Cell therapy for diabetes

Abstract

Disclosed herein are compositions, kits, and methods related to cell therapy for diseases characterized by high blood glucose levels over an extended period of time, such as diabetes. In some aspects, the methods provided herein relate to administration of non-native pancreatic cells to a subject having a disease characterized by high blood glucose levels over an extended period of time, such as diabetes. In some aspects, the disclosure provides pharmaceutical compositions comprising the non-native cells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 305,575, filed February 1, 2022, which is specifically incorporated by reference in its entirety into this specification.

[0002] Incorporation by reference of sequence listing

[0002] The sequence listing in XML file 41639_SequenceListing.xml (28 KB), created on January 19, 2023 and submitted via the Patent Center, is incorporated herein by reference. [Background technology]

[0003]

[0003] The production of stem cell-derived β-cells can provide a potentially useful step towards the production of pancreatic islets and pancreatic organs. One rapidly progressing disease that can be treated by stem cell-derived tissues is diabetes. Type 1 diabetes is caused by autoimmune destruction of β-cells in pancreatic islets. Type 2 diabetes is caused by insulin resistance and abnormal function of β-cells in peripheral tissues. Diabetic patients, especially those suffering from type 1 diabetes, could be cured by transplantation of new β-cells. Patients who receive transplants of cadaveric human islets can be free of insulin dependence for 5 years or more with this strategy, but the rarity and quality of donor islets limits this approach. Producing an unlimited supply of human β-cells from stem cells could extend this therapy to millions of new patients, which could be an important test case for translating stem cell biology to the clinic.

[0004] Incorporation by Reference

[0004] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Unless otherwise indicated, all publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety. Summary of the Invention

[0005] In some aspects, a method for treating a subject in need of treatment comprises administering to the subject by injection a first pharmaceutical composition comprising a cell population in a liquid suspension, wherein the cell population is greater than or equal to 1×10 8 ~10×10 8 (For example, 3×10 8 ~8.5×10 8Disclosed herein are methods for administering a subject a stimulated blood C-peptide level of at least 450 pmol / L, 460 pmol / L, 470 pmol / L, 480 pmol / L, 490 pmol / L, 500 pmol / L, 510 pmol / L, 520 pmol / L, 530 pmol / L, 540 pmol / L, 550 pmol / L, 700 pmol / L, or 800 pmol / L, as measured using a mixed meal tolerance test at least about 1, 2, 3, 4, 5, or 6 months after administration. , 800 pmol / L, 900 pmol / L, 1000 pmol / L, 1400 pmol / L, or increased to 150-350 pmol / L, 250-400 pmol / L, 400-1400 pmol / L, 400-1200 pmol / L, 400-1000 pmol / L, 400-800 pmol / L, 400-600 pmol / L, 600-800 pmol / L, 600-1400 pmol / L, or 900-1200 pmol / L. In some instances, the method includes increasing the subject's stimulated blood C-peptide level to at least 450 pmol / L, 460 pmol / L, 470 pmol / L, 480 pmol / L, 490 pmol / L, 500 pmol / L, 510 pmol / L, 520 pmol / L, 530 pmol / L, 540 pmol / L, or 550 pmol / L when measured using a mixed meal tolerance test at about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months after administration. Increase to 50 pmol / L, 700 pmol / L, 800 pmol / L, 900 pmol / L, 1000 pmol / L, 1400 pmol / L, or 150-350 pmol / L, 250-400 pmol / L, 400-1400 pmol / L, 400-1200 pmol / L, 400-1000 pmol / L, 400-800 pmol / L, 400-600 pmol / L, 600-800 pmol / L, 600-1400 pmol / L, or 900-1200 pmol / L.In some cases, the method increases the subject's stimulated blood C-peptide levels to at least 200 pmol / L, 300 pmol / L, 450 pmol / L, 460 pmol / L, 470 pmol / L, 480 pmol / L, 490 pmol / L, 500 pmol / L, 510 pmol / L, 520 pmol / L, 530 pmol / L. 540 pmol / L, 550 pmol / L, 600 pmol / L, or 650 pmol / L, or to 150-350 pmol / L, 200-700 pmol / L, 150-700 pmol / L, 300-650 pmol / L, or 500-700 pmol / L, as measured using a mixed meal tolerance test about three months after administration.

[0006] In some instances, the method provides for increasing the subject's stimulated blood C-peptide levels to at least about 450 pmol / L, 460 pmol / L, 470 pmol / L, 480 pmol / L, 490 pmol / L, 500 pmol / L, 510 pmol / L, 520 pmol / L, 530 pmol / L, 540 pmol / L, 550 pmol / L, 560 pmol / L, 700 pmol / L, 800 pmol / L, 900 pmol / L, 10 ... Increase to 800 pmol / L, 900 pmol / L, 1000 pmol / L, 1400 pmol / L, or 150-350 pmol / L, 250-400 pmol / L, 400-1400 pmol / L, 400-1200 pmol / L, 400-1000 pmol / L, 400-800 pmol / L, 400-600 pmol / L, 600-800 pmol / L, 600-1400 pmol / L, or 900-1200 pmol / L. In some cases, the method increases the subject's blood C-peptide level to at least 100 pmol / L, 120 pmol / L, 130 pmol / L, 150 pmol / L, 160 pmol / L, 180 pmol / L, 200 pmol / L, 210 pmol / L, 220 pmol / L, 230 pmol / L, 240 pmol / L, 250 pmol / L, 260 pmol / L, 270 pmol / L, or 280 pmol / L under fasting conditions when measured at least about 1, 2, 3, 4, 5, or 6 months after administration. In some cases, the method increases the subject's blood C-peptide levels under fasting conditions to at least 100 pmol / L, 120 pmol / L, 130 pmol / L, 150 pmol / L, 160 pmol / L, 180 pmol / L, 200 pmol / L, 210 pmol / L, 220 pmol / L, 230 pmol / L, 240 pmol / L, 250 pmol / L, 260 pmol / L, 270 pmol / L, or 280 pmol / L when measured about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after administration.In some cases, the method increases the subject's blood C-peptide level under fasting conditions to at least 100 pmol / L, 120 pmol / L, 130 pmol / L, 150 pmol / L, 160 pmol / L, 180 pmol / L, 200 pmol / L, 210 pmol / L, 220 pmol / L, 230 pmol / L, 240 pmol / L, 250 pmol / L, 260 pmol / L, 270 pmol / L, or 280 pmol / L when measured about 3 months after administration. In some cases, the method increases the subject's blood C-peptide level under fasting conditions to about 200 pmol / L, 220 pmol / L, 240 pmol / L, 260 pmol / L, or 280 pmol / L when measured about 3 months after administration.

[0007] In some cases, prior to administration, the subject has a stimulated blood C-peptide level of less than 100 pmol / L, 80 pmol / L, 60 pmol / L, 40 pmol / L, 30 pmol / L, 20 pmol / L, or 10 pmol / L, as measured using a mixed meal tolerance test. In some cases, prior to administration, the subject has an undetectable stimulated blood C-peptide level, as measured using a mixed meal tolerance test. In some cases, prior to administration, the subject has an undetectable stimulated blood C-peptide level, as measured under fasting conditions.

[0008]

[0008] In some cases, the method reduces the subject's hemoglobin A1c (Hb1Ac) level to less than 8%, 7.5%, 7.0%, 6.5%, 6.0%, 5.5%, or to 5-8%, 5-7.5%, 5-6.5%, 5-5.5%, 5.5-8%, 5.5-7%, 5.5-6.5%, 6-8%, 6-7%, or 6-6.5% when measured using an HbA1c test at least about 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months after administration. In some cases, the method reduces the subject's Hb1Ac level to less than 8%, 7.5%, 7.0%, 6.5%, 6.0%, 5.5%, or 5%, or between 5-8%, 5-7.5%, 5-6.5%, 5-5.5%, 5.5-8%, 5.5-7%, 5.5-6.5%, 6-8%, 6-7%, or 6-6.5%, as measured using an HbA1c test about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months after administration. In some cases, the method reduces the subject's Hb1Ac level to less than 8%, 7.5%, 7.0%, or 6.75%, or between 5-8.0%, 6.5-8.0%, 6.5-7.5%, 6.5-7.0%, or between 7-8%, when measured about 3 months after administration. In some cases, the method reduces the subject's Hb1Ac level to less than 8%, 7.5%, 7.0%, 6.5%, 6.0%, 5.5%, or 5%, or between 4.5-8%, 5-8%, 5-7.5%, 5-7%, 5-6.5%, 5-5.5%, 5.5-8%, 5.5-7%, 5.5-6.5%, 6-8%, 6-7%, or 6-6.5%, when measured about 9 months after administration.

[0009] In some cases, prior to administration, the subject has a baseline Hb1Ac level of more than 7.0%, 7.3%, 7.8%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.8%, or 9.0%. In some cases, prior to administration, the subject receives an infusion of insulin at a level of at least about 20 U / day, 22 U / day, 24 U / day, 26 U / day, 28 U / day, 30 U / day, 32 U / day, or 34 U / day. In some cases, prior to administration, the subject has a history of severe hypoglycemic events. In some cases, prior to administration, the subject has a history of impaired hypoglycemic awareness. In some cases, prior to administration, the subject demonstrates no residual endogenous islet cell function. In some cases, the absence of endogenous islet cell function is indicated by undetectable fasting C-peptide and undetectable stimulated C-peptide in a mixed meal tolerance test. In some cases, the absence of endogenous islet cell function is indicated by sustained stimulated glucose levels of greater than 350, 400, 450, 475, or 500 mg / dL in a mixed meal tolerance test. In some cases, prior to administration, the subject had an HbA1c level of greater than 7.7%, 8%, 8.2%, or 8.5%. In some cases, prior to administration, the subject was receiving greater than 10, 15, 20, 25, 30, or 35 units of insulin per day.

[0010]

[0010] In some cases, the subject has a disease characterized by high blood glucose levels over an extended period of time. In some cases, the disease is diabetes. In some cases, the diabetes is type 1 diabetes.

[0011] In some cases, the cell population is at most about 12×10 8 , 10×10 8 , 8×10 8 , 7×10 8 , 6.5×10 8 , 6×10 8 , 5.5×10 8 , 5×10 8 , 4.5×10 8 , 4×10 8 , 3.5×10 8 , 3×108 , 2.5×10 8 , or 2 × 10 8 In some cases, the cell population comprises about 1×10 cells. 8 ~Approx. 7×10 8 , about 1×10 8 ~about 6×10 8 , about 1×10 8 ~Approx. 5×10 8 , about 1×10 8 ~Approx. 4×10 8 , about 1×10 8 ~Approx. 3×10 8 , about 1×10 8 ~about 2×10 8 , about 1.5×10 8 ~Approx. 6.5×10 8 , about 2×10 8 ~Approx. 7×10 8 , about 2×10 8 ~about 6×10 8 , about 2×10 8 ~Approx. 5×10 8 , about 2×10 8 ~Approx. 4×10 8 , about 2×10 8 ~Approx. 3×10 8 , about 2.5×10 8 ~Approx. 5.5×10 8 , about 3×10 8 ~Approx. 7×10 8 , about 3×10 8 ~about 6×10 8 , about 3×10 8 ~Approx. 5×10 8 , about 3×10 8 ~Approx. 4×10 8 , about 3.5×10 8 ~Approx. 4.5×10 8 , or about 3.8 × 10 8 ~Approx. 4.2×10 8 In some cases, the cell population comprises about 3.5×10 8 ~Approx. 4.5×10 8 In some cases, the cell population comprises about 3.5×10 8 ~Approx. 8.5×10 8 Contains cells.

[0012] In some cases, the subject is administered by injection a second pharmaceutical composition comprising the cell population in a liquid suspension, the cell population being greater than or equal to 1×10 8 ~10×10 8 pieces (e.g., 3×10 8 ~8.5×10 8 , 4×10 8 ~8.5×10 8 , or 5 × 10 8 ~8.5×10 8 cells), the cell population comprises non-native cells expressing C-peptide and ISL1, and the second pharmaceutical composition is administered to the subject at a later time than the first pharmaceutical composition. In some cases, the second pharmaceutical composition is administered to the subject at least 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, or 3-12 months, 3-10 months, 3-9 months, 3-7 months, 3-5 months, 5-12 months, 8-10 months, 7-12 months, 9-15 months, or 9-12 months after the subject is administered the first pharmaceutical composition. In some cases, the first pharmaceutical composition comprises 3.5×10 8 ~Approx. 8.5×10 8 cells, and the second pharmaceutical composition contains 3.5×10 8 ~Approx. 8.5×10 8 In some instances, the first pharmaceutical composition comprises 3.5×10 cells. 8 ~Approx. 8.5×10 8 cells, and the second pharmaceutical composition contains 3.5×10 8 ~Approx. 4.5×10 8 Contains cells.

[0013]

[0013] In some cases, the subject is also administered at least one immunosuppressant. In some cases, the at least one immunosuppressant comprises one selected from the group consisting of thymoglobulin, etanercept, basiliximab, tacrolimus, sirolimus, and mycophenolate mofetil. In some cases, the subject is also administered thymoglobulin and etanercept. In some cases, the subject is also administered thymoglobulin and basiliximab. In some cases, the subject is also administered tacrolimus and sirolimus. In some cases, the subject is also administered tacrolimus and basiliximab. In some cases, the subject is also administered sirolimus and mycophenolate.

[0014] In some cases, the first pharmaceutical composition includes a sugar. In some cases, the sugar is sucrose or glucose. In some cases, the liquid suspension includes a sugar at a concentration of about 0.05% to about 1.5%.

[0015] In some instances, the first pharmaceutical composition comprises CMRL medium. In some instances, the first pharmaceutical composition comprises HypoThermosol® FRS storage medium.

[0016] In some cases, the cell population in the first pharmaceutical composition further comprises non-naturally occurring cells that express glucagon but do not express somatostatin, and non-naturally occurring cells that express somatostatin but do not express glucagon.

[0016]

[0017] In some instances of the method, (a) between 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 30-40%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-90%, 50-80%, 50-70%, 50-60%, 60-90%, 60-80%, 60-70%, 70-90%, 70-80%, 70-90%, 70-80%, or 80-90% of the cells in the cell population in the first pharmaceutical composition express C-peptide and IS (b) expressing L1 but not VMAT1 in the cell population in the first pharmaceutical composition; (c) expressing L1 but not VMAT1 in the cell population in the first pharmaceutical composition; (d) expressing L1 but not VMAT1 in the cell population in the first pharmaceutical composition; and / or (c) between 1-20%, 1-15%, 1-12%, 1-10%, 1-8%, 1-5%, 2-20%, 2-15%, 2-12%, 2-10%, 2-8%, 2-5%, 3-20%, 3-15%, 3-12%, 3-10%, 3-8%, 3-5%, 4-20%, 4-15%, 4-12%, 4-10%, 4-8%, 4-5%, 5-20%, 5-15%, 5-12%, 5-10%, 5-8%, 7-20%, 7-15%, 7-12%, 7-10%, 9-20%, 9-15%, 9-12%, 8-10%, 8-12%, 8-15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15% or 15-20% of cells express somatostatin but do not express glucagon.

[0017]

[0018] In some instances of the method, (a) between 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 30-40%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-90%, 50-80%, 50-70%, 50-60%, 60-90%, 60-70%, 70-90%, 70-80%, or 80-90% of the cells in the cell population express C-peptide and ISL1 but do not express VMAT1. (b) 3-40%, 3-35%, 3-30%, 3-25%, 3-20%, 3-15%, 3-10%, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-40%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15-40%, 15-35%, 15-30%, 15-25%, 15-20%, 20-40%, 20-35%, 20-30%, 20- 25%, 25-40%, 25-35%, 25-30%, 30-40%, 30-35%, or 35-40% of the cells express glucagon but not somatostatin; and (c) 1-20%, 1-15%, 1-12%, 1-10%, 1-8%, 1-5%, 2-20%, 2-15%, 2-12%, 2-10%, 2-8%, 2-5%, 3-20%, 3-15%, 3-12%, 3-10%, 3-8%, 3-5%, 4-20%, 4-15%, 4-12%, 4-10%, 4-8%, 4-5%, 5-20%, 5-15%, 5-12%, 5-10%, 5-8%, 7-20%, 7-15%, 7-12%, 7-10%, 9-20%, 9-15%, 9-12%, 8-10%, 8-12%, 8-15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15% or 15-20% of cells express somatostatin but not glucagon.

[0018]

[0019] In some instances of the method, (a) 35-60% of the cells in the cell population express C-peptide and ISL1 but do not express VMAT1; (b) 4-25% of the cells in the cell population express glucagon but do not express somatostatin; and (c) 1-10% of the cells in the cell population express somatostatin but do not express glucagon. In some instances of the method, (a) 40-60% of the cells in the cell population express C-peptide and ISL1 but do not express VMAT1; (b) 10-25% of the cells in the cell population express glucagon but do not express somatostatin; and (c) 4-10% of the cells in the cell population express somatostatin but do not express glucagon.

