation. A lineage-specific marker refers to a characteristic specifically associated with the phenotype of cells of a lineage of interest and can be used to assess the differentiation of an uncommitted cell to the lineage of interest. The stem cells can be induced pluripotent stem cells (iPSC), pluripotent stem cells (PSC), embryonic stem cells (ESC), or adult stem cells (ASC). The stem cells can be mammalian cells. The mammalian cells can be human cells.

[0087] Provided herein is the use of stem cells for producing immature beta cells or precursors thereof. In some embodiments, germ cells can be used in place of, or with, the stem cells to provide at least one immature beta cell. In some embodiments, the disclosure provides for methods of generating cell populations that are enriched for immature beta cells.

[0088] Generally, the at least one immature beta 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 primitive gut tube cells, PDXl-positive pancreatic progenitors, PDX1 -positive, NKX6.1 -positive pancreatic progenitors, Ngn3 -positive endocrine progenitor cells, and / or other pluripotent or stem cells.

[0089] The at least one immature beta 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 immature beta 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 oneimmature beta or the precursor thereof.

[0090] In some embodiments, the at least one immature beta cell or precursor thereof is a substantially pure population of immature beta cell or precursors thereof. In some embodiments, a population of immature beta cells or precursors thereof comprises a mixture of pluripotent cells or differentiated cells. In some embodiments, a population of immature beta cells or precursors thereof are substantially free or devoid of embryonic stem cells or pluripotent cells or iPS cells.

[0091] In some embodiments, a somatic cell, e.g., fibroblast can be isolated from a subject, for example as a tissue biopsy, such as, for example, a skin biopsy, and reprogrammed into an induced pluripotent stem cell for further differentiation to produce the at least one beta cell (e.g., immature beta cell) or precursor thereof for use in the compositions and methods described herein. In some embodiments, a somatic cell, e.g., fibroblast is maintained in culture by methods known by one of ordinary skill in the art, and in some embodiments, propagated prior to being converted into immature beta cells by the methods as disclosed herein.

[0092] In some embodiments, the at least one immature beta cell or precursor thereof are maintained in culture by methods known by one of ordinary skill in the art, and in some embodiments, propagated prior to being converted into immature beta cells by the methods as disclosed herein.

[0093] Further, at least one immature beta cell or precursor thereof, e.g., pancreatic progenitor can be from any mammalian species, with non-limiting examples including a murine, bovine, simian, porcine, equine, ovine, or human cell. For clarity and simplicity, the description of the methods herein refers to a mammalian at least one immature beta cell or precursor thereof but it should be understood that all of the methods described herein can be readily applied to other cell types of at least one immature beta cell or precursor thereof. In some embodiments, the at least one immature beta cell or precursor thereof is derived from a human individual.Stem Cells

[0094] Embodiments of the present disclosure are related to use of stem cells for generation of immature beta cells or precursors thereof. The term "stem cell" as used herein can refer 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" can be 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(e.g., 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 progenies. In some embodiments, the stem cell is an adult stem cell, a somatic stem cell, a non-embryonic stem cell, an embryonic stem cell, hematopoietic stem cell, a pluripotent stem cell, or a trophoblast stem cell.

[0095] Stem cells of interest, e.g, that can be used in the methods provided herein, can include pluripotent stem cells (PSCs). The term "pluripotent stem cell" or "PSC" as used herein can refer to 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 can be capable of forming teratomas and of contributing to ectoderm, mesoderm, or endoderm tissues in a living organism. Pluripotent stem cells of plants can be capable of giving rise to all cell types of the plant (e.g., cells of the root, stem, leaves, etc.).

[0096] Embodiments of the present disclosure are related to use of PSCs for generation of immature beta cells or precursors thereof. PSCs of animals can be derived in a number of different ways. For example, embryonic stem cells (ESCs) can be derived from the inner cell mass of an embryo (Thomson et. al, Science. 1998 Nov. 6; 282(5391): 1145-7) whereas induced pluripotent stem cells (iPSCs) can be derived from somatic cells (Takahashi et. al, Cell. 2007 Nov. 30; 131(5):861-72; Takahashi et. al, NatProtoc. 2007; 2(12):3081-9; Yu et. al, Science. 2007 Dec. 21; 318(5858): 1917-20. Epub 2007 Nov. 20). Because the term PSC can refer to pluripotent stem cells regardless of their derivation, the term PSC can encompass 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.

[0097] Embodiments of the present disclosure are related to use of ESCs for generation of immature beta cells or precursors thereof. By "embryonic stem cell" (ESC) can be 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-01, hESBGN-02, hESBGN-03, hESBGN-04 (BresaGen, Inc ); HES-1, HES-2, HES-3, HES-4, HES-5, HES-6 (ES Cell International); Miz-hESl (MizMedi Hospital- Seoul National University); HSF-1, HSF-6 (University of California at San Francisco); and HI, 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 cellscan 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 can grow as flat colonies with large nucleo-cytoplasmic ratios, defined borders and prominent nucleoli. In addition, ESCs can express SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and Alkaline Phosphatase, but not SSEA-1. Examples of methods of generating and characterizing ESCs can 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 WO99 / 20741, WOOl / 51616, and W003 / 020920, each of which is incorporated herein by its entirety.

[0098] By “embryonic germ stem cell” (EGSC) or “embryonic germ cell” or “EG cell”, it can be meant a PSC that is derived from germ cells and / or germ cell progenitors, e.g., primordial germ cells, e.g., 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 in its entirety.

[0099] Embodiments of the present disclosure are related to use of iPSCs for generation of immature beta cells or precursors thereof. By “induced pluripotent stem cell” or “iPSC”, it can be meant a PSC that is derived from a cell that is not a PSC (e.g., from a cell this is differentiated relative to a PSC). iPSCs can be derived from multiple different cell types, including terminally differentiated cells. iPSCs can have an ES cell-like morphology, growing as flat colonies with large nucleo-cytoplasmic ratios, defined borders and prominent nuclei. In addition, iPSCs can 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.

[0100] Examples of methods of generating and characterizing iPSCs can be found in, for example, US20090047263, US20090068742, US20090191159, US20090227032, US20090246875, and US20090304646, each of which are incorporated herein in its entirety. Generally, to generate iPSCs, somatic cells are provided with reprogramming factors (e.g., Oct4, SOX2, KLF4, MYC, Nanog, Lin28) known in the art to reprogram the somatic cells to become pluripotent stem cells.

[0101] Embodiments of the present disclosure are related to use of somatic cells for generation of immature beta cells or precursors thereof. By "somatic cell", it can be 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 can be cells that have differentiated sufficiently that they may not naturally generate cells of all three germ layers of the body, e.g., 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.

[0102] In some embodiments, the stem cells can be undifferentiated (e.g., a cell not committed to a specific lineage) prior to exposure to at least one differentiation factor or composition according to the methods as disclosed herein, whereas in other examples it can be desirable to differentiate the stem cells to one or more intermediate cell types prior to exposure of the at least one differentiation factor or composition described herein. For example, the stems cells can 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 embodiments, undifferentiated cells can appear in the two dimensions of a microscopic view in colonies of cells with high nuclear / cytoplasmic ratios and prominent nucleoli. The stem cells can be themselves (for example, without substantially any undifferentiated cells being present) or can be used in the presence of differentiated cells. In some examples, the stem cells can 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 can be present in the culture to assist in the growth of the stem cells. The fibroblast can be present during one stage of stem cell growth but not necessarily at all stages. For example, the fibroblast can 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.

[0103] Stem cells used in all aspects of the present invention can be any cells derived from any kind of tissue (e.g., embryonic tissue such as fetal or pre-fetal tissue, or adult tissue), which stem cells can 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 can be provided in the form of an established cell line, or they can be obtained directly from primary embryonic tissue and used immediately for differentiation. Included are cells listed in the NIH Human Embryonic Stem Cell Registry, e.g., hESBGN-01, hESBGN-02, hESBGN-03, hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3, HES-4, HES-5, HES-6 (ES Cell International); Miz-hESl (MizMedi Hospital-Seoul National University); HSF-1, FISF-6 (University of California at San Francisco); and HI, 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. In some embodiments, the source of human stem cells or pluripotent stem cells used for chemically-induced differentiation into mature, insulin positive cells do not involve destroying a human embryo.

[0104] In some embodiments, 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.

[0105] Stem cells that can be used in the methods provided herein can also include embryonic cells of various types, exemplified by human embryonic stem (hES) cells, as 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 applicable to the methods provided herein can be 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):205-16; etc.) 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. In some embodiments, a human embryo was not destroyed for the source of pluripotent cell used on the methods and compositions as disclosed herein. In some embodiments, a human embryo is not destroyed for the source of pluripotent cell used on the methods and compositions as disclosed herein.

[0106] A mixture of cells from a suitable source of endothelial, muscle, and / or neural stem cells can be harvested from a mammalian donor for the purpose of the present disclosure. A suitable source is the hematopoietic microenvironment. For example, circulating peripheral blood, preferably mobilized (e.g., recruited), may be removed from a subject. In some embodiments, the stem cells can be reprogrammed stem cells, such as stem cells derived from somatic or differentiated cells. In such embodiments, 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.

[0107] In some embodiments, the immature beta cells can be derived from one or more of trichocytes, keratinocytes, gonadotropes, corticotropes, thyrotropes, somatotropes, lactotrophs,chromaffin cells, parafollicular cells, glomus cells melanocytes, nevus cells, Merkel cells, odontoblasts, cementoblasts comeal keratocytes, retina Muller cells, retinal pigment epithelium cells, neurons, glias (e.g., oligodendrocyte astrocytes), ependymocytes, pinealocytes, pneumocytes (e.g., type I pneumocytes, and type II pneumocytes), clara cells, goblet cells, G cells, D cells, ECL cells, gastric chief cells, parietal cells, foveolar cells, K cells, D cells, I cells, goblet cells, paneth cells, enterocytes, microfold cells, hepatocytes, hepatic stellate cells (e.g., Kupffer cells from mesoderm), cholecystocytes, centroacinar cells, pancreatic stellate cells, pancreatic a cells, pancreatic beta cells, pancreatic d cells, pancreatic F cells, pancreatic e cells, thyroid (e.g., follicular cells), parathyroid (e.g., parathyroid chief cells), oxyphil cells, urothelial cells, osteoblasts, osteocytes, chondroblasts, chondrocytes, fibroblasts, fibrocytes, myoblasts, myocytes, myosatellite cells, tendon cells, cardiac muscle cells, lipoblasts, adipocytes, interstitial cells of cajal, angioblasts, endothelial cells, mesangial cells (e.g., intraglomerular mesangial cells and extraglomerular mesangial cells), juxtaglomerular cells, macula densa cells, stromal cells, interstitial cells, telocytes simple epithelial cells, podocytes, kidney proximal tubule brush border cells, sertoli cells, leydig cells, granulosa cells, peg cells, germ cells, spermatozoon ovums, lymphocytes, myeloid cells, endothelial progenitor cells, endothelial stem cells, angioblasts, mesoangioblasts, pericyte mural cells, splenocytes (e.g., T lymphocytes, B lymphocytes, dendritic cells, microphages, leukocytes), trophoblast stem cells, or any combination thereof.Reprosrammins

[0108] The term “reprogramming” as used herein can refer to the process that alters or reverses the differentiation state of a somatic cell. The cell can either be partially or terminally differentiated prior to the reprogramming. Reprogramming can encompass complete reversion of the differentiation state of a somatic cell to a pluripotent cell. Such complete reversal of differentiation can produce an induced pluripotent (iPS) cell. Reprogramming as used herein can also encompass partial reversion of a cell differentiation state, for example to a multipotent state or to a somatic cell that is neither pluripotent or multipotent, but is a cell that has lost one or more specific characteristics of the differentiated cell from which it arises, e.g., direct reprogramming of a differentiated cell to a different somatic cell type. Reprogramming can involve alteration, e.g., reversal, of at least some of the heritable patterns of nucleic acid modification (e.g., methylation), chromatin condensation, epigenetic changes, genomic imprinting, etc., that occur during cellular differentiation as a zygote develops into an adult.

[0109] As used herein, the term “reprogramming factor” can refer to a molecule that is associated with cell “reprogramming,” that is, differentiation, and / or de-differentiation, and / or trans-differentiation, such that a cell converts to a different cell type or phenotype. Reprogramming factors generally affect expression of genes associated with cell differentiation,de-differentiation and / or trans-differentiation. Transcription factors are examples of reprogramming factors.

[0110] The term “differentiation” and their grammatical equivalents as used herein can refer to the process by which a less specialized cell (e.g., a more naive cell with a higher cell potency) becomes a more specialized cell type (e.g., a less naive cell with a lower cell potency); and that the term “de-differentiation” can refer to the process by which a more specialized cell becomes a less specialized cell type (e.g., a more naive cell with a higher cell potency); and that the term “trans-differentiation” refers to the process by which a cell of a particular cell type converts to another cell type without significantly changing its “cell potency” or “naivety” level. Without wishing to be bound by theory, it is thought that cells “transdifferentiate” when they convert from one lineage-committed cell type or terminally differentiated cell type to another lineage-committed cell type or terminally differentiated cell type, without significantly changing their “cell potency” or “naivety” level.[oni] As used herein, the term “cell potency” is to be understood as referring to the ability of a cell to differentiate into cells of different lineages. For example, a pluripotent cell (e.g., a stem cell) has the potential to differentiate into cells of any of the three germ layers, that is, endoderm (interior stomach lining, gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood, urogenital), or ectoderm (epidermal tissues and nervous system), and accordingly has high cell potency; a multipotent cell (e.g., a stem cell or an induced stem cell of a certain type) has the ability to give rise to cells from a multiple, but limited, number of lineages (such as hematopoietic stem cells, cardiac stem cells, or neural stem cells, etc.) comparatively has a lower cell potency than pluripotent cells. Cells that are committed to a particular lineage or are terminally differentiated can have yet a lower cell potency. Specific examples of trans-differentiation known in the art include the conversion of e.g., fibroblasts to beta cells or from pancreatic exocrine cells to beta cells, etc.

[0112] Accordingly, the cell can be caused to differentiate into a more naive cell (e.g., a terminally differentiated cell can be de-differentiated to be multipotent or pluripotent); or the cell may be caused to de-differentiate into a less naive cell (e.g., a multipotent or pluripotent cell can be differentiated into a lineage-committed cell or a terminally differentiated cell). However, in some embodiments, the cell is caused to convert or transdifferentiate from one cell type (or phenotype) to another cell type (or phenotype), for example, with a similar cell potency level. Accordingly, in some embodiments, the inducing steps of the present disclosure can reprogram the cells of the present disclosure to differentiate, de-differentiate and / or transdifferentiate. In some embodiments, the inducing steps of the present disclosure may reprogram the cells to transdifferentiate.

[0113] Methods of reprogramming or inducing a particular type of cell to become another type of cell, for example, by differentiation, de-differentiation and / or trans-differentiation using one or more exogenous polynucleotide or polypeptide reprogramming factors are known to the person skilled in the art. Such methods may rely on the introduction of genetic material encoding one or more transcription factor(s) or other polypeptide(s) associated with cell reprogramming. For example, PDX1, Ngn3 and MafA, or functional fragments thereof are all known to encode peptides that can induce cell differentiation, de-differentiation and / or trans- differentiation of the cells of the present disclosure. In some methods known to the person skilled in the art, exogenous polypeptides (e.g., recombinant polypeptides) encoded by reprogramming genes (such as the above genes) are contacted with the cells to induce, for example, cells of the present disclosure. The person skilled in the art will appreciate that other genes may be associated with reprogramming of cells, and exogenous molecules encoding such genes (or functional fragments thereof) and the encoded polypeptides are also considered to be polynucleotide or polypeptide reprogramming factors e.g., polynucleotides or polypeptides that in turn affect expression levels of another gene associated with cell reprogramming). For example, it has been shown that the introduction of exogenous polynucleotide or polypeptide epigenetic gene silencers that decrease p53 inactivation increase the efficiency of inducing induced pluripotent stem cells (iPSC).

[0114] Accordingly, exogenous polynucleotides or polypeptides encoding epigenetic silencers and other genes or proteins that may be directly or indirectly involved in cell reprogramming or increasing cell programming efficiency would be considered to constitute an exogenous polynucleotide or polypeptide reprogramming factor. The person skilled in the art will appreciate that other methods of influencing cell reprogramming exist, such as introducing RNAi molecules (or genetic material encoding RNAi molecules) that can knock down expression of genes involved in inhibiting cell reprogramming. Accordingly, any exogenous polynucleotide molecule or polypeptide molecule that is associated with cell reprogramming, or enhances cell reprogramming, is to be understood to be an exogenous polynucleotide or polypeptide reprogramming factor as described herein.

[0115] In some embodiments, the method excludes the use of reprogramming factor(s) that are not small molecules. In some embodiments, the method can utilize “routine” tissue culture components such as culture media, serum, serum substitutes, supplements, antibiotics, etc., such as RPMI, Renal Epithelial Basal Medium (REBM), Dulbecco's Modified Eagle Medium (DMEM), MCDB131 medium, CMRL 1066 medium, F12, foetal calf serum (FCS), foetal bovine serum (FBS), bovine serum albumin (BSA), D-glucose, L-glutamine, GlutaMAX.TM.-l (dipeptide, L-alanine-L-glutamine), B27, heparin, progesterone, putrescine, laminin,nicotinamide, insulin, transferrin, sodium selenite, selenium, ethanolamine, human epidermal growth factor (hEGF), basic fibroblast growth factor (bFGF), hydrocortisone, epinephrine, normacin, penicillin, streptomycin, gentamicin and amphotericin, etc. It is to be understood that these typical tissue culture components (and other similar tissue culture components that are routinely used in tissue culture) are not small molecule reprogramming molecules for the purposes of the present disclosure. These components are either not small molecules as defined herein and / or are not reprogramming factors as defined herein.

[0116] Accordingly, in some embodiments, the present disclosure does not involve a culturing step of the cell(s) with one or more exogenous polynucleotide or polypeptide reprogramming factor(s). Accordingly, in some embodiments, the method of the present disclosure does not involve the introduction of one or more exogenous polynucleotide or polypeptide reprogramming factor(s), e.g., by introducing transposons, viral transgenic vectors (such as retroviral vectors), plasmids, mRNA, miRNA, peptides, or fragments of any of these molecules, that are involved in producing induced differentiated cells (e.g., immature beta cells) or, otherwise, inducing cells of the present disclosure to differentiate, de-differentiation and / or transdifferentiate.

[0117] In some embodiments, the method occurs in the absence of one or more exogenous polynucleotide or polypeptide reprogramming factor(s). Accordingly, it is to be understood that in some embodiments, the method of the present disclosure utilizes small molecules (e.g., HD AC inhibitors) to reprogram cells, without the addition of polypeptide transcription factors; other polypeptide factors specifically associated with inducing differentiation, de-differentiation, and / or transdifferentiation; polynucleotide sequences encoding polypeptide transcription factors, polynucleotide sequences encoding other polypeptide factors specifically associated with inducing differentiation, de-differentiation, and / or transdifferentiation; mRNA; interference RNA; microRNA and fragments thereof.Genetic modifications

[0118] The population of stem cells can be a population of genetically modified cells. In some embodiments, the stem cells can be genetically modified by an RNA-guided endonuclease system, (e.g., a CRISPR system comprising a CRISPR nuclease and a guide RNA). In some embodiments, a cell described herein comprises at least one gene edit and is referred to as an “engineered cell.” In some embodiments, a stem or progenitor cell comprises at least one gene edit, and that edit is maintained through differentiation. In some embodiments, a stem or progenitor cell comprises at least one gene edit, and that edit is maintained through differentiation into an immature cell such that the immature beta cell comprises the gene edit.

[0119] Genome editing generally refers to the process of modifying the nucleotide sequence of a genome, preferably in a precise or pre -determined manner. In some embodiments, genome editing methods, e.g., the CRISPR-endonuclease system, are used to genetically modify a cell as described herein, e.g., to create a gene-edited iPSC cell. In some embodiments, genome editing methods e.g., the CRISPR-endonuclease system, are used to genetically modify a cell as described herein. Any genome editing method to known to one of skill in the art is useful for engineering the cells described herein.

[0120] Examples of methods of genome editing described herein include methods of using site-directed nucleases to cut deoxyribonucleic acid (DNA) at precise target locations in the genome, thereby creating single-strand or double-strand DNA breaks at particular locations within the genome. Such breaks can be and regularly are repaired by natural, endogenous cellular processes, such as homology-directed repair (HDR) and non-homologous end joining (NHEJ), as described in Cox et al., “Therapeutic genome editing: prospects and challenges,”, Nature Medicine, 2015, 21(2), 121-31. These two main DNA repair processes consist of a family of alternative pathways. NHEJ directly joins the DNA ends resulting from a double-strand break, sometimes with the loss or addition of nucleotide sequence, which may disrupt or enhance gene expression. HDR utilizes a homologous sequence, or donor sequence, as a template for inserting a defined DNA sequence at the break point. The homologous sequence can be in the endogenous genome, such as a sister chromatid. Alternatively, the donor sequence can be an exogenous polynucleotide, such as a plasmid, a single-strand oligonucleotide, a double-stranded oligonucleotide, a duplex oligonucleotide or a virus, that has regions (e.g., left and right homology arms) of high homology with the nuclease-cleaved locus, but which can also contain additional sequence or sequence changes including deletions that can be incorporated into the cleaved target locus. A third repair mechanism can be microhomology-mediated end joining (MMEJ), also referred to as "Alternative NHEJ,” in which the genetic outcome is similar to NHEJ in that small deletions and insertions can occur at the cleavage site. MMEJ can make use of homologous sequences of a few base pairs flanking the DNA break site to drive a more favored DNA end joining repair outcome, and recent reports have further elucidated the molecular mechanism of this process; see, e.g., Cho and Greenberg, Nature, 2015, 518, 174-76; Kent et al., Nature Structural and Molecular Biology, 2015, 22(3):230-7; Mateos-Gomez et al., Nature, 2015, 518, 254-57; Ceccaldi et al., Nature, 2015, 528, 258-62. In some instances, it may be possible to predict likely repair outcomes based on analysis of potential microhomologies at the site of the DNA break.

[0121] Each of these genome editing mechanisms can be used to create desired genetic modifications. A step in the genome editing process can be to create one or two DNA breaks, thelatter as double-strand breaks or as two single-stranded breaks, in the target locus as near the site of intended mutation. This can be achieved via the use of endonucleases.Exemplary Genetic Modifications

[0122] In some embodiments, an engineered cell (e.g., a beta cell derived from a gene- edited iPSC) evades an immune response and / or survives following engraftment into a subject at higher success rates than an unmodified cell. In some embodiments, an engineered cell is hypoimmunogenic. In some embodiments, an engineered cell has improved persistency and / or immune evasiveness, as compared to an unmodified or wild-type cell.

[0123] In some embodiments, any cells described herein can be gene-edited using any gene-editing method. In some embodiments, a disrupted gene is a gene that does not encode functional protein. In some embodiments, a cell that comprises a disrupted gene does not express (e.g., at the cell surface) a detectable level (e.g., by antibody, e.g., by flow cytometry) of the protein encoded by the gene. A cell that does not express a detectable level of the protein may be referred to as a knockout cell.

[0124] In some embodiments, the cell can comprise one or more genetic modifications. In some embodiments, the one or more additional genetic modifications enable genetically modified cells, to increase their survival or viability and / or evade immune response following engraftment into a subject. In some embodiments, these strategies enable the cells to survive and / or evade immune response at higher success rates than an unmodified cell. Methods and cells comprising one or more additional edits are disclosed herein and are also described in PCT publications W02020049535A1, WO2021044377A1, WO2021044379A1, and WO2022144855A1, which are hereby incorporated by reference in their entireties.

[0125] In some embodiments, genetically modified cells comprise the introduction of at least one genetic modification within or near at least one gene that encodes a survival factor. In some embodiments, genetically modified cells comprise the introduction of at least one genetic modification within or near at least one gene that encodes a survival factor, wherein the genetic modification comprises an insertion of a polynucleotide encoding a tolerogenic factor. The genetically modified cells may further comprise at least one genetic modification within or near a gene that encodes one or more MHC-I or MHC-II human leukocyte antigens or a component or a transcriptional regulator of a MHC-I or MHC-II complex. The genetically modified cells may further comprise at least one genetic modification within or near a gene that encodes one or more MHC-I or MHC-II human leukocyte antigens or a component or a transcriptional regulator of a MHC-I or MHC-II complex, wherein said genetic modification comprises an insertion of a polynucleotide encoding a second tolerogenic factor.

[0126] As used herein, the term “survival factor” generally refers to a factor that, whenincreased or decreased in a cell, enables the cell, e.g., a genetically modified cell, to survive after transplantation or engraftment into a host subject at higher survival rates relative to an unmodified cell. In some embodiments, a survival factor is a human survival factor. In some embodiments, a survival factor is a member of a critical pathway involved in cell survival. In some embodiments, a critical pathway involved in cell survival has implications on hypoxia, reactive oxygen species, nutrient deprivation, and / or oxidative stress. In some embodiments, the genetic modification, e.g., deletion or insertion, of at least one survival factor enables a genetically modified cell to survive for a longer time period, e.g., at least 1.05, at least 1.1, at least 1.25, at least 1.5, at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, or at least 50 times longer time period, than an unmodified cell following engraftment. In some embodiments, a survival factor is MANF (NCBI Gene ID No: 7873), ZNF143 (NCBI Gene ID No: 7702), TXNIP (NCBI Gene ID No: 10628), FOXO1 (NCBI Gene ID No: 2308), or JNK (NCBI Gene ID No: 5599). In some embodiments, a survival factor is inserted into a cell. In some embodiments, a survival factor is deleted from a cell. In some embodiments, an insertion of a polynucleotide that encodes MANF enables a cell to survive after transplantation or engraftment into a host subject at higher survival rates relative to an unmodified cell. In some embodiments, a deletion or insertion-deletion mutation within or near a TXNIP gene enables a cell to survive after transplantation or engraftment into a host subject at higher survival rates relative to an unmodified cell.

[0127] As used herein, the term “tolerogenic factor” generally refers to a protein (e.g., expressed by a polynucleotide as described herein) that, when increased or decreased in a cell, enables the cell to inhibit or evade immune rejection after transplantation or engraftment into a host subject at higher rates relative to an unmodified cell. In some embodiments, a tolerogenic factor is a human tolerogenic factor. In some embodiments, the genetic modification of at least one tolerogenic factor (e.g., the insertion or deletion of at least one tolerogenic factor) enables a cell to inhibit or evade immune rejection with rates at least 1.05, at least 1.1, at least 1.25, at least 1.5, at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, or at least 50 times higher than an unmodified cell following engraftment. In some embodiments, a tolerogenic factor is TNFAIP3 (NCBI Gene ID No: 7128), CD39 (NCBI Gene ID No: 953), CD73 (NCBI Gene ID No. 4907), PD-L-1 (NCBI Gene ID No: 29126), HLA-E (NCBI Gene ID No: 3133), HLA-G (NCBI Gene ID No: 3135), CTLA-4 (NCBI Gene ID No: 1493), or CD47 (NCBI Gene ID No: 961). In some embodiments, a tolerogenic factor is inserted into a cell. In some embodiments, a tolerogenic factor is deleted from a cell. In some embodiments, an insertion of a polynucleotide that encodes TNFAIP3, CD39, CD73, HLA-E, PD-L-1, HLA-G, CTLA-4, and / or CD47 enables a cell to inhibit or evade immune rejection after transplantation or engraftment into a host subject.

[0128] As used herein, the term “transcriptional regulator of MHC-I or MHC-II”generally refers to a biomolecule that modulates, e.g., increases or decreases, the expression of a MHC-I and / or MHC-II human leukocyte antigen. In some embodiments, a biomolecule is a polynucleotide, e.g., a gene, or a protein. In some embodiments, a transcriptional regulator of MHC-I or MHC-II will increase or decrease the cell surface expression of at least one MHC-I or MHC-II protein. In some embodiments, a transcriptional regulator of MHC-I or MHC-II will increase or decrease the

[0129] In some embodiments, genetically modified cells comprise the introduction of at least one genetic modification within or near at least one gene that decreases the expression of one or more MHC-I and MHC-II human leukocyte antigens relative to an unmodified cell; at least one genetic modification that increases the expression of at least one polynucleotide that encodes a tolerogenic factor relative to an unmodified cell; and at least one genetic modification that alters the expression of at least one gene that encodes a survival factor relative to an unmodified cell. In some embodiments, genetically modified cells comprise at least one deletion or insertion-deletion mutation within or near at least one gene that alters the expression of one or more MHC-I and MHC-II human leukocyte antigens relative to an unmodified cell. In other embodiments, genetically modified cells comprise at least one deletion or insertion-deletion mutation within or near at least one gene that alters the expression of one or more MHC-I and MHC-II human leukocyte antigens relative to an unmodified cell; and at least one insertion of a polynucleotide that encodes at least one tolerogenic factor at a site that partially overlaps, completely overlaps, or is contained within, the site of a deletion of a gene that alters the expression of one or more MHC-I and MHC-II HLAs. In some embodiments, genetically modified cells comprise at least one genetic modification that alters the expression of at least one gene that encodes a survival factor relative to an unmodified cell.

[0130] The genes that encode the major histocompatibility complex (MHC) are located on human Chr. 6p21. The resultant proteins coded by the MHC genes are a series of surface proteins that are essential in donor compatibility during cellular transplantation. MHC genes are divided into MHC class I (MHC-I) and MHC class II (MHC-II). MHC-I genes (HLA- A, HLA-B, and HLA-C) are expressed in almost all tissue cell types, presenting “non-self’ antigen-processed peptides to CD8+ T cells, thereby promoting their activation to cytolytic CD8+ T cells. Transplanted or engrafted cells expressing “non-self’ MHC-I molecules will cause a robust cellular immune response directed at these cells and ultimately resulting in their demise by activated cytolytic CD8+ T cells. MHC-I proteins are intimately associated with B2M in the endoplasmic reticulum, which is essential for forming functional MHC-I molecules on the cell surface. In addition, there are three non-classical MHC-Ib molecules (HLA-E, HLA-F, and HLA- G), which have immune regulatory functions. MHC-II biomolecule include HLA-DP, HLA-DM,HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR. Due to their primary function in the immune response, MHC-I and MHC-II biomolecules contribute to immune rejection following cellular engraftment of non-host cells, e.g., cellular engraftment for purposes of regenerative medicine.

[0131] MHC-I cell surface molecules are composed of MHC-encoded heavy chains (HLA-A, HLA-B, or HLA-C) and the invariant subunit B2M. Thus, a reduction in the concentration of B2M within a cell allows for an effective method of reducing the cell surface expression of MHC-I cell surface molecules. In some embodiments, the genome of a cell has been modified to disrupt or decrease the expression of beta-2-microglobulin (B2M), also known as P2 microglobulin, B2 microglubulin, or IMD43. B2M is a non-polymorphic gene that encodes a common protein subunit required for surface expression of all polymorphic MHC class I heavy chains. HLA-I proteins are intimately associated with B2M in the endoplasmic reticulum, which is essential for forming functional, cell-surface expressed HLA-I molecules. In some embodiments, the genetic modification is generated using a CRISPR / Cas9 system, using e.g., a gRNA targeting B2M gene.

[0132] The gRNA / CRISPR nuclease complex targets and cleaves a target site in the B2M locus. Repair of a double-stranded break by NHEJ can result in a deletion of at least on nucleotide and / or an insertion of at least one nucleotide, thereby disrupting or eliminating expression of B2M. Alternatively, the B2M locus can be targeted by at least two CRISPR systems each comprising a different gRNA, such that cleavage at two sites in the B2M locus leads to a deletion of the sequence between the two cuts, thereby eliminating expression of B2M.

[0133] In some embodiments, genetically modified cells comprise at least one genetic modification that disrupts the expression of at least one gene that encodes a survival factor, such as TXNIP, relative to an unmodified cell. In some embodiments, the genome of a cell has been modified to decrease the expression of thioredoxin interacting protein (TXNIP), which is also known as EST01027, HHCPA78, THIF, VDUP1, or ARRDC6. TXNIP is metabolic gene involved in redox regulation that can also function as a tumor suppressor. Downregulation or knockout of TXNIP can protect cells from metabolic stress. In some embodiments, the genetic modification is generated using a CRISPR / Cas9 system, using e.g., a gRNA targeting TXNIP gene. The gRNA / CRISPR nuclease complex targets and cleaves a target site in the TXNIP gene locus. Repair of a double-stranded break by NHEJ can result in a deletion of at least one nucleotide and / or an insertion of at least one nucleotide, thereby disrupting or eliminating expression of TXNIP. Alternatively, insertion of a polynucleotide encoding an exogenous gene into the TXNIP gene locus can disrupt or eliminate expression of TXNIP.

[0134] In some embodiments, the genetic modification comprises insertion of one or more nucleotide sequences each encoding a tolerogenic factor. In some embodiments, thetolerogenic factor is PD-L-1 (programmed death ligand 1) also known as cluster of differentiation 274 (CD274), B7 homolog (B7-H, B7H1), PDCD1L1, PDCD1LG1, or PDL1. PD-L-1 appears to play a major role in suppressing the adaptive arm of immune system and is considered to be a co- inhibitory factor of the immune response. In some embodiments, the tolerogenic factor is HLA- E, also known as EA1.2, EA2.1, HLA-6.2, MHC, QA1, or major histocompatibility complex, class I, E. HLA-E is an important modulator of natural killer (NK) and cytotoxic T lymphocyte (CTL) activation and inhibitory function. In some embodiments, the tolerogenic factor is Tumor Necrosis Factor Alpha-Induced Protein 3 (TNFAIP3, also referred to herein as A20). TNFAIP3 was identified as a gene whose expression is rapidly induced by the tumor necrosis factor (TNF). The protein encoded by this gene is a zinc finger protein and ubiquitin-editing enzyme, and has been shown to inhibit NF-kappa B activation as well as TNF-mediated apoptosis. The encoded protein, which has both ubiquitin ligase and de-ubiquitinase activities, is involved in the cytokine- mediated immune and inflammatory responses. Several transcript variants encoding the same protein have been found for this gene.

[0135] In some embodiments, a polynucleotide encoding one or more survival factors, such as MANF, can be inserted into genetically modified or genetically unmodified cells to create genetically modified cells having increased survival. In some embodiments, the survival factor is MANF, which is also known as arginine-rich, mutated in early-stage tumors (ARMET), arginine- rich protein (ARP), or mesencephalic astrocyte derived neurotrophic factor. MANF is an endoplasmic reticulum (ER) stress-inducible neurotrophic factor that promotes proliferation and survival of pancreatic beta cells, as well as survival of dopaminergic neurons. In some embodiments, insertion of a polynucleotide encoding one or more survival factors, such as MANF, enables a genetically modified cell to survive after transplantation or engraftment into a host subject with rates at least 1.05, at least 1.1, at least 1.25, at least 1.5, at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, or at least 50 times higher than an unmodified cell following transplantation or engraftment.

[0136] In some embodiments, the genetic modification, e.g., insertion of at least one polynucleotide encoding at least one tolerogenic factor enables a genetically modified cell to inhibit or evade immune rejection with rates at least 1.05, at least 1.1, at least 1.25, at least 1.5, at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, or at least 50 times higher than an unmodified cell following engraftment. In some embodiments, a cell can comprise one or more of the genetic modifications described above.

[0137] In some embodiments, one or more nucleotide sequences each encoding a protein selected from PD-L1, MANF, TNFAIP3, and HLA-E (e.g., HLA-E trimer) is inserted into a gene locus. In some embodiments, the gene locus is B2M or TXNIP. In some embodiments, apolynucleotide encoding PD-L-1 is inserted at a site within or near a B2M gene locus or within or near a TXNIP gene locus. In some embodiments, a polynucleotide encoding PD-L-1 is inserted at a site within or near a B2M gene locus. In some embodiments, a polynucleotide encoding PD-L- 1 is inserted at a site within or near a B2M gene locus concurrent with or following a deletion of all or part of a B2M gene or promoter. In some embodiments, a polynucleotide encoding PD-L-1 is inserted at a site within or near a TXNIP gene locus concurrent with or following a deletion of all or part of a TXNIP gene or promoter. The polynucleotide encoding PD-L-1 can be operably linked to an exogenous promoter. The exogenous promoter can be a CAG or CAGGS promoter.

[0138] In some embodiments, a polynucleotide encoding HLA-E is inserted at a site within or near a B2M gene locus or within or near a TXNIP gene locus. In some embodiments, a polynucleotide encoding HLA-E is inserted at a site within or near a TXNIP gene locus. In some embodiments, a polynucleotide encoding HLA-E is inserted at a site within or near a B2M gene locus concurrent with or following a deletion of all or part of a B2M gene or promoter. In other embodiments, a polynucleotide encoding HLA-E is inserted at a site within or near a TXNIP gene locus concurrent with or following a deletion of all or part of a TXNIP gene or promoter. In some embodiments, the polynucleotide encoding HLA-E comprises a sequence encoding an HLA-E trimer, the HLA-E trimer comprising a B2M signal peptide fused to an HLA-G presentation peptide fused to a B2M membrane protein fused to HLA-E without its signal peptide. In some embodiments, the polynucleotide encoding HLA-E is operably linked to an exogenous promoter. The exogenous promoter can be a CMV promoter.

[0139] In some embodiments, a polynucleotide encoding TNFAIP3 is inserted at a site within or near a B2M gene locus or within or near a TXNIP gene locus. In some embodiments, a polynucleotide encoding TNFAIP3 is inserted at a site within or near a B2M gene locus. In some embodiments, a polynucleotide encoding TNFAIP3 is inserted at a site within or near a B2M gene locus concurrent with or following a deletion of all or part of a B2M gene or promoter. In some embodiments, a polynucleotide encoding TNFAIP3 is inserted at a site within or near a TXNIP gene locus concurrent with or following a deletion of all or part of a TXNIP gene or promoter. In some embodiments, the polynucleotide encoding TNFAIP3 is operably linked to an exogenous promoter. The exogenous promoter can be a CAG or CAGGS promoter.

[0140] In some embodiments, a polynucleotide encoding MANF is inserted at a site within or near a B2M gene locus or within or near a TXNIP gene locus. In some embodiments, a polynucleotide encoding MANF is inserted at a site within or near a B2M gene locus. In some embodiments, a polynucleotide encoding MANF is inserted at a site within or near a B2M gene locus concurrent with or following a deletion of all or part of a B2M gene or promoter. In other embodiments, a polynucleotide encoding MANF is inserted at a site within or near a TXNIP genelocus concurrent with or following a deletion of all or part of a TXNIP gene or promoter. The polynucleotide encoding MANF can be operably linked to an exogenous promoter. The exogenous promoter can be a CAG or CAGGS promoter.

[0141] In some embodiments, a polynucleotide encoding TNFAIP3 and PD-L-1 is inserted at a site within or near a B2M gene locus or within or near a TXNIP gene locus. In some embodiments, a polynucleotide encoding TNFAIP3 and PD-L-1 is inserted at a site within or near a B2M gene locus. In some embodiments, a polynucleotide encoding TNFAIP3 and PD-L-1 is inserted at a site within or near a B2M gene locus concurrent with or following a deletion of all or part of a B2M gene or promoter. The polynucleotide encoding TNFAIP3 and PD-L-1 comprises sequence encoding TNFAIP3 that is linked to sequence encoding a ribosome skip that is linked to sequence encoding PD-L-1. The ribosome skip can be a 2 A sequence family member, such as P2A. In some embodiments, the polynucleotide comprises TNFAIP3-P2A-PD-L-1 coding sequence.

[0142] In some embodiments, a polynucleotide encoding MANF and HLA-E is inserted at a site within or near a B2M gene locus or within or near a TXNIP gene locus. In some embodiments, a polynucleotide encoding MANF and HLA-E is inserted at a site within or near a TXNIP gene locus. In some embodiments, a polynucleotide encoding MANF and HLA-E is inserted at a site within or near a TXNIP gene locus concurrent with, or following a deletion of all or part of a TXNIP gene or promoter. In some embodiments, the polynucleotide encoding MANF and HLA-E comprises sequence encoding MANF that is linked to sequence encoding a ribosome skip that is linked to sequence encoding HLA-E. The ribosome skip can be a 2A sequence family member, such as P2A. In some embodiments, the sequence encoding HLA-E comprises sequence encoding a HLA-E trimer, the HLA-E trimer comprising a B2M signal peptide fused to an HLA- G presentation peptide fused to a B2M membrane protein fused to HLA-E without its signal peptide. In some embodiments the polynucleotide comprises MANF-P2A-HLA-E coding sequence. In some embodiments, the polynucleotide encoding MANF-P2A-HLA-E is operably linked to an exogenous promoter. The exogenous promoter can be a CAG or CAGGS promoter.

[0143] The one or more additional genetic modifications can comprise: a disrupted beta-2-microglobulin (B2M) gene and insertion of a nucleotide sequence encoding programmed death-ligand 1 (PD-L1) into the disrupted B2M gene, wherein the cell expresses PD-L1 and has reduced or eliminated expression of B2M; and / or a disrupted thioredoxin interacting protein (TXNIP) gene and insertion of a nucleotide sequence encoding HLA class I histocompatibility antigen, alpha chain E (HLA-E); optionally the nucleotide sequence encoding HLA-E encodes an HLA-E trimer comprising a B2M signal peptide fused to an HLA-G presentation peptide fused to a B2M membrane protein fused to HLA-E without its signal peptide; wherein the cell expressesHLA-E or the HLA-E trimer and has reduced or eliminated expression of TXNIP.

[0144] The one or more additional genetic modifications can comprise: a disrupted beta-2-microglobulin (B2M) gene and insertion of a nucleotide sequence encoding PD-L1 and a nucleotide sequence encoding tumor necrosis factor alpha induced protein 3 (TNFAIP3) into the disrupted B2M gene, wherein the cell expresses PD-L1 and TNFAIP3 and has reduced or eliminated expression of B2M; and / or a disrupted thioredoxin interacting protein (TXNIP) gene and insertion of a nucleotide sequence encoding HLA class I histocompatibility antigen, alpha chain E (HLA-E) and a nucleotide sequence mesencephalic astrocyte derived neurotrophic factor (MANF) into the disrupted TXNIP gene; optionally the nucleotide sequence encoding HLA-E encodes an HLA-E trimer comprising a B2M signal peptide fused to an HLA-G presentation peptide fused to a B2M membrane protein fused to HLA-E without its signal peptide; wherein the cell expresses HLA-E or the HLA-E trimer and MANF and has disrupted expression of TXNIP.

[0145] The nucleotide sequence encoding TNFAIP3 and the nucleotide sequence encoding PD-L1 can be linked by a nucleotide sequence encoding a P2A peptide such that the insertion of comprises a nucleotide sequence encoding TNFAIP3-P2A-PD-L1. The nucleotide sequence encoding the HLA-E trimer and the nucleotide sequence encoding MANF can be linked by a nucleotide sequence encoding a P2A peptide such that the insertion of comprises a nucleotide sequence encoding MANF-P2A-HLA-E trimer.

[0146] In some embodiments, the cells are engineered to comprise a safety switch. As used herein, the term “safety switch” generally refers to a biomolecule that leads a cell to undergo apoptosis. In some embodiments, a safety switch is a protein or gene. In some embodiments, a safety switch is a suicide gene. In some embodiments, a safety switch, e.g., herpes simplex virus thymidine kinase (HSV-tk), leads a cell to undergo apoptosis by metabolizing a prodrug, e.g., ganciclovir. In some embodiments, the overexpressed presence of a safety switch on its own leads a cell to undergo apoptosis. In some embodiments, a safety switch is a p53-based molecule, HSV- tk, or inducible caspase-9. In some embodiments, the cells further comprise increased expression of one or more safety switch proteins relative to an unmodified cell. In some embodiments, the cells comprise increased expression of one or more additional genes that encode a safety switch protein. In some embodiments, a safety switch is also a suicide gene. In some embodiments, a safety switch is herpes simplex virus-1 thymidine kinase (HSV-tk) or inducible caspase-9. In some embodiments, a polynucleotide that encodes at least one safety switch is inserted into a genome, e.g., into a safe harbor locus. In some other embodiments, the one or more additional genes that are genetically modified encode one or more of safety switch proteins; targeting modalities; receptors; signaling molecules; transcription factors; pharmaceutically active proteins or peptides; drug target candidates; and proteins promoting engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival thereof integrated with the construct.CRISPR Endonuclease System

[0147] The CRISPR-endonuclease system is a naturally occurring defense mechanism in prokaryotes that has been repurposed as a RNA-guided DNA-targeting platform used for gene editing. Accordingly, in some embodiments, a CRISPR-endonuclease system is utilized to introduce a gene edit into a cell. CRISPR systems include Types I, II, III, IV, V, and VI systems. In some embodiments, the CRISPR system is a Type II CRISPR / Cas9 system. In some embodiments, the CRISPR system is a Type V CRISPR / Cprf system. CRISPR systems rely on a DNA endonuclease, e.g., Cas9, and two noncoding RNAs - crisprRNA (crRNA) and transactivating RNA (tracrRNA) - to target the cleavage of DNA.

[0148] The crRNA drives sequence recognition and specificity of the CRISPR- endonuclease complex through Watson-Crick base pairing, typically with a ~20 nucleotide (nt) sequence in the target DNA. Changing the sequence of the 5’ 20 nt in the crRNA allows targeting of the CRISPR-endonuclease complex to specific loci. The CRISPR-endonuclease complex only binds DNA sequences that contain a sequence match to the first 20 nt of the single-guide RNA (sgRNA) if the target sequence is followed by a specific short DNA motif (with the sequence NGG) referred to as a protospacer adjacent motif (PAM).

[0149] TracrRNA hybridizes with the 3’ end of crRNA to form an RNA-duplex structure that is bound by the endonuclease to form the catalytically active CRISPR-endonuclease complex, which can then cleave the target DNA.

[0150] Once the CRISPR-endonuclease complex is bound to DNA at a target site, two independent nuclease domains within the endonuclease each cleave one of the DNA strands three bases upstream of the PAM site, leaving a double-strand break (DSB) where both strands of the DNA terminate in a base pair (a blunt end).

[0151] In some embodiments, the endonuclease is a Cas9 (CRISPR associated protein 9). In some embodiments, the Cas9 endonuclease is from Streptococcus pyogenes, although other Cas9 homologs may be used, e.g., S. aureus Cas9, N. meningitidis Cas9, S. thermophilus CRISPR 1 Cas9, S. thermophilus CRISPR 3 Cas9, or T. denticola Cas9. In some embodiments, the CRISPR endonuclease is Cpfl, e.g, L. bacterium ND2006 Cpfl or Acidaminococcus sp. BV3L6 Cpfl. In some embodiments, the endonuclease is Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslOO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, or Cpfl endonuclease. In some embodiments, wild-type variants may be used. In some embodiments, modified versions (e.g, a homolog thereof, a recombination of the naturally occurring moleculethereof, codon-optimized thereof, or modified versions thereof) of the preceding endonucleases may be used.

[0152] The CRISPR nuclease can be linked to at least one nuclear localization signal (NLS). The at least one NLS can be located at or within 50 amino acids of the amino-terminus of the CRISPR nuclease and / or at least one NLS can be located at or within 50 amino acids of the carboxy -terminus of the CRISPR nuclease.

[0153] Exemplary CRISPR / Cas polypeptides include the Cas9 polypeptides as published in Fonfara et al., “Phylogeny of Cas9 determines functional exchangeability of dual- RNA and Cas9 among orthologous type II CRISPR-Cas systems,” Nucleic Acids Research, 2014, 42: 2577-2590. The CRISPR / Cas gene naming system has undergone extensive rewriting since the Cas genes were discovered. Fonfara et al. also provides PAM sequences for the Cas9 polypeptides from various species.Zinc Finger Nucleases

[0154] In some embodiments, a zinc finger nuclease (ZFN) is used to introduce a gene edit into a cell. ZFNs are modular proteins comprised of an engineered zinc finger DNA binding domain linked to the catalytic domain of the type II endonuclease Fokl. Because FokI functions only as a dimer, a pair of ZFNs must be engineered to bind to cognate target “half-site” sequences on opposite DNA strands and with precise spacing between them to enable the catalytically active Fokl dimer to form. Upon dimerization of the Fokl domain, which itself has no sequence specificity per se, a DNA double-strand break is generated between the ZFN half-sites as the initiating step in genome editing.

[0155] The DNA binding domain of each ZFN is typically comprised of 3-6 zinc fingers of the abundant Cys2-His2 architecture, with each finger primarily recognizing a triplet of nucleotides on one strand of the target DNA sequence, although cross-strand interaction with a fourth nucleotide also can be important. Alteration of the amino acids of a finger in positions that make key contacts with the DNA alters the sequence specificity of a given finger. Thus, a four- finger zinc finger protein will selectively recognize a 12 bp target sequence, where the target sequence is a composite of the triplet preferences contributed by each finger, although triplet preference can be influenced to varying degrees by neighboring fingers. An important aspect of ZFNs is that they can be readily re-targeted to almost any genomic address simply by modifying individual fingers. In most applications of ZFNs, proteins of 4-6 fingers are used, recognizing 12- 18 bp respectively. Hence, a pair of ZFNs will typically recognize a combined target sequence of 24-36 bp, not including the typical 5-7 bp spacer between half-sites. The binding sites can be separated further with larger spacers, including 15-17 bp. A target sequence of this length is likely to be unique in the human genome, assuming repetitive sequences or gene homologs are excludedduring the design process. Nevertheless, the ZFN protein-DNA interactions are not absolute in their specificity so off-target binding and cleavage events do occur, either as a heterodimer between the two ZFNs, or as a homodimer of one or the other of the ZFNs. The latter possibility has been effectively eliminated by engineering the dimerization interface of the FokI domain to create “plus” and “minus” variants, also known as obligate heterodimer variants, which can only dimerize with each other, and not with themselves. Forcing the obligate heterodimer prevents formation of the homodimer. This has greatly enhanced specificity of ZFNs, as well as any other nuclease that adopts these FokI variants.

[0156] A variety of ZFN-based systems have been described in the art, modifications thereof are regularly reported, and numerous references describe rules and parameters that are used to guide the design of ZFNs; see, e.g., Segal et al., Proc Natl Acad Sci, 1999 96(6):2758-63; Dreier B et al., J Mol Biol., 2000, 303(4):489-502; Liu Q et al., J Biol Chem., 2002, 277(6):3850- 6; Dreier et al., J Biol Chem., 2005, 280(42):35588-97; and Dreier et al., J Biol Chem. 2001, 276(3 l):29466-78.Transcription Activator-Like Effector Nucleases (TALENs)

[0157] TALENs can be utilized to introduce a gene edit into a cell. TALENs represent another format of modular nucleases whereby, as with ZFNs, an engineered DNA binding domain is linked to the FokI nuclease domain, and a pair of TALENs operate in tandem to achieve targeted DNA cleavage. The major difference from ZFNs is the nature of the DNA binding domain and the associated target DNA sequence recognition properties. The TALEN DNA binding domain derives from TALE proteins, which were originally described in the plant bacterial pathogen Xanthomonas sp. TALEs are comprised of tandem arrays of 33-35 amino acid repeats, with each repeat recognizing a single base pair in the target DNA sequence that is typically up to 20 bp in length, giving a total target sequence length of up to 40 bp. Nucleotide specificity of each repeat is determined by the repeat variable diresidue (RVD), which includes just two amino acids at positions 12 and 13. The bases guanine, adenine, cytosine and thymine are predominantly recognized by the four RVDs: Asn-Asn, Asn-Ile, His-Asp and Asn-Gly, respectively. This constitutes a much simpler recognition code than for zinc fingers, and thus represents an advantage over the latter for nuclease design. Nevertheless, as with ZFNs, the protein-DNA interactions of TALENs are not absolute in their specificity, and TALENs have also benefitted from the use of obligate heterodimer variants of the FokI domain to reduce off-target activity.

[0158] Additional variants of the FokI domain have been created that are deactivated in their catalytic function. If one half of either a TALEN or a ZFN pair contains an inactive FokI domain, then only single-strand DNA cleavage (nicking) will occur at the target site, rather than a DSB. The outcome is comparable to the use of CRISPR / Cas9 or CRISPR / Cpfl “nickase”mutants in which one of the Cas9 cleavage domains has been deactivated. DNA nicks can be used to drive genome editing by HDR, but at lower efficiency than with a DSB. The main benefit is that off-target nicks are quickly and accurately repaired, unlike the DSB, which is prone to NHEJ- mediated mis-repair.

[0159] A variety of TALEN-based systems have been described in the art, and modifications thereof are reported; see, e.g., Boch, Science, 2009 326(5959): 1509-12; Mak et al., Science, 2012, 335(6069):716-9; and Moscou et al., Science, 2009, 326(5959): 1501. The use of TALENs based on the "Golden Gate" platform, or cloning scheme, has been described by multiple groups; see, e.g., Cermak et al., Nucleic Acids Res., 2011, 39(12):e82; Li et al., Nucleic Acids Res., 2011, 39(14):6315-25; Weber et al., PLoS One., 2011, 6(2):el6765; Wang et al., J Genet Genomics, 2014, 41(6):339-47; and Cermak T et al., Methods Mol Biol., 2015 1239: 133-59.Homing Endonucleases

[0160] In some embodiments, a homing endonuclease (HE) is used to introduce a gene edit into a cell. HEs are sequence -specific endonucleases that have long recognition sequences (14-44 base pairs) and cleave DNA with high specificity - often at sites unique in the genome. There are at least six known families of HEs as classified by their structure, including GIY-YIG, His-Cis box, H-N-H, PD-(DZE)xK, and Vsr-like that are derived from a broad range of hosts, including eukarya, protists, bacteria, archaea, cyanobacteria and phage. As with ZFNs and TALENs, HEs can be used to create a DSB at a target locus as the initial step in genome editing. In addition, some natural and engineered HEs cut only a single strand of DNA, thereby functioning as site-specific nickases. The large target sequence of HEs and the specificity that they offer have made them attractive candidates to create site-specific DSBs.

[0161] A variety of HE-based systems have been described in the art, and modifications thereof are regularly reported; see, e.g., the reviews by Steentoft et al., Glycobiology, 2014, 24(8):663-80; Belfort and Bonocora, Methods Mol Biol., 2014, 1123: 1-26; and Hafez and Hausner, Genome, 2012, 55(8): 553 -69.MegaTAL / Tev-mTALEN / MegaTev

[0162] As further examples of hybrid nucleases, the MegaTAL platform and Tev- mTALEN platform use a fusion of TALE DNA binding domains and catalytically active HEs, taking advantage of both the tunable DNA binding and specificity of the TALE, as well as the cleavage sequence specificity of the HE; see, e.g., Boissel et al., Nucleic Acids Res., 2014, 42: 2591-2601; Kleinstiver et al., G3, 2014, 4: 1155-65; and Boissel and Scharenberg, Methods Mol. Biol., 2015,

[0163] In a further variation, the MegaTev architecture is the fusion of a meganuclease (Mega) with the nuclease domain derived from the GIY -YIG homing endonuclease LTevI (Tev).The two active sites are positioned ~30 bp apart on a DNA substrate and generate two DSBs with non-compatible cohesive ends; see, e.g., Wolfs et al., Nucleic Acids Res., 2014, 42, 8816-29. It is anticipated that other combinations of existing nuclease-based approaches will evolve and be useful in achieving the targeted genome modifications described herein. dCas9-FokI or dCpfl -Fokl and Other Nucleases

[0164] Combining the structural and functional properties of the nuclease platforms described above offers a further approach to genome editing that can potentially overcome some of the inherent deficiencies. As an example, the CRISPR genome editing system typically uses a single Cas9 endonuclease to create a DSB. The specificity of targeting is driven by a 20 or 24 nucleotide sequence in the guide RNA that undergoes Watson-Crick base-pairing with the target DNA (plus an additional 2 bases in the adjacent NAG or NGG PAM sequence in the case of Cas9 from S., pyogenes). Such a sequence is long enough to be unique in the human genome, however, the specificity of the RNA / DNA interaction is not absolute, with significant promiscuity sometimes tolerated, particularly in the 5’ half of the target sequence, effectively reducing the number of bases that drive specificity. One solution to this has been to completely deactivate the Cas9 or Cpfl catalytic function - retaining only the RNA-guided DNA binding function - and instead fusing a Fokl domain to the deactivated Cas9; see, e.g., Tsai et al., Nature Biotech, 2014, 32: 569-76; and Guilinger et al., Nature Biotech., 2014, 32: 577-82. Because Fokl must dimerize to become catalytically active, two guide RNAs are required to tether two Fokl fusions in close proximity to form the dimer and cleave DNA. This essentially doubles the number of bases in the combined target sites, thereby increasing the stringency of targeting by CRISPR-based systems.