[0019]

[0020] In some cases, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 18%, less than 15%, less than 12%, or less than 10% of the cells in the cell population express VMAT1 but do not express C-peptide. In some cases, 50%, 40%, 30%, or 20% or more of the cells in the first pharmaceutical composition express NKX6.1 as determined by flow cytometry. + / ISL1 + In some cases, 20% or more of the cells in the first pharmaceutical composition are NKX6.1 cells as determined by flow cytometry. + / ISL1 + In some cases, 40%, 35%, 30%, 26%, 25%, or 20% or more of the cells in the first pharmaceutical composition are NKX6.1 cells as determined by flow cytometry. - / ISL1 + In some cases, 26% or more of the cells in the first pharmaceutical composition are NKX6.1 cells as determined by flow cytometry. - / ISL1 + In some cases, between 5 and 25%, between 5 and 40%, between 5 and 35%, or between 8 and 20% of the cells in the first pharmaceutical composition are NKX6.1 cells as determined by flow cytometry. - / ISL1 +In some cases, no more than 50%, 45%, 40%, 35%, 30%, or 25% of the cells in the first pharmaceutical composition are NKX6.1 cells as determined by flow cytometry. + / ISL1 - In some cases, 50% or less of the cells in the first pharmaceutical composition are NKX6.1 cells as determined by flow cytometry. + / ISL1 - In some cases, 20-60%, 20-50%, 20-45%, 20-40%, 20-35%, 20-30%, 20-25%, 25-50%, 25-40%, 25-35%, 30-60%, 30-50%, 30-40%, 30-35%, 35-50%, or 40-50% of the cells in the pharmaceutical composition are NKX6.1 cells as determined by flow cytometry. + / ISL1 + In some cases, 20-60%, 20-50%, 20-45%, 20-40%, 20-35%, 20-30%, 20-25%, 25-50%, 25-40%, 25-35%, 30-60%, 30-50%, 30-40%, 30-35%, 35-50%, or 40-50% of the cells in the pharmaceutical composition are NKX6.1 cells as determined by flow cytometry. - / ISL1 + In some cases, 20-50%, 20-45%, 20-40%, 20-35%, 20-30%, 20-25%, 25-50%, 25-40%, 25-35%, 30-60%, 30-50%, 30-40%, 30-35%, 35-50%, 40-50%, 10-20%, or 10-25% of the cells in the pharmaceutical composition are NKX6.1 cells. + / ISL1 - In some embodiments of the method, at least 20% of the cells in the composition are NKX6.1 cells. + / ISL1 + at least 25% of the cells in the composition are NKX6.1 - / ISL1 + cells; 10-50% of the cells in the composition are NKX6.1 + / ISL1 -In some instances, the composition comprises an NKX6.1 cell that exhibits GSIS in vitro. + / ISL1 + In some cases, the composition comprises an NKX6.1 cell that exhibits GSIS in vivo. + / ISL1 + In some cases, the cell population is generated in vitro from stem cells. In some cases, the non-naturally occurring cells expressing C-peptide and ISL1 but not VMAT1 in the cell population exhibit a glucose-stimulated insulin secretion response in vitro. In some cases, the secretion of insulin by the non-naturally occurring cells expressing C-peptide and ISL1 but not VMAT1 in response to a glucose challenge is proportional to the glucose concentration of the glucose challenge. In some cases, the secretion of insulin by the non-naturally occurring cells expressing C-peptide and ISL1 but not VMAT1 secretes insulin in response to a first glucose challenge, a second glucose challenge, and a third glucose challenge, where the first glucose challenge, the second glucose challenge, and the third glucose challenge are applied sequentially.

[0020]

[0021] In some cases, at least a portion of the cells in the cell population are present in multiple cell clusters. In some cases, the cell clusters are about 50 μm to about 500 μm, about 50 μm to about 400 μm, about 50 μm to about 300 μm, about 60 μm to about 400 μm, about 60 μm to about 300 μm, about 60 μm to about 250 μm, about 75 μm to about 400 μm, about 75 μm to about 300 μm, about 75 μm to about 250 μm, about 125 μm to about 225 μm, about 130 μm to about 160 μm, about 170 μm to about 225 μm, about 140 μm to about 200 μm, about 140 μm to about 170 μm, about 160 μm to about 220 μm, about 170 μm to about 215 μm, or about 170 μm to about 200 μm in diameter. In some embodiments, the cell clusters are about 80-270 μm in diameter.

[0021]

[0022] In some cases, the population of cells is present in the first pharmaceutical composition as a single cell suspension.

[0023] In some cases, in the first pharmaceutical composition, the cell population is suspended in a serum-free solution.

[0022]

[0024] In some cases, the method further includes administering an immune response modulator to the subject. In some cases, the immune response modulator is administered simultaneously with the first pharmaceutical composition. In some cases, the immune response modulator is administered before or after the first pharmaceutical composition. In some cases, the immune response modulator is not a steroid. In some cases, the immune response modulator is azathioprine, mycophenolic acid, leflunomide, teriflunomide, methotrexate, tacrolimus, cyclosporine, pimecrolimus, avetimus, gusperimus, lenalidomide, pomalidomide, thalidomide, PDE4 inhibitors, apremilast, anakinra, sirolimus, everolimus, ridaforolimus, temsirolimus, umirolimus, zotarolimus, antithymocyte globulin anti. Anti-lymphocyte globulin antibody, CTLA-4, abatacept, belatacept, etanercept, pegsunercept, aflibercept, alefacept, rilonacept, eculizumab, adalimumab, afelimomab, certolizumab pegol, golimumab, infliximab, nerelimomab, mepolizumab, omalizumab, faralimomab, ersilimomab, lebrikizumab, ustekinumab, secukinumab, muromonab-CD3, o Telixizumab, Teplizumab, Bisilizumab, Clenoliximab, Keliximab, Zanolimumab, Efalizumab, Erlizumab, Obinutuzumab, Rituximab, Ocrelizumab, Pascolizumab, Gomiliximab, Rumiliximab, Teneliximab, Toralizumab, Acelizumab, Galiximab, Gavilimomab, Ruplizumab, Belimumab, Brisibimod, Ipilimumab, Tremelimumab, Bertilimumab, Lerdelimumab, Metformin, including terimumab, natalizumab, tocilizumab, odulimomab, basiliximab, daclizumab, inolimomab, atrolizumab, cedelizumab, fontolizumab, maslimomab, morolimumab, pexelizumab, reslizumab, lovelizumab, siplizumab, talizumab, terimomab alitox, bapaliximab, beparimomab, alemtuzumab, mycophenolate mofetil, FTY720, or any combination thereof.In some cases, the immune response modulator comprises tacrolimus, cyclosporine, mycophenolate mofetil, azathioprine, everolimus, sirolimus, abatacept, belatacept, antithymocyte globulin, alemtuzumab, rituximab, basiliximab, daclizumab, muromonab-CD3, efalizumab, FTY720, or any combination thereof. In some cases, the immune response modulator comprises a corticosteroid.

[0023]

[0025] In some cases, the first pharmaceutical composition is injected into the portal vein of the subject.

[0026] In some cases, the subject receives a single dose of the first pharmaceutical composition.

[0027] In some cases, the non-native cells expressing C-peptide and ISL1 cells were obtained from a culture of cells comprising PDX1-positive, NKX6.1-positive, insulin-positive cells that were treated with i) a TGF-β signaling pathway inhibitor (e.g., Alk5i), ii) a thyroid hormone signaling pathway activator (e.g., GC-1), iii) a BMP pathway inhibitor (e.g., LDN193189), iv) a ROCK inhibitor (e.g., thiazovivin), v) a protein kinase inhibitor (e.g., staurosporine), and / or vi) an epigenetic modifier (e.g., DZNEP). In some cases, the non-native cells expressing C-peptide and ISL1 cells were obtained from a culture of cells including PDX1-positive, NKX6.1-positive, insulin-positive cells that were treated with i) a TGF-β signaling pathway inhibitor (e.g., Alk5i), ii) a thyroid hormone signaling pathway activator (e.g., GC-1), iii) a BMP pathway inhibitor (e.g., LDN193189), iv) a ROCK inhibitor (e.g., thiazovivin), v) a protein kinase inhibitor (e.g., staurosporine), and / or vi) an epigenetic modifier (e.g., DZNEP) for at least 3 days. In some cases, the PDX1-positive, NKX6.1-positive, insulin-positive cells were also treated with human serum albumin. In some cases, the PDX1-positive, NKX6.1-positive, insulin-positive cells were also treated with human serum albumin for at least 6 days.In some cases, the PDX1 positive, NKX6.1 positive, insulin positive cells were obtained from a culture of cells comprising PDX1 positive, NKX6.1 positive, insulin negative cells that were treated with i) a TGF-β signaling pathway inhibitor (e.g., Alk5i), ii) a TH signaling pathway activator (e.g., GC-1), iii) at least one SHH pathway inhibitor (e.g., Sant1), iv) an RA signaling pathway activator (e.g., retinoic acid), v) a γ-secretase inhibitor (e.g., XXI), vi) at least one growth factor from the epidermal growth factor (EGF) family (e.g., betacellulin), vii) a BMP pathway inhibitor (e.g., LDN193189), viii) a ROCK inhibitor (e.g., thiazovivin), ix) a protein kinase inhibitor (e.g., staurosporine), and / or x) an epigenetic modifier (e.g., DZNEP).

[0024]

[0028] In some cases, the PDX1 positive, NKX6.1 positive, insulin positive cells are treated with i) a TGF-β signaling pathway inhibitor (e.g., Alk5i), ii) a TH signaling pathway activator (e.g., GC-1), iii) at least one SHH pathway inhibitor (e.g., Sant1), iv) an RA signaling pathway activator (e.g., retinoic acid), v) a γ-secretase inhibitor (e.g., XXI), vi) at least one growth factor from the epidermal growth factor (EGF) family (e.g., betacellulin), vii) a BMP pathway inhibitor (e.g., LDN193189), viii) a ROCK inhibitor (e.g., thiazovivin), ix) a protein kinase inhibitor (e.g., staurosporine), and and / or x) treated with an epigenetic modifier (e.g., DZNEP) for 2 or 3 days, followed by contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells with i) a TGF-β signaling pathway inhibitor (e.g., Alk5i), ii) a TH signaling pathway activator (e.g., GC-1), iii) a γ-secretase inhibitor (e.g., XXI), iv) a BMP pathway inhibitor (e.g., LDN193189), v) a ROCK inhibitor (e.g., thiazovivin), vi) a protein kinase inhibitor (e.g., staurosporine), and vii) an epigenetic modifier (e.g., DZNEP) for 4 or 5 days, from a culture of cells including PDX1-positive, NKX6.1-positive, insulin-negative cells.

[0025]

[0029] In some cases, the PDX1 positive, NKX6.1 positive, insulin positive cells are treated with i) a TGF-β signaling pathway inhibitor (e.g., Alk5i), ii) a TH signaling pathway activator (e.g., GC-1), iii) at least one SHH pathway inhibitor (e.g., Sant1), iv) an RA signaling pathway activator (e.g., retinoic acid), v) a γ-secretase inhibitor (e.g., XXI), vi) at least one growth factor from the epidermal growth factor (EGF) family (e.g., betacellulin), vii) a BMP pathway inhibitor (e.g., LDN193189), viii) a ROCK inhibitor (e.g., thiazovivin), ix) a protein kinase inhibitor (e.g., staurosporine), and / or x) an epigenetic modifier (e.g., DZNEP) for 2 or 3 days, followed by isolating the PDX1 positive , NKX6.1-positive pancreatic progenitor cells were contacted with i) a TGF-β signaling pathway inhibitor (e.g., Alk5i), ii) a TH signaling pathway activator (e.g., GC-1), iii) a γ-secretase inhibitor (e.g., XXI), iv) a BMP pathway inhibitor (e.g., LDN193189), v) a ROCK inhibitor (e.g., thiazovivin), vi) a protein kinase inhibitor (e.g., staurosporine), and vii) an epigenetic modifier (e.g., DZNEP) for 4 or 5 days in the absence of i) an SHH pathway inhibitor (e.g., Sant1), ii) an RA signaling pathway activator (e.g., retinoic acid), and iii) a growth factor from the epidermal growth factor (EGF) family (e.g., betacellulin), from a culture of cells including PDX1-positive, NKX6.1-positive, insulin-negative cells.

[0026]

[0030] In some cases, the PDX1 positive, NKX6.1 positive, insulin negative cells were obtained from a culture of cells comprising PDX1 positive, NKX6.1 negative cells that were treated with i) at least one growth factor from the FGF family (e.g., KGF), ii) at least one SHH pathway inhibitor (e.g., Sant1), iii) an RA signaling pathway activator (e.g., retinoic acid), iv) a ROCK inhibitor (e.g., thiazovivin), and v) at least one factor from the TGFβ superfamily (e.g., activin A).

[0027]

[0031] In some cases, the PDX1-positive, NKX6.1-positive, insulin-negative cells were obtained from a culture of cells comprising PDX1-positive, NKX6.1-negative cells that were treated for 5 days with i) at least one growth factor from the FGF family (e.g., KGF), ii) at least one SHH pathway inhibitor (e.g., Sant1), iii) an RA signaling pathway activator (e.g., retinoic acid), iv) a ROCK inhibitor (e.g., thiazovivin), and v) at least one factor from the TGFβ superfamily (e.g., activin A).

[0028]

[0032] In some cases, the PDX1 positive, NKX6.1 negative cells were obtained from a culture of cells comprising PDX1 negative gastrula cells that were treated with i) a retinoic acid signaling pathway activator, ii) at least one factor from the FGF family (e.g., KGF), iii) an SHH pathway inhibitor (e.g., Sant1), iv) a PKC activator (e.g., PDBU), v) a ROCK inhibitor (e.g., thiazovivin), and vi) a BMP pathway inhibitor (e.g., DMH-1).

[0029]

[0033] In some cases, the PDX1 positive, NKX6.1 negative cells were obtained from a culture of cells comprising PDX1 negative gastrula cells that had been treated with i) a retinoic acid signaling pathway activator, ii) at least one factor from the FGF family (e.g., KGF), iii) an SHH pathway inhibitor (e.g., Sant1), iv) a PKC activator (e.g., PDBU), v) a ROCK inhibitor (e.g., thiazovivin), and vi) a BMP pathway inhibitor (e.g., DMH-1) for one day, followed by contacting the cells with i) a retinoic acid signaling pathway activator, ii) at least one factor from the FGF family (e.g., KGF), iii) an SHH pathway inhibitor (e.g., Sant1), iv) a PKC activator (e.g., PDBU), v) a ROCK inhibitor (e.g., thiazovivin) for an additional day.

[0030]

[0034] In some cases, PDX1-positive, NKX6.1-negative cells were obtained from a culture of cells comprising PDX1-negative gastrula cells that had been treated with i) a retinoic acid signaling pathway activator, ii) at least one factor from the FGF family (e.g., KGF), iii) an SHH pathway inhibitor (e.g., Sant1), iv) a PKC activator (e.g., PDBU), v) a ROCK inhibitor (e.g., thiazovivin), and vi) a BMP pathway inhibitor (e.g., DMH-1) for one day, and subsequently contacted with i) a retinoic acid signaling pathway activator, ii) at least one factor from the FGF family (e.g., KGF), iii) an SHH pathway inhibitor (e.g., Sant1), iv) a PKC activator (e.g., PDBU), v) a ROCK inhibitor (e.g., thiazovivin) for an additional day in the absence of the BMP pathway inhibitor.

[0031]

[0035] In some cases, PDX1-negative gastrula cells were obtained from cultures containing definitive endoderm cells that were treated with factors from the FGF family (eg, KGF).

[0036] In some cases, PDX1-negative gastrula cells were obtained from cultures containing definitive endoderm cells that had been treated with factors from the FGF family (eg, KGF) for three days.

[0032]

[0037] In some cases, the definitive endoderm cells were obtained from a culture comprising pluripotent stem cells that were treated with a factor from the TGFβ superfamily (e.g., activin A) and a WNT signaling pathway activator (e.g., CHIR99021). In some cases, the definitive endoderm cells were obtained from a culture comprising pluripotent stem cells that were treated with a factor from the TGFβ superfamily (e.g., activin A) and a WNT signaling pathway activator (e.g., CHIR99021) for one day, followed by contacting the cells with a factor from the TGFβ superfamily (e.g., activin A) for an additional two days.

[0033]

[0038] In some aspects, provided herein is a pharmaceutical composition formulated for injection, comprising a population of cells in liquid suspension, the population of cells being about 1×10 8 ~About 10×10 8 pieces (e.g., 3×10 8 ~8.5×10 8 The cell population comprises non-native cells that express C-peptide and ISL1.