[0165] As further example, fusion of the TALE DNA binding domain to a catalytically active HE, such as I-TevI, takes advantage of both the tunable DNA binding and specificity of the TALE, as well as the cleavage sequence specificity of I-TevI, with the expectation that off-target cleavage can be further reduced.RNA-Guided Endonucleases

[0166] The RNA-guided endonuclease systems as used herein can comprise an amino acid sequence having at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% amino acid sequence identity to a wild-type exemplary endonuclease, e.g., Cas9 from S. pyogenes, US2014 / 0068797 Sequence ID No. 8 or Sapranauskas et al., Nucleic Acids Res, 39(21): 9275-9282 (2011). The endonuclease can comprise at least 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type endonuclease (e.g., Cas9 from S. pyogenes, supra) over 10 contiguous amino acids. The endonuclease can comprise at most: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type endonuclease (e.g., Cas9 from S. pyogenes, supra) over 10contiguous amino acids. The endonuclease can comprise at least: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type endonuclease (e.g., Cas9 from S. pyogenes, supra) over 10 contiguous amino acids in a HNH nuclease domain of the endonuclease. The endonuclease can comprise at most: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type endonuclease (e.g., Cas9 from S. pyogenes, supra) over 10 contiguous amino acids in a HNH nuclease domain of the endonuclease. The endonuclease can comprise at least: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wildtype endonuclease (e.g., Cas9 from S. pyogenes, supra) over 10 contiguous amino acids in a RuvC nuclease domain of the endonuclease. The endonuclease can comprise at most: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type endonuclease (e.g., Cas9 from S. pyogenes, supra) over 10 contiguous amino acids in a RuvC nuclease domain of the endonuclease.

[0167] The endonuclease can comprise a modified form of a wild-type exemplary endonuclease. The modified form of the wild-type exemplary endonuclease can comprise a mutation that reduces the nucleic acid-cleaving activity of the endonuclease. The modified form of the wild-type exemplary endonuclease can have less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid-cleaving activity of the wild-type exemplary endonuclease (e.g., Cas9 from S. pyogenes, supra). The modified form of the endonuclease can have no substantial nucleic acid-cleaving activity. When an endonuclease is a modified form that has no substantial nucleic acid-cleaving activity, it is referred to herein as "enzymatically inactive."

[0168] Mutations contemplated can include substitutions, additions, and deletions, or any combination thereof. The mutation converts the mutated amino acid to alanine. The mutation converts the mutated amino acid to another amino acid (e.g, glycine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, or arginine). The mutation converts the mutated amino acid to a non-natural amino acid (e.g, selenomethionine). The mutation converts the mutated amino acid to amino acid mimics (e.g., phosphomimics). The mutation can be a conservative mutation. For example, the mutation converts the mutated amino acid to amino acids that resemble the size, shape, charge, polarity, conformation, and / or retainers of the mutated amino acids (e.g., cysteine / serine mutation, lysine / asparagine mutation, histidine / phenylalanine mutation). The mutation can cause a shift in reading frame and / or the creation of a premature stop codon. Mutations can cause changes to regulatory regions of genes or loci that affect expression of one or more genes.Guide RNAs

[0169] In some embodiments, a guide RNA (gRNA) that can direct the activities of an associated endonuclease to a specific target site within a polynucleotide is used to introduce a gene edit into a cell. A guide RNA can comprise at least a spacer sequence that hybridizes to a target nucleic acid sequence of interest, and a CRISPR repeat sequence. In CRISPR Type II systems, the gRNA also comprises a second RNA called the tracrRNA sequence. In the CRISPR Type II guide RNA (gRNA), the CRISPR repeat sequence and tracrRNA sequence hybridize to each other to form a duplex. In CRISPR Type V systems, the gRNA comprises a crRNA that forms a duplex. In some embodiments, a gRNA can bind an endonuclease, such that the gRNA and endonuclease form a complex. The gRNA can provide target specificity to the complex by virtue of its association with the endonuclease. The genome-targeting nucleic acid thus can direct the activity of the endonuclease.

[0170] Exemplary guide RNAs include a spacer sequence that comprises 15-200 nucleotides wherein the gRNA targets a genome location based on the GRCh38 human genome assembly. As is understood by the person of ordinary skill in the art, each gRNA can be designed to include a spacer sequence complementary to its genomic target site or region. See Jinek et al., Science, 2012, 337, 816-821 and Deltcheva et al., Nature, 2011, 471, 602-607.

[0171] The gRNA can be a double-molecule guide RNA. The gRNA can be a single molecule guide RNA.

[0172] A double -molecule guide RNA can comprise two strands of RNA. The first strand comprises in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence and a minimum CRISPR repeat sequence. The second strand can comprise a minimum tracrRNA sequence (complementary to the minimum CRISPR repeat sequence), a 3’ tracrRNA sequence and an optional tracrRNA extension sequence.

[0173] A single -molecule guide RNA (sgRNA) can comprise, in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence, a minimum CRISPR repeat sequence, a single-molecule guide linker, a minimum tracrRNA sequence, a 3’ tracrRNA sequence and an optional tracrRNA extension sequence. The optional tracrRNA extension can comprise elements that contribute additional functionality (e.g., stability) to the guide RNA. The single-molecule guide linker can link the minimum CRISPR repeat and the minimum tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension can comprise one or more hairpins.

[0174] In some embodiments, a sgRNA comprises a 20 nucleotide spacer sequence at the 5’ end of the sgRNA sequence. In some embodiments, a sgRNA comprises a less than a 20 nucleotide spacer sequence at the 5’ end of the sgRNA sequence. In some embodiments, a sgRNA comprises a more than 20 nucleotide spacer sequence at the 5’ end of the sgRNA sequence. In some embodiments, a sgRNA comprises a variable length spacer sequence with 17-30 nucleotidesat the 5’ end of the sgRNA sequence. In some embodiments, a sgRNA comprises a spacer extension sequence with a length of more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, or 200 nucleotides. In some embodiments, a sgRNA comprises a spacer extension sequence with a length of less than 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides.

[0175] In some embodiments, a sgRNA comprises a spacer extension sequence that comprises another moiety (e.g., a stability control sequence, an endoribonuclease binding sequence, or a ribozyme). The moiety can decrease or increase the stability of a nucleic acid targeting nucleic acid. The moiety can be a transcriptional terminator segment (i.e., a transcription termination sequence). The moiety can function in a eukaryotic cell. The moiety can function in a prokaryotic cell. The moiety can function in both eukaryotic and prokaryotic cells. Non-limiting examples of suitable moi eties include: a 5' cap (e.g., a 7-methylguanylate cap (m7 G)), a riboswitch sequence (e.g., to allow for regulated stability and / or regulated accessibility by proteins and protein complexes), a sequence that forms a dsRNA duplex (i.e., a hairpin), a sequence that targets the RNA to a subcellular location (e.g., nucleus, mitochondria, chloroplasts, and the like), a modification or sequence that provides for tracking (e.g., direct conjugation to a fluorescent molecule, conjugation to a moiety that facilitates fluorescent detection, a sequence that allows for fluorescent detection, etc.), and / or a modification or sequence that provides a binding site for proteins (e.g., proteins that act on DNA, including transcriptional activators, transcriptional repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, and the like).

[0176] In some embodiments, a sgRNA comprises a spacer sequence that hybridizes to a sequence in a target polynucleotide. The spacer of a gRNA can interact with a target polynucleotide in a sequence-specific manner via hybridization (i.e., base pairing). The nucleotide sequence of the spacer can vary depending on the sequence of the target nucleic acid of interest.

[0177] In a CRISPR-endonuclease system, a spacer sequence can be designed to hybridize to a target polynucleotide that is located 5' of a PAM of the endonuclease used in the system. The spacer may perfectly match the target sequence or may have mismatches. Each endonuclease, e.g., Cas9 nuclease, has a particular PAM sequence that it recognizes in a target DNA. For example, S. pyogenes Cas9 recognizes a PAM that comprises the sequence 5'-NRG-3', where R comprises either A or G, where N is any nucleotide and N is immediately 3' of the target nucleic acid sequence targeted by the spacer sequence.

[0178] A target polynucleotide sequence can comprise 20 nucleotides. The target polynucleotide can comprise less than 20 nucleotides. The target polynucleotide can comprise more than 20 nucleotides. The target polynucleotide can comprise at least: 5, 10, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. The target polynucleotide can comprise at most: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. The target polynucleotide sequence can comprise 20 bases immediately 5' of the first nucleotide of the PAM.

[0179] A spacer sequence that hybridizes to a target polynucleotide can have a length of at least or at least about 6 nucleotides (nt). The spacer sequence can be at least or at least about 6 nt, at least or at least about 10 nt, at least or at least about 15 nt, at least or at least about 18 nt, at least or at least about 19 nt, at least or at least about 20 nt, at least or at least about 25 nt, at least or at least about 30 nt, at least or at least about 35 nt or at least or at least about 40 nt, from (or from about) 6 nt to (or to about) 80 nt, from (or from about) 6 nt to (or to about) 50 nt, from (or from about) 6 nt to (or to about) 45 nt, from (or from about) 6 nt to (or to about) 40 nt, from (or from about) 6 nt to (or to about) 35 nt, from (or from about) 6 nt to (or to about) 30 nt, from (or from about) 6 nt to (or to about) 25 nt, from (or from about) 6 nt to (or to about) 20 nt, from (or from about) 6 nt to (or to about) 19 nt, from (or from about) 10 nt to (or to about) 50 nt, from (or from about) 10 nt to (or to about) 45 nt, from (or from about) 10 nt to (or to about) 40 nt, from (or from about) 10 nt to (or to about) 35 nt, from (or from about) 10 nt to (or to about) 30 nt, from (or from about) 10 nt to (or to about) 25 nt, from (or from about) 10 nt to (or to about) 20 nt, from (or from about) 10 nt to (or to about) 19 nt, from (or from about) 19 nt to (or to about) 25 nt, from (or from about) 19 nt to (or to about) 30 nt, from (or from about) 19 nt to (or to about) 35 nt, from (or from about) 19 nt to (or to about) 40 nt, from (or from about) 19 nt to (or to about) 45 nt, from (or from about) 19 nt to (or to about) 50 nt, from (or from about) 19 nt to (or to about) 60 nt, from (or from about) 20 nt to (or to about) 25 nt, from (or from about) 20 nt to (or to about) 30 nt, from (or from about) 20 nt to (or to about) 35 nt, from (or from about) 20 nt to (or to about) 40 nt, from (or from about) 20 nt to (or to about) 45 nt, from (or from about) 20 nt to (or to about) 50 nt, or from (or from about) 20 nt to (or to about) 60 nt. In some examples, the spacer sequence can comprise 20 nucleotides. In some examples, the spacer can comprise 19 nucleotides. In some examples, the spacer can comprise 18 nucleotides. In some examples, the spacer can comprise 22 nucleotides.

[0180] In some examples, the percent complementarity between the spacer sequence and the target nucleic acid is at least or at least about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some examples, the percent complementarity between the spacer sequence and the target nucleic acid is at most or at most about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some examples, the percent complementarity between the spacer sequence and the target nucleic acid is 100% over the six contiguous 5 '-most nucleotides of the target sequence of the complementary strand of the target nucleic acid. The percent complementarity between the spacer sequence and the target nucleic acid can be at least 60% over about 20 contiguous nucleotides. The length of the spacersequence and the target nucleic acid can differ by 1 to 6 nucleotides, which may be thought of as a bulge or bulges.

[0181] A tracrRNA sequence can comprise nucleotides that hybridize to a minimum CRISPR repeat sequence in a cell. A minimum tracrRNA sequence and a minimum CRISPR repeat sequence may form a duplex, i.e. a base-paired double-stranded structure. Together, the minimum tracrRNA sequence and the minimum CRISPR repeat can bind to an RNA-guided endonuclease. At least a part of the minimum tracrRNA sequence can hybridize to the minimum CRISPR repeat sequence. The minimum tracrRNA sequence can be at least or at least about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the minimum CRISPR repeat sequence.

[0182] The minimum tracrRNA sequence can have a length from (or from about) 7 nucleotides to (or to about) 100 nucleotides. For example, the minimum tracrRNA sequence can be from (or from about) 7 nucleotides (nt) to (or to about) 50 nt, from (or from about) 7 nt to (or to about) 40 nt, from (or from about) 7 nt to (or to about) 30 nt, from (or from about) 7 nt to (or to about) 25 nt, from (or from about) 7 nt to (or to about) 20 nt, from (or from about) 7 nt to (or to about) 15 nt, from (or from about) 8 nt to (or to about) 40 nt, from (or from about) 8 nt to (or to about) 30 nt, from (or from about) 8 nt to (or to about) 25 nt, from (or from about) 8 nt to (or to about) 20 nt, from (or from about) 8 nt to (or to about) 15 nt, from (or from about) 15 nt to (or to about) 100 nt, from (or from about) 15 nt to (or to about) 80 nt, from (or from about) 15 nt to (or to about) 50 nt, from (or from about) 15 nt to (or to about) 40 nt, from (or from about) 15 nt to (or to about) 30 nt or from (or from about) 15 nt to (or to about) 25 nt long. The minimum tracrRNA sequence can be approximately 9 nucleotides in length. The minimum tracrRNA sequence can be approximately 12 nucleotides. The minimum tracrRNA can consist of tracrRNA nt 23-48 described in Jinek et al., supra.

[0183] The minimum tracrRNA sequence can be at least or at least about 60% identical to a reference minimum tracrRNA (e.g., wild type, tracrRNA from S. pyogenes) sequence over a stretch of at least 6, 7, or 8 contiguous nucleotides. For example, the minimum tracrRNA sequence can be at least or at least about 65% identical, 70% identical, 75% identical, 80% identical, 85% identical, 90% identical, 95% identical, 98% identical, 99% identical or 100% identical to a reference minimum tracrRNA sequence over a stretch of at least 6, 7, or 8 contiguous nucleotides.

[0184] The duplex between the minimum CRISPR RNA and the minimum tracrRNA can comprise a double helix. The duplex between the minimum CRISPR RNA and the minimum tracrRNA can comprise at least or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides. The duplex between the minimum CRISPR RNA and the minimum tracrRNA can comprise at most or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides.

[0185] The duplex can comprise a mismatch (i.e., the two strands of the duplex are not 100% complementary). The duplex can comprise at least or at least about 1, 2, 3, 4, or 5 or mismatches. The duplex can comprise at most or at most about 1, 2, 3, 4, or 5 or mismatches. The duplex can comprise no more than 2 mismatches.

[0186] In some embodiments, a tracrRNA may be a 3' tracrRNA. In some embodiments, a 3’ tracrRNA sequence can comprise a sequence with at least or at least about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to a reference tracrRNA sequence (e.g. a tracrRNA from S. pyogenes).

[0187] In some embodiments, a gRNA may comprise a tracrRNA extension sequence. A tracrRNA extension sequence can have a length from (or from about) 1 nucleotide to (or to about) 400 nucleotides. The tracrRNA extension sequence can have a length of more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, or 200 nucleotides. The tracrRNA extension sequence can have a length from (or from about) 20 to (or to about) 5000 or more nucleotides. The tracrRNA extension sequence can have a length of less than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides. The tracrRNA extension sequence can comprise less than 10 nucleotides in length. The tracrRNA extension sequence can be 10-30 nucleotides in length. The tracrRNA extension sequence can be 30-70 nucleotides in length.

[0188] The tracrRNA extension sequence can comprise a functional moiety (e.g., a stability control sequence, ribozyme, endoribonuclease binding sequence). The functional moiety can comprise a transcriptional terminator segment (i.e., a transcription termination sequence). The functional moiety can have a total length from (or from about) 10 nucleotides (nt) to (or to about) 100 nucleotides, from (or from about) 10 nt to (or to about) 20 nt, from (or from about) 20 nt to (or to about) 30 nt, from (or from about) 30 nt to (or to about) 40 nt, from (or from about) 40 nt to(or to about) 50 nt, from (or from about) 50 nt to (or to about) 60 nt, from (or from about) 60 nt to(or to about) 70 nt, from (or from about) 70 nt to (or to about) 80 nt, from (or from about) 80 nt to(or to about) 90 nt, or from (or from about) 90 nt to (or to about) 100 nt, from (or from about) 15 nt to (or to about) 80 nt, from (or from about) 15 nt to (or to about) 50 nt, from (or from about) 15 nt to (or to about) 40 nt, from (or from about) 15 nt to (or to about) 30 nt, or from (or from about) 15 nt to (or to about) 25 nt.

[0189] In some embodiments, a sgRNA may comprise a linker sequence with a length from (or from about) 3 nucleotides to (or to about) 100 nucleotides. In Jinek et al., supra, for example, a simple 4 nucleotide "tetraloop" (-GAAA-) was used (Jinek et al., Science, 2012, 337(6096): 816-821). An illustrative linker has a length from (or from about) 3 nucleotides (nt) to (or to about) 90 nt, from (or from about) 3 nt to (or to about) 80 nt, from (or from about) 3 nt to (or to about) 70 nt, from (or from about) 3 nt to (or to about) 60 nt, from (or from about) 3 nt to(or to about) 50 nt, from (or from about) 3 nt to (or to about) 40 nt, from (or from about) 3 nt to (or to about) 30 nt, from (or from about) 3 nt to (or to about) 20 nt, from (or from about) 3 nt to (or to about) 10 nt. For example, the linker can have a length from (or from about) 3 nt to (or to about) 5 nt, from (or from about) 5 nt to (or to about) 10 nt, from (or from about) 10 nt to (or to about) 15 nt, from (or from about) 15 nt to (or to about) 20 nt, from (or from about) 20 nt to (or to about) 25 nt, from (or from about) 25 nt to (or to about) 30 nt, from (or from about) 30 nt to (or to about) 35 nt, from (or from about) 35 nt to (or to about) 40 nt, from (or from about) 40 nt to (or to about) 50 nt, from (or from about) 50 nt to (or to about) 60 nt, from (or from about) 60 nt to (or to about) 70 nt, from (or from about) 70 nt to (or to about) 80 nt, from (or from about) 80 nt to (or to about) 90 nt, or from (or from about) 90 nt to (or to about) 100 nt. The linker of a single-molecule guide nucleic acid can be between 4 and 40 nucleotides. The linker can be at least or at least about 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, or 7000 or more nucleotides. The linker can be at most or at most about 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, or 7000 or more nucleotides.

[0190] Linkers can comprise any of a variety of sequences, although in some examples the linker will not comprise sequences that have extensive regions of homology with other portions of the guide RNA, which might cause intramolecular binding that could interfere with other functional regions of the guide. In Jinek et al., supra, a simple 4 nucleotide sequence - GAAA- was used (Jinek et al., Science, 2012, 337(6096):816-821), but numerous other sequences, including longer sequences can likewise be used.

[0191] The linker sequence can comprise a functional moiety. For example, the linker sequence can comprise one or more features, including an aptamer, a ribozyme, a proteininteracting hairpin, a protein binding site, a CRISPR array, an intron, or an exon. The linker sequence can comprise at least or at least about 1, 2, 3, 4, or 5 or more functional moieties. In some examples, the linker sequence can comprise at most or at most about 1, 2, 3, 4, or 5 or more functional moieties.

[0192] In some embodiments, a sgRNA does not comprise a uracil, e.g., at the 3 ’end of the sgRNA sequence. In some embodiments, a sgRNA does comprise one or more uracils, e.g., at the 3’end of the sgRNA sequence. In some embodiments, a sgRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 uracils (U) at the 3’ end of the sgRNA sequence.

[0193] An sgRNA may be chemically modified. In some embodiments, a chemically modified gRNA is a gRNA that comprises at least one nucleotide with a chemical modification, e.g., a 2'-O-methyl sugar modification. In some embodiments, a chemically modified gRNA comprises a modified nucleic acid backbone. In some embodiments, a chemically modified gRNA comprises a 2'-O-methyl-phosphorothioate residue. In some embodiments, chemicalmodifications enhance stability, reduce the likelihood or degree of innate immune response, and / or enhance other attributes, as described in the art.

[0194] In some embodiments, a modified gRNA may comprise a modified backbones, for example, phosphorothioates, phosphotriesters, morpholines, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages.

[0195] Morpholino-based compounds are described in Braasch and David Corey, Biochemistry, 2002, 41(14): 4503-4510; Genesis, 2001, Volume 30, Issue 3; Heasman, Dev. Biol., 2002, 243: 209-214; Nasevicius et al., Nat. Genet., 2000, 26:216-220; Lacerra et al., Proc. Natl. Acad. Sci., 2000, 97: 9591-9596.; and U.S. Pat. No. 5,034,506, issued Jul. 23, 1991.

[0196] Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al., J. Am. Chem. Soc., 2000, 122: 8595-8602.

[0197] In some embodiments, a modified gRNA may comprise one or more substituted sugar moi eties, e.g., one of the following at the 2' position: OH, SH, SCH3, F, OCN, OCH3, OCH3O(CH2)nCH3, O(CH2)nNH2, or O(CH2)nCH3, where n is from 1 to about 10; Cl to CIO lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; 2'-O-(2-methoxy ethyl); 2'-methoxy (2'-O- CH3); 2'-propoxy (2'-OCH2CH2CH3); and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on the gRNA, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' terminal nucleotide. In some examples, both a sugar and an inter-nucleoside linkage, i.e., the backbone, of the nucleotide units can be replaced with novel groups.

[0198] Guide RNAs can also include, additionally or alternatively, nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5 -methylcytosine (also referred to as 5 -methyl -2' deoxy cytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2-(methylamino)adenine, 2- (imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine, 5 -bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7- deazaguanine, N6 (6-aminohexyl)adenine, and 2,6-diaminopurine. Kornberg, A., DNAReplication, W. H. Freeman & Co., San Francisco, pp75-77, 1980; Gebeyehu et al., Nucl. Acids Res. 1997, 15:4513. A "universal" base known in the art, e.g., inosine, can also be included. 5- Me-C substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 °C. (Sanghvi, Y. S., in Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are aspects of base substitutions.

[0199] Modified nucleobases can comprise other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5- halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5- uracil (pseudo-uracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8- substituted adenines and guanines, 5-halo particularly 5-bromo, 5 -trifluoromethyl and other 5- substituted uracils and cytosines, 7-methylquanine and 7-methyladenine, 8-azaguanine and 8- azaadenine, 7-deazaguanine and 7-deazaadenine, and 3 -deazaguanine and 3 -deazaadenine.Complexes of a Genome-targeting Nucleic Acid and an Endonuclease

[0200] A gRNA interacts with an endonuclease (e.g., a RNA-guided nuclease such as Cas9), thereby forming a complex. The gRNA guides the endonuclease to a target polynucleotide.

[0201] The endonuclease and gRNA can each be administered separately to a cell or a subject. In some embodiments, the endonuclease can be pre-complexed with one or more guide RNAs, or one or more crRNA together with a tracrRNA. The pre-complexed material can then be administered to a cell or a subject. Such pre-complexed material is known as a ribonucleoprotein particle (RNP). The endonuclease in the RNP can be, for example, a Cas9 endonuclease or a Cpfl endonuclease. The endonuclease can be flanked at the N-terminus, the C-terminus, or both the N- terminus and C-terminus by one or more nuclear localization signals (NLSs). For example, a Cas9 endonuclease can be flanked by two NLSs, one NLS located at the N-terminus and the second NLS located at the C-terminus. The NLS can be any NLS known in the art, such as a SV40 NLS. The weight ratio of genome-targeting nucleic acid to endonuclease in the RNP can be 1 : 1. For example, the weight ratio of sgRNA to Cas9 endonuclease in the RNP can be 1 : 1.Base editing

[0202] In some embodiments, a gene can be edited using base editing. Base editing is a genome editing method that directly generates point mutations within a specific region of the genomic DNA without causing double-stranded breaks (DSB). DNA base editors (BEs) comprise fusions between a catalytically impaired Cas nuclease and a base-modification enzyme. Nucleobase editors typically include a polynucleotide programmable nucleotide binding domain and a nucleobase editing domain (e.g., adenosine deaminase, cytidine deaminase). Apolynucleotide programmable nucleotide binding domain, when in conjunction with a bound guide polynucleotide (e.g., gRNA), can specifically bind to a target polynucleotide sequence and thereby localize the base editor to the target nucleic acid sequence desired to be edited. In some embodiments, base editing can be used to introduce a loss-of-function mutation (e.g., premature stop codons, destabilizing mutations, altering splicing, etc.). In other embodiments, base editing can be used to correct, a mutation (e.g., a disease-causing mutation).

[0203] In some embodiments, base editors comprising a polynucleotide programmable nucleotide binding domain comprise all or a portion (e.g., a functional portion) of a CRISPR protein. In some embodiments, the polynucleotide programmable nucleotide binding domain comprises a nickase domain. Herein the term “nickase” shall be given its ordinary meaning, and shall also refer to a polynucleotide programmable nucleotide binding domain comprising a nuclease domain that is capable of cleaving only one strand of the two strands in a double-stranded nucleic acid molecule (e.g., DNA). For example, where a polynucleotide programmable nucleotide binding domain comprises a nickase domain derived from Cas9, the Cas9-derived nickase domain can include a D10A mutation and a histidine at position 840. In another example, a Cas9-derived nickase domain comprises an H840A mutation, while the amino acid residue at position 10 remains a D. In some embodiments, a Cas9 nuclease has an inactive (e.g, an inactivated) DNA cleavage domain (e.g, the Cas9 is a nickase, referred to as an “nCas9” protein). Suitable Cas9 nickases will be apparent to those of skill in the art based on this disclosure and knowledge in the field and are within the scope of this disclosure. In some embodiments, base editors comprise a polynucleotide programmable nucleotide binding domain which is catalytically dead (e.g., incapable of cleaving a target polynucleotide sequence). For example, in the case of a base editor comprising a Cas9 domain, the Cas9 can comprise both a D10A mutation and an H840A mutation. In further embodiments, a catalytically dead polynucleotide programmable nucleotide binding domain comprises a point mutation (e.g., D10A or H840A) as well as a deletion of all or a portion (e.g., a functional portion) of a nuclease domain.

[0204] In some embodiments, a base editor comprises an adenosine deaminase domain. Such an adenosine deaminase domain of a base editor can facilitate the editing of an adenine (A) nucleobase to a guanine (G) nucleobase by deaminating the A to form inosine (I), which exhibits base pairing properties of G. In some embodiments, an A-to-G base editor further comprises an inhibitor of inosine base excision repair, for example, a uracil glycosylase inhibitor (UGI) domain or a catalytically inactive inosine specific nuclease. Without wishing to be bound by any particular theory, the UGI domain or catalytically inactive inosine specific nuclease can inhibit or prevent base excision repair of a deaminated adenosine residue (e.g., inosine), which can improve the activity or efficiency of the base editor. The adenosine deaminase can be derivedfrom any suitable organism (e.g., E. coli, e.g., ecTadA deaminase). In some embodiments, the adenine deaminase is a naturally-occurring adenosine deaminase that includes one or more mutations. Details of A to G nucleobase editing proteins are described W02018 / 027078 and Gaudelli, N.M., et al., “Programmable base editing of A»T to G»C in genomic DNA without DNA cleavage” Nature, 551, 464-471 (2017), the entire contents of which are hereby incorporated by reference.

[0205] In some embodiments, a base editor comprises a fusion protein or complex comprising cytidine deaminase capable of deaminating a target cytidine (C) base of a polynucleotide to produce uridine (U), which has the base pairing properties of thymine. In some embodiments, for example where the polynucleotide is double-stranded (e.g., DNA), the uridine base can then be substituted with a thymidine base (e.g., by cellular repair machinery) to give rise to a C:G to a T:A transition. In other embodiments, deamination of a C to U in a nucleic acid by a base editor cannot be accompanied by substitution of the U to a T. The deamination of a target C in a polynucleotide to give rise to a U is a non-limiting example of a type of base editing that can be executed by a base editor described herein. In another example, a base editor comprising a cytidine deaminase domain can mediate conversion of a cytosine (C) base to a guanine (G) base. For example, a U of a polynucleotide produced by deamination of a cytidine by a cytidine deaminase domain of a base editor can be excised from the polynucleotide by a base excision repair mechanism (e.g., by a uracil DNA glycosylase (UDG) domain), producing an abasic site. The nucleobase opposite the abasic site can then be substituted (e.g., by base repair machinery) with another base, such as a C, by for example a translesion polymerase. Although it is typical for a nucleobase opposite an abasic site to be replaced with a C, other substitutions e.g., A, G or T) can also occur.

[0206] Accordingly, in some embodiments a base editor described herein comprises a deamination domain e.g., cytidine deaminase domain) capable of deaminating a target C to a U in a polynucleotide. Further, as described below, the base editor can comprise additional domains which facilitate conversion of the U resulting from deamination to, in some embodiments, a T or a G. For example, a base editor comprising a cytidine deaminase domain can further comprise a uracil glycosylase inhibitor (UGI) domain to mediate substitution of a U by a T, completing a C- to-T base editing event. In another example, the base editor can comprise a uracil stabilizing protein as described herein. In another example, a base editor can incorporate a translesion polymerase to improve the efficiency of C-to-G base editing, since a translesion polymerase can facilitate incorporation of a C opposite an abasic site (i.e., resulting in incorporation of a G at the abasic site, completing the C-to-G base editing event). A base editor comprising a cytidine deaminase as a domain can deaminate a target C in any polynucleotide, including DNA, RNA andDNA-RNA hybrids.

[0207] In some embodiments, a cytidine deaminase of a base editor comprises all or a portion (e.g., a functional portion) of an apolipoprotein B mRNA editing complex (APOB EC) family deaminase. APOBEC is a family of evolutionarily conserved cytidine deaminases. Members of this family are C-to-U editing enzymes. The N-terminal domain of APOBEC like proteins is the catalytic domain, while the C-terminal domain is a pseudocatalytic domain. More specifically, the catalytic domain is a zinc dependent cytidine deaminase domain and is important for cytidine deamination. APOBEC family members include APOBEC 1, AP0BEC2, AP0BEC3 A, AP0BEC3B, APOBEC3C, AP0BEC3D (“AP0BEC3E” now refers to this), APOBEC3F, AP0BEC3G, AP0BEC3H, AP0BEC4, and Activation-induced (cytidine) deaminase. In some embodiments, the deaminases are activation-induced deaminases (AID). In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise one or more mutations selected from the group consisting of H121R, H122R, R126A, R126E, R118A, W90A, W90Y, and R132E of rAPOBECl; D316R, D317R, R320A, R320E, R313 A, W285A, W285 Y, and R326E of hAPOBEC3G; and any alternative mutation at the corresponding position, or one or more corresponding mutations in another APOBEC deaminase. A number of modified cytidine deaminases are commercially available, including, but not limited to, SaBE3, SaKKH-BE3, VQR- BE3, EQR-BE3, VRER-BE3, YE1-BE3, EE-BE3, YE2-BE3, and YEE-BE3, which are available from Addgene (plasmids 85169, 85170, 85171, 85172, 85173, 85174, 85175, 85176, 85177). In some embodiments, a deaminase incorporated into a base editor comprises all or a portion (e.g., a functional portion) of an APOBEC 1 deaminase.

[0208] Details of C to T nucleobase editing proteins are described in W02017 / 070632 and Komor, A.C., et al., “Programmable editing of a target base in genomic DNA without doublestranded DNA cleavage” Nature 533, 420-424 (2016), the entire contents of which are hereby incorporated by reference.

[0209] A polynucleotide programmable nucleotide binding domain, when in conjunction with a bound guide polynucleotide (e.g., gRNA), can specifically bind to a target polynucleotide sequence (i.e., via complementary base pairing between bases of the bound guide nucleic acid and bases of the target polynucleotide sequence) and thereby localize the base editor to the target nucleic acid sequence desired to be edited (e.g., a double-stranded DNA target). In one embodiment, the guide polynucleotide is a gRNA. In some embodiments, the guide polynucleotide is at least one single guide RNA (“sgRNA” or “gRNA”). In some embodiments, the methods described herein can utilize an engineered Cas protein. A guide RNA (gRNA) is a short synthetic RNA composed of a scaffold sequence necessary for Cas-binding and a user- defined ~20 nucleotide spacer that defines the genomic target to be modified. Thus, the specificityof the Cas protein for the genomic target of the Cas protein is partially determined by how specific the gRNA targeting sequence is for the genomic target compared to the rest of the genome. In some embodiments, the spacer is or is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more nucleotides in length. The spacer of a gRNA can be or can be about 19, 20, or 21 nucleotides in length.Additional exemplary editing

[0210] In some embodiments, a gene can be edited using additional exemplary editing. In some embodiments, additional editing can be used to introduce a loss-of-function mutation (e.g., premature stop codons, destabilizing mutations, altering splicing, etc.). In other embodiments, the editing can be used to correct a mutation (e.g., a disease-causing mutation). The editing systems generally include an editor comprising a polynucleotide programmable nucleotide binding domain (e.g. , a nickase Cas9) and a DNA-polymerase domain (e.g. , a reverse transcriptase (RT), such as a Moloney murine leukemia virus reverse transcriptase (M-MLV RT)). The guide nucleic acid can contain an editing template. The gRNA can also include a primer-binding site (PBS). The PBS may be designed to hybridize with the displaced strand on the 5’ side of the introduced cut generated by the nickase. The PBS may be complementary to a portion of the protospacer sequence. The editing template sequence includes the edit to be installed and is typically located between the tracr region (e.g., scaffold or core region) and the PBS. The length of the edit to be installed may vary, e.g., from deletions of 10 or fewer nucleotides to insertions of more than 80 nucleotides. In some embodiments, the edit comprises a substitution of 1 or more nucleotides.

[0211] In some embodiments, the target sequence is bound by the nickase Cas9 (e.g., Cas9-H840A domain) via the spacer region of the guide RNA (gRNA). The hybridization of the spacer sequence to the complementary target sequences can result in displacement of the other strand (e.g., the PAM-strand or the edit strand). The Cas9-H840A domain can cut the displaced strand, and the displaced strand then may pair with the PBS. The RT may recognize the RNA- DNA duplex formed by the displaced strand and PBS and extend the DNA of the displaced strand in the 3’ direction, using the editing template (e.g., RT template) of the gRNA as a template. This can create a “flap” of single-stranded DNA on the displaced strand including the desired edit. The editor may then dissociate from the DNA, leaving two redundant “flaps” on the displaced strand, wherein one flap is the original sequence, and one flap is the edited sequence. Through a process called “flap equilibration”, one of the sequences will bind the target sequence, and the other will remain attached to the displaced strand as a single-stranded flap. If the flap with the edited sequence is bound by the target sequence, the complex may be called a “DNA heteroduplex”, in view of the mismatch caused by the edit. Cellular DNA repair machinery may then act on theDNA heteroduplex, incorporating the edit.

[0212] In some embodiments, where an editor comprises a nickase domain derived from Cas9, the Cas9-derived nickase domain can include a D10A mutation and a histidine at position 840. In another example, a Cas9-derived nickase domain comprises an H840A mutation, while the amino acid residue at position 10 remains a D. In some embodiments, a Cas9 nuclease has an inactive (e.g., an inactivated) DNA cleavage domain, that is, the Cas9 is a nickase, referred to as an “nCas9” protein. The Cas9 nickase may be a Cas9 protein that is capable of cleaving only one strand of a duplexed nucleic acid molecule (e.g., a duplexed DNA molecule). Additional suitable Cas9 nickases will be apparent to those of skill in the art based on this disclosure and knowledge in the field and are within the scope of this disclosure. In some embodiments, an editor comprises an RNA-dependent DNA polymerase domain, such as a reverse transcriptase (RT). In some embodiments, an editor comprises a virus RT, such as a retrovirus RT (e.g., Moloney murine leukemia virus (M-MLV or MLVRT)). In some embodiments, an editor may comprise a fusion of an S. pyogenes Cas9 polypeptide and a Moloney murine leukemia virus (M-MLV) reverse transcriptase polypeptide.

[0213] The gRNA can refer to a guide polynucleotide that comprises one or more intended nucleotide edits for incorporation into the target DNA. In some embodiments, the gRNA associates with and directs an editor to incorporate the one or more intended nucleotide edits into the target gene via editing. “Nucleotide edit” or “intended nucleotide edit” shall be given their ordinary meaning, and shall also refer to a specified deletion of one or more nucleotides at one specific position, insertion of one or more nucleotides at one specific position, substitution of a single nucleotide, or other alterations at one specific position to be incorporated into the sequence of the target gene. Intended nucleotide edit may refer to the edit on the editing template as compared to the sequence on the target strand of the target gene or may refer to the edit encoded by the editing template on the newly synthesized single stranded DNA. In some embodiments, a gRNA comprises a spacer sequence that is complementary or substantially complementary to a sequence on a target strand of the target gene. In some embodiments, the gRNA comprises a gRNA core that associates with a DNA binding domain, e.g., a CRISPR-Cas protein domain, of an editor. In some embodiments, the gRNA further comprises an extended nucleotide sequence comprising one or more intended nucleotide edits compared to the endogenous sequence of the target gene, wherein the extended nucleotide sequence may be referred to as an extension arm.

[0214] The extension arm can comprise a primer binding site sequence (PBS) that can initiate target-primed DNA synthesis. In some embodiments, the PBS is complementary or substantially complementary to a free 3’ end on the edit strand of the target gene at a nick site generated by the editor. In some embodiments, the extension arm further comprises an editingtemplate that comprises one or more intended nucleotide edits to be incorporated in the target gene by editing. In some embodiments, the editing template is a template for an RNA-dependent DNA polymerase domain or polypeptide of the editor, for example, a reverse transcriptase domain. In some embodiments, the editing template comprises partial complementarity to an editing target sequence in the target gene. In some embodiments, the editing template comprises substantial or partial complementarity to the editing target sequence except at the position(s) of the intended nucleotide edit(s) to be incorporated into the target gene.

[0215] Some editors include a Cas9 variant comprising an H840A mutation (i.e., a Cas9 nickase) and an M-MLV RT wild type, as well as an N-terminal NLS sequence (19 amino acids) and an amino acid linker (32 amino acids) that joins the C-terminus of the Cas9 nickase domain to the N-terminus of the RT domain. The fusion protein can have the following structure: [NLS]-[Cas9(H840A)]-[linker]-[MMLV_RT(wt)]. The editor proteins can, in some instances, include a Cas9 variant comprising an H840A mutation (i.e., a Cas9 nickase) and an M-MLV RT comprising mutations D200N, T330P, L603W, T306K, and W313F, as well as an N-terminal NLS sequence (19 amino acids) and an amino acid linker (33 amino acids) that joins the C-terminus of the Cas9 nickase domain to the N-terminus of the RT domain. The fusion protein can have the following structure: [NLS]-[Cas9(H840A)]-[linker]-[MML V_RT(D200N)(T330P)(L603 W)(T306K)(W313 F)] .

[0216] In some embodiments, the editing system or composition further comprises a nick guide polynucleotide, such as a nick guide RNA (ngRNA). Without wishing to be bound by any particular theory, the non-edit strand of a double stranded target DNA in the target gene may be nicked by a CRISPR-Cas nickase directed by an ngRNA. In some embodiments, the nick on the non-edit strand directs endogenous DNA repair machinery to use the edit strand as a template for repair of the non-edit strand, which may increase efficiency of editing. Some editor systems have an editor plus a second-strand nicking guide RNA that complexes with the editor and introduces a nick in the non-edited DNA strand in order to induce preferential replacement of the edited strand. In some editors, the second-strand nicking guide RNA is designed for temporal control such that the second strand nick is not introduced until after the installation of the desired edit. This is achieved by designing a gRNA with a spacer sequence that matches only the edited strand, but not the original allele. Using this strategy, mismatches between the protospacer and the unedited allele should disfavor nicking by the sgRNA until after the editing event on the PAM strand takes place. Some additional editors comprise a fusion protein comprising Cas9(R221K N39K H840A) and a variant MMLV RT pentamutant (D200N T306K W313F T330P L603W) having the following structure: [bipartite NLS]-[Cas9(R221K)(N394K)(H840A)]-[linker]- [MMLV_RT(D200N)(T330P)(L603W)]-[bipartite NLS]-[NLS] + a desired gRNA.

[0217] Some of the methods and compositions related to the editing disclosed herein are also described in W02023015309, W02022150790, W02022067130, WO2020191233, WO2020191234, WO2020191239, W02020191241, WO2020191242, WO2020191243, WO2020191245, WO2020191246, WO2020191248, WO2020191249, W02020191153, and W02020191171, the contents of which are incorporated herein by reference in their entireties.Methods of generating stem cell-derived immature beta cells

[0218] Provided herein are methods of generating immature beta cells ex vivo or in vitro.

[0219] The endoderm can give rise to digestive and respiratory tracts, thyroid, liver, and pancreas. Representative disease of endoderm lineages is type 1 diabetes resulting from destruction of the insulin-producing beta cells. Pancreatic beta cells are critical for maintaining glucose homeostasis via insulin secretion. Generation of functional beta cells from human pluripotent stem cells or induced pluripotent stem cells (hPSC, iPSC) in vitro can be practical, renewable cell source for replacement cell therapy for type 1 diabetes or other diseases related to glucose homeostasis. Other diseases related to abnormal glucose homeostasis include, but are not limited to, hyperglycemia, metabolic syndrome, obesity, hypercholesterolemia, and / or hypertension. In some embodiments, a subject can have one or more diseases related to abnormal glucose homeostasis.

[0220] 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 immature beta 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, and directing ES cells into the endoderm lineage is a prerequisite for generating therapeutic endoderm derivatives.

[0221] Precise patterning of anterior-posterior axis of the definitive endoderm can eventually form the primitive gut tube. The definitive endoderm-derived primitive gut tube develops into 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 andlarge 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).

[0222] The successful differentiation to beta cells requires, in some embodiments, that differentiated cells synthesize insulin. An exemplary stepwise protocol directing pancreatic cell differentiation is described herein, which entails differentiation processes that recapitulates the major stages of normal pancreatic endocrine development. The differentiation of stem cells to hormone-expressing pancreatic endocrine cells is conducted by transiting the 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, cells can be obtained that possess or have the potential to become cells that have a pancreatic endocrine phenotype and ability of glucose responsive insulin secretion in vitro.

[0223] Generally, the at least one immature beta 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 PDXl-positive pancreatic progenitors, pancreatic progenitors co-expressing PDX1 and NKX6-1, Ngn3 -positive endocrine progenitor cells, insulin-positive endocrine cells (e.g., NKX6.1 -positive, ISLl-positive cells, or beta-like cells), and / or other pluripotent or stem cells.

[0224] In some embodiments, the at least one immature beta cell or precursor thereof is a substantially pure population of immature beta cells or precursors thereof. In some embodiments, a population of immature beta cells or precursors thereof comprises a mixture of pluripotent cells or differentiated cells. In some embodiments, a population of immature beta cells or precursors thereof are substantially free or devoid of embryonic stem cells or pluripotent cells or iPS cells.

[0225] In some embodiments, a somatic cell, e.g., fibroblast can be isolated from a subject, for example as a tissue biopsy, such as, for example, a skin biopsy, and reprogrammed into an induced pluripotent stem cell for further differentiation to produce the at least one immature beta cell or precursor thereof for use in the compositions and methods described herein. In some embodiments, a somatic cell, e.g., fibroblast is maintained in culture by methods known by one of ordinary skill in the art, and in some embodiments, propagated prior to being converted into pancreatic beta cells by the methods as disclosed herein.

[0226] Further, at least one immature beta cell or precursor thereof, e.g., pancreatic progenitor can be from any mammalian species, with non-limiting examples including a murine, bovine, simian, porcine, equine, ovine, or human cell. For clarity and simplicity, the description of the methods herein refers to a mammalian at least one immature beta cell or precursor thereof but it should be understood that all of the methods described herein can be readily applied to other cell types of at least one pancreatic beta cell or precursor thereof. In some embodiments, the at least one pancreatic beta cell or precursor thereof is derived from a human individual.

[0227] Provided herein are methods and compositions for generating immature P cells from stem cells. The addition of FGF2 to culture media for generating Stage 1 definitive endoderm can, for example, result in improved generation of immature P cells (e.g., as assessed by gene marker expression). The addition of Thiazovivin to culture media for generating Stage 1 definitive endoderm can result in the generation of immature P cells with increased potency as compared to previous methods (e.g., increased glucose-stimulated insulin secretion). The addition of a defined, non-serum serum replacement to culture media for generating Stage 1 definitive endoderm can result in increased yield of immature P cells.

[0228] Disclosed herein include methods for generating immature P cells from stem cells. In some embodiments, the method comprises: (a) culturing a population of stem cells, (i) in a first medium comprising AA and a WNT pathway activator to generate a first population of progenitor cells, and (ii) culturing the first population of progenitor cells in a second medium comprising AA and FGF2 for a time sufficient to generate a second population of progenitor cells comprising definitive endoderm cells; (b) culturing the second population of progenitor cells in a third medium comprising FGF7 for a time sufficient to generate a third population of progenitor cells comprising gut tube cells; (c) culturing the third population of progenitor cells in a fourth medium comprising FGF7, retinoic acid, a Sonic Hedgehog (Shh) pathway inhibitor, and a protein kinase C (PKC) activator for a time sufficient to generate a fourth population of progenitor cells comprising posterior foregut cells; (d) culturing the fourth population of progenitor cells in a fifth medium comprising FGF7, retinoic acid, a Shh pathway inhibitor, and a PKC activator, wherein the fifth medium comprises a lower concentration of FGF7 and a lower concentration of retinoic acid than the fourth medium, for a time sufficient to generate a fifth population of progenitor cells comprising pancreatic endoderm cells, optionally the fifth medium comprises a bone morphogenetic protein (BMP) antagonist; (e) culturing the fifth population of progenitor cells in a sixth medium comprising retinoic acid, a Shh pathway inhibitor, a bone morphogenetic protein (BMP) antagonist, an anaplastic lymphoma kinase 5 (ALK-5) inhibitor, a thyroid hormone, and an anti-coagulant for a time sufficient to generate a sixth population of progenitor cells comprising endocrine precursor cells; and (f) culturing the sixth population of progenitor cells in a seventhmedium comprising a BMP antagonist, an ALK-5 inhibitor, a thyroid hormone, an anti-coagulant, and a y-secretase inhibitor, for a time sufficient to generate a seventh population of cells comprising immature P cells. In some embodiments, the first, second, third, fourth, fifth, sixth, and / or seventh media comprise B27 supplement. In some embodiments, the first, second, third, fourth, fifth, sixth, and / or seventh media comprise about 1% B27 supplement.

[0229] The population of stem cells can be cultured in the first culture medium for at least 12 hours. The population of stem cells can be cultured in the first culture medium for at least 24 hours. The WNT pathway activator of the first medium can be CHIR. The Shh pathway inhibitor of the fourth, fifth, and / or sixth medium can be SANT. The PKC activator of the fourth and / or fifth medium can be (2E,4E)-N-[(2S,5S)-l,2,3,4,5,6-Hexahydro-5-(hydroxymethyl)-l- methyl-2-(l-methylethyl)-3-oxo-l,4-benzodiazocin-8-yl]-5-[4-(trifluoromethyl)phenyl]-2,4- pentadienamide (TPPB). The BMP antagonist of the fifth, sixth and / or seventh medium can be LDN-193189-HCL. The ALK-5 inhibitor of the sixth and / or seventh medium can be RepSox. The thyroid hormone of the sixth and / or seventh medium can be 3,3’,5-Triiodo-L-thyronine sodium salt (T3). The y-secretase inhibitor of the seventh medium can be DBZ. The anti-coagulant of the sixth and / or seventh medium can be Heparin.

[0230] In some embodiments, the first medium, the second medium, or both comprise a ROCK inhibitor. The ROCK inhibitor can be Thiazovivin. In some embodiments, the first and / or second media comprise about 2 pM Thiazovivin. In some embodiments, the first medium, the second medium, or both, comprise a defined, serum-free serum replacement. In some embodiments, the first medium, the second medium, or both comprise about 0.1% the defined, serum-free serum replacement. In some embodiments, the first medium, the second medium, or both comprise about 0.001%,-l% (e.g., 0.001%, 0.01%, 0.1%, 1% or a number or a range between any two of these values) the defined, serum-free serum replacement. In some embodiments, the defined, serum-free serum replacement comprises one or more of insulin, transferrin, ascorbic acid, a plurality of amino acids, a plurality of trace elements, and albumin. The albumin can be AlbuMAX. In some embodiments, the plurality of amino acids comprises two or more of Glycine, L-Histidine, L-Isoleucine, L-Methionine, L-Phenylalanine, L-Proline, L-Hydroxyproline, L- Serine, L-Threonine, L-Tryptophan, L-Tyrosine, and L-Valine. In some embodiments, the plurality of trace elements comprises two or more of Ag+, Al3+, Ba2+, Cd2+, Co2+, Cr+, Ge4+, Se4+, Br“, E, Mn2+, F“, Si4+, V5+, Mo6+, Ni2+, Rb+, Sn2+and Zr4+. In some embodiments, the defined, serum-free serum replacement is KnockOut serum replacement (KSR). As provided herein, addition of a defined, serum-free serum replacement to the first and / or second media can improve the yield of S6 cells from stem cells.

[0231] In some embodiments, the first medium comprises (i) about 100 ng / mL of AAand about 3 pM CHIR or (ii) about 100 ng / mL of AA and about 6 pM CHIR. The second medium can comprise about 20-30 ng / mL FGF2. The second medium can comprise about 25 ng / mL FGF2. The second medium can comprise about 100 ng / mL of AA. The first and / or second media can comprise about 2 pM Thiazovivin. The third medium can comprise about 50 ng / mL FGF7. The fourth medium can comprise about 25 ng / mL FGF7, about 1 pM Retinoic Acid, about 0.25 pM SANT, and about 300 nM TPPB. In some embodiments, the fifth medium comprises about 2 ng / mL FGF7 or about 4 ng / mL FGF7, about 50 nM Retinoic Acid or about 100 nM Retinoic Acid, about 0.25 pM SANT, and about 300 nM TPPB. In some embodiments, the fifth medium comprises about 200 nM LDN. The sixth medium can comprise about 50 nM Retinoic Acid, about 0.25 pM SANT, about 100 nM LDN-193189-HCL, about 10 pM RepSox, about IpM T3, and about 10 pg / mL Heparin. The seventh medium can comprise about 100 nM LDN-193189-HCL, about 10 pM RepSox, about 1 pM T3, about 10 pg / mL Heparin, and about 100 nM DBZ. The first and / or second medium can comprise about 2 pM Thiazovivin. The first and / or second medium can comprise about 0.1% KSR.

[0232] The population of stem cells can be generated by culturing a plurality of stem cells in a 3D culture medium comprising a ROCK inhibitor. The ROCK inhibitor can be thiazovivin. The 3D culture medium can comprise 1 pM to 10 pM thiazovivin. The 3D culture medium can comprise about 5 pM thiazovivin. The starting concentration of the plurality of stem cells added to the 3D culture medium can be about 2 * 105cells / mL. The 3D culture medium can comprise StemScale media. The plurality of stem cells can be cultured in the 3D culture medium for at least 3 days. At least half of the media can be replaced with fresh 3D culture medium at Day 2.

[0233] In some embodiments, the seventh medium further comprises a DNA- damaging agent. In some embodiments, the DNA-damaging agent is selected from the group comprising: radiation, cisplatin, oxaliplatin, carboplatin, nedaplatin, lobaplatin, triplatin, tetranitrate, picoplatin, satraplatin, prolindac, aroplatin, camptothecin, topotecan, irinotecan / sn38, rubitecan, belotecan, idarubicin, amrubicin, pirarubicin, valrubicin, zorubicin, teniposide, aminopterin, methotrexate, pemetrexed, raltitrexed, pentostatin, cladribine, clofarabine, fludarabine, thioguanine, mercaptopurine, fluorouracil, capecitabine, tegafur, carmofur, floxuridine, cytarabine, gemcitabine, azacitidine, hydroxyurea, mechlorethamine, cyclophosphamide, ifosfamide, trofosfamide, chlorambucil, melphalan, prednimustine, bendamustine, uramustine, estramustine, carmustine, lomustine, semustine, fotemustine, nimustine, ranimustine, streptozocin, busulfan, mannosulfan, treosulfan, carboquone, thiotepa, triaziquone, triethylenemelamine, procarbazine, dacarbazine, etopside, temozolomide, altretamine, mitobronitol, actinomycin, bleomycin, mitomycin and plicamycin. In someembodiments, the DNA-damaging agent is bleomycin. In some embodiments, the seventh medium comprises about 50 nM bleomycin. In some embodiments, the sixth population of cells is cultured in the seventh medium comprising the DNA-damaging agent for two days. In some embodiments, the sixth population of cells is cultured in the seventh medium for at least seven days. In some embodiments, the method comprises removing the DNA-damaging agent from the seventh medium following the first two days of culture in the seventh medium comprising the DNA- damaging agent.

[0234] The time sufficient to generate the first population of progenitor cells can comprise culturing for at least 12 hours. The time sufficient to generate the first population of progenitor cells can comprise culturing for at least 24 hours. The time sufficient to generate the second population of progenitor cells can comprise culturing for at least 12 hours. The time sufficient to generate the second population of progenitor cells can comprise culturing for at least 24 hours. The time sufficient to generate the third population of progenitor cells can comprise culturing for at least three days. The time sufficient to generate the fourth population of progenitor cells can comprise culturing for at least two days. The time sufficient to generate the fifth population of progenitor cells can comprise culturing for at least three days. The time sufficient to generate the sixth population of progenitor cells can comprise culturing for at least three days. The time sufficient to generate the seventh population of cells can comprise culturing for at least seven days. In some embodiments, obtaining or generating the seventh population of cells can occur in 20 days or less. In some embodiments, obtaining or generating the seventh population can occur in more than 20 days.

[0235] The method can be carried out under suspension agitation. Suspension agitation can comprise rotation. The rotation speed can be at least or at least about 35 RPM to 45 RPM. The rotation speed of steps (a)-(e) above can be at least 45 RPM, and the rotation speed of step (f) above can be at least 35 RPM.

[0236] The first, second, third, fourth, fifth, sixth, and / or seventh media can comprise MCDB131 medium or S3S6 (BLAR001) medium. The first, second, third, fourth, fifth, six and / or seventh media can comprise GlutaMax, non-human serum, glucose, sodium bicarbonate, or any combination thereof. The non-human serum can be BSA or fatty acid free BSA (FAF BSA). The concentration of non-human serum can be 0.05%-3%, the concentration of glucose can be 5 mM to 30 mM, the concentration of sodium bicarbonate can be 0.005%-0.02%, and any combination thereof. ITS-X (insulin, transferrin, selenium, ethanolamine solution) can be added to the first, second, third, fourth, fifth, sixth, and / or seventh media at a v:v ratio of (or of about) 1 :50000 to (or to about) 1 :200. The third, fourth, and / or fifth media can comprise vitamin C. The concentration of vitamin C can be 0.1 mM to 0.5 mM. The sixth and / or seventh media cancomprise zinc sulfate. The concentration of zinc sulfate can be 5 pM to 15 pM.

[0237] The first medium can comprise about 0.1% FAF-BSA, about 14.4 mM glucose, and / or about 0.01% sodium bicarbonate. The first medium can comprise about 0.5% FAF-BSA, about 10 mM glucose, and / or about 0.015% sodium bicarbonate. The second medium can comprise about 0.1% FAF-BSA, about 14.4 mM glucose, and / or about 0.01% sodium bicarbonate. The second medium can comprise about 0.5% FAF-BSA, about 10 mM glucose, and / or about 0.015% sodium bicarbonate. The third and / or fourth media can comprise about 0.5% FAF-BSA, about 10 mM glucose, about 0.015% sodium bicarbonate, and / or about 0.25 mM vitamin C. The fifth medium can comprise about 0.25% FAF-BSA, about 10 mM glucose, about 0.015% sodium bicarbonate, and / or about 0.25 mM vitamin C. The sixth and / or seventh media can comprise about 2% FAF-BSA, about 20 mM glucose, about 0.015% sodium bicarbonate, and / or about 10 pM zinc sulfate. Any of the first, second, third, fourth, fifth, sixth, and seventh media can comprise an antibiotic.

[0238] In some embodiments, the first medium comprises MCDB 131 medium or S3 S6 (BLAR001) medium, GlutaMax, 0.1% FAF-BSA, 14.4 mM glucose, 0.01% sodium bicarbonate, 100 ng / mL of AA, and 3 pM CHIR. In some embodiments, the first medium comprises MCDB 131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.015% sodium bicarbonate, 100 ng / mL of AA, 3 pM CHIR, and 2 pM Thiazovivin. In some embodiments, the first medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.015% sodium bicarbonate, 0.1% KSR, 100 ng / mL of AA, 3 pM CHIR, and 2 pM Thiazovivin. In some embodiments, the first medium comprises MCDB 131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.01% sodium bicarbonate, 1% B27, 100 ng / mL of AA, 6 pM CHIR, and 2 pM Thiazovivin.

[0239] In some embodiments, the second medium comprises MCDB 131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.1% FAF-BSA, 14.4 mM glucose, 0.01% sodium bicarbonate, 100 ng / mL AA and 25 ng / mL FGF2. In some embodiments, the second medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.015% sodium bicarbonate, 100 ng / mL AA, 2 pM Thiazovivin, and 25 ng / mL FGF2. In some embodiments, the second medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.015% sodium bicarbonate, 0.1% KSR, 100 ng / mL AA, 2 pM Thiazovivin, and 25 ng / mL FGF2. In some embodiments, the second medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.01% sodium bicarbonate, 1% B27, 100 ng / mL AA, 2 pM Thiazovivin, and 25 ng / mL FGF2.

[0240] In some embodiments, the third medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.015% sodium bicarbonate, 0.25 mM Vitamin C, and 50 ng / mL FGF7. In some embodiments, the third medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.015% sodium bicarbonate, 0.25 mM Vitamin C, trace elements, and 50 ng / mL FGF7. In some embodiments, the third medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.015% sodium bicarbonate, 0.25 mM Vitamin C, trace elements, 1% B27, and 50 ng / mL FGF7.

[0241] In some embodiments, the fourth medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :200 (v:v) ITS-X, 0.015% sodium bicarbonate, 0.25 mM Vitamin C, 25 ng / mL FGF7, 1 pM retinoic acid, 0.25 pM SANT, and 300 nM TPPB. In some embodiments, the fourth medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :200 (v:v) ITS-X, 0.015% sodium bicarbonate, 0.25 mM Vitamin C, 1% B27, 25 ng / mL FGF7, 1 pM retinoic acid, 0.25 pM SANT, and 300 nM TPPB.