[0034]

[0039] In some cases, the cell population is at most about 12×10 8 , 10×10 8 , 8×10 8 , 7×10 8 , 6.5×10 8 , 6×10 8 , 5.5×10 8 , 5×10 8 , 4.5×10 8 , 4×10 8 , 3.5×10 8 , 3×10 8 , 2.5×10 8 , or 2 × 10 8 In some cases, the cell population comprises about 1×10 cells. 8 ~Approx. 7×10 8 , about 1×10 8 ~about 6×10 8 , about 1×10 8 ~Approx. 5×10 8 , about 1×10 8 ~Approx. 4×10 8, about 1×10 8 ~Approx. 3×10 8 , about 1×10 8 ~about 2×10 8 , about 1.5×10 8 ~Approx. 6.5×10 8 , about 2×10 8 ~Approx. 7×10 8 , about 2×10 8 ~about 6×10 8 , about 2×10 8 ~Approx. 5×10 8 , about 2×10 8 ~Approx. 4×10 8 , about 2×10 8 ~Approx. 3×10 8 , about 2.5×10 8 ~Approx. 5.5×10 8 , about 3×10 8 ~Approx. 7×10 8 , about 3×10 8 ~about 6×10 8 , about 3×10 8 ~Approx. 5×10 8 , about 3×10 8 ~Approx. 4×10 8 , about 3.5×10 8 ~Approx. 4.5×10 8 , or about 3.8 × 10 8 ~Approx. 4.2×10 8 In some cases, the cell population comprises about 3.5×10 8 ~Approx. 4.5×10 8 In some cases, the cell population comprises about 3.5×10 cells. 8 ~Approx. 8.5×10 8 In some cases, the cell population further comprises non-naturally occurring cells that express glucagon but do not express somatostatin, and non-naturally occurring cells that express somatostatin but do not express glucagon.

[0035]

[0040] In some instances of the pharmaceutical composition, (a) between 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 30-40%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-90%, 50-80%, 50-70%, 50-60%, 60-90%, 60-80%, 60-70%, 70-90%, 70-80%, or 80-90% of the cells in the cell population express C-peptide and ISL1, but not VMA (b) not expressing T1 in the cell population; (c) 3-40%, 3-35%, 3-30%, 3-25%, 3-20%, 3-15%, 3-10%, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-40%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15-40%, 15-35%, 15-30%, 15-25%, 15-20%, 20-40%, 20-35%, 20-30%, 20-25%, 25-40%, 25-35%, 25-30%, 30-40%, 30-35% or 35-40% of the cells in the cell population express glucagon but not somatostatin; and / or (c) 1-20%, 1-15%, 1-12%, 1-10%, 1-8%, 1-5%, 2-20%, 2-15%, 2-12%, 2-10%, 2-8%, 2-5%, 3-20%, 3-15%, 3-12%, 3-10%, 3-8%, 3-5%, 4- 20%, 4-15%, 4-12%, 4-10%, 4-8%, 4-5%, 5-20%, 5-15%, 5-12%, 5-10%, 5-8%, 7-20%, 7-15%, 7-12%, 7-10%, 9-20%, 9-15%, 9-12%, 8-10%, 8-12%, 8-15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15% or 15-20% of cells express somatostatin but do not express glucagon.

[0036]

[0041] In some instances of the pharmaceutical composition, (a) between 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 30-40%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-90%, 50-80%, 50-70%, 50-60%, 60-90%, 60-80%, 60-70%, 70-90%, 70-80%, or 80-90% of the cells in the cell population express C-peptide and ISL1, but not VM do not express AT1; (b) 3-40%, 3-35%, 3-30%, 3-25%, 3-20%, 3-15%, 3-10%, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-40%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15-40%, 15-35%, 15-30%, 15-25%, 15-20%, 20-40%, 20-35%, 20-30% of the cell population; %, 20-25%, 25-40%, 25-35%, 25-30%, 30-40%, 30-35% or 35-40% of the cells in the cell population express glucagon but not somatostatin; and (c) 1-20%, 1-15%, 1-12%, 1-10%, 1-8%, 1-5%, 2-20%, 2-15%, 2-12%, 2-10%, 2-8%, 2-5%, 3-20%, 3-15%, 3-12%, 3-10%, 3-8%, 3-5%, 4-2 0%, 4-15%, 4-12%, 4-10%, 4-8%, 4-5%, 5-20%, 5-15%, 5-12%, 5-10%, 5-8%, 7-20%, 7-15%, 7-12%, 7-10%, 9-20%, 9-15%, 9-12%, 8-10%, 8-12%, 8-15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15% or 15-20% of cells express somatostatin but do not express glucagon.

[0037]

[0042] In some instances of the pharmaceutical composition, (a) 35-60% of the cells in the cell population express C-peptide and ISL1 but do not express VMAT1; (b) 4-25% of the cells in the cell population express glucagon but do not express somatostatin; and (c) 1-10% of the cells in the cell population express somatostatin but do not express glucagon.

[0038]

[0043] In some instances of the pharmaceutical composition, (a) 40-60% of the cells in the cell population express C-peptide and ISL1 but do not express VMAT1; (b) 10-25% of the cells in the cell population express glucagon but do not express somatostatin; and (c) 4-10% of the cells in the cell population express somatostatin but do not express glucagon.

[0039]

[0044] In some cases, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 18%, less than 15%, less than 12%, or less than 10% of the cells in the cell population express VMAT1 but do not express C-peptide. In some cases, 50%, 40%, 30%, or 20% or more of the cells in the pharmaceutical composition express NKX6.1 as determined by flow cytometry. + / ISL1 + In some cases, 30% or more of the cells in the composition are NKX6.1 positive, ISL1 positive cells, 25% or more of the cells in the composition are NKX6.1 negative, ISL1 positive cells, less than 12% of the cells in the composition are NKX6.1 negative, ISL1 negative cells, or 9-25% of the cells in the composition are NKX6.1 positive, ISL1 negative cells, as determined by flow cytometry. In some cases, 40%, 35%, 30%, 26%, 25%, or 20% or more of the cells in the pharmaceutical composition are NKX6.1 positive, ISL1 negative cells, as determined by flow cytometry. - / ISL1 + In some cases, 26% or more of the cells in the pharmaceutical composition are NKX6.1 cells as determined by flow cytometry. - / ISL1 + In some cases, between 5% and 25%, between 5% and 40%, between 5% and 35%, or between 8% and 20% of the cells in the pharmaceutical composition are NKX6.1 cells as determined by flow cytometry. - / ISL1 + In some cases, no more than 50%, 45%, 40%, 35%, 30%, or 25% of the cells in the pharmaceutical composition are NKX6.1 cells as determined by flow cytometry.+ / ISL1 - In some cases, 50% or less of the cells in the pharmaceutical composition are NKX6.1 cells as determined by flow cytometry. + / ISL1 - It is a cell.

[0040]

[0045] In some cases, the cell population is generated from stem cells in vitro. In some cases, the non-naturally occurring cells expressing C-peptide and ISL1 but not VMAT1 exhibit a glucose-stimulated insulin secretion response in vitro. In some cases, the secretion of insulin by the non-naturally occurring cells expressing C-peptide and ISL1 but not VMAT1 in response to a glucose challenge is proportional to the glucose concentration of the glucose challenge. In some cases, the secretion of insulin by the non-naturally occurring cells expressing C-peptide and ISL1 but not VMAT1 secretes insulin in response to a first glucose challenge, a second glucose challenge, and a third glucose challenge, where the first glucose challenge, the second glucose challenge, and the third glucose challenge are applied sequentially.

[0041]

[0046] In some cases, at least a portion of the cells in the cell population are present in multiple cell clusters. In some cases, the cell clusters are about 50 μm to about 500 μm, about 50 μm to about 400 μm, about 50 μm to about 300 μm, about 60 μm to about 400 μm, about 60 μm to about 300 μm, about 60 μm to about 250 μm, about 75 μm to about 400 μm, about 75 μm to about 300 μm, about 75 μm to about 250 μm, about 125 μm to about 225 μm, about 130 μm to about 160 μm, about 170 μm to about 225 μm, about 140 μm to about 200 μm, about 140 μm to about 170 μm, about 160 μm to about 220 μm, about 170 μm to about 215 μm, or about 170 μm to about 200 μm in diameter. In some cases, the cell population is present in the pharmaceutical composition as a single cell suspension. In some cases, the cell population is suspended in a serum-free solution.

[0042]

[0047] In some aspects, disclosed herein are kits comprising a pharmaceutical composition disclosed herein and instructions for administering the pharmaceutical composition to a subject in need thereof.

[0048] In some aspects, (a) a pharmaceutical composition formulated for injection, the pharmaceutical composition comprising a population of cells in suspension, the population of cells being at least about 1×10 8 Disclosed herein is a kit comprising: (a) a pharmaceutical composition comprising non-naturally occurring cells, the cell population expressing C-peptide and ISL1; and (b) instructions for administering the pharmaceutical composition to a subject in need thereof.

[0043]

[0049] In some cases, the kit further comprises an immune response modulator. In some cases, the immune response modulator is not a steroid. In some cases, the immune response modulator is azathioprine, mycophenolic acid, leflunomide, teriflunomide, methotrexate, tacrolimus, cyclosporine, pimecrolimus, avetimus, gusperimus, lenalidomide, pomalidomide, thalidomide, PDE4 inhibitors, apremilast, anakinra, sirolimus, everolimus, ridaforolimus, temsirolimus, umirolimus, zotarolimus, antithymocyte globulin anti. Anti-lymphocyte globulin antibody, CTLA-4, abatacept, belatacept, etanercept, pegsunercept, aflibercept, alefacept, rilonacept, eculizumab, adalimumab, afelimomab, certolizumab pegol, golimumab, infliximab, nerelimomab, mepolizumab, omalizumab, faralimomab, ersilimomab, lebrikizumab, ustekinumab, secukinumab, muromonab-CD3, o Telixizumab, Teplizumab, Bisilizumab, Clenoliximab, Keliximab, Zanolimumab, Efalizumab, Erlizumab, Obinutuzumab, Rituximab, Ocrelizumab, Pascolizumab, Gomiliximab, Rumiliximab, Teneliximab, Toralizumab, Acelizumab, Galiximab, Gavilimomab, Ruplizumab, Belimumab, Brisibimod, Ipilimumab, Tremelimumab, Bertilimumab, Lerdelimumab, Metformin, including terimumab, natalizumab, tocilizumab, odulimomab, basiliximab, daclizumab, inolimomab, atrolizumab, cedelizumab, fontolizumab, maslimomab, morolimumab, pexelizumab, reslizumab, lovelizumab, siplizumab, talizumab, terimomab alitox, bapaliximab, beparimomab, alemtuzumab, mycophenolate mofetil, FTY720, or any combination thereof.In some cases, the immune response modulator comprises tacrolimus, cyclosporine, mycophenolate mofetil, azathioprine, everolimus, sirolimus, abatacept, belatacept, antithymocyte globulin, alemtuzumab, rituximab, basiliximab, daclizumab, muromonab-CD3, efalizumab, FTY720, or any combination thereof. In some cases, the immune response modulator comprises a corticosteroid.

[0044]

[0050] In some cases, the kit includes instructions for administering the pharmaceutical composition to a subject with a history of severe hypoglycemic events. In some cases, the kit includes instructions for administering the pharmaceutical composition to a subject with a history of hypoglycemic awareness disorder. In some cases, the kit includes instructions for administering the pharmaceutical composition to a subject with no remaining endogenous islet cell function. In some cases, no remaining endogenous islet cell function is indicated by undetectable fasting C-peptide and undetectable stimulated C-peptide in a mixed meal tolerance test. In some cases, no remaining endogenous islet cell function is indicated by sustained stimulated glucose levels of more than 350, 400, 450, 475, 500 mg / dL in a mixed meal tolerance test.

[0045]

[0051] In some cases, the kit includes instructions for administering the pharmaceutical composition to a subject with an HbA1c level of more than 7.7%, 8%, 8.2%, or 8.5%. In some cases, the kit includes instructions for administering the pharmaceutical composition to a subject receiving more than 10, 15, 20, 25, 30, or 35 units of insulin per day. In some cases, the kit includes instructions for administering the pharmaceutical composition to a subject with a disease characterized by high blood glucose levels over an extended period of time. In some cases, the disease is diabetes. In some cases, the disease is type 1 diabetes.

[0046]

[0052] The features of the present disclosure are pointed out with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings. [Brief description of the drawings]

[0047] [Figure 1A-1]

[0053] FIG. 1A shows stimulated blood C-peptide levels (top panel) and stimulated glucose levels (bottom panel) of subject A1 measured by MMTT test during a screening period prior to infusion of non-native pancreatic cells according to an embodiment of the present disclosure. LLOQ: lower limit of quantification; MMTT: mixed meal tolerance test. C-peptide values ​​below the LLOQ (13 pmol / L [0.04 ng / mL]) were entered as ½LLOQ (7 pmol / L [0.02 ng / mL]). [Figure 1A-2] A portion of FIG. 1A is shown. [Figure 1A-3] A portion of FIG. 1A is shown. [Figure 1B] Shown are stimulated blood C-peptide levels (top panel) and stimulated glucose levels (bottom panel) for subject A2 / B2 measured by MMTT test during a screening period prior to infusion of non-native pancreatic cells according to embodiments of the present disclosure. LLOQ: lower limit of quantification; MMTT: mixed meal tolerance test. C-peptide values ​​below the LLOQ (13 pmol / L [0.04 ng / mL]) were entered as ½ LLOQ (7 pmol / L [0.02 ng / mL]). [Figure 1C] Shown are stimulated blood C-peptide levels (top panel) and stimulated glucose levels (bottom panel) for subject B1 measured by MMTT test during a screening period prior to infusion of non-native pancreatic cells according to embodiments of the present disclosure. LLOQ: lower limiting quantity; MMTT: mixed meal tolerance test. C-peptide values ​​below the LLOQ (13 pmol / L [0.04 ng / mL]) were entered as ½ LLOQ (7 pmol / L [0.02 ng / mL]). [Figure 2A]

[0054] 1 is a plot summarizing the change in HbA1c for subject A1 before and after receiving an infusion of non-native pancreatic cells according to an embodiment of the present disclosure. B: Baseline; D: Days; HbA1c: Hemoglobin A1c; M: Months; SCR: Screening; Unsch: Unscheduled. [Figure 2B] 1 is a plot summarizing the change in HbA1c for subjects A2 / B2 before and after receiving an infusion of non-native pancreatic cells according to an embodiment of the present disclosure. B: baseline; D: days; HbA1c: hemoglobin A1c; INF1: 1st infusion of SC-islets; INF2: 2nd infusion of SC-islets; SCR: screening; Unsch: unscheduled. [Figure 2C] 1 is a plot summarizing the change in HbA1c for subject B1 before and after receiving an infusion of non-native pancreatic cells according to an embodiment of the present disclosure. B: baseline; D: days; HbA1c: hemoglobin A1c; SCR: screening. [Figure 3A]

[0055] 1 is a bar graph summarizing time in target range for subject A1 as measured by CGM according to an embodiment of the present disclosure. The percentages of time for [301, 330] days and [426, 455] days are set as missing values ​​because less than 70% data was available and there was no 14 consecutive day period with more than 70% data available. [Figure 3B] 1 is a bar graph summarizing time in target range for subject A2 / B2 as measured by CGM according to an embodiment of the present disclosure. INF1: 1st SC-islet infusion; INF2: 2nd SC-islet infusion. 249 days after the 1st infusion, subject A2 / B2 stopped using CGM and insulin pump for insulin delivery and started using a hybrid closed loop system (HCLS). [Figure 3C] 13 is a bar graph summarizing time in target range for subject B1 as measured by a CGM according to an embodiment of the present disclosure. [Figure 4A]

[0056] FIG. 13 is a plot summarizing the change over time in total daily exogenous insulin dose for subject A1. [Figure 4B]Summary plots of change over time in total daily exogenous insulin dose for subject A2 / B2. Total daily exogenous insulin dose data from baseline to day 57 after first infusion and from days 150 to 210 after first infusion are based on insulin pump data. Data for days 90 and 120 after first infusion are based on eDiary. On study day 249, after the first infusion, subject A2 / B2 discontinued use of CGM and insulin pump for insulin delivery and began using a hybrid closed-loop system (HCLS). [Figure 4C] 1 is a plot summarizing the change over time in total daily exogenous insulin dose for subject B1. Total daily exogenous insulin dose data from baseline to day 150 are based on insulin pump data. Day 180 data are based on eDiary. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048]

[0057] The following description and examples will describe the embodiments of the present disclosure in detail.It should be understood that the present disclosure is not limited to the specific embodiments described herein and can therefore vary.Those skilled in the art will recognize that there are many variations and modifications to the present disclosure, which are included within the scope of the present disclosure.

[0049]

[0058] All terms are intended to be understood as understood by one of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0050]

[0059] The headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0060] Although various features of the present disclosure may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although for clarity the present disclosure may be described herein in the context of separate embodiments, the present disclosure may also be implemented in a single embodiment.

[0051]

[0061] The following definitions supplement the definitions in the art and are directed to this application, and are not attributable to any related or unrelated examples, such as any commonly owned patents or patent applications. Any methods and materials similar or equivalent to those described herein can be used in the practice of testing the present disclosure, but preferred materials and methods are described herein.Therefore, the terms used herein are only for the purpose of describing specific embodiments, and are not intended to be limiting.

[0052]

[0062] In this application, the use of the singular includes the plural unless specifically stated otherwise. It should be noted that as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0053]

[0063] The use of "or" in this application means "and / or" unless otherwise stated. As used herein, the terms "and / or" and "any combination thereof" and their grammatical equivalents may be used interchangeably. These terms may convey that any combination is specifically intended. For illustrative purposes only, the following phrases "A, B, and / or C" or "A, B, C, or any combination thereof" may mean "A individually, B individually, C individually, A and B, B and C, A and C, and A, B, and C." The term "or" may be used conjunctively or disjunctively, unless the context specifically indicates a disjunctive use.

[0054]

[0064] Furthermore, use of the term "including" as well as other forms such as "include," "includes," and "included" is not limiting.