[0242] In some embodiments, the fifth medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.25% FAF-BSA, 10 mM glucose, 1 :200 (v:v) ITS-X, 0.015% sodium bicarbonate, 0.25 mM Vitamin C, 2 ng / mL FGF7, 50 nM retinoic acid, 0.25 pM SANT, and 300 nM TPPB. In some embodiments, the fifth medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.25% FAF-BSA, 10 mM glucose, 1 :200 (v:v) ITS-X, 0.015% sodium bicarbonate, 0.25 mM Vitamin C, 1% B27, 4 ng / mL FGF7, 100 nM retinoic acid, 0.25 pM SANT, 300 nM TPPB, and 200 nM LDN.

[0243] In some embodiments, the sixth medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 2% FAF-BSA, 20 mM glucose, 1 :200 (v:v) ITS-X, 0.015% sodium bicarbonate, 10 pM Zinc Sulfate, 50 nM retinoic acid, 0.25 pM SANT, 100 nM LDN, 10 pM RepSOX, 1 pM T3, and 10 pg / mL Heparin. In some embodiments, the sixth medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 2% FAF-BSA, 20 mM glucose, 1 :200 (v:v) ITS-X, 0.015% sodium bicarbonate, 10 pM Zinc Sulfate, 1% B27, 50 nM retinoic acid, 0.25 pM SANT, 100 nM LDN, 10 pM RepSOX, 1 pM T3, and 10 pg / mL Heparin.

[0244] In some embodiments, the seventh medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 2% FAF-BSA, 20 mM glucose, 1 :200 (v:v) ITS-X, 0.015% sodium bicarbonate, 10 pM Zinc Sulfate, 100 nM LDN, 10 pM RepSOX, 1 pM T3, 10 pg / mL Heparin, and 100 nM DBZ. In some embodiments, the seventh medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 2% FAF-BSA, 20 mM glucose, 1 :200 (v:v)ITS-X, 0.015% sodium bicarbonate, 10 pMZinc Sulfate, 1% B27, 100 nMLDN, 10 pM RepSOX, 1 pM T3, 10 pg / mL Heparin, and 100 nM DBZ.

[0245] In some embodiments, at least 25% of the seventh population of cells express PDX1 and NKX6.1, at least 20% of the seventh population of cells express ISL1 and NKX6.1, at least 30% of the seventh population of cells express NKX6.1 and INS, less than 1% of the seventh population of cells express GCG and INS, or any combination thereof. The proportion of cells expressing FOXA2, PDX1, NKX6.1, INS, ISL1, or any combination thereof, can be increased by at least 0.5-fold in the seventh population of cells relative to a population of stem cells. In some embodiments, at least a portion of the fifth population of progenitor cells and / or the seventh population of cells express one or more markers of differentiation. Each of the one or more markers can comprise CHGA, FOXA2, PDX1, NKX6.1, orNGN3. In some embodiments, at least 25% of the fifth population of progenitor cells express NKX6.1 and PDX1. In some embodiments, at least 0.5-fold more cells of the fifth population of progenitor cells and / or the seventh population of cells express one or more markers of differentiation each selected from the group consisting of CHGA, FOXA2, PDX1, NKX6.1 and NGN3; as compared to a fifth population of progenitor cells and / or a seventh population of cells generated without culturing in a medium comprising FGF2 in step (a). In some embodiments, at least 75% of the second population of progenitor cells express FOXA2 and SOX17.

[0246] In some embodiments, the yield of the seventh population of cells is increased by at least 10% when culturing in a medium comprising the defined, serum-free serum replacement as compared to culturing in a medium that does not comprise the defined, serum-free serum replacement in step (a). In some embodiments, the yield of the seventh population of cells is increased by at least 2-fold when culturing in: a first medium comprising B27 and 6 pM CHIR, a second medium comprising B27, a third medium comprising B27, a fourth medium comprising B27, a fifth medium comprising B27, 4 ng / mL FGF7, 100 nM retinoic acid, and 200 nM LDN, a sixth medium comprising B27, and a seventh medium comprising B27; e.g., as compared to culturing in a first medium that does not comprise B27 and 6 pM CHIR, a second medium that does not comprise B27, a third medium that does not comprise B27, a fourth medium that does not comprise B27, a fifth medium that does not comprise B27, 4 ng / mL FGF7, 100 nM retinoic acid, and 200 nM LDN, a sixth medium that does not comprise B27, and a seventh medium that does not comprise B27.

[0247] In some embodiments, one or more of the first, third, fourth, fifth, sixth, and seventh media do not comprise FGF2. In some embodiments, FGF2 is not added to one or more of the first, third, fourth, fifth, sixth, and seventh media. In some embodiments, one or more of the third, fourth, fifth, sixth, and seventh media comprise only residual amounts of FGF2 (e.g.,FGF2 carried over from culture of cells in the second medium comprising FGF2). In some embodiments, one or more of the third, fourth, fifth, sixth, and seventh media do not comprise a ROCK inhibitor (e.g., Thiazovivin). In some embodiments, a ROCK inhibitor is not added to one or more of the third, fourth, fifth, sixth, and seventh media. In some embodiments, the third, fourth, fifth, sixth, and / or seventh media comprise only residual amounts of a ROCK inhibitor (e.g., ROCK inhibitor carried over from culture of cells in the first and / or second media comprising a ROCK inhibitor). In some embodiments, one or more of the third, fourth, fifth, sixth, and seventh media do not comprise a defined, serum-free serum replacement (e.g., KSR). In some embodiments, a defined, serum-free serum replacement is not added to one or more of the third, fourth, fifth, sixth, and seventh media. In some embodiments, the third, fourth, fifth, sixth, and / or seventh media comprise only residual amounts of a defined, serum-free serum replacement (e.g., defined, serum-free serum replacement carried over from culture of cells in the first and / or second media comprising a defined, serum-free serum replacement).

[0248] The immature P cells can be capable of differentiating into mature P cells capable of secreting insulin in response to glucose. About 60 * 106to about 80 * 106immature P cells can be produced from a population of stem cells comprising about 20 * 106cells.

[0249] The population of stem cells can be a population of genetically modified cells. In some embodiments, the stem cells can be genetically modified by an RNA-guided endonuclease system. The RNA-guided endonuclease system can be a CRISPR system comprising a CRISPR nuclease and a guide RNA. The stem cells can be induced pluripotent stem cells (iPSC), pluripotent stem cells (PSC), embryonic stem cells (ESC), or adult stem cells (ASC). The stem cells can be mammalian cells. The mammalian cells can be human cells.

[0250] The method can comprise differentiating the immature P cells into mature P cells capable of secreting insulin in response to glucose.Stem cell cultures

[0251] In some embodiments, stem cells (e.g., iPSCs) are cultured in a 3D culture system prior to culture in the first culture medium, to, e.g., obtain the population of stem cells. The population of stem cells can be generated by culturing a plurality of stem cells (e.g., iPSCs) in a 3D culture medium comprising a rho-associated protein kinase (ROCK) inhibitor. Any ROCK inhibitor can be used in the 3D culture medium. In some cases, the ROCK inhibitor comprises Thiazovivin, Y-27632, Fasudil / HA1077, or H-l 152. The ROCK inhibitor can be thiazovivin. The 3D culture medium can comprise 1 pM to 10 pM (e.g., 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, or a number or a range between any two of the values) thiazovivin. The 3D culture medium can comprise about 5 pM thiazovivin. The starting concentration of the plurality of stem cells added to the 3D culture medium can be about, at least or at most 2 * 105cells / mL.

[0252] The 3D culture medium can comprise any basal media known in the art for culture of stem cells. The medium that can be used is not particularly limited, and, in some embodiments, any basal medium can be used. In one aspect, the following media can be preferably used: Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12 Medium, DMEM / F12 Medium, McCoy's 5 A Medium, Eagle MEM Medium (Eagle's Minimum Essential Medium; EMEM), aMEM medium (alpha Modified Eagle's Minimum Essential Medium; aMEM), MEM medium (Minimum Essential Medium), RPMI1640 medium, Iscove's Modified Dulbecco's Medium (IMDM), MCDB131 medium or S3S6 (BLAR001) medium, William medium E, IPL41 medium, Fischer's medium, StemPro34 (Invitrogen), X-VIVO 10 (Cambrex), X-VIVO 15 (Cambrex), HPGM (Cambrex), StemSpan H3000 (Stemcell Technology), StemSpanSFEM (Stemcell Technology) company), Stemlinell (Sigma- Aldrich), QBSF-60 (Quality Biological), StemProhESCSFM (Invitrogen), Essential6 medium (Gibco), Essentials medium (Gibco (Thermo Fisher), Essentials Flex medium (Thermo Fisher), StemFlex medium (Thermo Fisher), StemScale PSC Suspension Medium (Thermo Fisher), mTeSRl or 2 or Plus medium (Stemcell technology), Repro FF or Repro FF2 (Reprocell), PSGro hESC / iPSC medium (System Bioscience), NutriStem medium (Biological Industries), MSC NutriStem XF Medium (Biological Industries), CSTI-7 medium (Cell Science Research Institute), MesenPRO RS medium (Gibco), MF-Medium mesenchymal stem cell growth medium (Toyobo Co., Ltd.), Mesenchymal stem cell serum-free medium (Fukoku), Mesenchymal Stem Cell Growth Medium2 (PromoCell), Sf-900II (Invitrogen), Opti-Pro (Invitrogen), StemFit AK02N Alternatively, Basic02, AK03N, Basic03, or Basic04 medium (Ajinomoto Healthy Supply Co., Ltd.), STEMUP medium (Nissan Chemical Industries, Ltd.), LI 5 medium, and the like. The 3D culture medium can comprise StemScale media.

[0253] The plurality of stem cells can be cultured in the 3D culture medium for at least3 days. At least half the of media can be replaced with fresh 3D culture medium at Day 2. The 3D culture medium can also comprise an antibiotic. Exemplary antibiotics that can be used in the methods of disclosure include: Amphotericin B, Ampicillin, Antibiotic Antimycotic Solution (comprising, penicillin, streptomycin, and / or amphotericin B), Cephalothin, Dihydrostreptomycin, Erythromycin, Gentamicin Sulfate, L-Glutamine-Penicillin-Streptomycin, Kanamycin Sulfate, Lincomycin HCI, Neomycin Sulfate, Nystatin, Paromomycin Sulfate, Penicillin-G, Penicillin-Streptomycin, Phenoxymethylpenicillinic Acid, Polymyxin B Sulfate, Spectinomycin dihydrochloride pentahydrate, Streptomycin sulfate, Tetracycline Hydrochloride, and Tylosin Tartrate.Definitive Endoderm Cells

[0254] Aspects of the disclosure involve definitive endoderm cells. In some embodiments, pluripotent stem cells, e.g., iPSCs or hESCs, are differentiated to definitive endoderm cells. In some aspects, the endoderm cells (Stage 1, e.g., definitive endoderm) are further differentiated, e.g., to primitive gut tube cells (Stage 2, e.g., gut tube cells), PDX1 -positive pancreatic progenitor cells (Stage 3, e.g., foregut or posterior foregut), NKX6.1 -positive pancreatic progenitor cells (Stage 4, e.g., pancreatic endoderm), or Ngn3-positive endocrine progenitor cells or insulin-positive endocrine cells (Stage 5, e.g., endocrine precursor cells), followed by induction or maturation to immature beta cells (Stage 6).

[0255] As disclosed herein, it was surprisingly found that Stage 1 culture in the presence of FGF2, following culture in a medium comprising, e.g., a WNT pathway activator, can improve the yield of immature beta cells generated from stem cells.

[0256] Disclosed herein include methods for generating immature P (beta) cells from stem cells. In some embodiments, the method comprises: (a) culturing a population of stem cells, (i) in a first medium comprising AA and a WNT pathway activator to generate a first population of progenitor cells, and (ii) culturing the first population of progenitor cells in a second medium comprising AA and FGF2 for a time sufficient to generate a second population of progenitor cells comprising definitive endoderm cells. In some embodiments, the definitive endoderm cells express at least one marker characteristic of definitive endoderm. The population of stem cells can be cultured in the first culture medium for at least 12 hours (e.g., 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, or more). The population of stem cells can be cultured in the first culture medium for at least 24 hours (e.g., 1-day). The culture time can comprise an “overnight culture” as understood by a person of skill in the art.

[0257] In some embodiments, the first medium comprises, comprises at most, comprises at least, comprises about, comprises at most about, or comprises at least about, 50 ng / mL, 75 ng / mL, 80 ng / mL, 90 ng / mL, 95 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL, or a number or a range between any two of these values, of Activin A (AA). In some embodiments, the first medium comprises, comprises at most, comprises at least, comprises about, comprises at most about, or comprises at least about, 0.2 pM, 0.5 pM, 0.75 pM, 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 7.5 pM, 8 pM, 9 pM, 10 pM, 11 pM, 12 pM, 13 pM, 14 pM, 15 pM, 16 pM, 17 pM, 18 pM, 19 pM, 20 pM, 21 pM, 22 pM, 23 pM, 24 pM, 25 pM, 26 pM, 27 pM, 28 pM, 29 pM, 30 pM, 35 pM, 40 pM, 50 pM, or a number or a range between any two of these values, of a Wnt pathway activator (c.g, CHIR-99021 (CHIR)). Any WNT pathway activator may be used. The WNT pathway activator of the first medium can be CHIR. The first medium can comprise or comprise about 100 ng / mL of AA and about 3 pMCHIR. The first medium can comprise or comprise about 100 ng / mL of AA and about 6 pM CHIR.

[0258] In some embodiments, the second medium comprises, comprises at most, comprises at least, comprises about, comprises at most about, or comprises at least about, 5 ng / mL, 7.5 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 50 ng / mL, 100 ng / mL or a number or a range between any two of these values of FGF2. The second medium can comprise or comprise about 20-30 ng / mL FGF2 (e.g., (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, or (about) 30 ng / mL). The second medium can comprise or comprise about 25 ng / mL FGF2.

[0259] In some embodiments, the second medium comprises, comprises at most, comprises at least, ocomprises about, comprises at most about, or comprises at least about, 50 ng / mL, 75 ng / mL, 80 ng / mL, 90 ng / mL, 95 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL, or a number or a range between any two of these values, of AA. The second medium can comprise or comprise about 100 ng / mL of AA. The population of stem cells can be cultured in the second culture medium for or for at least 12 hours (e.g., 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours,23 hours, 1 day, or more). The population of stem cells can be cultured in the second culture medium for at least 24 hours. The time sufficient to generate the second population of progenitor cells (comprising definitive endoderm cells) can comprise culturing for at least 12 hours. The time sufficient to generate the second population of progenitor cells can comprise culturing for at least24 hours. The culture time can comprise an “overnight culture” as understood by a person of skill in the art.

[0260] In some embodiments, the first and / or second media comprise a ROCK inhibitor, e.g., Thiazovivin. In some cases, the ROCK inhibitor comprises Thiazovivin, Y-27632, Fasudil / HA1077, or H-l 152. In some embodiments, the first and / or second media comprise, comprise at most, comprise at least, comprise about, comprise at most about, or comprise at least about, 0.2 pM, 0.5 pM, 0.75 pM, 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 7.5 pM, 8 pM, 9 pM, 10 pM, 11 pM, 12 pM, 13 pM, 14 pM, 15 pM, 16 pM, 17 pM, 18 pM, 19 pM, 20 pM, 21 pM, 22 pM, 23 pM, 24 pM, 25 pM, 26 pM, 27 pM, 28 pM, 29 pM, 30 pM, 35 pM, 40 pM, 50 pM or a number or a range between any two of these values of Thiazovivin.

[0261] In some embodiments, the first and / or second media comprise a defined, serum-free serum replacement. In some embodiments, the defined, serum-free serum replacementis KnockOut Serum Replacement (KSR). In some embodiments, the first and / or second media comprise, comprise at most, comprise at least, comprise about, comprise at most about, or comprise at least about, 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or a number or a range between any two of these values of KSR.

[0262] In some embodiments, the first and / or second media comprise a supplement. The supplement can be B27 supplement. In some embodiments, the first and / or second media comprise, comprise at most, comprise at least, comprise about, comprise at most about, or comprise at least about, 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or a number or a range between any two of these values of B27 supplement. In some embodiments, the first and / or second media comprises or comprises about 1% B27 supplement.

[0263] In some cases, 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, Stl4, Spink3, Sh3gl2, Ripk4, Rabi 5, Npnt, Clic6, Cldn8, Cacnalb, Bnipl, Anxa4, Emb, FoxAl, Soxl7, and Rbm35a, wherein the expression of at least one marker is upregulated by a statistically significant amount in the definitive endoderm cell relative to the pluripotent stem cell from which it was derived. In some cases, 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 cases, 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 Zicl, Pax6, Flkl and CD31 relative to the pluripotent stem cell from which it was derived. In some cases, 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.

[0264] In some cases, a definitive endoderm cell produced by the methods as disclosed herein has the capacity to form gut tube in vivo, in vitro, and / or ex vivo. In some cases, 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, e.g., an immature beta cell.

[0265] In some cases, a population of pluripotent stem cells are cultured in the presence of at least one beta cell differentiation factor prior to any differentiation or during the first stage of differentiation. One can use any pluripotent 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 suitablepluripotent stem cells. In some cases, a beta cell differentiation factor as described herein can be present in the culture medium of a population of 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 some examples, a population of pluripotent stem cells can be exposed to at least one beta cell differentiation factor prior to any differentiation. In other examples, a population of pluripotent stem cells may be exposed to at least one beta cell differentiation factor during the first stage of differentiation.Primitive Gut Tube Cells

[0266] In some embodiments, definitive endoderm cells are differentiated to primitive gut tube cells (e.g. , Stage 2). In some aspects, the primitive gut tube cells are further differentiated, e.g., to PDXl-positive pancreatic progenitor cells (e.g., Stage 3), NKX6.1 -positive pancreatic progenitor cells (e.g., Stage 4), Ngn3-positive endocrine progenitor cells (e.g., Stage 5), insulinpositive beta cells (e.g., Stage 6).

[0267] In some embodiments, the method comprises: culturing the second population of progenitor cells in a third medium comprising FGF7 (e.g. , KGF) for a time sufficient to generate a third population of progenitor cells comprising gut tube cells. The third medium can comprise about 50 ng / mL FGF7.

[0268] In some cases, 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 FGF7 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. The time sufficient to generate the third population of progenitor cells (e.g., comprising gut tube cells) can comprise culturing for at least three days.

[0269] In some embodiments, the third medium comprises, comprises at most, comprises at least, comprises about, comprises at most about, or comprises at least about, 10 ng / mL, 20 ng / mL, 50 ng / mL, 75 ng / mL, 80 ng / mL, 90 ng / mL, 95 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL, 175 ng / mL, 180 ng / mL, 200 ng / mL, 250 ng / mL, 300 ng / mL, or a number or a range between any two of these values, of FGF7. In some cases, the method comprises use of or of about 50 ng / mL FGF7 for differentiation of definitive endoderm cells into primitive gut tube cells. In some embodiments, the third medium comprises a supplement. The supplement can be B27 supplement. In some embodiments, the third medium comprises, comprises at most, comprises at least, comprises about, comprises at most about, or comprises at least about, 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or a number or a range between any two of these values of B27 supplement. In someembodiments, the third medium comprises or comprises about 1% B27 supplement.

[0270] Gut tube cells may be characterized by their substantially increased expression of HNF4a over that expressed by definitive endoderm cells. For example, a ten- to forty-fold increase in mRNA expression of HNF4a may be observed during Stage 2.Foregut cells

[0271] Aspects of the disclosure involve generation of PDXl-positive pancreatic progenitor cells (e.g., foregut cells or posterior foregut cells, Stage 3). In some embodiments, primitive gut tube cells are differentiated to PDX1 -positive pancreatic progenitor cells (e.g., posterior foregut cells. In some aspects, the PDX1 -positive pancreatic progenitor cells are further differentiated, e.g., to NKX6.1 -positive pancreatic progenitor cells (Stage 4) and Ngn3-positive endocrine progenitor cells (e.g., endocrine precursor cells, Stage 5), and to insulin-positive beta cells (Stage 6).

[0272] In some embodiments, the method comprises: culturing the third population of progenitor cells in a fourth medium comprising FGF7, retinoic acid, a Sonic Hedgehog (Shh) pathway inhibitor, and a protein kinase C (PKC) activator for a time sufficient to generate a fourth population of progenitor cells comprising posterior foregut cells. Any Shh pathway inhibitor can be used in the disclosed methods. Any PKC activator can be used in the disclosed methods. The Shh pathway inhibitor of the fourth medium can be SANT. The PKC activator of the fourth medium can be (2E,4E)-N-[(2S,5S)-l,2,3,4,5,6-Hexahydro-5-(hydroxymethyl)-l-methyl-2-(l- methylethyl)-3-oxo-l,4-benzodiazocin-8-yl]-5-[4-(trifluoromethyl)phenyl]-2,4-pentadienamide (TPPB). The fourth medium can comprise about 25 ng / mL FGF7, about 1 pM Retinoic Acid, about 0.25 pM SANT, and about 300 nM TPPB. The time sufficient to generate the fourth population of progenitor cells can comprise culturing for at least two days (e.g., two, three or more days).

[0273] In some embodiments, the fourth medium comprises, comprises at most, comprises at least, comprises about, comprises at most about, or comprises at least about, 5 ng / mL, 7.5 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 20 ng / mL, 25 ng / mL FGF7, 50 ng / mL, 75 ng / mL, 80 ng / mL, 90 ng / mL, 95 ng / mL, 100 ng / mL, or a number or a range between any two of these values, of FGF7. In some embodiments, the concentration of FGF7 in the fourth medium is less than the concentration of FGF7 in the third medium. In some embodiments, the fourth medium comprises, comprises at most, comprises at least, comprises about, comprises at most about, or comprises at least about, 0.2 pM, 0.5 pM, 0.75 pM, 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 7.5 pM, 8 pM, 9 pM, 10 pM, or a number or a range between any two of these values, of retinoic acid. In some embodiments, the fourth medium comprises, comprises at most, comprises at least, comprises about, comprises at most about, or comprises at least about, 10 nM, 20 nM, 30 nM, 40nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 0.1 pM, 0.2 pM, 0.25 pM, 0.5 pM, 0.75 pM, 1 pM, 1.2 pM, 1.5 pM, 1.75 pM, 2 pM, or a number or a range between any two of these values, of a Shh pathway inhibitor (e.g., SANT-1 (SANT)). In some embodiments, the fourth medium comprises, comprises at most, comprises at least, comprises about, comprises at most about, or comprises at least about, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, 200 nM, 210 nM, 220 nM, 230 nM, 240 nM, 250 nM, 280 nM, 300 nM, 400 nM, 500 nM, or 1 pM, or a number or a range between any two of these values, of a PKC activator (e.g., TPPB).

[0274] In some embodiments, the fourth medium comprises a supplement. The supplement can be B27 supplement. In some embodiments, the fourth medium comprises, comprises at most, comprises at least, comprises about, comprises at most about, or comprises at least about, 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or a number or a range between any two of these values of B27 supplement. In some embodiments, the fourth medium comprises or comprises about 1% B27 supplement.

[0275] Posterior foregut may be characterized by an increase in expression of PDX1 compared to gut tube cells. For example, greater than fifty percent of the cells in Stage 3 cultures can express PDX1.Pancreatic Endoderm Precursors

[0276] Aspects of the disclosure involve NKX6.1 -positive pancreatic progenitor cells (e.g., pancreatic endoderm, Stage 4). In some aspects, the NKX6.1 -positive pancreatic progenitor cells are further differentiated, e.g., to Ngn3-positive endocrine progenitor cells (Stage 5), or insulin-positive beta cells (Stage 6).

[0277] In some embodiments, the method comprises: culturing the fourth population of progenitor cells in a fifth medium comprising FGF7, retinoic acid, a Shh pathway inhibitor, and a PKC activator, wherein the fifth medium comprises a lower concentration of FGF7 and a lower concentration of retinoic acid than the fourth medium, for a time sufficient to generate a fifth population of progenitor cells comprising pancreatic endoderm cells. The fifth medium can comprise optionally wherein the fifth medium comprises a bone morphogenetic protein (BMP) antagonist. Any Shh pathway inhibitor or PKC activator known in the art can be used. The Shh pathway inhibitor of the fifth medium can be SANT. The PKC activator of the fifth medium can be (2E,4E)-N-[(2S, 5 S)- 1 ,2,3 ,4, 5,6-Hexahydro-5-(hydroxymethyl)- 1 -methyl-2-( 1 -methylethyl)- 3-oxo-l,4-benzodiazocin-8-yl]-5-[4-(trifluoromethyl)phenyl]-2,4-pentadienamide (TPPB). The fifth medium can comprise about 2 ng / mL FGF7, about 50 nM Retinoic Acid, about 0.25 pM SANT, and about 300 nM TPPB. The time sufficient to generate the fifth population of progenitor cells can comprise culturing for at least three days (e.g., three, four or more days). The fifth-n-medium can comprise about 4 ng / mL FGF7, about 100 nM Retinoic Acid, about 0.25 pM SANT, about 300 nM TPPB, and about 200 nM LDN.

[0278] In some embodiments, the fifth medium comprises, comprises at most, comprises at least, comprises about, comprises at most about, or comprises at least about, 0.5 ng / mL, 1 ng / mL, 1.5 ng / mL, 2 ng / mL, 2.5 ng / mL, 3 ng / mL, 4ng / mL, 5 ng / mL, or a number or a range between any two of these values, of FGF7. In some embodiments, the fifth medium comprises, comprises at most, comprises at least, comprises about, comprises at most about, or comprises at least about, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, 200 nM, or a number or a range between any two of these values, of retinoic acid. In some embodiments, the fifth medium comprises, comprises at most, comprises at least, comprises about, comprises at most about, or comprises at least about, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 0.1 pM, 0.2 pM, 0.25 pM, 0.5 pM, 0.75 pM, 1 pM, 1.2 pM, 1.5 pM, 1.75 pM, 2 pM, or a number or a range between any two of these values, of a Shh pathway inhibitor (e.g., SANT-1 (SANT)). In some embodiments, the fifth medium comprises, comprises at most, comprises at least, comprises about, comprises at most about, or comprises at least about, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, 200 nM, 210 nM, 220 nM, 230 nM, 240 nM, 250 nM, 280 nM, 300 nM, 400 nM, 500 nM, 1 pM, or a number or a range between any two of these values, of a PKC activator (e.g., TPPB).

[0279] In some embodiments, the fifth medium comprises, comprises at most, comprises at least, comprises about, comprises at most about, or comprises at least about, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, 200 nM, 250 nM, 300 nM, 400 nM or 500 nM of a BMP antagonist (e.g, LDN- 193189-HCL).

[0280] In some embodiments, the fifth medium comprises a supplement. The supplement can be B27 supplement. In some embodiments, the third medium comprises, comprises at most, comprises at least, comprises about, comprises at most about, or comprises at least about, 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or a number or a range between any two of these values of B27 supplement. In some embodiments, the fifth medium comprises or comprises about 1% B27 supplement.

[0281] The pancreatic endoderm cells can express at least one of the following markers: PDX1, NKX6.1, HNFip, PTFla, HNF6, HNF4a, SOX9, NGN3, gastrin; HB9, or PROX1. Pancreatic endoderm cells may be characterized by their lack of substantial expression of CDX2 or SOX2.Endocrine precursor cells

[0282] Aspects of the disclosure involve differentiation of the NKX6.1 -positive pancreatic progenitor cells (Stage 4) to Ngn3-positive endocrine progenitor cells (Stage 5), which can be differentiated into insulin-positive beta cells (Stage 6).

[0283] The method disclosed herein can comprise generation of endocrine precursor cells from pancreatic progenitor cells. In some embodiments, the method comprises: culturing the fifth population of progenitor cells in a sixth medium comprising retinoic acid, a Shh pathway inhibitor, a bone morphogenetic protein (BMP) antagonist, an anaplastic lymphoma kinase 5 (ALK-5) inhibitor, a thyroid hormone, and an anti-coagulant for a time sufficient to generate a sixth population of progenitor cells comprising endocrine precursor cells. Any Shh pathway inhibitor, BMP antagonist, ALK-5 inhibitor, thyroid hormone, and / or anti -coagulant can be used. The Shh pathway inhibitor of the sixth medium can be SANT. The BMP antagonist of the sixth medium can be LDN-193189-HCL. The ALK-5 inhibitor of the sixth medium can be RepSox. The thyroid hormone of the sixth medium can be 3,3’,5-Triiodo-L-thyronine sodium salt (T3). The anti-coagulant of the sixth medium can be Heparin. The sixth medium can comprise about 50 nM Retinoic Acid, about 0.25 pM SANT, about 100 nM LDN-193189-HCL, about 10 pM RepSox, about IpM T3, and about 10 pg / mL Heparin. The time sufficient to generate the sixth population of progenitor cells of can comprise culturing for at least three days (e.g., three, four, or more days).

[0284] In some embodiments, the sixth medium comprises, comprises at most, comprises at least, comprises about, comprises at most about, or comprises at least about, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, 200 nM of retinoic acid. In some embodiments, the sixth medium comprises, comprises at most, comprises at least, comprises about, comprises at most about, or comprises at least about, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 0.1 pM, 0.2 pM, 0.25 pM, 0.5 pM, 0.75 pM, 1 pM, 1.2 pM, 1.5 pM, 1.75 pM, 2 pM of a Shh pathway inhibitor (e.g., SANT-1 (SANT)). In some embodiments, the sixth medium comprises, comprises at most, comprises at least, comprises about, comprises at most about, or comprises at least about, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, 200 nM, 250 nM, 300 nM, 400 nM or 500 nM of aBMP antagonist e.g., LDN-193189-HCL). In some embodiments, the sixth medium comprises, comprises at most, comprises at least, comprises about, comprises at most about, or comprises at least about, 0.2 pM, 0.5 pM, 0.75 pM, 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 7.5 pM, 8 pM, 9 pM, 10 pM, 11 pM, 12 pM, 13 pM, 14 pM, 15 pM, 16 pM, 17 pM, 18 pM, 19 pM, 20 pM, 21 pM, 22 pM, 23 pM, 24 pM, 25 pM, 26 pM, 27 pM, 28 pM, 29 pM, 30 pM, 35 pM, 40 pM, 50 pM of an ALK-5 inhibitor (e.g., RepSox). In some embodiments, the sixth medium comprises, comprisesat most, comprises at least, comprises about, comprises at most about, or comprises at least about, 0.2 pM, 0.5 pM, 0.75 pM, 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 7.5 pM, 8 pM, 9 pM, or 10 pM of a thyroid hormone (e.g., T3). In some embodiments, the sixth medium comprises, comprises at most, comprises at least, comprises about, comprises at most about, or comprises at least about, 1 pg / mL, 2.5 pg / mL, 5 pg / mL, 7.5 pg / mL, 8 pg / mL, 9 pg / mL, 10 pg / mL, 20 pg / mL, 50 pg / mL, or a number or a range between any two of these values, of an anti-coagulant (e.g., Heparin).

[0285] In some embodiments, the sixth medium comprises a supplement. The supplement can be B27 supplement. In some embodiments, the sixth medium comprises, comprises at most, comprises at least, comprises about, comprises at most about, or comprises at least about, 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or a number or a range between any two of these values of B27 supplement. In some embodiments, the sixth medium comprises or comprises about 1% B27 supplement.

[0286] In some embodiments, the endocrine precursor cells are capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin, ghrelin, and pancreatic polypeptide. In addition to these hormones, markers characteristic of pancreatic endocrine cells include one or more of NeuroDl, ISL1, PDX1, NKX6.1, PAX4, ARX, NKX2.2, HB9 and PAX6.Beta cells (e.g., immature beta cells)

[0287] Aspects of the disclosure involve insulin-positive endocrine cells (e.g., NKX6.1 -positive, ISLl-positive cells, or beta-like cells). In some aspects, NKX6.1 -positive pancreatic progenitor cells are differentiated to insulin-positive endocrine cells (e.g., NKX6.1- positive, ISLl-positive cells, or beta-like cells). In some embodiments, the method comprises: culturing the sixth population of progenitor cells in a seventh medium comprising a BMP antagonist, an ALK-5 inhibitor, a thyroid hormone, an anti-coagulant, and a y-secretase inhibitor, for a time sufficient to generate a seventh population of cells comprising immature P cells. Any BMP antagonist, ALK-5 inhibitor, thyroid hormone, anti-coagulant, or gamma secretase inhibitor can be used. The BMP antagonist of the seventh medium can be LDN-193189-HCL. The ALK-5 inhibitor of the seventh medium can be RepSox. The thyroid hormone of the seventh medium can be 3,3’,5-Triiodo-L-thyronine sodium salt (T3). The y-secretase inhibitor of the seventh medium can be Dibenzazepine (DBZ). The anti-coagulant of the seventh medium can be Heparin. The seventh medium can comprise about 100 nM LDN-193189-HCL, about 10 pM RepSox, about 1 pM T3, about 10 pg / mL Heparin, and about 100 nM DBZ. The time sufficient to generate the seventh population of cells can comprise culturing for at least seven days (e.g., 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks or more).

[0288] In some embodiments, the seventh medium comprises, comprises at most,comprises at least, comprises about, comprises at most about, or comprises at least about, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, 200 nM, 250 nM, 300 nM, 400 nM or 500 nM of a BMP antagonist (e.g., LDN-193189-HCL). In some embodiments, the seventh medium comprises, comprises at most, comprises at least, comprises about, comprises at most about, or comprises at least about, 0.2 pM, 0.5 pM, 0.75 pM, 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 7.5 pM, 8 pM, 9 pM, 10 pM, 11 pM, 12 pM, 13 pM, 14 pM, 15 pM, 16 pM, 17 pM, 18 pM, 19 pM, 20 pM, 21 pM, 22 pM, 23 pM, 24 pM, 25 pM, 26 pM, 27 pM, 28 pM, 29 pM, 30 pM, 35 pM, 40 pM, 50 pM of an ALK-5 inhibitor (e.g., RepSox). In some embodiments, the seventh medium comprises, comprises at most, comprises at least, comprises about, comprises at most about, or comprises at least about, 0.2 pM, 0.5 pM, 0.75 pM, 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 7.5 pM, 8 pM, 9 pM, 10 pM of a thyroid hormone (e.g., T3). In some embodiments, the seventh medium comprises, comprises at most, comprises at least, comprises about, comprises at most about, or comprises at least about, 1 pg / mL, 2.5 pg / mL, 5 pg / mL, 7.5 pg / mL, 8 pg / mL, 9 pg / mL, 10 pg / mL, 20 pg / mL, 50 pg / mL, or a number or a range between any two of these values, of an anti -coagulant (e.g., Heparin). In some embodiments, the seventh medium comprises, comprises at most, comprises at least, comprises about, comprises at most about, or comprises at least about, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, 200 nM of a gamma secretase inhibitor (e.g., Dibenzazepine (DBZ)).

[0289] In some embodiments, the seventh medium comprises a supplement. The supplement can be B27 supplement. In some embodiments, the seventh medium comprises, comprises at most, comprises at least, comprises about, comprises at most about, or comprises at least about, 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or a number or a range between any two of these values of B27 supplement. In some embodiments, the seventh medium comprises or comprises about 1% B27 supplement.

[0290] In some embodiments, the seventh medium further comprises a DNA- damaging agent. In some embodiments, the DNA-damaging agent is selected from the group comprising: radiation, cisplatin, oxaliplatin, carboplatin, nedaplatin, lobaplatin, triplatin, tetranitrate, picoplatin, satraplatin, prolindac, aroplatin, camptothecin, topotecan, irinotecan / sn38, rubitecan, belotecan, idarubicin, amrubicin, pirarubicin, valrubicin, zorubicin, teniposide, aminopterin, methotrexate, pemetrexed, raltitrexed, pentostatin, cladribine, clofarabine, fludarabine, thioguanine, mercaptopurine, fluorouracil, capecitabine, tegafur, carmofur, floxuridine, cytarabine, gemcitabine, azacitidine, hydroxyurea, mechlorethamine, cyclophosphamide, ifosfamide, trofosfamide, chlorambucil, melphalan, prednimustine,bendamustine, uramustine, estramustine, carmustine, lomustine, semustine, fotemustine, nimustine, ranimustine, streptozocin, busulfan, mannosulfan, treosulfan, carboquone, thiotepa, triaziquone, triethylenemelamine, procarbazine, dacarbazine, etopside, temozolomide, altretamine, mitobronitol, actinomycin, bleomycin, mitomycin and plicamycin. In some embodiments, the DNA-damaging agent is bleomycin. In some embodiments, the seventh medium comprises about 50 nM bleomycin. In some embodiments, the seventh medium comprises, comprises at most, comprises at least, comprises about, comprises at most about, or comprises at least about, 20 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, or 200 nM of a DNA-damaging agent. In some embodiments, the seventh medium comprises, comprises at most, comprises at least, comprises about, comprises at most about, or comprises at least about, 0.2 pM, 0.5 pM, 0.75 pM, 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 7.5 pM, 8 pM, 9 pM, 10 pM, 11 pM, 12 pM, 13 pM, 14 pM, 15 pM, 16 pM, 17 pM, 18 pM, 19 pM, 20 pM, 21 pM, 22 pM, 23 pM, 24 pM, 25 pM, 26 pM, 27 pM, 28 pM, 29 pM, 30 pM, 35 pM, 40 pM, 50 pM or a number or a range between any two of these values of a DNA-damaging agent. In some embodiments, the sixth population of cells is cultured in the seventh medium comprising the DNA-damaging agent for two days. In some embodiments, the sixth population of cells is cultured in the seventh medium for at least seven days. In some embodiments, the method comprises removing the DNA- damaging agent from the seventh medium following the first two days of culture in the seventh medium comprising the DNA-damaging agent.

[0291] The time needed or sufficient for producing any of the cell populations of the disclosure can vary. The population of stem cells can be cultured in the first culture medium for at least 12 hours (e.g., 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, or more). The population of stem cells can be cultured in the first culture medium for at least 24 hours (e.g., 1-day). The culture time can comprise an “overnight culture” as understood by a person of skill in the art. The time sufficient to generate the second population of progenitor cells can comprise culturing for at least 12 hours. The time sufficient to generate the second population of progenitor cells of can comprise culturing for at least 24 hours. The time sufficient to generate the third population of progenitor cells can comprise culturing for at least three days. The time sufficient to generate the fourth population of progenitor cells can comprise culturing for at least two days. The time sufficient to generate the fifth population of progenitor cells can comprise culturing for at least three days. The time sufficient to generate the sixth population of progenitor cells can comprise culturing for at least three days. The time sufficient to generate the seventh population of cells can comprise culturing for at least seven days. In some embodiments, obtaining the seventh population of cells comprisingimmature P cells from a population of stem cells can occur in 20 days or less. In some embodiments, obtaining the seventh population of cells comprising immature P cells from a population of stem cells can occur in more than 20 days.

[0292] The immature beta cells generated or obtained by the methods of the disclosure can express one or more markers of immature beta cells. The one or more markers can comprise PDX1, NKX6.1, INS, and ISL. The populations of cells comprising immature beta cells (e.g., the seventh population of cells) can comprise at least 20% (e.g., 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%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) of cells that express one or more of PDX1, NKX6.1, INS, and ISL. In some embodiments, at least 25% (e.g., 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) of the seventh population of cells express PDX1 and NKX6.1, at least 10% (e.g., 10%, 11%, 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%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) of the seventh population of cells express ISL1 and NKX6.1, at least 30% (e.g., 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) of the seventh population of cells express NKX6.1 and INS, less than 1% (e.g., 0.000000001%, 0.00000001%, 0.0000001%, 0.000001%, 0.00001%, 0.0001%, 0.001%, 0.01%, 0.1% or a number or a range between any two of these values) of the seventh population of cells express GCG and INS, or any combination thereof. The proportion of cells expressing FOXA2, PDX1, NKX6.1, INS, ISL1, or anycombination thereof, can be increased by at least 0.5-fold (e.g., 0.5-fold, 1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60- fold, 70-fold, 80-fold, 90-fold, 100-fold, or a number or a range between any of these values) in the seventh population of cells relative to a population of stem cells.

[0293] The methods provided herein can improve the yield of pancreatic progenitor cells and / or immature beta cells, generated from a population stem cells, as compared to previous methods. In some embodiments, at least a portion of the fifth population of progenitor cells and / or the seventh population of cells express one or more markers of differentiation. Each of the one or more markers can comprise CHGA, FOXA2, PDX1, or NGN3. In some embodiments, at least 0.5-fold (e.g., 0.5-fold, 1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or a number or a range between any of these values) more cells of the fifth population of progenitor cells and / or the seventh population of cells express one or more markers of differentiation each selected from the group consisting of CHGA, FOXA2, PDX1, and NGN3; as compared to a fifth population of progenitor cells and / or a seventh population of cells generated without culturing in a medium comprising FGF2 to generate a population of progenitor cells comprising definitive endoderm cells. The immature P cells can be capable of differentiating into mature P cells capable of secreting insulin in response to glucose. About 60 * 106to about 80 * 106immature P cells can be produced from a population of stem cells comprising about 20 x io6cells. In some embodiments, at least, at most, or about 60 x io6to about 80 x io6immature P cells (e.g., 60 x106, 61 x 106, 62 x 106, 63 x 106, 64 x 106, 65 x 106, 66 x 106, 67 x 106, 68 x 106, 69 x 106, 70 x106, 71 x 106, 72 x 106, 73 x 106, 74 x 106, 75 x 106, 76 x 106, 77 x 106, 78 x 106, 79 x 106, 80 x106or a number or a range between any two of these values) can be produced from a population of stem cells, e.g., comprising about 20 x 106cells.

[0294] In some embodiments, the average C-peptide levels capable of being secreted from the sixth population of progenitor cells or seventh population of cells (e.g., populations of cells comprising Stage 5 endocrine precursor cells or Stage 6 immature P cells) is increased by at least 5-fold (e.g., 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or a number or a range between any of these values) as compared to a sixth population of progenitor cells or seventh population of cells generated without culturing in a medium comprising FGF2 to generate a population of progenitor cells comprising definitive endoderm cells.

[0295] The first, second, third, fourth, fifth, sixth, and seventh media can comprise a basal media and / or one or more supplements. Several basal media suitable for culture of stem cells and / or progenitor cells are known in the art. However, it will be appreciated that the method canutilize “routine” tissue culture components such as culture media, serum, serum substitutes, supplements, antibiotics, etc., such as RPMI, Renal Epithelial Basal Medium (REBM), Dulbecco's Modified Eagle Medium (DMEM), MCDB131 medium, CMRL 1066 medium, F12, foetal calf serum (FCS), foetal bovine serum (FBS), bovine serum albumin (BSA), D-glucose, L-glutamine, GlutaMAX.TM.-l (dipeptide, L-alanine-L-glutamine), heparin, progesterone, putrescine, laminin, nicotinamide, insulin, transferrin, sodium selenite, selenium, ethanolamine, human epidermal growth factor (hEGF), basic fibroblast growth factor (bFGF), hydrocortisone, epinephrine, normacin, penicillin, streptomycin, gentamicin and amphotericin, etc. It is to be understood that these typical tissue culture components (and other similar tissue culture components that are routinely used in tissue culture) are not small molecule reprogramming molecules for the purposes of the present disclosure. These components are either not small molecules as defined herein and / or are not reprogramming factors as defined herein.

[0296] The basal media used canbe MCDB131 medium or S3S6 (BLAR001) medium. In some embodiments, the basal media is MCDB131 medium. MCDB 131 medium was originally developed by Knedler and Ham as reduced serum-supplemented medium for the culture of human microvascular endothelial cells (HMVEC). Knedler and Ham, “Optimized medium for clonal growth of human microvascular endothelial cells with minimal serum” In Vitro Cell Dev Biol. 1987 Jul;23(7):481-91. MCDB 131 medium is also used with other cell types, including human omental microvascular cells, hepatocytes, myocytes, and smooth muscle cells. MCDB131 medium can comprise one or more amino acids, one or more vitamins, and / or one or more inorganic salts. See Table 1 for exemplary formulation of MCDB131 medium. MCDB131 medium is commercially available from, e.g., Thermo Fisher Scientific (Cat# 10372019).Table 1 : Exemplary MCDB131 Media Formulation

[0297] In some embodiments, the basal media is S3S6 (BLAR001) medium. BLAR media is described in WO2014105543A1 and US20140186305A1 and in Rezania et al. (2014) Nature Biotech, 32 1121- 1133 (e.g., Supplemental Table 3), which are incorporated herein in their entireties. See Table 2 for exemplary formulation of BLAR001 medium.Table 2: Exemplary BLAR001 Media Formulation

[0298] The first, second, third, fourth, fifth, sixth and / or seventh media can comprise MCDB131 medium or S3S6 (BLAR001) medium. The first, second, third, fourth, fifth, six and seventh media can comprise GlutaMax, non-human serum, glucose, sodium bicarbonate, or any combination thereof. The non-human serum can be BSA or fatty acid free BSA (FAF BSA). The concentration of non-human serum can be 0.05%-3% (e.g., 0.05%, 0.1%, 1%, 2%, 3%, or a number or a range between any two of these values), the concentration of glucose can be 5 mM to 30 mM (e.g., 5 mM, 5.5 mM, 6 mM, 6.5 mM, 7 mM, 7.5 mM, 8 mM, 8.5 mM, 9 mM, 9.5 mM,10 mM, 10.5 mM, 11 mM, 11.5 mM, 12 mM, 12.5 mM, 13 mM, 13.5 mM, 14 mM, 14.5 mM, 15mM, 15.5 mM, 16 mM, 16.5 mM, 17 mM, 17.5 mM, 18 mM, 18.5 mM, 19 mM, 19.5 mM, 20 mM, 20.5 mM, 21 mM, 21.5 mM, 22 mM, 22.5 mM, 23 mM, 23.5 mM, 24 mM, 24.5 mM, 25 mM, 25.5 mM, 26 mM, 26.5 mM, 27 mM, 27.5 mM, 28 mM, 28.5 mM, 29 mM, 29.5 mM, 30 mM or a number or a range between any two of these values), the concentration of sodium bicarbonate can be 0.005%-0.02% (0.005%, 0.0075%, 0.001%, 0.01%, 0.015%, 0.02%, or a number or a range between any two of these values), and any combination thereof.

[0299] ITS-X can be added to the first, second, third, fourth, fifth, sixth, and / or seventh media at a v:v ratio of (or of about) 1 :50000 to (or to about) 1 :200 (e.g., (about) 1 :200, 1 :300, 1 :400, 1 :500, 1 :600, 1 :700, 1 :800, 1 :900, 1 : 1000, 1 :2000, 1 :3000, 1 :4000, 1 :5000, 1 :6000, 1 :7000, 1 :8000, 1 :9000, 1 : 10000, 1 :50000 or a number or a range between any two of the values).

[0300] In some embodiments, the third medium comprises a plurality of trace elements comprising two or more elements selected from the group consisting of Cupric Sulfate, Ferric Citrate, Sodium Selenite, Zinc Sulfate, Ammonium Molybdate, Ammonium Vanadate, Manganese Sulfate, Nickel Sulfate, Sodium Silicate, Stannous Chloride and Hydrochloric Acid.

[0301] The third, fourth, and / or fifth media can comprise vitamin C (e.g., ascorbate). The concentration of vitamin C can be 0.1 mM to 0.5 mM (e.g., 0.1 mM, 0.11 mM, 0.12 mM, 0.13 mM, 0.14 mM, 0.15 mM, 0.16 mM, 0.17 mM, 0.18 mM, 0.19 mM, 0.2 mM, 0.21 mM, 0.22 mM, 0.23 mM, 0.24 mM, 0.25 mM, 0.26 mM, 0.27 mM, 0.28 mM, 0.29 mM, 0.3 mM, 0.31 mM,0.32 mM, 0.33 mM, 0.34 mM, 0.35 mM, 0.36 mM, 0.37 mM, 0.38 mM, 0.39 mM, 0.4 mM, 0.41 mM, 0.42 mM, 0.43 mM, 0.44 mM, 0.45 mM, 0.46 mM, 0.47 mM, 0.48 mM, 0.49 mM, 0.5 mM, or a number or a range between any two of the values). The sixth and / or seventh media can comprise zinc sulfate. The concentration of zinc sulfate can be 5 pM to 15 pM (e.g, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 11 pM, 12 pM, 13 pM, 14 pM, 15 pM, or a number or a range between any two of the values).

[0302] The first medium can comprise about 0.1% FAF-BSA, about 14.4 mM glucose, and / or about 0.01% sodium bicarbonate. The first medium can comprise about 0.5% FAF-BSA, about 10 mM glucose, and / or about 0.015% sodium bicarbonate. The second medium can comprise about 0.1% FAF-BSA, about 14.4 mM glucose, and / or about 0.01% sodium bicarbonate. The second medium can comprise about 0.5% FAF-BSA, about 10 mM glucose, and / or about 0.015% sodium bicarbonate. The third and / or fourth media can comprise about 0.5% FAF-BSA, about 10 mM glucose, about 0.015% sodium bicarbonate, and / or about 0.25 mM vitamin C. The fifth medium can comprise about 0.25% FAF-BSA, about 10 mM glucose, about 0.015% sodium bicarbonate, and / or about 0.25 mM vitamin C. The sixth and / or seventh media can comprise about 2% FAF-BSA, about 20 mM glucose, about 0.015% sodium bicarbonate, and / or about 10 pM zinc sulfate. Any of the first, second, third, fourth, fifth, sixth, and seventh media can comprise anantibiotic. Exemplary antibiotics that can be used in the methods of disclosure include: Amphotericin B, Ampicillin, Antibiotic Antimycotic Solution (comprising, penicillin, streptomycin, and / or amphotericin B), Cephalothin, Dihydrostreptomycin, Erythromycin, Gentamicin Sulfate, L-Glutamine-Penicillin-Streptomycin, Kanamycin Sulfate, Lincomycin HCI, Neomycin Sulfate, Nystatin, Paromomycin Sulfate, Penicillin-G, Penicillin-Streptomycin, Phenoxymethylpenicillinic Acid, Polymyxin B Sulfate, Spectinomycin dihydrochloride pentahydrate, Streptomycin sulfate, Tetracycline Hydrochloride, and Tylosin Tartrate.

[0303] The medium can be changed periodically in the culture, e.g., to provide optimal environment for the cells in the medium. For example, any of the medium can be change daily, or every two days, three days, four days, etc. In some embodiments, the media can be changed every 12 hours.

[0304] In some embodiments, the first medium comprises MCDB 131 medium or S3 S6 (BLAR001) medium, GlutaMax, 0.1% FAF-BSA, 14.4 mM glucose, 0.01% sodium bicarbonate, 100 ng / mL of AA, and 3 pM CHIR. In some embodiments, the first medium comprises MCDB 131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.015% sodium bicarbonate, 100 ng / mL of AA, 3 pM CHIR, and 2 pM Thiazovivin. In some embodiments, the first medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.015% sodium bicarbonate, 0.1% KSR, 100 ng / mL of AA, 3 pM CHIR, and 2 pM Thiazovivin. In some embodiments, the first medium comprises MCDB 131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.01% sodium bicarbonate, 1% B27, 100 ng / mL of AA, 6 pM CHIR, and 2 pM Thiazovivin. In some embodiments, the method comprises culturing a population of stem cells in the first culture medium thereby generating a first population of progenitor cells.

[0305] In some embodiments, the second medium comprises MCDB 131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.1% FAF-BSA, 14.4 mM glucose, 0.01% sodium bicarbonate, 100 ng / mL AA and 25 ng / mL FGF2. In some embodiments, the second medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.015% sodium bicarbonate, 100 ng / mL AA, 2 pM Thiazovivin, and 25 ng / mL FGF2. In some embodiments, the second medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.015% sodium bicarbonate, 0.1% KSR, 100 ng / mL AA, 2 pM Thiazovivin, and 25 ng / mL FGF2. In some embodiments, the second medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.01% sodium bicarbonate, 1% B27, 100 ng / mL AA, 2 pM Thiazovivin, and 25 ng / mL FGF2. In someembodiments, the method comprises culturing the first population of progenitor cells in the second culture medium thereby generating a second population of progenitor cells comprising definitive endoderm cells.

[0306] In some embodiments, the third culture medium comprises: MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BASA, 10 mM glucose, 1 :50000 ITS-X, 0.015% sodium bicarbonate, 0.25 mM Vitamin C, and 50 ng / mL FGF7. In some embodiments, the third culture medium comprises: MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.015% sodium bicarbonate, 0.25 mM Vitamin C, trace elements, and 50 ng / mL FGF7. In some embodiments, the third medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.015% sodium bicarbonate, 0.25 mM Vitamin C, trace elements, 1% B27, and 50 ng / mL FGF7. In some embodiments, the method comprises culturing the second population of progenitor cells in the third culture medium thereby generating a third population of progenitor cells comprising gut tube cells.

[0307] In some embodiments, the fourth culture medium comprises: MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BASA, 10 mM glucose, 1 :200 ITS- X, 0.015% sodium bicarbonate, 0.25 mM Vitamin C, 25 ng / mL FGF7, 1 pM retinoic acid, 0.25 pM SANT, and 300 nM TPPB. In some embodiments, the fourth medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :200 (v:v) ITS-X, 0.015% sodium bicarbonate, 0.25 mM Vitamin C, 1% B27, 25 ng / mL FGF7, 1 pM retinoic acid, 0.25 pM SANT, and 300 nM TPPB. In some embodiments, the method comprises culturing the third population of progenitor cells in the fourth culture medium thereby generating a fourth population of progenitor cells comprising posterior foregut cells.

[0308] In some embodiments, the fifth culture medium comprises: MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.25% FAF-BASA, 10 mM glucose, 1 :200 ITS-X, 0.015% sodium bicarbonate, 0.25 mM Vitamin C, 2 ng / mL FGF7, 50 nM retinoic acid, 0.25 pM of a Shh pathway inhibitor (e.g., SANT-1 (SANT)), and 300 nM of a PKC activator (e.g., TPPB). In some embodiments, the fifth culture medium comprises: MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.25% FAF-BASA, 10 mM glucose, 1 :200 ITS-X, 0.015% sodium bicarbonate, 0.25 mM Vitamin C, 1% B27, 4 ng / mL FGF7, 100 nM retinoic acid, 0.25 pM of a Shh pathway inhibitor (e.g., SANT-1 (SANT)), 200 nM of a BMP antagonist (e.g., LDN) and 300 nM of a PKC activator (e.g., TPPB). In some embodiments, the method comprises culturing the fourth population of progenitor cells in the fifth culture medium thereby generating a fifth population of progenitor cells comprising pancreatic endoderm cells.

[0309] In some embodiments, the sixth culture medium comprises: MCDB131medium or S3S6 (BLAR001) medium, GlutaMax, 2% FAF-BASA, 20 mM glucose, 0.015% sodium bicarbonate, 1 :200 ITS-X, 10 pM zinc sulfate, 50 nM retinoic acid, 0.25 pM of a Shh pathway inhibitor e.g., SANT-1 (SANT)), 100 nM of a BMP antagonist (e.g., LDN), 10 pM of an ALK-5 inhibitor (e.g., RepSox), 1 pM of a thyroid hormone (e.g., T3), and 10 pg / mL of an anti-coagulant (e.g., Heparin). In some embodiments, the sixth culture medium comprises: MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 2% FAF-BASA, 20 mM glucose, 0.015% sodium bicarbonate, 1 :200 ITS-X, 10 pM zinc sulfate, 1% B27, 50 nM retinoic acid, 0.25 pM of a Shh pathway inhibitor (e.g., SANT-1 (SANT)), 100 nM of a BMP antagonist (e.g., LDN), 10 pM of an ALK-5 inhibitor (e.g., RepSox), 1 pM of a thyroid hormone (e.g., T3), and 10 pg / mL of an anti-coagulant (e.g., Heparin). In some embodiments, the method comprises culturing the fifth population of progenitor cells in the sixth culture medium thereby generating a sixth population of progenitor cells comprising endocrine precursor cells.

[0310] In some embodiments, the seventh culture medium comprises: MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 2% FAF-BASA, 20 mM glucose, 0.015% sodium bicarbonate, 1 :200 ITS-X, 10 pM zinc sulfate, 100 nM of a BMP antagonist (e.g., LDN), 10 pM of an ALK-5 inhibitor (e.g., RepSox), 1 pM of a thyroid hormone (e.g., T3), 10 pg / mL of an anti -coagulant (e.g., Heparin), and 100 nM of a gamma secretase inhibitor (e.g., DBZ). In some embodiments, the seventh culture medium comprises: MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 2% FAF-BASA, 20 mM glucose, 0.015% sodium bicarbonate, 1 :200 ITS-X, 10 pM zinc sulfate, 1% B27, 100 nM of a BMP antagonist (e.g., LDN), 10 pM of an ALK-5 inhibitor (e.g., RepSox), 1 pM of a thyroid hormone (e.g., T3), 10 pg / mL of an anti-coagulant (e.g., Heparin), and 100 nM of a gamma secretase inhibitor (e.g., DBZ).Culture conditions

[0311] In some cases, cells can be cultured under dynamic conditions (e.g., under conditions in which the cells are subject to constant movement or stirring while in the suspension culture). For dynamic culturing of cells, the cells can be cultured in a container (e.g., an nonadhesive container such as a spinner flask (e.g., of 200 mL to 3000 mL, for example 250 mL; of 100 mL; or in 125 mL Erlenmeyer), which can be connected to a control unit and thus present a controlled culturing system. The method can be carried out under suspension agitation. Suspension agitation can comprise rotation. The rotation speed can be at least or at least about 35 RPM to 45 RPM (e.g., 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 RPM, or a number or a range between any two of these values). The rotation speed for generating the first, second, third, fourth, fifth, and sixth populations is 45 RPM, and the rotation speed for generating the seventh population of cells can be at least 35 RPM. In some embodiments, the method can be carried out using a commercially available bioreactor unit. An exemplary bioreactor unit is the PBS minibioreactor available from STEMCELL Technologies.