[0055]

[0065] References in the specification to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least some embodiments of the present disclosure, but not necessarily in all embodiments.

[0056]

[0066] As used in the specification and claims, the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is intended that any embodiment discussed herein can be implemented with respect to any method or composition of the disclosure, and vice versa. Additionally, the compositions of the disclosure can be used to achieve the methods of the disclosure.

[0057]

[0067] As used herein, the term "about" in connection with a reference numerical value and grammatical equivalents thereof may include the numerical value itself, as well as a range of values ​​of plus or minus 10% of that numerical value.

[0068] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within or more than 1 standard deviation as is customary in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. In another example, the amount "about 10" includes 10 and any amount between 9 and 11. In yet another example, the term "about" in relation to a reference numerical value can also include a range of plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of that value. Alternatively, particularly with respect to biological systems or processes, the term "about" can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value. When a particular value is described in the application and claims, unless otherwise stated, the term "about" should be assumed to mean within an acceptable error range for the particular value.

[0058]

[0069] The term "diabetes" and its grammatical equivalents used herein can refer to disease characterized by high blood glucose level for a long period of time.For example, the term "diabetes" and its grammatical equivalents used herein can refer to all or any type of diabetes, including but not limited to type 1 diabetes, type 2 diabetes, cystic fibrosis-related diabetes, surgical diabetes, gestational diabetes, and mitochondrial diabetes.In some cases, diabetes can be a form of hereditary diabetes.

[0059]

[0070] The term "endocrine cells", unless otherwise specified, may refer to hormone-producing cells present in the pancreas of an organism, such as "pancreatic islets", "pancreatic islet cells", "pancreatic islet equivalents", "pancreatic islet-like cells", "pancreatic islets", and grammatical equivalents thereof. In one embodiment, endocrine cells can be differentiated from pancreatic progenitor or precursor cells. Pancreatic islet cells may include various types of cells, including, but not limited to, pancreatic alpha cells, pancreatic beta cells, pancreatic delta cells, pancreatic F cells, and / or pancreatic epsilon cells. Pancreatic islet cells may also refer to groups of cells, cell clusters, and the like.

[0060]

[0071] As used herein, the term "pharmacologically acceptable" can refer to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, and commensurate with a reasonable benefit / risk ratio.

[0061]

[0072] As used herein, the term "pharmaceutical acceptable carrier" can refer to a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium stearate, calcium or zinc stearate, or stearic acid), or solvent encapsulating material, involved in carrying or transporting a compound of interest from one organ or body part to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which may serve as pharma- ceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; and (10) glycols, such as propylene glycol. (11) polyols such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) bulking agents such as polypeptides and amino acids; (23) serum components such as serum albumin, HDL and LDL; (22) C2-C12 alcohols such as ethanol; and (23) other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, coloring agents, releasing agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives and antioxidants may be present in the formulation.For example, the terms "excipient," "carrier," "pharmaceutical acceptable carrier," etc. are used interchangeably herein.

[0062]

[0073] The terms "progenitor cell" and "precursor" cell are used interchangeably herein to refer to cells that have a more primitive cellular phenotype (e.g., at an earlier step in the developmental pathway or development than a fully differentiated cell) compared to cells that can result from differentiation. Progenitor cells can also often have significant or extremely high proliferation potential. Progenitor cells can give rise to many different differentiated cell types or a single differentiated cell type, depending on the developmental pathway and the environment in which the cells develop and differentiate.

[0063]

[0074] The term "progenitor thereof" in relation to an insulin-positive endocrine cell may refer to any cell, including, for example, a pluripotent stem cell, a definitive endoderm cell, a primitive gut cell, a pancreatic progenitor cell, or an endocrine precursor cell, that can differentiate into an insulin-positive endocrine cell when cultured under conditions suitable for differentiating the precursor cell into an insulin-positive endocrine cell.

[0064]

[0075] The terms "stem cell-derived beta cells", "SC-beta cells", "functional beta cells", "functional pancreatic beta cells", "mature SC-beta cells", and their grammatical equivalents may refer to cells (e.g., non-native pancreatic beta cells) that exhibit at least one marker indicative of a pancreatic beta cell (e.g., PDX-1 or NKX6.1) and secrete insulin. In some embodiments, SC-beta cells exhibit a glucose-stimulated insulin secretion (GSIS) response characteristic of endogenous mature beta cells. In some embodiments, the terms "SC-beta cells" and "non-native beta cells" as used herein are interchangeable. In some embodiments, "SC-beta cells" include mature pancreatic cells. It is understood that SC-beta cells need not be derived (e.g., directly) from stem cells. This is because the method of the present disclosure can derive SC-β cells from any insulin-positive endocrine cell or its precursor using any cell as a starting point (e.g., embryonic stem cells, induced pluripotent stem cells, progenitor cells, partially reprogrammed somatic cells (e.g., somatic cells partially reprogrammed to an intermediate state between induced pluripotent stem cells and the somatic cells from which they were derived), pluripotent cells, totipotent cells, transdifferentiated versions of any of the above cells, and others, as the disclosure is not intended to be limited in this manner). In some embodiments, the SC-β cells exhibit a response to multiple glucose challenges (e.g., at least one, at least two, or at least three or more series of glucose challenges). In some embodiments, the response is similar to the response of endogenous pancreatic islets (e.g., human pancreatic islets) to multiple glucose challenges. In some embodiments, the morphology of the SC-β cells is similar to that of endogenous β cells. In some embodiments, the SC-β cells exhibit an in vitro GSIS response similar to that of endogenous β cells. In some embodiments, SC-β cells exhibit in vivo GSIS response similar to that of endogenous β cells. In some embodiments, SC-β cells exhibit both in vitro and in vivo GSIS response similar to that of endogenous β cells. The GSIS response of SC-β cells can be observed within 2 weeks after transplanting SC-β cells into a host (e.g., human or animal).In some embodiments, the SC-β cells package insulin into secretory granules. In some embodiments, the SC-β cells exhibit encapsulated crystalline insulin granules. In some embodiments, the SC-β cells exhibit a stimulation index greater than 1. In some embodiments, the SC-β cells exhibit a stimulation index greater than 1.1. In some embodiments, the SC-β cells exhibit a stimulation index greater than 2. In some embodiments, the SC-β cells exhibit cytokine-induced apoptosis in response to cytokines. In some embodiments, the secretion of insulin from SC-β cells is enhanced in response to known antidiabetic drugs (e.g., secretagogues). In some embodiments, the SC-β cells are monohormonal. In some embodiments, the SC-β cells do not abnormally co-express other hormones, such as glucagon, somatostatin, or pancreatic polypeptide. In some embodiments, the SC-β cells exhibit a low replication rate. In some embodiments, the SC-β cells increase intracellular Ca2+ in response to glucose. In some embodiments, the SC-β cells express lower levels of MAFA than β cells from the pancreas of healthy control adult subjects. In some embodiments, the SC-β cells express higher levels of MAFB than β cells from the pancreas of healthy control adult subjects. In some embodiments, the SC-β cells express higher levels of SIX2, HOPX, IAPP and / or UCN3 than β cells from the pancreas of healthy control adult subjects. In some embodiments, the SC-β cells do not express MAFA. In some embodiments, the SC-β cells express MAFB. In some embodiments, any of the cell markers disclosed herein (e.g., MAFA, MAFB, SIX2, HOPX, IAPP and / or UCN3) are detected by flow cytometry. In some embodiments, the population includes one or more NKX6.1-positive, ISL1-positive cells that express CHGA, MAFB, and / or ESRRG at a higher level (e.g., at least 10%, 30%, 50%, 70%, 100%, 125%, 150%, or 200% higher) than NKX6.1-positive, ISL1-positive cells derived from the pancreas of a healthy control adult subject.In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the NKX6.1-positive, ISL1-positive cells in the cell population express CHGA, MAFB, and / or ESRRG at a level greater than at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the NKX6.1-positive, ISL1-positive cells derived from the pancreas of a healthy control adult subject. In some embodiments, the population comprises one or more NKX6.1-positive, ISL1-positive cells that express SIX3, MAFA, CHGB, RBP4 and / or FXYD2 at a lower level (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% lower) than NKX6.1-positive, ISL1-positive cells from the pancreas of a healthy control adult subject. In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the NKX6.1-positive, ISL1-positive cells in the cell population express SIX3, MAFA, CHGB, RBP4, and / or FXYD2 at a level lower than at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the NKX6.1-positive, ISL1-positive cells from the pancreas of a healthy control adult subject. In some embodiments, the population comprises NKX6.1-positive, ISL1-positive cells that express a lower level of SIX3 than the NKX6.1-positive, ISL1-positive cells from the pancreas of a healthy control adult subject. In some embodiments, the population comprises NKX6.1-positive, ISL1-positive cells that express a lower level of CHGB than the NKX6.1-positive, ISL1-positive cells from the pancreas of a healthy control adult subject. In some embodiments, the population comprises NKX6.1 positive, ISL1 positive cells that express lower levels of RBP4 than NKX6.1 positive, ISL1 positive cells from the pancreas of a healthy control adult subject. In some embodiments, the population comprises NKX6.1 positive, ISL1 positive cells that express lower levels of FXYD2 than NKX6.1 positive, ISL1 positive cells from the pancreas of a healthy control adult subject.

[0065]

[0076] In some embodiments, any of the NKX6.1-positive, ISL1-positive cells disclosed herein also express any one or more of the following genes: PC2, MNX1, or ABCC8.

[0066]

[0077] The terms "stem cell-derived α-cells", "SC-α-cells", "functional α-cells", "functional pancreatic α-cells", "mature SC-α-cells" and their grammatical equivalents can refer to cells (e.g., non-native pancreatic α-cells) that exhibit at least one marker indicative of a pancreatic α-cell (e.g., express glucagon, ISL1 but not NKX6.1), express glucagon, and secrete functional glucagon. In some embodiments, "SC-α-cells" do not express somatostatin. In some embodiments, "SC-α-cells" do not express insulin. In some embodiments, the terms "SC-α-cells" and "non-native α-cells" as used herein are interchangeable. In some embodiments, "SC-α-cells" include mature pancreatic cells.

[0067]

[0078] The terms "stem cell-derived δ cells", "SC-δ cells", "functional δ cells", "functional pancreatic δ cells", "mature SC-δ cells" and their grammatical equivalents can refer to cells (e.g., non-native pancreatic δ cells) that exhibit at least one marker indicative of a pancreatic δ cell (e.g., somatostatin) and that express and secrete somatostatin. In some embodiments, "SC-δ cells" do not express glucagon. In some embodiments, "SC-δ cells" do not express insulin. In some embodiments, the terms "SC-δ cells" and "non-native δ cells" as used herein are interchangeable. In some embodiments, "SC-δ cells" include mature pancreatic cells.

[0068]

[0079] The terms "stem cell-derived enterochromaffin (EC) cells", "SC-EC cells", and their grammatical equivalents can refer to cells (e.g., non-native pancreatic EC cells) that exhibit at least one marker indicative of a pancreatic EC cell (e.g., express VMAT1, NKX6.1, but do not express ISL1). In some embodiments, the terms "SC-EC cells" and "non-native EC cells" used herein are interchangeable.

[0069]

[0080] It should be understood that, like SC-β cells, SC-α, SC-δ, and SC-EC cells need not be derived (e.g., directly) from stem cells, as the disclosed methods can derive SC-α cells from other progenitor cells produced during in vitro differentiation of SC-β cells as a starting point (e.g., embryonic stem cells, induced pluripotent stem cells, progenitor cells, partially reprogrammed somatic cells (e.g., somatic cells partially reprogrammed to an intermediate state between an induced pluripotent stem cell and the somatic cell from which it was derived), multipotent cells, totipotent cells, transdifferentiated versions of any of the foregoing cells, etc. can be used, as the disclosure is not intended to be so limited).

[0070]

[0081] "Stem cell-derived islet cells" or "SC-islet cells" are islet cells derived from stem cells. Examples of SC-islet cells include SC-β cells, SC-α cells, SC-δ cells, and SC-EC cells. SC islet cells are not mature islet cells obtained from cadavers or human subjects.

[0071]

[0082] The phrase "therapeutically effective amount" as used herein with respect to a cell population refers to an amount of cells of the present disclosure, e.g., a population of SC-β cells or mature pancreatic β cells, or related cells in a composition comprising SC-β cells, that is effective to produce some desired therapeutic effect in at least a subpopulation of cells in an animal, at a reasonable benefit / risk ratio applicable to any medical treatment. For example, the amount of SC-β cell population administered to a subject is sufficient to produce a statistically significant, measurable change in at least one symptom of type 1, type 1.5 or type 2 diabetes, such as glycated hemoglobin level, fasting blood glucose level, hypoinsulinemia, etc. The determination of a therapeutically effective amount is well within the capabilities of a person skilled in the art. In general, a therapeutically effective amount may vary depending on the subject's medical history, age, condition, sex, and the severity and type of the subject's medical condition, as well as the administration of other pharmacologic active agents.

[0072]

[0083] As used herein, the term "insulin-producing cells" and its grammatical equivalents refer to cells that differentiate from pancreatic precursor cells or their precursors and secrete insulin. Insulin-producing cells, as that term is described herein, can include pancreatic β cells, as well as pancreatic β-like cells (e.g., insulin-positive endocrine cells) that synthesize (e.g., transcribe insulin gene, translate proinsulin mRNA, and modify proinsulin mRNA to insulin protein), express (e.g., realize the phenotypic trait conveyed by insulin gene), or secrete (release insulin into extracellular space) insulin in a constitutive or inducible manner. For example, the insulin-producing cell population generated by differentiating insulin-positive endocrine cells or their precursors into SC-β cells according to the method of the present disclosure can be pancreatic β cells, or β-like cells (e.g., cells having at least one, or at least two) characteristic of endogenous β cells, and can exhibit glucose-stimulated insulin secretion (GSIS) responses similar to endogenous adult β cells. For example, the population of insulin-producing cells generated by the methods disclosed herein can include mature pancreatic β cells or SC-β cells, and can also include non-insulin-producing cells (e.g., cells that have a cell-like phenotype but do not produce or secrete insulin).

[0073]

[0084] The terms "insulin-positive β-like cells", "insulin-positive endocrine cells" and their grammatical equivalents may refer to cells (e.g., pancreatic endocrine cells) that exhibit at least one marker indicative of pancreatic β cells and also express insulin, but lack the glucose-stimulated insulin secretion (GSIS) response characteristic of endogenous β cells. Exemplary markers of "insulin-positive endocrine cells" include, but are not limited to, NKX6.1, ISL1, and insulin. In some instances, the terms "insulin-positive endocrine cells" and "NKX6.1-positive, ISL1-positive cells" are used interchangeably.

[0074]

[0085] As used herein, "cell marker" refers to, but is not limited to, proteins, peptides, nucleic acids, polymorphisms of proteins and nucleic acids, splice variants, fragments of proteins or nucleic acids, elements, and other analytes that are specifically expressed or present in one or more specific cell types and not expressed or present in other specific cell types. For example, some cells of a cell population may express PDX1, while other cell populations do not. In certain embodiments, the presence or absence of one or more cell markers is determined using commercially available antibodies, for example, by flow cytometry and / or immunohistochemistry. In other embodiments, the presence or absence of one or more cell markers is detected using RT-PCR. Examples of cell markers include PDX1, NKX6.1, NGN-3, Neuro-D, ISL1, insulin, C-peptide, glucagon, somatostatin, VMAT1, MAFA, MAFB. Other examples of specific cell markers are referenced throughout this disclosure.

[0075]

[0086] The term "β-cell marker" refers to, but is not limited to, proteins, peptides, nucleic acids, polymorphisms of proteins and nucleic acids, splice variants, fragments of proteins or nucleic acids, elements, and other analytes that are specifically expressed or present in pancreatic β-cells. Exemplary β-cell markers may include, but are not limited to, pancreatic and duodenal homeobox 1 (PDX1) polypeptide, insulin, c-peptide, amylin, E-cadherin, Hnf3β, PCI / 3, B2, Nkx2.2, GLUT2, PC2, ZnT-8, ISL1, Pax6, Pax4, NeuroD, 1 Inf1b, Hnf-6, Hnf-3β, and MafA, as well as those described in Zhang et al., Diabetes. 50(10):2231-6 (2001). In some embodiments, the β-cell marker is a nuclear β-cell marker. In some embodiments, the β-cell marker is PDX1 or PH3.

[0076]

[0087] The term "pancreatic endocrine marker" can refer, without limitation, to proteins, peptides, nucleic acids, protein and nucleic acid polymorphisms, splice variants, protein or nucleic acid fragments, elements, and other analytes that are specifically expressed or present in pancreatic endocrine cells. Exemplary pancreatic endocrine cell markers include, but are not limited to, Ngn-3, NeuroD, and Islet-1.

[0077]

[0088] The terms "pancreatic progenitor cells," "pancreatic endocrine precursor cells," "pancreatic precursors," "pancreatic endocrine precursors," and their grammatical equivalents are used interchangeably herein and may refer to stem cells that can become pancreatic hormone-expressing cells that can form pancreatic endocrine cells, pancreatic exocrine cells, or pancreatic duct cells. These cells are committed to differentiation toward at least one type of pancreatic cell, such as beta cells that produce insulin, alpha cells that produce glucagon, delta cells (or D cells) that produce somatostatin, and / or F cells that produce pancreatic polypeptide. Such cells may express at least one of the following markers: NGN3, NKX2.2, NeuroD, ISL-1, Pax4, Pax6, or ARX.

[0078]

[0089] The term "PDX1-positive pancreatic progenitor cells" or "PDX1-positive, NKX6.1-negative pancreatic progenitor cells" as used herein can refer to cells that are pancreatic endoderm (PE) cells that have the ability to differentiate into SC-β cells, such as pancreatic β cells. PDX1-positive pancreatic progenitor cells express the marker PDX1. Other markers include, but are not limited to, Cdcp1, or Ptf1a, or HNF6, or NRx2.2. Expression of PDX1 can be evaluated by any method known to those skilled in the art, such as flow cytometry, immunochemistry using anti-PDX1 antibodies, or quantitative RT-PCR. In some examples, PDX1-positive pancreatic progenitor cells lack expression of NKX6.1. In some examples, PDX1-positive pancreatic progenitor cells can also be referred to as PDX1-positive, NKX6.1-negative pancreatic progenitor cells, since they lack expression of NKX6.1. In some examples, PDX1-positive pancreatic progenitor cells can also be named "pancreatic foregut endoderm cells."

[0079]

[0090] The terms "PDX1 positive, NKX6.1 positive pancreatic progenitor cells" and "NKX6.1 positive pancreatic precursors" are used interchangeably herein and can refer to cells that are pancreatic endoderm (PE) cells that have the ability to differentiate into insulin-producing cells, such as pancreatic β cells. PDX1 positive, NKX6-1 positive pancreatic progenitor cells express markers PDX1 and NKX6-1. Other markers include, but are not limited to, Cdcp1, or Ptf1a, or HNF6 or NRx2.2. NKX6-1 expression can be evaluated by any method known to those skilled in the art, such as flow cytometry, immunochemistry using anti-NKX6-1 antibody, or quantitative RT-PCR. As used herein, the terms "NKX6.1" and "NKX6-1" are equivalent and interchangeable. In some examples, PDX1 positive, NKX6-1 positive pancreatic progenitor cells can also be named "pancreatic foregut precursor cells".

[0080]

[0091] The terms "NeuroD" and "NeuroD1" are used interchangeably to identify a protein and its encoding gene expressed in pancreatic endocrine precursor cells.

[0092] The term "epigenetics" refers to heritable changes in gene function that do not involve changes in the DNA sequence. Epigenetics most often refers to chromosomal changes that affect gene activity and expression, but can also be used to describe any heritable phenotypic change that does not result from modifications of the genome. Such effects on cellular and physiological phenotypic traits can result from external or environmental factors, or can be part of the normal developmental program. Epigenetics can also refer to functionally relevant changes in the genome that do not involve changes in the nucleotide sequence. Examples of mechanisms that generate such changes are DNA methylation and histone modifications, each of which alters the way genes are expressed without modifying the underlying DNA sequence. Expression of genes can be controlled by the action of repressor proteins that bind to silencer regions of DNA. These epigenetics can persist through cell divisions during the life of a cell, and can persist for many generations, even though they do not involve changes in the underlying DNA sequence of the organism. One example of epigenetics in eukaryotic cell biology is the process of cell differentiation. During morphogenesis, totipotent stem cells give rise to a variety of pluripotent cells, which can then become fully differentiated cells.

[0081]

[0093] The term "epigenetic modifying compound" refers to a compound that causes epigenetics in a gene, i.e., changes the expression of the gene without changing the DNA sequence. Epigenetics helps determine whether a gene is turned on or off and can affect the production of proteins in certain cells, e.g., beta cells. Epigenetic modifications, such as DNA methylation and histone modifications, alter DNA accessibility and the structure of chromatin, thereby regulating patterns of gene expression. These processes are important for the normal development and differentiation of unique cell lineages in adult organisms. They can be modified by exogenous influences and thus may contribute to or be the result of environmental modifications of phenotypes or pathophenotypes. Importantly, epigenetic modifications have a key role in the regulation of pluripotency genes, which are inactivated during differentiation. Non-limiting examples of epigenetic modifying compounds include DNA methylation inhibitors, histone acetyltransferase inhibitors, histone deacetylase inhibitors, histone methyltransferase inhibitors, bromodomain inhibitors, or any combination thereof.

[0082]

[0094] The term "differentiated cell" or its grammatical equivalents refers to any primary cell that is not pluripotent in its natural form as that term is defined herein. In another way, the term "differentiated cell" can refer to a cell of a more specialized cell type derived from a cell of a less specialized cell type (e.g., a stem cell such as an induced pluripotent stem cell) during the cell differentiation process. Without wishing to be limited by theory, pluripotent stem cells during normal ontogeny can initially differentiate into endodermal cells that can form pancreatic cells and other endodermal cell types. Further differentiation of endodermal cells can lead to the pancreatic pathway, in some embodiments, about 98% of the cells become exocrine, ductal, or matrix cells, and about 2% become endocrine cells. Early endocrine cells are pancreatic islet precursor cells, which can then further differentiate into insulin-producing cells (e.g., functional endocrine cells) that secrete insulin, glucagon, somatostatin, or pancreatic polypeptide. Endodermal cells can also differentiate into other cells of endodermal origin, such as lung, liver, intestine, thymus, etc.

[0083]

[0095] As used herein, the term "somatic cell" can refer to any cell that forms an organism, as opposed to a germline cell. In mammals, germline cells (also known as "gametes") are the sperm and eggs that fuse during fertilization to produce a cell called a zygote, from which the entire mammalian embryo develops. Apart from sperm and eggs (gametocytes) and undifferentiated stem cells, which are the cells from which somatic cells are made, all other cell types in the mammalian body are somatic cells. Internal organs, skin, bone, blood, and connective tissue are all made from somatic cells. In some embodiments, the somatic cell is a "non-embryonic somatic cell", which means a somatic cell that is not present in or obtained from an embryo and does not result from the in vitro propagation of such a cell. In some embodiments, the somatic cell is an "adult somatic cell", which means a cell that is present in or obtained from an organism other than an embryo or fetus, or that results from the in vitro propagation of such a cell. Unless otherwise indicated, the method for converting at least one insulin-positive endocrine cell or precursor thereof into an insulin-producing, glucose-responsive cell can be performed both in vivo and in vitro (performed in vivo when at least one insulin-positive endocrine cell or precursor thereof is present in the subject, and performed in vitro with at least one insulin-positive endocrine cell or precursor thereof isolated and maintained in culture).

[0084]

[0096] As used herein, the term "adult cell" may refer to cells found throughout the body after embryonic development.

[0097] As used herein, the term "endodermal cells" may refer to cells that are from one of the three primary germ cell layers in the very early embryo (the other two being mesoderm and ectoderm). Endoderm is the innermost of the three layers. Endoderm cells can differentiate to give rise first to the embryonic gut, then to the respiratory and digestive tracts (e.g., intestine), liver, and the lining of the pancreas.

[0085]

[0098] As used herein, the term "cells of endodermal origin" may refer to any cell that develops or differentiates from endodermal cells. For example, cells of endodermal origin include cells of the liver, lung, pancreas, thymus, intestine, stomach, and thyroid. Without wishing to be bound by theory, hepatic and pancreatic progenitor cells (also called pancreatic progenitor cells) can develop from endodermal cells in the embryonic foregut. Soon after their identification, hepatic and pancreatic progenitor cells rapidly acquire distinct cellular functions and regenerative capabilities. These changes are driven by inductive signals and genetic regulators that are highly conserved among vertebrates. Interest in organ development and regeneration has been fueled by the intense need for hepatic and pancreatic β cells in the therapeutic treatment of liver failure and type I diabetes. Studies in various model organisms and humans have revealed a network of evolutionarily conserved inductive signals and transcription factors that trigger hepatic and pancreatic cell differentiation and provide guidance on how to promote hepatic and β cell differentiation from various stem and progenitor cell types.

[0086]

[0099] The term "definitive endoderm" as used herein may refer to cells that can differentiate from endoderm cells and differentiate into SC-β cells (e.g., pancreatic β cells). Definitive endoderm cells express the marker Sox17. Other markers characteristic of definitive endoderm cells include, but are not limited to, MIXL2, GATA4, HNF3B, GSC, FGF17, VWF, CALCR, FOXQ1, CXCR4, Cerberus, OTX2, Goosecoid, C-Kit, CD99, CMKOR1, and CRIP1. In particular, the definitive endoderm cells herein express Sox17 and in some embodiments Sox17 and HNF3B, and do not express significant levels of GATA4, SPARC, APF, or DAB. Definitive endoderm cells are not positive for the marker PDX1 (e.g., they are PDX1 negative). Definitive endoderm cells have the capacity to differentiate into cells including liver, lung, pancreatic, thymus, intestine, stomach, and thyroid cells. Expression of Sox17 and other markers of definitive endoderm can be assessed by any method known to those skilled in the art, such as, for example, immunochemistry using anti-Sox17 antibodies or quantitative RT-PCR.

[0087]

[0100] The term "pancreatic endoderm" may refer to cells of endodermal origin that can differentiate into multiple pancreatic lineages, including pancreatic β cells, but no longer have the capacity to differentiate into non-pancreatic lineages.

[0101] The term "primitive gut cells" or "intestinal cells" as used herein can refer to cells that can differentiate from endoderm cells and differentiate into SC-β cells (e.g., pancreatic β cells). Primitive gut cells express at least one of the following markers: HNP1-β, HNF3-β, or HNF4-α. In some examples, primitive gut cells are FOXA2-positive and SOX2-positive, i.e., express both FOXA2 (also known as HNF3-β) and SOX2. In some examples, primitive gut cells are FOXA2-positive and PDX1-negative, i.e., express FOXA2 but not PDX1. Primitive gut cells have the ability to differentiate into cells including lung, liver, pancreas, stomach, and intestinal cells. Expression of HNF1-β and other primitive gut markers can be assessed by any method known to those skilled in the art, such as, for example, immunochemistry using anti-HNF1-β antibodies.

[0088]

[0102] As used herein, the term "stem cell" may refer to an undifferentiated cell that can proliferate and give rise to more progenitor cells that have the capacity to produce a large number of mother cells that can then give rise to differentiated or differentiable daughter cells. The daughter cells themselves can be induced to proliferate and generate progeny that subsequently differentiate into one or more mature cell types while retaining one or more cells with the developmental potential of the parent. The term "stem cell" may refer to a subset of progenitor cells that have the capacity or potential to differentiate into a more specialized or differentiated phenotype under certain circumstances and that retain the capacity to proliferate without substantially differentiating under certain circumstances. In one embodiment, the term stem cell generally refers to a naturally occurring mother cell whose descendants (descendants, progeny) specialize by differentiation, often in different directions, e.g., by acquiring completely individual characteristics, as occurs in the gradual diversification of cells and tissues of an embryo. Differentiation of cells is a complex process that typically occurs through many cell divisions. Differentiated cells may be derived from pluripotent cells, which are themselves derived from pluripotent cells, and so on. Each of these pluripotent cells is considered a stem cell, although the range of cell types each can give rise to can vary considerably. Some differentiated cells also have the ability to give rise to cells with greater developmental potential. Such ability may be natural or may be artificially induced by treatment with various factors. In many biological examples, stem cells are also "pluripotent" because they can generate progeny with two or more different cell types, but this is not necessary to be "stemness". "Self-renewal" is the other classical part of the definition of a stem cell. In theory, self-renewal can occur by either of two main mechanisms. Stem cells divide asymmetrically, with one daughter retaining the stem cell state and the other daughter expressing some different other specific function and phenotype. Alternatively, some of the stem cells in a population can divide symmetrically into two stem cells, whereby some stem cells in the population are maintained as a whole, while other cells in the population give rise only to differentiated progeny.Formally, cells that begin as stem cells progress toward a differentiated phenotype, but can also "reverse" and reverse express the stem cell phenotype. This is often referred to by those skilled in the art as "dedifferentiation" or "reprogramming" or "reverse differentiation." As used herein, the term "pluripotent stem cells" includes embryonic stem cells, induced pluripotent stem cells, placental stem cells, and the like.

[0089]

[0103] As used herein, the term "pluripotent" may refer to cells that have the ability to differentiate under various conditions into two or more differentiated cell types, preferably into cell types characteristic of all three germ layers. Pluripotent cells are primarily characterized by their ability to differentiate into two or more cell types, preferably into all three germ layers, using, for example, a nude mouse teratoma formation assay. Although pluripotency can also be demonstrated by expression of embryonic stem (ES) cell markers, the preferred test for pluripotency is to demonstrate the ability to differentiate into cells of each of the three germ layers. It should be noted that merely culturing such cells does not, in and of itself, render them pluripotent. Reprogrammed pluripotent cells (e.g., iPS cells, as that term is defined herein) are also characterized by their ability to be passaged for an extended period without loss of growth potential, as compared to primary parent cells, which generally have the ability to divide only a limited number of times in culture.

[0090]

[0104] As used herein, the terms "iPS cells" and "induced pluripotent stem cells" are used interchangeably and may refer to pluripotent stem cells that have been artificially derived (e.g., induced or by complete reversal) from non-pluripotent cells, typically adult somatic cells, for example by inducing forced expression of one or more genes.

[0091]

[0105] The term "phenotype" can refer to all of the biological characteristics, or several, that define a cell or organism under a particular set of environmental conditions and factors, regardless of the actual genotype.

[0092]

[0106] The terms "subject", "patient", or "individual" are used interchangeably herein and may refer to an animal, e.g., a human, from which cells are obtained and / or to which treatment, including prophylactic treatment, with cells as described herein is provided. For treatment of an infection, condition, or disease state specific to a particular animal, such as a human subject, the term subject may refer to that particular animal. "Non-human animals" and "non-human mammals", as used interchangeably herein, include mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The term "subject" also encompasses any vertebrate, including, but not limited to, mammals, reptiles, amphibians, and fish. Advantageously, however, the subject is a mammal, such as a human, or other mammal, such as, for example, dogs, cats, horses, other domestic animals, or cows, sheep, pigs, and other production mammals. "Patient in need thereof" or "subject in need thereof" as used herein refers to a patient diagnosed with or suspected of having a disease or disorder, such as, but not limited to, diabetes.

[0093]

[0107] As used herein, "administering" may mean providing one or more compositions described herein to a patient or subject. By way of example and not limitation, administration, e.g., injection, of a composition may be performed by intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im) injection. One or more such routes may be employed. Parenteral administration may be, for example, by bolus injection or gradual perfusion over time. Alternatively, or concurrently, administration may be by oral route. Additionally, administration may involve surgically depositing a bolus or pellet of cells or positioning a medical device. In some embodiments, administration is administering one or more devices containing a cell population to a subject (e.g., subcutaneously and / or preperitoneally). In one embodiment, the composition of the present disclosure may comprise engineered or host cells expressing a nucleic acid sequence described herein or a vector comprising at least one nucleic acid sequence described herein in an amount effective to treat or prevent a proliferative disorder. Pharmaceutical compositions may include the cell populations described herein in combination with one or more pharma- ceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline, phosphate buffered saline, carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives.

[0094] Treatment

[0108] In some embodiments, methods are provided herein for treating a disease characterized by high blood glucose levels over an extended period of time, such as diabetes, e.g., type 1 diabetes in a subject. In some embodiments, methods are provided herein for preventing a disease characterized by high blood glucose levels over an extended period of time, such as diabetes, e.g., type 1 diabetes in a subject, e.g., in a subject at risk of developing such a disease. In some embodiments, methods are provided herein for reducing the likelihood of developing a disease characterized by high blood glucose levels over an extended period of time, such as diabetes, e.g., type 1 diabetes in a subject. A composition comprising a cell population (e.g., cell clusters and / or cells) provided herein or generated according to a method provided herein can be administered to a subject to restore some pancreatic function in the subject. For example, cell clusters similar to endogenous pancreatic islets, or cells similar to endogenous pancreatic alpha, beta and / or delta cells (e.g., non-native pancreatic alpha, beta and / or delta cells) or precursors thereof can be administered to a subject to treat, prevent, or reduce the likelihood of developing a disease characterized by high blood glucose levels over an extended period of time, such as diabetes, e.g., type 1 diabetes.

[0095]

[0109] The method can include administering the cell clusters or cells disclosed in the present application to a subject, e.g., a subject in need thereof, via implantation of the cells in an encapsulation composition (e.g., a device). In some embodiments, the subject is administered the cells / cell clusters in the device (e.g., subcutaneously or intraperitoneally).

[0096]

[0110] The method can include administering a cell cluster or cell disclosed in the present application to a subject, e.g., a subject in need thereof, via injection. The term "injection" can refer to the delivery of a cell or cell cluster, a cell or any portion of a cell cluster, or any composition comprising a cell, cell cluster, or any portion thereof, to a subject's bloodstream, e.g., a vein of the subject, e.g., the hepatic portal vein, or an infusion port inserted into the bloodstream.