[0312] In some cases, cells can be cultured under non-dynamic conditions (e.g. , a static culture) while preserving their proliferative capacity. For non-dynamic culturing of cells, the cells can be cultured in an adherent culture vessel. An adhesive culture vessel can be coated with any of a number of substrates for cell adhesion such as extracellular matrix (ECM) to improve the adhesiveness of the vessel surface to the cells. The substrate for cell adhesion can be any material intended to attach stem cells or feeder cells (if used). The substrate for cell adhesion includes collagen, gelatin, poly-L-lysine, poly-D-lysine, vitronectin, laminin, fibronectin, PLO laminin, fibrin, thrombin, and RetroNectin and mixtures thereof, for example, Matrigel™, and lysed cell membrane preparations.Storage of cells generated by the disclosed methods

[0313] In some embodiments, the present disclosure relates to cry opreservation of the beta cells or precursors thereof obtained using the methods provided herein. In some embodiments, the cell population comprising pancreatic beta cells can be stored via cryopreservation. For instance, the cell population comprising beta cells, e.g, Stage 6 cells in some cases, can be dissociated into cell suspension, e.g, single cell suspension, and the cell suspension can be cryopreserved, e.g., frozen in a cry opreservation solution. The dissociation of the cells can be conducted by any of the techniques provided herein, for example, by enzymatic treatment. The cells can be frozen at a temperature of at highest -20 °C, at highest -30 °C, at highest -40 °C, at highest -50 °C, at highest -60 °C, at highest -70 °C, at highest -80 °C, at highest -90 °C, at highest -100 °C, at highest -110 °C, at highest -120 °C, at highest -130 °C, at highest - 140 °C, at highest -150 °C, at highest -160 °C, at highest -170 °C, at highest -180 °C, at highest - 190 °C, or at highest -200 °C. In some cases, the cells are frozen at a temperature of or of about - 80 °C. In some cases, the cells are frozen at a temperature of or of about -195°C. Any cooling methods can be used for providing the low temperature needed for cry opreservation, such as, but not limited to, electric freezer, solid carbon dioxide, and liquid nitrogen. In some cases, any cry opreservation solution available to one skilled in the art can be used for incubating the cells for storage at low temperature, including both custom made and commercial solutions. For example, a solution containing a cryoprotectant can be used. The cryoprotectant can be an agent that is configured to protect the cell from freezing damage. For instance, a cryoprotectant can be a substance that can lower the glass transition temperature of the cryopreservation solution. Exemplary cryoprotectants that can be used include DMSO (dimethyl sulfoxide), glycols (e.g., ethylene glycol, propylene glycol and glycerol), dextran (e.g., dextran-40), and trehalose.

[0314] Additional agents can be added into the cryopreservation solution for other effects. In some cases, commercially available cryopreservation solutions can be used in themethod provided herein, for instance, FrostaLife™, pZerve™, Prime-XV®, Gibco Synth-a- Freeze Cryopreservation Medium, STEM-CELLB ANKER®, CryoStor® Freezing Media, HypoThermosol® FRS Preservation Media, and CryoDefend® Stem Cells Media.

[0315] During the differentiation process, the cells can be subject to irradiation treatment as provided herein. In some cases, the cell population at Stage 6, e.g., the cell population that has cells being differentiated from insulin-positive endocrine cells into pancreatic beta cells, is irradiated for a period of time. In some cases, the cell population at Stage 6 after re-aggregation following the recovery from cry opreservation is irradiated for a period of time. In some cases, the cryopreserved cells (e.g., the cells that are cryopreserved at the end of Stage 5) are irradiated for a certain period of time prior to thawing and recovery for subsequent differentiation process.Differentiation Factors

[0316] Aspects of the disclosure relate to contacting progenitor cells (e.g., stem cells, e.g., iPS cells, definitive endoderm cells, primitive gut tube cells, PDX1 -positive pancreatic progenitor cells, NKX6.1 -positive pancreatic progenitor cells, insulin-positive endocrine cells) with cell differentiation factors, for example, to induce the maturation of the stem cells into definitive endoderm (e.g, Stage 1).

[0317] In some embodiments, the differentiation factor can induce the differentiation of pluripotent cells (e.g, iPSCs or hESCs) into definitive endoderm cells, e.g., in accordance with a method described herein. In some embodiments, the differentiation factor can induce the differentiation of definitive endoderm cells into primitive gut tube cells, e.g., in accordance with a method described herein. In some embodiments, the differentiation factor can induce the differentiation of primitive gut tube cells into PDX1 -positive posterior foregut, e.g., in accordance with a method described herein. In some embodiments, the differentiation factor can induce the differentiation of PDX1 -positive posterior foregut cells into NKX6-1 -positive pancreatic progenitor cells, e.g., in accordance with a method described herein. In some embodiments, the differentiation factor can induce the differentiation of NKX6-1 -positive posterior foregut cells into NKX2.2-positive endocrine precursor cells. In some embodiments, the differentiation factor induces the NKX2.2-positive endocrine precursor cells to insulin-positive endocrine cells, e.g, in accordance with a method described herein.

[0318] At least one differentiation factor described herein can be used alone, or in combination with other differentiation actors, to beta cells (e.g., immature beta cells) according to the methods as disclosed herein. In some embodiments, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten differentiation factors described herein are used in the methods of generating beta cells.Fibroblast Growth Factor (FGF) Family

[0319] FGFs are multifunctional proteins with a wide variety of effects; they are most commonly mitogens but also have regulatory, morphological, and endocrine effects. They have been alternately referred to as "pluripotent" growth factors and as "promiscuous" growth factors due to their multiple actions on multiple cell types. Promiscuous refers to the biochemistry and pharmacology concept of how a variety of molecules can bind to and elicit a response from single receptor. In the case of FGF, four receptor subtypes can be activated by more than twenty different FGF ligands. Thus the functions of FGFs in developmental processes include mesoderm induction, anterior-posterior patterning, limb development, neural induction and neural development, and in mature tissues / systems angiogenesis, keratinocyte organization, and wound healing processes. FGF is critical during normal development of both vertebrates and invertebrates and any irregularities in their function leads to a range of developmental defects.

[0320] Aspects of the disclosure relate to the use of growth factors from the FGF family as beta cell differentiation factors. In particular, it was surprisingly found that culture of cells with FGF2 at the Stage 1 step result in much improved efficiency of obtaining, e.g., Stage 4 and / or Stage 6 cells. The polypeptide sequences of FGF2 are available to the skilled artisan. In some embodiments, the FGF2 comprises a polypeptide having an amino acid sequence at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, or greater identical to the human FGF2 polypeptide sequence (GenBank Accession P09038).

[0321] In some embodiments, a non-FGF2 FGF can be used, e.g., to induce differentiation of definitive endoderm to gut tube cells. In some embodiments, a non-FGF2 FGF can be used, e.g., to induce differentiation of gut tube cells to posterior foregut. In some embodiments, a non-FGF2 FGF can be used, e.g., to induce differentiation of posterior foregut to pancreatic endoderm. In some embodiments, the growth factor from the FGF family in the methods and compositions provided herein comprises keratinocyte growth factor (KGF), also referred to herein as FGF7. The polypeptide sequences of FGF7 are available to the skilled artisan. In some embodiments, the FGF7 comprises a polypeptide having an amino acid sequence at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, or greater identical to the human FGF7 polypeptide sequence.Bone Morphogenetic Protein (BMP) Signaling Pathway Antagonists

[0322] Aspects of the disclosure relate to the use of BMP signaling pathway antagonists (or inhibitors) as beta cell differentiation factors. The BMP signaling family is a diverse subset of the TGF-beta superfamily (Sebald et al. Biol. Chem. 385:697-710, 2004). Over twenty known BMP ligands are recognized by three distinct type II (BMPRII, ActRIIa, andActRIIb) and at least three type I (ALK2, ALK3, and ALK6) receptors. Dimeric ligands facilitate assembly of receptor heteromers, allowing the constitutively-active type II receptor serine / threonine kinases to phosphorylate type I receptor serine / threonine kinases. Activated type I receptors phosphorylate BMP -responsive (BR-) SMAD effectors (SMADs 1, 5, and 8) to facilitate nuclear translocation in complex with SMAD4, a co-SMAD that also facilitates TGF signaling. In addition, BMP signals can activate intracellular effectors such as MAPK p38 in a SMAD-independent manner (Nohe et al. Cell Signal 16:291-299, 2004). Soluble BMP antagonists such as noggin, chordin, gremlin, and follistatin limit BMP signaling by ligand sequestration.

[0323] In some embodiments, the BMP signaling pathway inhibitor or antagonist in the methods and composition provided herein comprises DMH-1, or a derivative, analogue, or variant thereof.

[0324] In some embodiments, the BMP signaling pathway inhibitor or antagonist comprises LDN193189 (also known as LDN193189, 1062368-24-4, LDN-193189, DM 3189, DM-3189, IUPAC Name: 4-[6-(4-piperazin-l-ylphenyl)pyrazolo[l,5-a]pyrimidin-3- yl]quinolone). In some embodiments, the BMP signaling pathway inhibitor or antagonist in the methods and compositions provided herein comprises the following compound or a derivative, analogue, or variant of the following compound:N"' - H

[0325] In some embodiments, the BMP signaling pathway inhibitor in the methods and composition provided herein comprise an analog or derivative of LDN193189, e.g., a salt, hydrate, solvent, ester, or prodrug of LDN193189. In some embodiments, a derivative (e.g., salt) of LDN193189 comprises LDN193189 hydrochloride.ALK-5 Inhibitors

[0326] In some embodiments, ALK-5 (also referred to as TGF-P type I receptor) inhibitors are used. In some embodiments, the ALK-5 inhibitor comprises ALK5 inhibitor II (CAS 446859-33-2, an ATP-competitive inhibitor of TGF -B RI kinase, also known as RepSox, IUPAC Name: 2-[5-(6-methylpyridin-2-yl)-lH-pyrazol-4-yl]-l,5-naphthyridine. In some embodiments,the TGF-b signaling pathway inhibitor is an analog or derivative of ALK5 inhibitor II. In some embodiments, the ALK-5 inhibitor is selected from any of LY2157299 GW788388, LY364947, R268712, RepSox, SB525334, and SD208. Useful ALK5 inhibitors include: ALK5 inhibitor II (Enzo Life Sciences, Inc., Farmingdale, New York), which is also the preferred ALK5 inhibitor; ALK5i (Axxora, Inc., San Diego, California), SD208 (R&D Systems); TGF-P inhibitor SB431542 (Xcess Biosciences, Inc., San Diego, California); ITD-1 (Xcess Biosciences); LY2109761 (Xcess Biosciences); A83-01 (Xcess Biosciences); LY2157299 (Xcess Biosciences); TGF-P receptor inhV (EMD Millipore Chemical, Gibstown, New Jersey);TGF-P receptor inh I (EMD Millipore); TGF-P receptor inh IV (EMD Millipore); TGF-P receptor inh VII (EMD Millipore); TGF-P receptor inh VIII (EMD Millipore); TGF-P receptor inh II (EMD Millipore); TGF-P receptor inhVI (EMD Millipore); and TGF-P receptor inh VI (EMD Millipore). In some embodiments, the ALK-5 inhibitor comprises the following compound or a derivative, analogue, or variant of the following compound:WNT Signaling Pathway

[0327] Aspects of the disclosure relate to the use of activators of the WNT signaling pathway as cell differentiation factors.

[0328] In some embodiments, the WNT signaling pathway activator comprises CHIR99021. In some embodiments, the WNT signaling pathway activator comprises Wnt3a recombinant protein. In some embodiments, the WNT signaling pathway activator comprises a glycogen synthase kinase 3 (GSK3) inhibitor. Exemplary GSK3 inhibitors include, without limitation, 3F8, A 1070722, AR-A 014418, BIO, BlO-acetoxime, FRATide, lOZ-Hymenial disine, Indimbin-3 'oxime, kenpaullone, L803, L803-mts, lithium carbonate, NSC 693868, SB 216763, SB 415286, TC-G 24, TCS 2002, TCS 21311, TWS 119, and analogs or derivatives of any of these. Exemplary GSK3 inhibitors include CHIR-99021 (CT99021) HCI, SB216763, CHIR- 98014, TWS119, Tideglusib, SB415286, CHIR-99021 (CT99021), AZD2858, AZD1080, AR- A014418, TDZD-8, LY2090314, BlO-acetoxime, IM-12, 1- Azakenpaullone, Indirubin and 6- BIO. In some embodiments, the GSK-3 inhibitor is CHIR-99021 (CHIR). In some embodiments, the WNT pathway activator comprises the following compound or a derivative, analogue, orvariant of the following compound:Sonic Hedgehog (SHH) Signaling Pathway

[0329] Aspects of the disclosure relate to the use of Sonic Hedgehog (SHH, or Shh) signaling pathway inhibitors as cell differentiation factors.

[0330] In some embodiments, the SHH signaling pathway inhibitor comprises SANT- 1 (SANT). In some embodiments, the SHH signaling pathway inhibitor comprises SANT2. In some embodiments, the SHH signaling pathway inhibitor in the methods and composition provided herein comprises SANT3. In some embodiments, the SHH signaling pathway inhibitor in the methods and composition provided herein comprises SANT4. In some embodiments, the SHH signaling pathway inhibitor comprises Cur61414. In some embodiments, the SHH signaling pathway inhibitor in the methods and composition provided herein comprises forskolin. In some embodiments, the SHH signaling pathway inhibitor in the methods and composition provided herein comprises tomatidine. In some embodiments, the SHH signaling pathway inhibitor in the methods and composition provided herein comprises AY9944. In some embodiments, the SHH signaling pathway inhibitor in the methods and composition provided herein comprises triparanol. In some embodiments, the SHH signaling pathway inhibitor comprises compound A or compound B (as disclosed in U.S. Pub. No. 2004 / 0060568). In some embodiments, the SHH signaling pathway inhibitor comprises a steroidal alkaloid that antagonizes hedgehog signaling (e.g., cyclopamine or a derivative thereof) as disclosed in U.S. Pub. No. 2006 / 0276391. In some embodiments, the Shh signaling pathway inhibitor comprises the following compound or a derivative, analogue, or variant of the following compound:Protein Kinase C

[0331] Aspects of the disclosure relate to the use of protein kinase C activators for beta cell differentiation factors. Protein kinase C is one of the largest families of protein kinase enzymes and is composed of a variety of isoforms. Conventional isoforms include a, 1, 011, y; novel isoforms include 5, s, q, 9; and atypical isoforms includeand X / t. PKC enzymes are primarily cytosolic but translocate to the membrane when activated. In the cytoplasm, PKC is phosphorylated by other kinases or auto-phosphorylated. In order to be activated, some PKC isoforms (e.g., PKC-s) require a molecule to bind to the diacylglycerol (“DAG”) binding site or the phosphatidylserine (“PS”) binding site. Others are able to be activated without any secondary binding messengers at all. PKC activators that bind to the DAG site include, but are not limited to, bryostatin, picologues, phorbol esters, aplysiatoxin, and gnidimacrin. PKC activators that bind to the PS site include, but are not limited to, polyunsaturated fatty acids and their derivatives.

[0332] In some embodiments, the PKC activator comprises PdbU. In some embodiments, the PKC activator comprises TPB. In some embodiments, the PKC activator comprises cyclopropanated polyunsaturated fatty acids, cyclopropanated monounsaturated fatty acids, cyclopropanated polyunsaturated fatty alcohols, cyclopropanated monounsaturated fatty alcohols, cyclopropanated polyunsaturated fatty acid esters, cyclopropanated monounsaturated fatty acid esters, cyclopropanated polyunsaturated fatty acid sulfates, cyclopropanated monounsaturated fatty acid sulfates, cyclopropanated polyunsaturated fatty acid phosphates, cyclopropanated monounsaturated fatty acid phosphates, macrocyclic lactones, DAG derivatives, isoprenoids, octylindolactam V, gnidimacrin, iripallidal, ingenol, napthalenesulfonamides, diacylglycerol kinase inhibitors, fibroblast growth factor 18 (FGF-18), insulin growth factor, hormones, and growth factor activators, as described in PCT Pub. No. WO / 2013 / 071282. In some embodiments, the bryostain comprises bryostatin-1, bryostatin-2, bryostatin-3, bryostatin-4, bryostatin-5, bryostatin-6, bryostatin-7, bryostatin-8, bryostatin-9, bryostatin- 10, bryo statin- 11, bryostatin- 12, bryo statin- 13, bryostatin- 14, bryostatin-15, bryostatin- 16, bryo statin- 17, or bryostatin-18. In some embodiments, the PKC activator is (2E,4E)-N-[(2S,5S)-1,2,3,4,5,6- Hexahydro-5-(hydroxymethyl)-l-methyl-2-(l-methylethyl)-3-oxo-l,4-benzodiazocin-8-yl]-5-[4- (trifluoromethyl)phenyl]-2,4-pentadienamide, e.g., TPPB. In some embodiments, the PKC activator is (2S,5S)-(E,E)-8-(5-(4- 27 (trifluoromethyl)phenyl)-2,4-pentadienoylamino) benzolactam, e.g., TPPB. In some embodiments, the PKC activator comprises the following compound or a derivative, analogue, or variant of the following compound:gamma-Secretase Inhibitors

[0333] Aspects of the disclosure relate to the use of gamma (g, y-secretase inhibitors) as cell differentiation factors. In some embodiments, the g-secretase inhibitor comprises N-[(1S)- 2-[[(7S)-6,7-Dihydro-5-methyl-6-oxo-5H-dibenz[b,d]azepin-7-yl]amino]-l-methyl-2-oxoethyl]- 3,5-difluorobenzeneacetamide, e.g., Dibenzazepine (DBZ).

[0334] In some embodiments, the g-secretase inhibitor in the methods and composition provided herein comprises XXI. In some embodiments, the g-secretase inhibitor in the methods and composition provided herein comprises DAPT. In some embodiments of any one of the methods provided herein, the gamma secretase inhibitor is selected from LY3039478, secretase inhibitor I (GSI I) Z-Leu-Leu-Norleucine; y-secretase inhibitor II (GSI II); y-secretase inhibitor III (GSI III), N-Benzyloxycarbonyl-Leu-leucinal, N-(2-Naphthoyl)-Val-phenylalaninal; y-secretase inhibitor III (GSI IV); y-secretase inhibitor III (GSI V), N-Benzyloxycarbonyl-Leu- phenylalaninal; y-secretase inhibitor III (GSI VI), l-(S)-endo-N-(l,3,3)- Trimethylbicyclo[2.2.1]hept-2-yl)-4-fluorophenyl Sulfonamide; y-secretase inhibitor III (GSI VII), Menthyloxycarbonyl-LL-CHO; y-secretase inhibitor III (GSI IX), (DAPT), N-[N-(3,5- Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-Butyl Ester; y-secretase inhibitor X (GSI X), { 1 S-Benzyl-4R-[1-(1 S-carbamoyl-2-phenethylcarbamoyl)-l S-3-methylbutylcarb-amoyl]-2R- hydroxy-5-phenylpentyl}carbamic Acid tert-butyl Ester; y-secretase inhibitor XI (GSI XI), 7- Amino-4-chl oro-3 -methoxyisocoumarin; y-secretase inhibitor XII (GSI XII), Z-Ile-Leu-CHO; y- secretase inhibitor XIII (GSI XIII), Z-Tyr-Ile-Leu-CHO; y-secretase inhibitor XIV (GSI XIV), Z- Cys(t-Bu)-Ile-Leu-CHO; y-secretase inhibitor XVI (GSI XVI), N-[N-3,5-Difluorophenacetyl]-L- alanyl-S-phenylglycine Methyl Ester; y-secretase inhibitor XVII (GSI XVII); y-secretase inhibitor XIX (GSI XIX), benzo[e][l,4]diazepin-3-yl)-butyramide; y-secretase inhibitor XX (GSI XX), (S,S)-2-[2-(3,5-Difluorophenyl)acetylamino]-N-(5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl)propionamide; y-secretase inhibitor XXI (GSI XXI), (S,S)-2-[2-(3,5- Difluorophenyl)-acetylamino]-N-(l-methyl-2-oxo-5-phenyl-2-,3-dihydro-lH- benzo[e][l,4]diazepin-3-yl)-propionamide; Gamma40 secretase inhibitor I, N-trans-3,5- Dimethoxycinnamoyl-Ile-leucinal; Gamma40 secretase inhibitor II, N-tert-Butyloxycarbonyl- Gly-Val-Valinal Isovaleryl-V V-Sta-A-Sta-OCH3; MK-0752; MRK-003 (Merck); semagacestat / LY450139; RO4929097; PF-03084,014; BMS-708163; MPC-7869 (y-secretase modifier), YO-01027 (Dibenzazepine), Compound E ([(2S)-2-{[(3,5- Difluorophenyl)acetyl]amino}-N-[(3S)-l-methyl-2-oxo-5-phenyl-2,3-dihydro-lH-l,4- benzodiazepin-3-yl]propanamide], LY411575, L-685,458, BMS-289948 (4-chloro-N-(2,5- difluorophenyl)-N-(( 1 R)- { 4-fluoro-2- [3 -( 1 H-imidazol- 1 - yl)propyl]phenyl}ethyl)benzenesulfonamide hydrochloride) and BMS-299897 (4-[2-((lR)-l- {[(4-chlorophenyl)sulfonyl]-2,5-difluoroanilino}ethyl)-5-fluorophenyljbutanoic acid). In some embodiments, the gamma secretase inhibitor in the methods and composition provided herein comprises the following compound or a derivative, analogue, or variant of the following compound:Thyroid Hormone Signaling Pathway Activators

[0335] Aspects of the disclosure relate to the use of thyroid hormone signaling pathway activators as differentiation factors. The thyroid hormone signaling pathway activators can comprise a thyroid hormone, analog, or derivative thereof, e.g., T3.

[0336] In some embodiments, the thyroid hormone signaling pathway activator in the methods and composition provided herein comprises triiodothyronine (T3), or analogs, derivatives or salts thereof. In some embodiments, the thyroid hormone signaling pathway activator in the methods and composition provided herein comprises GC-1. In some embodiments, the thyroid hormone signaling pathway activator in the methods and composition provided herein comprises an analog or derivative of T3 or GC-1. Exemplary analogs of T3 in the methods and composition provided herein include, but are not limited to, selective and non-selective thyromimetics, Tllb selective agonist-GC-1, GC-24,4-Hydroxy-PCB 106, MB07811, MB07344,3,5-diiodothyropropionic acid (DITPA); the selective TR-b agonist GC-1; 3- lodothyronamine (T(l)AM) and 3,3',5-triiodothyroacetic acid (Triac) (bioactive metabolites of thehormone thyroxine (T(4)); KB-2115 and KB-141; thyronamines; SKF L-94901; DIBIT; 3'-AC- T2; tetraiodothyroacetic acid (Tetrac) and triiodothyroacetic acid (Triac) (via oxidative deamination and decarboxylation of thyroxine [T4] and triiodothyronine [T3] alanine chain), 3,3 ',5 '-triiodothyronine (rT3) (via T4 and T3 deiodination), 3,3'-diiodothyronine (3,3 '-T2) and 3,5- diiodothyronine (T2) (via T4, T3, and rT3 deiodination), and 3-iodothyronamine (T1AM) and thyronamine (TOAM) (via T4 and T3 deiodination and amino acid decarboxylation), as well as for TH structural analogs, such as 3,5,3 '-triiodothyropropionic acid (Triprop), 3,5-dibromo-3- pyridazinone-l-thyronine (L-940901), N-[3,5-dimethyl-4-(4'-hydroxy-3'-isopropylphenoxy)- phenyl] -oxami c acid (CGS 23425), 3,5-dimethyl-4-[(4'-hydroxy-3 '-isopropylbenzyl)-phenoxy] acetic acid (GC-1), 3,5-dichloro-4-[(4-hydroxy-3-isopropylphenoxy)phenyl] acetic acid (KB- 141), and 3,5-diiodothyropropionic acid (DITPA). In some embodiments, the T3 hormone or hormone mimetics is 3,3',5-triiodothyroacetic sodium salt.

[0337] In some embodiments, the thyroid hormone signaling pathway activator in the methods and composition provided herein comprises a prodrug or prohormone of T3, such as T4 thyroid hormone (e.g., thyroxine or L-3,5,3',5'-tetraiodothyronine). In some embodiments, the gamma secretase inhibitor in the methods and compositions provided herein comprises the following compound or a derivative, analogue, or variant of the following compound:ROCK Inhibitors

[0338] In some embodiments, a culture medium (e.g., the first and / or second media) comprises an effective amount of an inhibitor of rho-associated protein kinase (ROCK) (also referred to herein as ROCK inhibitor). As described herein, addition of a Rock inhibitor to S1D1 and S1D2 media surprisingly improves potency of obtained S6 cells. Exemplary ROCK inhibitors include, but are not limited to N-[(l S)-2-Hydroxy-l-phenylethyl]-N'-[4-(4-pyridinyl)phenyl]-urea (AS1892802), fasudil hydrochloride (also known as HA 1077), -[3-[[2-(4-Amino-l,2,5- oxadiazol-3-yl)-I-ethyl-lH-imidazo[4,5-c]pyridin-6-yl]oxy]phenyl]-4-[2-(4- morpholinyl)ethoxy]benzamide (GSK269962), 4-[4-(Trifluoromethyl)phenyl]-N-(6-Fluoro-lH- indazol-5-yl)-2-methyl-6-oxo-l,4,5,6-tetrahydro-3-pyridinecarboxamide (GSK 429286), (S)-(+)- 2-Methyl-l-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-lH-l,4-diazepine dihydrochloride (H 1152 dihydrochloride), (S)-(+)-4-Glycyl-2-methyl-l-[(4-methyl-5-isoquinolinyl)sulfonyl]- hexahydro-lH-l,4-diazepine dihydrochloride (glycyl-H 1152 dihydrochloride), N-[(3-Hydroxyphenyl)methyl]-N ' -[4-(4-pyridinyl)-2-thiazolyl]urea dihydrochloride (RKI 1447 dihydrochloride), (3S)-l-[[2-(4-Amino-l,2,5-oxadiazol-3-yl)-l-ethyl-lH-imidazo[4,5-c]pyridin- 7-yl]carbonyl]-3-pyrrolidinamine dihydrochloride (SB772077B dihydrochloride), N-[2-[2- (Dimethylamino)ethoxy]-4-(lH-pyrazol-4-yl)phenyl-2,3-dihydro-l,4-benzodi oxin-2- carboxamide dihydrochloride (SR 3677 dihydrochloride), and trans-4-[(R)-l-Aminoethyl]-N-4- pyridinylcyclohexanecarboxamide dihydrochloride (Y-27632 dihydrochloride), N-Benzyl-[2- (pyrimidin-4-yl)amino]thiazole-4-carboxamide (Thiazovivin), Rock Inhibitor, a isoquinolinesulfonamide compound (Rho Kinase Inhibitor), N-(4-Pyridyl)-N ' -(2,4,6- trichlorophenyl)urea (Rho Kinase Inhibitor II), 3-(4-Pyridyl)-lH-indole (Rho Kinase Inhibitor III, Rockout), and 4-pyrazoleboronic acid pinacol ester; a Rock antibody commercially available from Santa Cruz Biotechnology selected from the group consisting of Rock-1 (B 1), Rock-1 (C-19), Rock-1 (H-l l), Rock-1 (G-6), Rock-1 (H-85), Rock-1 (K-18), Rock-2 (C-20), Rock-2 (D-2), Rock-2 (D-l 1), Rock-2 (N-19), Rock-2 (H-85), Rock-2 (30- J); a ROCK CRISPR / Cas9 knockout plasmid selected from the group consisting of Rock-1 CRISPR / Cas9 KO plasmid (h), Rock-2 CRISPR / Cas9 KO plasmid (h), Rock-1 CRISPR / Cas9 KO plasmid (m), Rock-2 CRISPR / Cas9 KO plasmid (m); a ROCK siRNA, shRNA plasmid and / or shRNA lentiviral particle gene silencer selected from the group consisting of Rock-1 siRNA (h): sc-29473, Rock-1 siRNA (m): sc-36432, Rock-1 siRNA (r): sc-72179, Rock-2 siRNA (h): sc-29474, Rock-2 siRNA (m): sc-36433, Rock- 2 siRNA (r): sc-108088. In some embodiments, the ROCK inhibitor comprises Thiazovivin. In some embodiments, the ROCK inhibitor comprises the following compound or a derivative, analogue, or variant of the following compound:Serum Replacement and Supplement

[0339] In some embodiments, the first and / or second medium can comprise a defined, non-serum serum replacement supplement. It was surprisingly found that addition of defined, nonserum serum replacement supplement to the first and / or second medium can improve yield of S6 cells. The defined, non-serum serum replacement supplement can comprise one or more of insulin, transferrin, ascorbic acid, a plurality of amino acids, a plurality of trace elements, and albumin. The albumin can comprise lipid-rich bovine serum albumin or albumin substitute (Albumax®). The plurality of amino acids can comprise two or more of Glycine, L-Histidine, L-Isoleucine, L-Methionine, L-Phenylalanine, L-Proline, L-Hydroxyproline, L-Serine, L-Threonine, L- Tryptophan, L-Tyrosine, and L-Valine. The plurality of trace elements can comprise two or more of Ag+, Al3+, Ba2+, Cd2+, Co2+, Cr+, Ge4+, Se4+, Br“, F, Mn2+, F“, Si4+, V5+, Mo6+, Ni2+, Rb+, Sn2+and Zr4+.