[0097]

[0111] In some embodiments, the methods disclosed herein include administering a pharmaceutical composition disclosed herein to a subject via injection. The pharmaceutical composition can include a population of cells (e.g., SC-islet cells) in a liquid suspension. The cell population can include cell clusters similar to endogenous pancreatic islets, or cells similar to endogenous pancreatic alpha, beta and / or delta cells (e.g., non-native pancreatic alpha, beta and / or delta cells) or their precursors. For example, the cell population can include non-native cells that express C-peptide and ISL1. In some embodiments, the cell population includes non-native cells that express C-peptide and ISL1 but do not express VMAT1. Additionally, the cell population can include non-native cells that express glucagon but do not express somatostatin (e.g., non-native alpha cells), and non-native cells that express somatostatin but do not express glucagon (e.g., non-native delta cells).

[0098]

[0112] As used herein, the terms "treat" and "treatment" can refer to administering an effective amount of a composition (e.g., a cell cluster or a portion thereof) to a subject such that the subject has a reduction in at least one symptom of the disease or an improvement in the disease, e.g., a beneficial or desired clinical outcome. In some embodiments, the subject becomes insulin independent after treatment. For purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, alleviation of one or more symptoms, whether detectable or undetectable, attenuation of the extent of the disease, stabilization of the disease state (e.g., not worsening), delay or slowing of disease progression, improvement or relief of the disease state, and remission (e.g., partial or total). Treating can refer to a prolongation of survival compared to the expected survival in the absence of treatment. Thus, one skilled in the art will understand that treatment can improve the disease state, but may not be a complete cure for the disease. As used herein, the term "treatment" includes prevention.

[0099]

[0113] "Treatment," "prevention," or "amelioration" of a disease or disorder means delaying or preventing the onset of such disease or disorder, reversing, alleviating, ameliorating, inhibiting, slowing, or halting the progression, aggravation, progression, or severity of the conditions associated with such disease or disorder. In one embodiment, symptoms of the disease or disorder are alleviated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%.

[0100]

[0114] The goal of diabetes treatment is to bring sugar levels as close to normal as safely possible. Commonly set goals are 80-120 milligrams per deciliter (mg / dl) before meals and 100-140 mg / dl at bedtime. A particular physician may set different targets for a patient depending on other factors such as how often the patient has hypoglycemic or hyperglycemic reactions. Useful medical tests include tests on the patient's blood and urine to determine blood glucose levels, tests for glycosylated hemoglobin levels (HbA1c; a measurement of the average blood glucose level over the past 2-3 months, normal range is 4-6%), tests for cholesterol and fat levels, and tests for urine protein levels. Such tests are standard tests known to those skilled in the art (see, e.g., American Diabetes Association, 1998). The success of a treatment program may also be determined by fewer patients in the program having diabetes-related complications, such as severe hypoglycemic events, eye disease, kidney disease, or neurological disease.

[0101]

[0115] The treatment methods disclosed herein can result in the improvement of one or more physiological parameters related to blood glucose regulation. In some cases, the methods disclosed herein can result in at least partial restoration of glucose-responsive insulin secretion in a subject. Without wishing to be bound by a particular theory, the glucose-responsive insulin secretion restored in a subject by the methods and compositions disclosed herein can lead to the improvement of one or more symptoms or complications related to diabetes in the subject. In some aspects, the present disclosure relates to a method comprising administering to a subject a pharmaceutical composition comprising a population of cells (e.g., SC-islet cells) provided herein, wherein the administered cells release a sufficient amount of insulin to reduce the blood glucose level of the subject.

[0102]

[0116] After administration of a pharmaceutical composition according to an embodiment of the present disclosure, the blood C-peptide level in the subject can be increased to a level that at least partially ameliorates one or more symptoms of the disease, such as more stable, reduced blood glucose levels, reduced hemoglobin A1c levels, less reliance on exogenous insulin intake (e.g., to stabilize blood glucose levels), or fewer severe hypoglycemic events. In some cases, the method can increase the subject's stimulated blood C-peptide level to at least 450 pmol / L, 460 pmol / L, 470 pmol / L, 480 pmol / L, 490 pmol / L, 500 pmol / L, 510 pmol / L, 520 pmol / L, 530 pmol / L, 540 pmol / L, 550 pmol / L, 700 pmol / L, 800 pmol / L, or 900 pmol / L when measured at least about 1, 2, 3, 4, 5, or 6 months after administration. ol / L, 900 pmol / L, 1000 pmol / L, 1400 pmol / L, or 150-350 pmol / L, 250-400 pmol / L, 400-1400 pmol / L, 400-1200 pmol / L, 400-1000 pmol / L, 400-800 pmol / L, 400-600 pmol / L, 600-800 pmol / L, 600-1400 pmol / L, or 900-1200 pmol / L. Measurement of stimulated blood C-peptide levels can be performed using standardized tests, such as a mixed meal tolerance test, an oral glucose tolerance test, etc.In some instances, the method provides for increasing the subject's stimulated blood C-peptide level to at least 450 pmol / L, 460 pmol / L, 470 pmol / L, 480 pmol / L, 490 pmol / L, 500 pmol / L, 510 pmol / L, 520 pmol / L, 530 pmol / L, 540 pmol / L, or 550 pmol / L, 700 pmol / L, when measured at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after administration. ol / L, 800pmol / L, 900pmol / L, 1000pmol / L, 1400pmol / L, or increased to 150-350pmol / L, 250-400pmol / L, 400-1400pmol / L, 400-1200pmol / L, 400-1000pmol / L, 400-800pmol / L, 400-600pmol / L, 600-800pmol / L, 600-1400pmol / L, or 900-1200pmol / L. In some cases, the method increases the subject's stimulated blood C-peptide level by at least 200 pmol / L, 300 pmol / L, 450 pmol / L, 460 pmol / L, 470 pmol / L, 480 pmol / L, 490 pmol / L, 500 pmol / L, 510 pmol / L, 520 pmol / L. 530 pmol / L, 540 pmol / L, 550 pmol / L, 600 pmol / L, or 650 pmol / L, or by 150-350 pmol / L, 200-700 pmol / L, 150-700 pmol / L, 300-650 pmol / L, or 500-700 pmol / L when measured at least about three months after administration. In some cases, the method increases the subject's stimulated blood C-peptide level to at least 450 pmol / L, 460 pmol / L, 470 pmol / L, 480 pmol / L, 490 pmol / L, 500 pmol / L, 510 pmol / L, 520 pmol / L, 530 pmol / L, 540 pmol / L, or 550 pmol / L when measured about 5 months after administration. In some embodiments, the method increases the subject's stimulated blood C-peptide level to about 480 pmol / L, about 500 pmol / L, about 520 pmol / L, about 540 pmol / L, or about 560 pmol / L when measured about 3 months after administration.In some embodiments, the method increases the subject's stimulated blood C-peptide level to about 480 pmol / L, about 500 pmol / L, about 520 pmol / L, about 540 pmol / L, or about 560 pmol / L when measured about 5 months after administration. In some embodiments, the treated subject's stimulated blood C-peptide level is between 480 pmol / L and 560 pmol / L when measured about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after administration (e.g., 3 or 5 months after administration).

[0103]

[0117] In some cases, oral glucose tolerance tests or mixed meal tolerance tests are performed in the morning after an overnight fast of more than 8 hours. Subjects may be restricted from strenuous exercise, alcohol, caffeine and tobacco use, all of which may affect insulin sensitivity. Mixed meal tolerance tests can be performed according to protocols provided with standardized meals of specific macronutrient content or proprietary meal replacements, such as Ensure® (Abbott) or Boost® Complete Nutritional Drink High Protein (Nestle Health Science). For oral glucose tolerance tests, 75 g of anhydrous glucose dissolved in 250 ml of water can be used as the reference method.

[0104]

[0118] In some cases, the method increases the subject's blood C-peptide level to at least 100 pmol / L, 120 pmol / L, 130 pmol / L, 150 pmol / L, 160 pmol / L, 180 pmol / L, 200 pmol / L, 210 pmol / L, 220 pmol / L, 230 pmol / L, 240 pmol / L, 250 pmol / L, 260 pmol / L, 270 pmol / L, or 280 pmol / L under fasting conditions when measured at least about 1, 2, 3, 4, 5, or 6 months after administration. In some embodiments, the method increases the subject's blood C-peptide levels under fasting conditions to at least 100 pmol / L, 120 pmol / L, 130 pmol / L, 150 pmol / L, 160 pmol / L, 180 pmol / L, 200 pmol / L. 210 pmol / L, 220 pmol / L, 230 pmol / L, 240 pmol / L, 250 pmol / L, 260 pmol / L, 270 pmol / L, or 280 pmol / L when measured about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after administration. In some cases, the method increases the subject's blood C-peptide levels under fasting conditions to at least 100 pmol / L, 120 pmol / L, 130 pmol / L, 150 pmol / L, 160 pmol / L, 180 pmol / L, 200 pmol / L, 210 pmol / L, 220 pmol / L, 230 pmol / L, 240 pmol / L, 250 pmol / L, 260 pmol / L, 270 pmol / L, or 280 pmol / L when measured about 3 months after administration. In some cases, the method increases the subject's blood C-peptide levels under fasting conditions to at least 100 pmol / L, 120 pmol / L, 130 pmol / L, 150 pmol / L, 160 pmol / L, 180 pmol / L, 200 pmol / L, 210 pmol / L, 220 pmol / L, 230 pmol / L, 240 pmol / L, 250 pmol / L, 260 pmol / L, 270 pmol / L, or 280 pmol / L when measured about 5 months after administration.In some cases, the method increases the subject's blood C-peptide level to about 200 pmol / L, 220 pmol / L, 240 pmol / L, 260 pmol / L, or 280 pmol / L under fasting conditions when measured about 3 months after administration. In some cases, the method increases the subject's blood C-peptide level to about 200 pmol / L, 220 pmol / L, 240 pmol / L, 260 pmol / L, or 280 pmol / L under fasting conditions when measured about 5 months after administration. In some cases, administration of a pharmaceutical composition according to the present disclosure reduces the subject's hemoglobin A1c level. For example, the subject's Hb1Ac level can be reduced to less than 8%, less than 7.5%, less than 7.0%, less than 6.5%, less than 6.0%, less than 5.5%, or between 5 and 8%, 5 and 7.5%, 5 and 6.5%, 5 and 5.5%, 5.5 and 8%, 5.5 and 7%, 5.5 and 6.5%, 6 and 8%, 6 and 7%, or 6 and 6.5% when measured at least about 1, 2, 3, 4, 5, or 6 months after administration. In some cases, the subject's Hb1Ac level is reduced to less than 8%, less than 7.5%, less than 7.0%, less than 6.5%, less than 6.0%, less than 5.5%, or less than 5%, or between 5-8%, 5-7.5%, 5-6.5%, 5-5.5%, 5.5-8%, 5.5-7%, 5.5-6.5%, 6-8%, 6-7%, or 6-6.5% when measured about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after administration. In some cases, the treated subject's Hb1Ac level is between 7-7.9% when measured about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after administration. In some cases, the reduction is less than 8%, 7.5%, 7.0%, or 6.75%, or between 5-8.0%, 6.5-8.0%, 6.5-7.5%, 6.5-7.0%, or between 7-8% when measured at about 3 months after administration. In some cases, the reduction is less than 8%, 7.9%, 7.8%, 7.7%, 7.6%, 7.5%, 7.3%, 7.2%, 7.1%, or 7.0% when measured at about 5 months after administration.In some cases, the reduction is 8%, 7.5%, 7.0%, 6.5%, 6.0%, 5.5%, or less than 5%, or 4.5-8%, 5-8%, 5-7.5%, 5-7%, 5-6.5%, 5-5.5%, 5.5-8%, 5.5-7%, 5.5-6.5%, 6-8%, 6-7%, or 6-6.5% when measured at about 9 months after administration. In some cases, the reduction is about 7.6%, 7.5%, 7.4%, 7.3%, 7.2%, 7.1%, or 7.0% when measured at about 5 months after administration.

[0105]

[0119] Patients with diseases characterized by high blood glucose levels, such as diabetes, have a medical need to inject external insulin to manage blood glucose levels ("glycemic control"). Typically, such patients are administered external insulin with a predefined goal to achieve glycemic control, e.g., to maintain blood glucose levels within a certain range. For example, goals discussed in Louis Monnier and Claude Colette, Target for Glycemic Control, Diabetes Care Nov 2009, 32(suppl 2)S199-S204; DOI:10.2337 / dc09-S310, incorporated herein in its entirety. In some cases, subjects may be prescribed personalized glycemic control goals by their physician to guide their insulin intake. The methods disclosed herein can reduce a subject's reliance on external insulin intake while achieving glycemic control goals. In some cases, at least about 1, 2, 3, 4, 5, or 6 months after administration, the subject's daily insulin is reduced to at most 10 units (U), 9U, 8U, 7U, 6U, 5U, 4U, 3U, or 2U on average for 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days, or at least 10 days, 15 days, 20 days, or 1 month. In some cases, at least about 1, 2, 3, 4, 5, or 6 months after administration, the subject's daily insulin is reduced to at most about 10 units (U), 9U, 8U, 7U, 6U, 5U, 4U, 3U, 2U, or 1U on average for 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days, or at least 10 days, 15 days, 20 days, or 1 month. In some cases, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after administration, the subject does not need to receive insulin infusions for 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days, or for at least 10 days, 15 days, 20 days, or 1 month.

[0106]

[0120] In some embodiments, the reduction in blood glucose level in a subject induced by administration of the pharmaceutical composition provided herein results in a glucose level lower than the diabetic threshold.In some embodiments, the subject is a mammalian subject.In some embodiments, the mammalian subject is a human.In some embodiments, the glucose level is reduced to below the diabetic threshold 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after administration.

[0107]

[0121] The subject that can be treated by the method of the present specification can be a human or a non-human animal. In some cases, the subject can be a mammal. Examples of subjects include, but are not limited to, primates, such as monkeys, chimpanzees, baboons, and humans. In some cases, the subject is a human. The subject can also be a non-primate animal, including, but not limited to, dogs, cats, horses, cows, pigs, sheep, goats, rabbits, etc. In some cases, the subject receiving treatment is a subject in need thereof, such as a human in need thereof.

[0108]

[0122] In certain embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms "patient" and "subject" are used interchangeably herein. Preferably, the subject is a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. A non-human mammal may be advantageously used as a subject representing an animal model of type 1 diabetes, type 2 diabetes mellitus, or prediabetes. In addition, the methods described herein may be used to treat livestock and / or pets. The subject may be male or female. The subject may be a person who has previously been diagnosed with or identified as having or having diabetes (e.g., type 1 or type 2), one or more complications associated with diabetes, or prediabetes, but optionally does not have to have already been treated for diabetes, one or more complications associated with diabetes, or prediabetes. The subject may not be one who has diabetes or prediabetes. The subject may also be a person who has been diagnosed with or identified as having diabetes, one or more complications associated with diabetes, or prediabetes, but who shows improvement in known diabetes risk factors as a result of receiving one or more treatments for diabetes, one or more complications associated with diabetes, or prediabetes. Alternatively, the subject may not have been previously diagnosed with diabetes, one or more complications associated with diabetes, or prediabetes. For example, the subject may be a person who exhibits one or more risk factors for diabetes, one or more complications associated with diabetes, or prediabetes, or a subject who does not exhibit risk factors for diabetes, or a subject who is asymptomatic for diabetes, one or more complications associated with diabetes, or prediabetes. The subject may also be a person who suffers from or is at risk of developing diabetes or prediabetes.A subject may also be one who has been diagnosed with or identified as having one or more complications associated with diabetes or pre-diabetes as defined herein, or, alternatively, a subject may not have been previously diagnosed with or identified as having one or more complications associated with diabetes or pre-diabetes.

[0109]

[0123] In some embodiments, prior to administration of any of the pharmaceutical compositions disclosed herein, the subject has a stimulated blood C-peptide level of less than 100 pmol / L, e.g., less than 80 pmol / L, 60 pmol / L, 40 pmol / L, 30 pmol / L, 20 pmol / L, or 10 pmol / L, as measured using a mixed meal tolerance test. In some embodiments, prior to administration of the pharmaceutical compositions disclosed herein, the subject has a stimulated blood C-peptide level of less than 100 pmol / L, e.g., less than 80 pmol / L, 60 pmol / L, 40 pmol / L, 30 pmol / L, 20 pmol / L, or 10 pmol / L, as measured using an oral glucose tolerance test. In some cases, prior to administration, the subject has an undetectable stimulated blood C-peptide level, e.g., as measured using a mixed meal tolerance test or an oral glucose tolerance test. In some cases, prior to administration, the subject has an undetectable stimulated blood C-peptide level, as measured under fasting conditions. In some embodiments, prior to administration, the subject demonstrates no residual endogenous islet cell function. In some embodiments, no residual endogenous islet cell function is indicated by undetectable fasting C-peptide and undetectable stimulated C-peptide in a mixed meal tolerance test. In some embodiments, no residual endogenous islet cell function is indicated by sustained stimulated glucose levels of greater than 350, 400, 450, 475, or 500 mg / dL. In some embodiments, prior to administration, the subject received more than 10, 15, 20, 25, 30, or 35 units of insulin per day. In some cases, prior to administration, the subject has a baseline plasma level of hemoglobin A1c greater than 8.0%, e.g., greater than 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.8%, or 9.0%. In some embodiments, prior to administration, the subject has an HbA1c level of greater than 7.7%, 8%, 8.2%, or 8.5%. In some cases, prior to administration, the subject receives an infusion of insulin at a level of at least about 20 U / day, e.g., at least about 22 U / day, 24 U / day, 26 U / day, 28 U / day, 30 U / day, 32 U / day, or 34 U / day.In some cases, prior to administration, the subject has a history of severe hypoglycemic events, for example, the subject may have at least one severe hypoglycemic event per month, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 20 severe hypoglycemic events. A "severe hypoglycemic event" may refer to an episode in which a patient with diabetes requires assistance from others to raise blood glucose levels, usually by administering glucagon or contacting a medical professional. By depleting the brain of glucose, severe hypoglycemia can acutely alter brain function, leading to neuroglycopenic symptoms, seizures, or even death. In some cases, prior to administration, the subject has a history of hypoglycemic cognitive impairment.

[0110]

[0124] In some cases, the treatment methods provided herein may include administering one or more immune response modulators to modulate or reduce transplant rejection or other immune responses to the administered cells. In some embodiments, the immune response modulator is not a steroid or does not include a steroid. In some cases, the immune response modulator includes a steroid, such as a corticosteroid. Examples of immune response modulators that may be used in the method include purine synthesis inhibitors, such as azathioprine and mycophenolic acid, pyrimidine synthesis inhibitors, such as leflunomide and teriflunomide, antifolates, such as methotrexate, tacrolimus, cyclosporine, pimecrolimus, avetimus, gasperimus, lenalidomide, pomalidomide, thalidomide, PDE4 inhibitors, apremilast, anakinra, sirolimus, everolimus, ridaforolimus, temsirolimus, umirolimus, zotarolimus, antithoracic acid, ... Lymphocyte globulin antibodies, antilymphocyte globulin antibodies, CTLA-4, fragments thereof, and fusion proteins thereof, e.g., abatacept and belatacept, TNF inhibitors, e.g., etanercept and pegsunercept, aflibercept, alefecept, rilonacept, antibodies against complement component 5, e.g., eculizumab, anti-TNF antibodies, e.g., adalimumab, afelimomab, certolizumab pegol, golimumab, infliximab, and nerelimomab, antibodies against interleukin 5, e.g., mepolizumab, anti-IgE antibodies, e.g., omalizumab, anti-interferon antibodies, e.g., faralimomab, anti-IL-6 antibodies, e.g., elsilimomab, antibodies against IL-12 and IL-23, e.g., lebrikizumab and ustekinumab, anti-IL-17A antibodies, e.g., secukinumab, anti-CD3 antibodies, e.g., muromonab-CD3, otelixizumab, teplizumab, visilizumab, anti-C D4 antibodies, such as clenoliximab, keliximab, and zanolimumab, anti-CD11a antibodies, such as efalizumab, anti-CD18 antibodies, such as erlizumab, anti-CD20 antibodies, such as obinutuzumab, rituximab, ocrelizumab, and pascolizumab, anti-CD23 antibodies, such as gomiliximab and lumiliximab, anti-CD40 antibodies, such as teneliximab and toralizumab, antibodies against CD62L / L-selectin, e.g., acelizumab, anti-CD80 antibodies, e.g., galiximab, anti-CD147 / basigin antibodies, e.g., gavilimomab, anti-CD154 antibodies, e.g., ruplizumab, anti-BLyS antibodies, e.g., belimumab and blisibimod, anti-CTLA-4 antibodies, e.g., ipilimumab and tremelimumab, anti-CAT antibodies, e.g., vellimumab, lerdelimumab, and methelimumab, anti-integrin antibodies, e.g., natalizumab, antibodies against interleukin-6 receptor, e.g., tocilizumab, anti-LFA-1 antibodies, e.g., odulimomab, antibodies against IL-2 receptor / CD25, e.g., basiliximab, daclizumab, and inolimomab, antibodies against T lymphocytes (zolimomab alitox, such as atrolizumab, cedelizumab, fontolizumab, maslimomab, morolimumab, pexelizumab, reslizumab, rovelizumab, siplizumab, talizumab, terimomab alitox, baparikimab, and beparimomab, antibodies against CD52, such as alemtuzumab, blockers of inosine monophosphate dehydrogenase (IMPDH), such as mycophenolate mofetil, and cell migration inhibitors, such as FTY720.

[0111]

[0125] In some embodiments, the subject receiving any of the pharmaceutical compositions disclosed herein according to any of the methods disclosed herein is also administered an immunosuppressant. In some embodiments, the immunosuppressant is selected from the group consisting of thymoglobulin, etanercept, basiliximab, tacrolimus, sirolimus, and mycophenolate mofetil. In some embodiments, the subject receiving SC-islet cells is also administered thymoglobulin and etanercept. In some embodiments, the subject receiving SC-islet cells is also administered thymoglobulin and basiliximab. In some embodiments, the subject receiving SC-islet cells is also administered tacrolimus and sirolimus. In some embodiments, the subject receiving SC-islet cells is also administered tacrolimus and basiliximab. In some embodiments, the subject receiving SC-islet cells is also administered sirolimus and mycophenolate. In some embodiments, the subject receiving SC-islet cells is also administered mycophenolate mofetil or mycophenolate sodium.

[0112]

[0126] In some embodiments, the present disclosure provides a method for the treatment of 1×10 8 cells ~ 12 × 10 8 (For example, 8×10 8 ~12×10 8 ) SC islet cells. In some embodiments, the present disclosure provides for administering to a subject a first pharmaceutical composition comprising 1×10 8 cells ~ 12 × 10 8 (For example, 8×10 8 ~12×10 8 In some embodiments, the present disclosure provides for administering to a subject a first pharmaceutical composition comprising 1×10 SC-islet cells in a single dose (i.e., in a single injection or in a single surgical procedure in which one or more devices are implanted into one or more incision sites in the subject). 8 cells ~ 12 × 10 8 pieces (e.g., 8×10 8 ~12×10 8In some embodiments, the present disclosure provides for administering to a subject multiple doses (i.e., two or more injections, or multiple surgical procedures in which one or more devices are implanted into one or more incision sites in the subject) of a first pharmaceutical composition comprising 1×10 SC-islet cells. 8 cells ~ 10 x 10 8 pieces (e.g., 3×10 8 ~8.5×10 8 In some embodiments, the first pharmaceutical composition comprises at least about 1×10 SC-islet cells. 8 SC islet cells, e.g., at least about 1.5 x 10 8 , at least about 2 × 10 8 , at least about 2.5 × 10 8 , at least about 3 × 10 8 , at least about 3.5 × 10 8 , at least about 4 × 10 8 , at least about 4.5 × 10 8 , at least about 5 × 10 8 , at least about 5.5 × 10 8 , at least about 6 × 10 8 , at least about 6.5 × 10 8 , or at least about 7 × 10 8 In some cases, the first pharmaceutical composition disclosed herein comprises at most about 8×10 SC-islet cells. 8 , at most about 7 × 10 8 , at most about 6.5 × 10 8 , at most about 6 × 10 8 , at most about 5.5 × 10 8 , at most about 5 × 10 8 , at most about 4.5 × 10 8 , at most about 4 × 10 8 , at most about 3.5 × 10 8 , at most about 3 × 10 8 , at most about 2.5 × 10 8 , or at most about 2 × 10 8 In some embodiments, the first pharmaceutical composition comprises about 1×10 SC-islet cells. 8 ~Approx. 8×108 , about 1×10 8 ~Approx. 7×10 8 , about 1×10 8 ~about 6×10 8 , about 1×10 8 ~Approx. 5×10 8 , about 1×10 8 ~Approx. 4×10 8 , about 1×10 8 ~Approx. 3×10 8 , about 1×10 8 ~about 2×10 8 , about 1.5×10 8 ~Approx. 6.5×10 8 , about 2×10 8 ~Approx. 7×10 8 , about 2×10 8 ~about 6×10 8 , about 2×10 8 ~Approx. 5×10 8 , about 2×10 8 ~Approx. 4×10 8 , about 2×10 8 ~Approx. 3×10 8 , about 2.5×10 8 ~Approx. 5.5×10 8 , about 3×10 8 ~Approx. 7×10 8 , about 3×10 8 ~about 6×10 8 , about 3×10 8 ~Approx. 5×10 8 , about 3×10 8 ~Approx. 4×10 8 , about 3.5×10 8 ~Approx. 4.5×10 8 , or about 3.8 × 10 8 ~Approx. 4.2×10 8 In some instances, the first pharmaceutical composition comprises about 3.5×10 SC-islet cells. 8 ~Approx. 4.5×10 8 In some embodiments, the first pharmaceutical composition comprises about 3.5×10 SC-islet cells. 8 ~Approx. 8.5×10 8 In some embodiments, the first pharmaceutical composition comprises about 3.9×10 SC-islet cells. 8 ~Approx. 4.1×10 8In some embodiments, the first pharmaceutical composition comprises about 7.9×10 SC-islet cells. 8 ~Approx. 8.1×10 8 In some embodiments, the first pharmaceutical composition comprises about 7.5×10 SC-islet cells. 8 ~Approx. 12×10 8 10 SC-islet cells (e.g., 7.5 x 10 8 ~10×10 8 In some embodiments, the subject receives only a single administration of the pharmaceutical composition (e.g., via injection or via any one or more of the devices disclosed herein), and the composition contains 7×10 8 ~14×10 8 , 8×10 8 ~13×10 8 , 9×10 8 ~12×10 8 , 9×10 8 ~11×10 8 , or 9×10 8 ~10×10 8 In some embodiments, the subject is administered a single dose of the cells (i.e., a single injection or a single surgical procedure in which one or more devices are implanted into one or more incision sites in the subject).

[0113]

[0127] In some embodiments, the subject is administered a first pharmaceutical composition comprising SC-islet cells, and the subject is not administered an additional pharmaceutical composition comprising SC-islet cells (i.e., the first pharmaceutical composition comprising SC-islet cells administered to the subject is the only pharmaceutical composition comprising SC-islet cells administered to the subject). 8 ~Approx. 4.5×10 8 SC-islet cells, approximately 3.9 x 10 8 ~Approx. 4.1×10 8 SC-islet cells, 7.5 x 10 8 ~Approx. 8.5×10 8 SC-islet cells, approximately 7.5 x 10 8 ~Approx. 9×10 8 SC-islet cells, approximately 7.5 x 10 8~About 10×10 8 SC-islet cells, approximately 10 x 10 8 ~Approx. 12×10 8 SC-islet cells, approximately 7.9 x 10 8 ~Approx. 8.1×10 8 SC-islet cells, approximately 4.0 x 10 8 10 SC-islet cells, or approximately 8.0 x 10 8 In some embodiments, the subject is administered a first pharmaceutical composition comprising SC-islet cells, and the subject is not administered a further pharmaceutical composition comprising SC-islet cells within 3, 4, 5, 6, 7, 8, 9 or 10 years after receiving the first pharmaceutical composition comprising SC-islet cells. In some embodiments, the subject is administered a first pharmaceutical composition comprising SC-islet cells, and the subject is not administered a further pharmaceutical composition comprising SC-islet cells within 5 years after receiving the first pharmaceutical composition comprising SC-islet cells. In some embodiments, the subject is administered a first pharmaceutical composition comprising SC-islet cells, and the subject is not administered a further pharmaceutical composition comprising SC-islet cells within 10 years after receiving the first pharmaceutical composition comprising SC-islet cells.

[0114]

[0128] In some embodiments, the subject is administered a first pharmaceutical composition comprising SC-islet cells, and the subject is not administered an additional pharmaceutical composition comprising SC-islet cells. In some embodiments, the subject is administered one or more pharmaceutical compositions comprising SC-islet cells in one surgical procedure (e.g., to implant one or more devices containing SC-islet cells), and the subject is not administered any additional pharmaceutical composition comprising SC-islet cells. In some embodiments, the subject is administered a first pharmaceutical composition comprising SC-islet cells, and the subject is administered at least a second pharmaceutical composition comprising SC-islet cells. In some embodiments, the subject is administered one or more pharmaceutical compositions comprising SC-islet cells in a first surgical procedure (e.g., to implant one or more devices containing SC-islet cells), and the subject is further administered one or more pharmaceutical compositions comprising SC-islet cells in a second surgical procedure (e.g., to implant one or more devices containing SC-islet cells). In some embodiments, the subject is administered a second pharmaceutical composition comprising SC-islet cells, but is not administered a third pharmaceutical composition comprising SC-islet cells for one year. In some embodiments, the subject is administered one or more pharmaceutical compositions comprising SC-islet cells in a first surgical procedure (e.g., to implant one or more devices containing SC-islet cells), and the subject is further administered one or more pharmaceutical compositions comprising SC-islet cells in a second surgical procedure (e.g., to implant one or more devices containing SC-islet cells), but the subject is not further administered any pharmaceutical composition comprising SC-islet cells for one year. In some embodiments, the subject is administered about 3.5×10 8 ~Approx. 4.5×10 8 SC-islet cells, approximately 3.9 x 10 8 ~Approx. 4.1×10 8 SC-islet cells, or approximately 4.0 x 10 8 10 SC-islet cells, and the subject receives at a later time point approximately 3.5×10 8 SC-islet cells, approximately 3.9 x 10 8 ~Approx. 4.1×10 8 SC-islet cells, or approximately 4.0 x 108 In some embodiments, the subject is administered a second pharmaceutical composition comprising SC- islet cells. In some embodiments, the second pharmaceutical composition is administered to the subject at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 15 months, or 3-12, 3-10, 3-9, 3-7, 3-5, 5-12, 8-10, 7-12, 9-15, or 9-12 months after the subject is administered the first pharmaceutical composition.

[0115]

[0129] In some embodiments, the subject receives about 3.5×10 8 ~Approx. 8.5×10 8 SC-islet cells, approximately 3.5 x 10 8 ~Approx. 4.5×10 8 SC-islet cells, approximately 3.9 x 10 8 ~Approx. 4.1×10 8 SC-islet cells, 7.5 x 10 8 ~Approx. 8.5×10 8 SC-islet cells, approximately 7.9 x 10 8 ~Approx. 8.1×10 8 SC-islet cells, approximately 4.0 x 10 8 10 SC-islet cells, or approximately 8.0 x 10 8 The subject is administered a first pharmaceutical composition comprising about 3.5×10 SC-islet cells. 8 ~Approx. 8.5×10 8 SC-islet cells, approximately 3.5 x 10 8 ~Approx. 4.5×10 8 SC-islet cells, approximately 3.9 x 10 8 ~Approx. 4.1×10 8 SC-islet cells, 7.5 x 10 8 ~Approx. 8.5×10 8 SC-islet cells, approximately 7.9 x 10 8 ~Approx. 8.1×10 8 SC-islet cells, approximately 4.0 x 10 8 10 SC-islet cells, or approximately 8.0 x 10 8In some embodiments, the second pharmaceutical composition is administered at least 3, 4, 5, 6, 7, 8, 9, or 10 years, or 3-20, 10-20, 15-20, 5-15, 3-5, 5-8, 8-10, 10-12, 12-15, or 20-25 years after the first pharmaceutical composition is administered to the subject.

[0116] Pharmaceutical Compositions

[0130] In some aspects, provided herein is a pharmaceutical composition comprising a cell population in liquid suspension. In some cases, the pharmaceutical composition disclosed herein is formulated for injection. In some cases, the cell population comprises non-native cells expressing C-peptide and ISL, e.g., SC-β cells. In some cases, the cell population also comprises non-native cells expressing glucagon but not somatostatin, e.g., SC-α cells. In some cases, the cell population also comprises non-native cells expressing somatostatin but not glucagon, e.g., SC-δ cells. In some cases, the cell population also comprises non-native cells expressing VMAT1, e.g., SC-EC cells. In some cases, the cellular composition of the pharmaceutical composition disclosed herein is similar to natural pancreatic islets. The non-native pancreatic cells in the pharmaceutical composition disclosed herein can be obtained by an in vitro differentiation process according to the differentiation method according to some embodiments of the present disclosure.

[0117]

[0131] The pharmaceutical composition can be used to treat, prevent, or stabilize diabetes. For example, somatic or stem cells can be obtained from an individual in need of treatment or from a healthy individual and reprogrammed into stem cell-derived beta cells by the methods of the present disclosure. In one embodiment of the present disclosure, stem cell-derived beta cells, and in some cases other stem cell-derived pancreatic cells (e.g., SC-α cells, SC-δ cells, SC-EC cells), are formulated according to the present disclosure and administered to an individual to treat a condition. In another embodiment, the stem cells are cultured under conditions suitable for differentiation into beta cells prior to introduction into the individual and can be used to replace or support the normal function of diseased or damaged tissues. A major advantage of the present disclosure is that it provides an essentially limitless supply of patient-specific human beta cells, or matched stem cell-derived beta cells from healthy individuals suitable for transplantation. The use of autologous and / or compatible cells in cell therapy offers a greater advantage over the use of non-autologous cells, which are prone to immune rejection. In contrast, autologous cells are less likely to induce significant immune responses.