[0340] A defined, non-serum serum replacement supplement (for adding to a culture media described herein) can comprise: about 150 mg / L Glycine, about 940 mg / L L-Histidine, about 3400 mg / L L-Isoleucine, about 90 mg / L L-Methionine, about 1800 mg / L L-Phenylalanine, about 4000 mg / L L-Proline, about 100 mg / L L-Hydroxyproline, about 800 mg / L L-Serine, about 2200 mg / L L-Threonine, about 440 mg / L L-Tryptophan, about 77 mg / L L-Tyrosine, about 2400 mg / L L-Valine, about 33 mg / L Thiamine, about 10 mg / L reduced glutathione, about 330 mg / L Ascorbic acid-2-PO4, about 55 mg / L Transferrin, about 100 mg / L insulin, about 0.07 mg / L Sodium selenite, about 83000 mg / L albumin or albumin substitute, and any combination of the above components. The amount of each trace element in the defined, non-serum serum replacement supplement can be, be about, be at least, or be at most, 0.0004-0.01 mg / L or any number or range between these two values.

[0341] Defined, non-serum serum replacement supplements are commercially available, and include, e.g., KnockOut™ Serum Replacement (KSR) available from Thermo Fisher Scientific. Non-limiting methods and compositions related to defined, non-serum serum replacement supplement are also described in US patent application publication US20020076747, which is hereby incorporated by refe...

Claims

WHAT IS CLAIMED IS:

1. A method for generating immature P cells from stem cells, the method comprising: (a) culturing a population of stem cells,(i) in a first medium comprising Activin A (AA) and a WNT pathway activator to generate a first population of progenitor cells, and(ii) culturing the first population of progenitor cells in a second medium comprising AA and FGF2 for a time sufficient to generate a second population of progenitor cells comprising definitive endoderm cells; and culturing the second population of progenitor cells under conditions sufficient to generate a population of cells comprising immature P cells.

2. The method of claim 1, comprising (b) culturing the second population of progenitor cells in a third medium comprising FGF7 for a time sufficient to generate a third population of progenitor cells comprising gut tube cells.

3. The method of claim 2, comprising (c) culturing the third population of progenitor cells in a fourth medium comprising FGF7, retinoic acid, a Sonic Hedgehog (Shh) pathway inhibitor, and a protein kinase C (PKC) activator for a time sufficient to generate a fourth population of progenitor cells comprising posterior foregut cells.

4. The method of claim 3, comprising (d) culturing the fourth population of progenitor cells in a fifth medium comprising FGF7, retinoic acid, a Shh pathway inhibitor, and a PKC activator, wherein the fifth medium comprises a lower concentration of FGF7 and a lower concentration of retinoic acid than the fourth medium, for a time sufficient to generate a fifth population of progenitor cells comprising pancreatic endoderm cells, optionally the fifth medium comprises a bone morphogenetic protein (BMP) antagonist.

5. The method of claim 4, comprising (e) culturing the fifth population of progenitor cells in a sixth medium comprising retinoic acid, a Shh pathway inhibitor, a BMP antagonist, an anaplastic lymphoma kinase 5 (ALK-5) inhibitor, a thyroid hormone, and an anti-coagulant for a time sufficient to generate a sixth population of progenitor cells comprising endocrine precursor cells.

6. The method of claim 5, comprising (f) culturing the sixth population of progenitor cells in a seventh medium comprising a BMP antagonist, an ALK-5 inhibitor, a thyroid hormone, an anti-coagulant, and a y-secretase inhibitor, for a time sufficient to generate a seventh population of cells comprising immature P cells.

7. The method of any one of claims 1-6, wherein the first, second, third, fourth, fifth, sixth, and / or seventh media comprise B27 supplement, optionally the first, second, third, fourth, fifth, sixth, and / or seventh media comprise about 1% B27 supplement.

8. The method of any one of claims 1-7, wherein the population of stem cells is cultured in the first culture medium for at least 12 hours, optionally at least 24 hours.

9. The method of any one of claims 1-8, wherein the WNT pathway activator of the first medium is CHIR-99021 (CHIR).

10. The method of any one of claims 3-9, wherein the Shh pathway inhibitor of the fourth, fifth, and / or sixth medium is SANT-1 (SANT).

11. The method of any one of claims 3-10, wherein the PKC activator of the fourth and / or fifth medium is (2E,4E)-N-[(2S,5S)-l,2,3,4,5,6-Hexahydro-5-(hydroxymethyl)-l-methyl- 2-(l-methylethyl)-3-oxo-l,4-benzodiazocin-8-yl]-5-[4-(trifluoromethyl)phenyl]-2,4- pentadienamide (TPPB).

12. The method of any one of claims 4-11, wherein the BMP antagonist of the fifth, sixth and / or seventh medium is LDN-193189-HCL (LDN).

13. The method of any one of claims 5-12, wherein the ALK-5 inhibitor of the sixth and / or seventh medium is RepSox.

14. The method of any one of claims 5-13, wherein the thyroid hormone of the sixth and / or seventh medium is 3,3’,5-Triiodo-L-thyronine sodium salt (T3).

15. The method of any one of claims 6-14, wherein the y-secretase inhibitor of the seventh medium is Dibenzazepine (DBZ).

16. The method of any one of claims 5-15, wherein the anti-coagulant of the sixth and / or seventh medium is Heparin.

17. The method of any one of claims 1-16, wherein the first medium comprises (i) about 100 ng / mL of AA and about 3 pM CHIR or (ii) about 100 ng / mL of AA and about 6 pM CHIR.

18. The method of any one of claims 1-17, wherein the second medium comprises about 20-30 ng / mL FGF2.

19. The method of any one of claims 1-18, wherein the second medium comprises about 25 ng / mL FGF2.

20. The method of any one of claims 18-19, wherein the second medium further comprises about 100 ng / mL of AA.

21. The method of any one of claims 1-20, wherein the first medium, the second medium, or both, comprise a ROCK inhibitor, optionally the ROCK inhibitor is Thiazovivin.

22. The method of claim 21, wherein the first medium, the second medium, or both comprise about 2 pM Thiazovivin.

23. The method of any one of claims 1-22, wherein the first medium, the second medium, or both, comprise a defined, serum-free serum replacement.

24. The method of claim 23, wherein the first medium, the second medium, or both comprise about 0.1% the defined, serum-free serum replacement.

25. The method of any one of claims 23-24, wherein the defined, serum-free serum replacement comprises one or more of insulin, transferrin, ascorbic acid, a plurality of amino acids, a plurality of trace elements, and albumin; and optionally the albumin is AlbuMAX.

26. The method of claim 25, wherein the plurality of amino acids comprises two or more of Glycine, L-Histidine, L-Isoleucine, L-Methionine, L-Phenylalanine, L-Proline, L- Hydroxyproline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine, and L-Valine.

27. The method of any one of claims 25-26, wherein the plurality of trace elements comprises two or more of Ag+, Al3+, Ba2+, Cd2+, Co2+, Cr+, Ge4+, Se4+, Br“, I“, Mn2+, F“, Si4+, V5+, MO6+, Ni2+, Rb+, Sn2+and Zr4+.

28. The method of any one of claims 23-27, wherein the defined, serum-free serum replacement is KnockOut serum replacement (KSR).

29. The method of any one of claims 2-28, wherein the third medium comprises about 50 ng / mL FGF7.

30. The method of any one of claims 3-29, wherein the fourth medium comprises about 25 ng / mL FGF7, about 1 pM Retinoic Acid, about 0.25 pM SANT, and about 300 nM TPPB.

31. The method of any one of claims 4-30, wherein the fifth medium comprises about 2 ng / mL FGF7 or about 4 ng / mL FGF7, about 50 nM Retinoic Acid or about 100 nM Retinoic Acid, about 0.25 pM SANT, and about 300 nM TPPB, optionally the fifth medium comprises about 200 nM LDN.

32. The method of any one of claims 5-31, wherein the sixth medium comprises about 50 nM Retinoic Acid, about 0.25 pM SANT, about 100 nM LDN, about 10 pM RepSox, about IpM T3, and about 10 pg / mL Heparin.

33. The method of any one of claims 6-32, wherein the seventh medium comprises about 100 nM LDN, about 10 pM RepSox, about 1 pM T3, about 10 pg / mL Heparin, and about lOO nM DBZ.

34. The method of any one of claims 1-33, wherein the population of stem cells is generated by culturing a plurality of stem cells in a 3D culture medium comprising a ROCK inhibitor, optionally, the ROCK inhibitor is thiazovivin.

35. The method of claim 34, wherein the 3D culture medium comprises 1 pM to 10 pM thiazovivin, optionally about 5 pM thiazovivin.

36. The method of any one of claims 34-35, wherein the starting concentration of the plurality of stem cells added to the 3D culture medium is about 2 * 105cells / mL.

37. The method of any one of claims 34-36, wherein the 3D culture medium furthercomprises StemScale media.

38. The method of any one of claims 34-37, wherein the plurality of stem cells are cultured in the 3D culture medium for at least 3 days, optionally wherein at least half of the media is replaced with fresh 3D culture medium at Day 2.

39. The method of any one of claims 6-38, wherein the seventh medium further comprises a DNA-damaging agent, optionally wherein the DNA-damaging agent is selected from the group comprising: radiation, cisplatin, oxaliplatin, carboplatin, nedaplatin, lobaplatin, triplatin, tetranitrate, picoplatin, satraplatin, prolindac, aroplatin, camptothecin, topotecan, irinotecan / sn38, rubitecan, belotecan, idarubicin, amrubicin, pirarubicin, valrubicin, zorubicin, teniposide, aminopterin, methotrexate, pemetrexed, raltitrexed, pentostatin, cladribine, clofarabine, fludarabine, thioguanine, mercaptopurine, fluorouracil, capecitabine, tegafur, carmofur, floxuridine, cytarabine, gemcitabine, azacitidine, hydroxyurea, mechlorethamine, cyclophosphamide, ifosfamide, trofosfamide, chlorambucil, melphalan, prednimustine, bendamustine, uramustine, estramustine, carmustine, lomustine, semustine, fotemustine, nimustine, ranimustine, streptozocin, busulfan, mannosulfan, treosulfan, carboquone, thiotepa, triaziquone, triethylenemelamine, procarbazine, dacarbazine, etopside, temozolomide, altretamine, mitobronitol, actinomycin, bleomycin, mitomycin and plicamycin.

40. The method of claim 39, wherein the DNA-damaging agent is bleomycin, optionally the seventh medium comprises about 50 nM bleomycin.

41. The method of any one of claims 39-40, wherein the sixth population of cells is cultured in the seventh medium comprising the DNA-damaging agent for two days.

42. The method of claim 41, wherein the sixth population of cells is cultured in the seventh medium for at least seven days and wherein the method comprises removing the DNA- damaging agent from the seventh medium following the first two days of culture in the seventh medium comprising the DNA-damaging agent.

43. The method of any one of claims 1-42, wherein the time sufficient to generate the first population of progenitor cells of step (a) (i) comprises culturing for at least 12 hours, optionally at least 24 hours and wherein the time sufficient to generate the second population of progenitor cells of step (a) (ii) comprises culturing for at least 12 hours, optionally at least 24 hours.

44. The method of any one of claims 2-43, wherein the time sufficient to generate the third population of progenitor cells of step (b) comprises culturing for at least three days.

45. The method of any one of claims 3-44, wherein the time sufficient to generate the fourth population of progenitor cells of step (c) comprises culturing for at least two days.

46. The method of any one of claims 4-45, wherein the time sufficient to generate thefifth population of progenitor cells of step (d) comprises culturing for at least three days.

47. The method of any one of claims 5-46, wherein the time sufficient to generate the sixth population of progenitor cells of step (e) comprises culturing for at least three days.

48. The method of any one of claims 6-47, wherein the time sufficient to generate the seventh population of cells of step (f) comprises culturing for at least seven days.

49. The method of any one of claims 6-48, wherein steps (a) - (f) occur in 20 days or less.

50. The method of any one of claims 6-48, wherein steps (a) - (f) occur in more than 20 days.

51. The method of any one of claims 1-50, wherein the method is carried out under suspension agitation.

52. The method of claim 51, wherein the suspension agitation comprises rotation, optionally wherein the rotation speed is at least about 35 RPM to about 45 RPM.

53. The method of claim 52, wherein the rotation speed of steps (a)-(e) is at least 45 RPM, and the rotation speed of step (f) is at least 35 RPM.

54. The method of any one of claims 1-53, wherein the first, second, third, fourth, fifth, sixth and / or seventh media comprise MCDB131 medium or S3S6 (BLAR001) medium.

55. The method of any one of claims 1-54, wherein the first, second, third, fourth, fifth, sixth and / or seventh media comprise GlutaMax, non-human serum, glucose, sodium bicarbonate, or any combination thereof; optionally the non-human serum is BSA or fatty acid free BSA (FAF BSA).

56. The method of claim 55, wherein the concentration of non-human serum is 0.05%- 3%, the concentration of glucose is 5 mM to 30 mM, the concentration of sodium bicarbonate is 0.005%-0.02%, and any combination thereof.

57. The method of any one of claims 55-56, wherein ITS-X is added to the first, second, third, fourth, fifth, sixth, and / or seventh media at a v:v ratio of about 1 :50000 to about 1 :200.

58. The method of any one of claims 2-57, wherein the third medium comprises a plurality of trace elements comprising two or more elements selected from the group consisting of Cupric Sulfate, Ferric Citrate, Sodium Selenite, Zinc Sulfate, Ammonium Molybdate, Ammonium Vanadate, Manganese Sulfate, Nickel Sulfate, Sodium Silicate, Stannous Chloride and Hydrochloric Acid.

59. The method of any one of claims 2-57, wherein the third, fourth, and / or fifth media comprise vitamin C.

60. The method of claim 59, wherein the concentration of vitamin C is 0.1 mM to 0.5mM.

61. The method of any one of claims 5-60, wherein the sixth and / or seventh media comprise zinc sulfate.

62. The method of claim 61, wherein the concentration of zinc sulfate is 5 pM to 15 pM.

63. The method of any one of claims 1-62, wherein the first medium comprises about 0.1% FAF-BSA, about 14.4 mM glucose, and / or about 0.01% sodium bicarbonate.

64. The method of any one of claims 1-62, wherein the first medium comprises about 0.5% FAF-BSA, about 10 mM glucose, and / or about 0.015% sodium bicarbonate, optionally about 0.01% sodium bicarbonate.

65. The method of any one of claims 1-64, wherein the second medium comprises about 0.1% FAF-BSA, about 14.4 mM glucose, and / or about 0.01% sodium bicarbonate.

66. The method of any one of claims 1-64, wherein the second medium comprises about 0.5% FAF-BSA, about 10 mM glucose, and / or about 0.015% sodium bicarbonate.

67. The method of any one of claims 2-66, wherein the third and / or fourth media comprise about 0.5% FAF-BSA, about 10 mM glucose, about 0.015% sodium bicarbonate, and / or about 0.25 mM vitamin C.

68. The method of any one of claims 4-67, wherein the fifth medium comprises about 0.25% FAF-BSA, about 10 mM glucose, about 0.015% sodium bicarbonate, and / or about 0.25 mM vitamin C.

69. The method of any one of claims 5-68, wherein the sixth and / or seventh media comprise about 2% FAF-BSA, about 20 mM glucose, about 0.015% sodium bicarbonate, and / or about 10 pM zinc sulfate.

70. The method of any one of claims 1-69, wherein any of the first, second, third, fourth, fifth, sixth, and seventh media further comprise an antibiotic.

71. The method of any one of claims 1-70, wherein the first medium comprises MCDB13 1 medium or S3S6 (BLAR001) medium, GlutaMax, 0.1% FAF-BSA, 14.4 mM glucose, 0.01% sodium bicarbonate, 100 ng / mL of AA, and 3 pM CHIR.

72. The method of any one of claims 1-70, wherein the first medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.015% sodium bicarbonate, 100 ng / mL of AA, 3 pM CHIR, and 2 pM Thiazovivin.

73. The method of any one of claims 1-70, wherein the first medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.015% sodium bicarbonate, 0.1% KSR, 100 ng / mL of AA, 3 pM CHIR,and 2 pM Thiazovivin.

74. The method of any one of claims 1-70, wherein the first medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.01% sodium bicarbonate, 1% B27, 100 ng / mL of AA, 6 pM CHIR, and 2 pM Thiazovivin.

75. The method of any one of claims 1-74, wherein the second medium comprises MCDB13 1 medium or S3S6 (BLAR001) medium, GlutaMax, 0.1% FAF-BSA, 14.4 mM glucose, 0.01% sodium bicarbonate, 100 ng / mL AA and 25 ng / mL FGF2.

76. The method of any one of claims 1-74, wherein the second medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.015% sodium bicarbonate, 100 ng / mL AA, 2 pM Thiazovivin, and 25 ng / mL FGF2.

77. The method of any one of claims 1-74, wherein the second medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.015% sodium bicarbonate, 0.1% KSR, 100 ng / mL AA, 2 pM Thiazovivin, and 25 ng / mL FGF2.

78. The method of any one of claims 1-74, wherein the second medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.01% sodium bicarbonate, 1% B27, 100 ng / mL AA, 2 pM Thiazovivin, and 25 ng / mL FGF2.

79. The method of any one of claims 2-78, wherein the third medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.015% sodium bicarbonate, 0.25 mM Vitamin C, and 50 ng / mL FGF7.

80. The method of any one of claims 2-78, wherein the third medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.015% sodium bicarbonate, 0.25 mM Vitamin C, trace elements, and 50 ng / mL FGF7.

81. The method of any one of claims 2-78, wherein the third medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :50000 (v:v) ITS-X, 0.015% sodium bicarbonate, 0.25 mM Vitamin C, trace elements, 1% B27, and 50 ng / mL FGF7.

82. The method of any one of claims 3-81, wherein the fourth medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :200 (v:v) ITS-X, 0.015% sodium bicarbonate, 0.25 mM Vitamin C, 25 ng / mL FGF7, 1 pM retinoic acid, 0.25 pM SANT, and 300 nM TPPB.

83. The method of any one of claims 3-81, wherein the fourth medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.5% FAF-BSA, 10 mM glucose, 1 :200 (v:v) ITS-X, 0.015% sodium bicarbonate, 0.25 mM Vitamin C, 1% B27, 25 ng / mL FGF7, 1 pM retinoic acid, 0.25 pM SANT, and 300 nM TPPB.

84. The method of any one of claims 4-83, wherein the fifth medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.25% FAF-BSA, 10 mM glucose, 1 :200 (v:v) ITS-X, 0.015% sodium bicarbonate, 0.25 mM Vitamin C, 2 ng / mL FGF7, 50 nM retinoic acid, 0.25 pM SANT, and 300 nM TPPB.

85. The method of any one of claims 4-83, wherein the fifth medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 0.25% FAF-BSA, 10 mM glucose, 1 :200 (v:v) ITS-X, 0.015% sodium bicarbonate, 0.25 mM Vitamin C, 1% B27, 4 ng / mL FGF7, 100 nM retinoic acid, 0.25 pM SANT, 300 nM TPPB, and 200 nM LDN.

86. The method of any one of claims 5-85, wherein the sixth medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 2% FAF-BSA, 20 mM glucose, 1 :200 (v:v) ITS-X, 0.015% sodium bicarbonate, 10 pM Zinc Sulfate, 50 nM retinoic acid, 0.25 pM SANT, 100 nM LDN, 10 pM RepSOX, 1 pM T3, and 10 pg / mL Heparin.

87. The method of any one of claims 5-85, wherein the sixth medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 2% FAF-BSA, 20 mM glucose, 1 :200 (v:v) ITS-X, 0.015% sodium bicarbonate, 10 pM Zinc Sulfate, 1% B27, 50 nM retinoic acid, 0.25 pM SANT, 100 nM LDN, 10 pM RepSOX, 1 pM T3, and 10 pg / mL Heparin.

88. The method of any one of claims 6-87, wherein the seventh medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 2% FAF-BSA, 20 mM glucose, 1 :200 (v:v) ITS-X, 0.015% sodium bicarbonate, 10 pM Zinc Sulfate, 100 nM LDN, 10 pM RepSOX, 1 pM T3, 10 pg / mL Heparin, and 100 nM DBZ.

89. The method of any one of claims 6-87, wherein the seventh medium comprises MCDB131 medium or S3S6 (BLAR001) medium, GlutaMax, 2% FAF-BSA, 20 mM glucose, 1 :200 (v:v) ITS-X, 0.015% sodium bicarbonate, 10 pM Zinc Sulfate, 1% B27, 100 nM LDN, 10 pM RepSOX, 1 pM T3, 10 pg / mL Heparin, and 100 nM DBZ.

90. The method of any one of claims 6-89, wherein at least 25% of the seventh population of cells express PDX1 and NKX6.1, at least 20% of the seventh population of cells express ISL1 and NKX6.1, at least 30% of the seventh population of cells express NKX6.1 and INS, less than 1% of the seventh population of cells express GCG and INS, or any combination thereof.

91. The method of any one of claims 6-89, wherein the proportion of cells expressing FOXA2, PDX1, NKX6.1, INS, ISL1, or any combination thereof, is increased by at least 0.5-foldin the seventh population of cells relative to the sixth, fifth, fourth or third population of progenitor cells.

92. The method of any one of claims 4-91, wherein at least a portion of the fifth population of progenitor cells and / or the seventh population of cells express one or more markers of differentiation, optionally each of the one or more markers comprise CHGA, F0XA2, PDX1, NKX6.1, or NGN3.

93. The method of claim 92, wherein at least 25% of the fifth population of progenitor cells express NKX6.1 and PDX1.

94. The method of any one of claims 92-93, wherein at least 0.5-fold more cells of the fifth population of progenitor cells and / or the seventh population of cells express one or more markers of differentiation each selected from the group consisting of CHGA, F0XA2, PDX1, NKX6.1 and NGN3; as compared to a fifth population of progenitor cells and / or a seventh population of cells generated without culturing in a medium comprising FGF2 in step (a).

95. The method of any one of claims 5-93, wherein the average C-peptide levels capable of being secreted from the sixth population of progenitor cells or seventh population of cells is increased by at least 5-fold as compared to a sixth population of progenitor cells or seventh population of cells generated without culturing in a medium comprising FGF2 in step (a).

96. The method of any one of claims 23-95, wherein the yield of the seventh population of cells is increased by at least 10% when culturing in a medium comprising the defined, serum- free serum replacement as compared to culturing in a medium that does not comprise the defined, serum-free serum replacement in step (a).

97. The method of any one of claims 31 -96, wherein the yield of the seventh population of cells is increased by at least 2-fold when culturing in: a first medium comprising B27 and 6 pM CHIR, a second medium comprising B27, a third medium comprising B27, a fourth medium comprising B27, a fifth medium comprising B27, 4 ng / mL FGF7, 100 nM retinoic acid, and 200 nM LDN, a sixth medium comprising B27, and a seventh medium comprising B27; optionally as compared to culturing in a first medium that does not comprise B27 and 6 pM CHIR, a second medium that does not comprise B27, a third medium that does not comprise B27, a fourth medium that does not comprise B27, a fifth medium that does not comprise B27, 4 ng / mL FGF7, 100 nM retinoic acid, and 200 nM LDN, a sixth medium that does not comprise B27, and a seventh medium that does not comprise B27.

98. The method of any one of claims 1-97, wherein at least 75% of the second population of progenitor cells express FOXA2 and SOX17.

99. The method of any one of claims 6-97, wherein the immature P cells are capableof differentiating into mature P cells capable of secreting insulin in response to glucose.

100. The method of any one of claims 6-98, wherein about 60 * 106to about 80 * 106immature P cells are produced from a population of stem cells comprising about 20 * 106cells.

101. The method of any one of claims 1-100, wherein the population of stem cells is a population of genetically modified cells.

102. The method of claim 101, wherein the stem cells are genetically modified by an RNA-guided endonuclease system.

103. The method of claim 102, wherein the RNA-guided endonuclease system is a CRISPR system comprising a CRISPR nuclease and a guide RNA.

104. The method of any one of claims 1-103, wherein the stem cells are induced pluripotent stem cells (iPSC), pluripotent stem cells (PSC), embryonic stem cells (ESC), or adult stem cells (ASC).

105. The method of any one of claims 1-104, wherein the stem cells are mammalian cells, optionally wherein the mammalian cells are human cells.

106. The method of any one of claims 6-105, further comprising differentiating the immature P cells into mature P cells capable of secreting insulin in response to glucose.

107. A population of immature P cells differentiated by or obtainable by the method of any one of claims 1-106.

108. A population of cells, wherein at least 20% of the cells in the population are pancreatic progenitor cells expressing one or more markers each selected from the group consisting of ISL1, NKX6.1, PDX1, INS, CHGA, FOXA2, PDX1, and NGN3; optionally wherein at least 10% of the population of cells express ISL1 and NKX6.1.

109. The population of cells of any one of claims 108-108, wherein at least 25% of the population of cells express PDX1 and NKX6.1.

110. The population of cells of any one of claims 108-109, wherein at least 30% of the population of cells express NKX6.1 and INS.

111. The population of cells of any one of claims 108-110, wherein the pancreatic progenitor cells are immature P cells.

112. The population of cells of any one of claims 107-111, wherein the population of cells are capable of differentiating into mature P cells capable of secreting insulin in response to glucose, following administration to a subject.

113. The population of cells of claim 112, wherein at least 800 pmol / L of C-peptide is detected in the blood of the subject following glucose stimulation, wherein the subject is administered about 3.5 million cells, optionally C-peptide is measured 8 weeks following the administration, further optionally C-peptide is measured 12 weeks following the administration.

114. A composition comprising the population of immature P cells of claim 107 or the population of cells of any one of claims 108-113, for use as a medicament, optionally wherein the composition is a pharmaceutical composition.

115. The population of immature P cells of claim 107, the population of cells of any one of claims 108-113, or the composition of claim 114 for use in treating a subject in need thereof.

116. The population of immature P cells of claim 107, the population of cells of any one of claims 108-113, or the composition of claim 114 for use in treating a disease related to abnormal glucose homeostasis, optionally, the disease hyperglycemia, metabolic syndrome, obesity, hypercholesterolemia, and / or hypertension, further optionally the disease is Type I or Type II diabetes.

117. A method for treating a subject in need thereof, the method comprising administering to the subject the population of immature P cells of claim 107 or the population of cells of any one of claims 108-113, optionally wherein the population of immature P cells or the population of cells are administered as one or more pharmaceutical compositions.

118. The method of claim 117, wherein glucose-stimulated insulin secretion in the subject increases by at least 1.5-fold after about 20 weeks following the administration; optionally as compared to insulin secretion after about 8 weeks following administration.

119. The method of any one of claims 117-118, wherein body weight of the subject increases after about two weeks following administration.

120. The method of any one of claims 117-119, wherein fasting blood glucose levels of the subject are decreased by at least 25% after about one month following administration.

121. The method of any one of claims 117-120, wherein fasting blood glucose levels of the subject are decreased by at least 50% after about two months following administration.

122. The method of any one of claims 117-121, wherein at least 3.0 x 106cells are administered to the subject, optionally about 7.0 x io6cells are administered to the subject, further optionally the subject is a mouse.

123. The method of any one of claims 117-122, wherein the subject has, is suspected of having, or is at risk for a disease related to abnormal glucose homeostasis.

124. The method of any one of claims 117-123, wherein the subject has, is suspected of having, oris at risk for hyperglycemia, metabolic syndrome, obesity, hypercholesterolemia, and / or hypertension.

125. The method of any one of claims 117-124, wherein the subject has, is suspected of having, or is at risk for diabetes; optionally wherein the diabetes is type I or type II diabetes.

126. The method of any one of claims 117-125, wherein the subject is a mammal, optionally the subject is a human.

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