[0118]

[0132] In some embodiments, the pharmaceutical compositions disclosed herein comprise about 1×10 8 ~ approx. 12 x 10 cells 8 pieces (e.g., 3×10 8 ~8.5×10 8 In some embodiments, the pharmaceutical composition comprises at least about 1 x 10 cells. 8 cells, e.g., at least about 1.5 x 10 8 , at least about 2 × 10 8 , at least about 2.5 × 10 8 , at least about 3 × 10 8 , at least about 3.5 × 10 8 , at least about 4 × 10 8 , at least about 4.5 × 10 8 , at least about 5 × 10 8 , at least about 5.5 × 10 8 , at least about 6 × 10 8 , at least about 6.5 × 10 8 , or at least about 7 × 10 8In some cases, the pharmaceutical compositions disclosed herein comprise a population of at most about 8×10 cells. 8 , at most about 7 × 10 8 , at most about 6.5 × 10 8 , at most about 6 × 10 8 , at most about 5.5 × 10 8 , at most about 5 × 10 8 , at most about 4.5 × 10 8 , at most about 4 × 10 8 , at most about 3.5 × 10 8 , at most about 3 × 10 8 , at most about 2.5 × 10 8 , or at most about 2 × 10 8 In some embodiments, the cell population in the pharmaceutical composition comprises about 1 x 10 8 ~Approx. 8×10 8 , about 1×10 8 ~Approx. 7×10 8 , about 1×10 8 ~about 6×10 8 , about 1×10 8 ~Approx. 5×10 8 , about 1×10 8 ~Approx. 4×10 8 , about 1×10 8 ~Approx. 3×10 8 , about 1×10 8 ~about 2×10 8 , about 1.5×10 8 ~Approx. 6.5×10 8 , about 2×10 8 ~Approx. 7×10 8 , about 2×10 8 ~about 6×10 8 , about 2×10 8 ~Approx. 5×10 8 , about 2×10 8 ~Approx. 4×10 8 , about 2×10 8 ~Approx. 3×10 8 , about 2.5×10 8 ~Approx. 5.5×10 8 , about 3×10 8 ~Approx. 7×10 8 , about 3×10 8 ~about 6×10 8 , about 3×10 8 ~Approx. 5×108 , about 3×10 8 ~Approx. 4×10 8 , about 3.5×10 8 ~Approx. 4.5×10 8 , about 3.8×10 8 ~Approx. 4.2×10 8 , about 7.5×10 8 ~About 10×10 8 , or about 8 × 10 8 ~Approx. 12×10 8 In some cases, the cell population in the pharmaceutical composition comprises about 3.5×10 cells. 8 ~Approx. 4.5×10 8 In some embodiments, the cell population in the pharmaceutical composition comprises about 3.5×10 cells. 8 ~Approx. 8.5×10 8 In some embodiments, the cell population in the pharmaceutical composition comprises about 3.9 x 10 cells. 8 ~Approx. 4.1×10 8 In some embodiments, the cell population in the pharmaceutical composition comprises about 7.9 x 10 cells. 8 ~Approx. 8.1×10 8 Contains cells.

[0119]

[0133] In some embodiments, to formulate a dose, the cell population suspension is first counted as dissociated single cells using an optical method that detects individual cells by phase contrast. After counting the cells in suspension, the pelleted stock of cells can be adjusted to the appropriate volume and formulated into a solution (e.g., HyopThermasol) to the desired concentration (e.g., 2 million cells per ml for the total volume). For example, if the target dose is 430e6 cells, 215 mL of HyopThermasol can be added.

[0120]

[0134] In one embodiment, two 0.5 mL samples are taken from the preformulated and harvested drug substance for cell count and viability to achieve the target dose. The total weight of the pooled cell suspension is calculated from the preformulated drug substance once placed in the injection medium, and the total volume of the cell suspension is calculated with the starting mass of the tread tube. For material release, the cells can be measured for cell count and viability with a hemocytometer. Trypan exclusion method combined with a disposable Neubauer hemocytometer can also be utilized to assess the cell density of the pooled drug substance suspension. This method is suitable for accurately counting cells in single cell suspensions within a concentration range of 0.4e6 to 2.5e6. Dosage can be assessed with a manual cell counting process that relies on the exclusion method (trypan blue) after TrypLE enzyme dissociation. When stained, live cells do not transmit trypan blue and retain a white appearance, while dead cells transmit the dye and turn blue. This method provides reportable values ​​for cell count (total cells / mL or viable cells / mL) and viability of the pooled cell suspension. In some embodiments, cell counts are assessed in two biological replicates, with three technical replicates per biological replicate, and two operators perform the counts. Final cell concentration can be determined in the following manner: cells / mL = average manual count per quadrant x final dilution factor x 10e4. In some embodiments, for each count to be valid, each technical replicate ranges from 80-500 cells and is within 35% of the average of the technical replicates counted for each biological replicate. In some embodiments, the % difference between biological replicates must be 25% or less to calculate reportable results for cell concentration and viability. In some embodiments, cell concentration (viable cells / mL) may be reported, and viable cells may be calculated by multiplying the volume measured in the total weight assessment of the pooled cell suspension by the cell concentration disclosed herein.

[0121] Total viable cells = pooled drug substance suspension volume x viable cell concentration cell composition

[0135] In some cases, the SC-β cells of the present disclosure share many characteristics of β cells that are important for normal β cell function. In some embodiments, the SC-β cells exhibit a glucose stimulated insulin secretion (GSIS) response in vitro. In some embodiments, the SC-β cells exhibit a GSIS response in vivo. In some embodiments, the SC-β cells exhibit a GSIS response in vitro and in vivo. In some embodiments, the GSIS response resembles the GSIS response of endogenous mature pancreatic β cells. In some embodiments, the SC-β cells exhibit a GSIS response to at least one glucose challenge. In some embodiments, the SC-β cells exhibit a GSIS response to at least two consecutive glucose challenges. In some embodiments, the SC-β cells exhibit a GSIS response to at least three consecutive glucose challenges. In some embodiments, the GSIS response resembles the GSIS response of endogenous human pancreatic islets to multiple glucose challenges. In some embodiments, the GSIS response is observed immediately after the cells are transplanted into a human or animal. In some embodiments, the GSIS response is observed within about 24 hours after the cells are transplanted into a human or animal. In some embodiments, the GSIS response is observed within about one week after the cells are implanted into a human or animal. In some embodiments, the GSIS response is observed within about two weeks after the cells are implanted into a human or animal. In some embodiments, the stimulation index of the cells, 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 endogenous mature pancreatic beta cells. In some embodiments, the SC-beta cells exhibit a stimulation index of greater than 1. In some embodiments, the SC-beta cells exhibit a stimulation index of 1 or greater. In some embodiments, the SC-beta cells exhibit a stimulation index of 1.1 or greater. In some embodiments, the SC-beta cells exhibit a stimulation index of 1.1 or greater. In some embodiments, the SC-beta cells exhibit a stimulation index of greater than 2. In some embodiments, the SC-beta cells exhibit a stimulation index of 1 or greater.In some embodiments, the SC-β cells exhibit 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 more.

[0122]

[0136] In some cases, the cell composition in the pharmaceutical composition disclosed herein has at least about 30% cells that express C-peptide and do not express VMAT1 as measured by flow cytometry. In some cases, the cell composition in the pharmaceutical composition disclosed herein has at least about 35% cells that express C-peptide and do not express VMAT1 as measured by flow cytometry. In some cases, the expression of C-peptide and the absence of VMAT1 in a cell in the cell composition suggests that the cell is an SC-β cell. In some cases, the cell composition has at least about 30%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% cells that express C-peptide and do not express VMAT1 as measured by flow cytometry. In some cases, the cell composition has about 30%, 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%, or 60% cells that express C-peptide and do not express VMAT1 as measured by flow cytometry. In some cases, the cell composition has about 30% to about 60%, about 35% to about 55%, or about 40% to about 50% cells that express C-peptide and do not express VMAT1 as measured by flow cytometry.

[0123]

[0137] In some cases, the cell composition in the pharmaceutical composition disclosed herein has at most about 35% cells expressing VMAT1 when measured by flow cytometry. In some cases, the cell composition in the pharmaceutical composition disclosed herein has at most about 35% cells expressing VMAT1 and not expressing C-peptide when measured by flow cytometry. In some cases, the expression of VMAT1 and the absence of C-peptide in a cell of the cell composition suggests that the cell is an SC-EC cell. In some cases, the cell composition has at most about 45%, 40%, 35%, 32%, 31%, 30%, 28%, 25%, 24%, 23%, 22%, 21%, or 20% cells expressing VMAT1 and not expressing C-peptide when measured by flow cytometry. In some cases, the cell composition has at most about 35%, 32%, 31%, 30%, 28%, 25%, 24%, 23%, 22%, 21%, or 20% cells that express VMAT1 and do not express C-peptide, as measured by flow cytometry. In some cases, the cell composition has at most about 35%, 32%, 31%, 30%, 28%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, or 15% cells that express VMAT1 and do not express C-peptide, as measured by flow cytometry. In some cases, the cell composition has at most about 15% to about 30%, at most about 16% to 25%, at most about 17% to about 22%, or at most about 18% to about 20% cells that express VMAT1 and do not express C-peptide, as measured by flow cytometry. In some cases, the cell composition has 5-25%, 5-15%, 10-25%, 10-15%, or 15-25% cells that express VMAT1 and do not express C-peptide, as measured by flow cytometry.

[0124]

[0138] In some cases, the cell composition comprises at least about 3% cells that express glucagon, as measured by flow cytometry. In some cases, the cell composition comprises at least about 5% cells that express glucagon, as measured by flow cytometry. In some cases, the cell composition comprises at least about 10% cells that express glucagon, as measured by flow cytometry. In some cases, the cell composition comprises at least about 20% cells that express glucagon, as measured by flow cytometry. In some cases, the cell composition comprises at least about 15% cells that express glucagon and do not express somatostatin, as measured by flow cytometry. In some cases, expression of glucagon and no expression of somatostatin in a cell of the cell composition indicates that the cell is an SC-α cell. In some cases, the cell composition comprises at least about 3%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, or 22% cells that express glucagon and do not express somatostatin as measured by flow cytometry. In some cases, the cell composition comprises at least about 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, or 22% cells that express glucagon and do not express somatostatin as measured by flow cytometry. In some cases, the cell composition comprises about 10% to about 30%, about 12% to about 25%, about 13% to about 22%, about 15% to about 20%, or about 16% to about 18% cells that express glucagon and do not express somatostatin as measured by flow cytometry. In some cases, the cell composition comprises about 3%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, or 22% cells that express glucagon and do not express somatostatin as measured by flow cytometry.

[0125]

[0139] In some cases, the cell composition comprises at least about 1% cells that express somatostatin and do not express glucagon, as measured by flow cytometry. In some cases, the cell composition comprises at least about 2% cells that express somatostatin and do not express glucagon, as measured by flow cytometry. In some cases, the cell composition comprises at least about 3% cells that express somatostatin and do not express glucagon, as measured by flow cytometry. In some cases, the cell composition comprises at least about 4% cells that express somatostatin and do not express glucagon, as measured by flow cytometry. In some cases, expression of glucagon and no expression of somatostatin in a cell of the cell composition suggests that the cell is an SC-δ cell. In some cases, the cell composition comprises at least about 2%, 3%, 4%, 5%, 6%, 7%, or 8% cells that express somatostatin and do not express glucagon, as measured by flow cytometry. In some cases, the cell composition comprises about 1% to about 9%, about 2% to about 8%, about 3% to about 7%, or about 4% to about 6% cells that express somatostatin and do not express glucagon as measured by flow cytometry. In some cases, the cell composition comprises about 1%, 2%, 3%, 4%, 5%, 6%, 7%, or 8% cells that express somatostatin and do not express glucagon as measured by flow cytometry.

[0126]

[0140] In some cases, the cell composition has at least about 35% cells that express C-peptide and do not express VMAT1, at most about 50% cells that express VMAT1, at least about 4% cells that express glucagon and do not express somatostatin, and at least about 1% cells that express somatostatin and do not express glucagon, as measured by flow cytometry. In some cases, the cell composition has at least about 35% cells that express C-peptide and do not express VMAT1, at most about 30% cells that express VMAT1, and at least about 20% cells that express glucagon, as measured by flow cytometry. In some cases, the cell composition has at least about 35% cells that express C-peptide and do not express VMAT1, at most about 30% cells that express VMAT1, at least about 20% cells that express glucagon, and at least 4% cells that express somatostatin and do not express glucagon, as measured by flow cytometry.

[0127]

[0141] In some cases, the cell compositions provided herein comprise (a) at least about 35% of cells that express C-peptide and do not express VMAT1; and (b) at least about 10% of cells that express somatostatin, as measured by flow cytometry. In some cases, at least about 15% of cells express somatostatin in the cell composition, as measured by flow cytometry.

[0128]

[0142] In some cases, provided herein are compositions that include a cell population, wherein (a) 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 30-40%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-90%, 50-80%, 50-70%, 50-60%, 60-90%, 60-80%, 60-70%, 70-90%, 70-80%, or 80-90% of the cells in the cell population express C-peptide and ISL 1 but not VMAT1; (b) 3–40%, 3–35%, 3–30%, 3–25%, 3–20%, 3–15%, 3–10%, 5–40%, 5–35%, 5–30%, 5–25%, 5–20%, 5–15%, 5–10%, 10–40%, 10–35%, 10–30%, 10–25%, 10–20%, 10–15%, 15–40%, 15–35%, 15–30%, 15–25%, 15–20%, 20–40%, 20–35%, 20-30%, 20-25%, 25-40%, 25-35%, 25-30%, 30-40%, 30-35% or 35-40% of the cells express glucagon but not somatostatin; and / or (c) 1-20%, 1-15%, 1-12%, 1-10%, 1-8%, 1-5%, 2-20%, 2-15%, 2-12%, 2-10%, 2-8%, 2-5%, 3-20%, 3-15%, 3-12%, 3-10%, 3-8%, 3-5% of the cells in the cell population express glucagon but not somatostatin; , 4-20%, 4-15%, 4-12%, 4-10%, 4-8%, 4-5%, 5-20%, 5-15%, 5-12%, 5-10%, 5-8%, 7-20%, 7-15%, 7-12%, 7-10%, 9-20%, 9-15%, 9-12%, 8-10%, 8-12%, 8-15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15% or 15-20% of cells express somatostatin but do not express glucagon.

[0129]

[0143] In some cases, provided herein are compositions that include a cell population, wherein (a) 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 30-40%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-90%, 50-80%, 50-70%, 50-60%, 60-90%, 60-80%, 60-70%, 70-90%, 70-80%, or 80-90% of the cells in the cell population express C-peptide and ISL 1 but not VMAT1; (b) 3–40%, 3–35%, 3–30%, 3–25%, 3–20%, 3–15%, 3–10%, 5–40%, 5–35%, 5–30%, 5–25%, 5–20%, 5–15%, 5–10%, 10–40%, 10–35%, 10–30%, 10–25%, 10–20%, 10–15%, 15–40%, 15–35%, 15–30%, 15–25%, 15–20%, 20–40%, 20–35%, 20-30%, 20-25%, 25-40%, 25-35%, 25-30%, 30-40%, 30-35% or 35-40% of the cells express glucagon but not somatostatin; and / or (c) 1-20%, 1-15%, 1-12%, 1-10%, 1-8%, 1-5%, 2-20%, 2-15%, 2-12%, 2-10%, 2-8%, 2-5%, 3-20%, 3-15%, 3-12%, 3-10%, 3-8%, 3-5% of the cells in the cell population express glucagon but not somatostatin; , 4-20%, 4-15%, 4-12%, 4-10%, 4-8%, 4-5%, 5-20%, 5-15%, 5-12%, 5-10%, 5-8%, 7-20%, 7-15%, 7-12%, 7-10%, 9-20%, 9-15%, 9-12%, 8-10%, 8-12%, 8-15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15% or 15-20% of cells express somatostatin but do not express glucagon.

[0130]

[0144] In some cases, in the cell populations provided herein, 35-60% of the cells express C-peptide and ISL1 but do not express VMAT1; 4-25% of the cells express glucagon but do not express somatostatin; 1-10% of the cells express somatostatin but do not express glucagon. In some cases, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 18%, less than 15%, less than 12%, or less than 10% of the cells in the cell populations provided herein express VMAT1 but do not express C-peptide.

[0131]

[0145] In some cases, in the cell populations provided herein, 40-60% of the cells express C-peptide and ISL1 but do not express VMAT1; 10-25% of the cells express glucagon but do not express somatostatin; 4-10% of the cells express somatostatin but do not express glucagon. In some cases, less than 25%, less than 20%, less than 18%, less than 15%, less than 12%, or less than 10% of the cells in the cell populations provided herein express VMAT1 but do not express C-peptide.

[0132]

[0146] In some embodiments, any of the pharmaceutical compositions disclosed herein contain 50% or more, 40% or more, 30% or more, or 20% or more NKX6.1 + / ISL1 + In some embodiments, 30% or more of the cells in the composition are NKX6.1 positive, ISL1 positive cells, 25% or more of the cells in the composition are NKX6.1 negative, ISL1 positive cells, less than 12% of the cells in the composition are NKX6.1 negative, ISL1 negative cells, or 9-25% of the cells in the composition are NKX6.1 positive, ISL1 negative cells (e.g., as determined by flow cytometry). In some embodiments, 40%, 35%, 30%, 26%, 25%, or 20% or more of the cells in the pharmaceutical composition are NKX6.1 positive, ISL1 negative cells. - / ISL1 +In some embodiments, 26% or more of the cells in the pharmaceutical composition are NKX6.1 cells (e.g., as determined by flow cytometry). - / ISL1 + In some embodiments, 5-25%, 5-40%, 5-35%, or 8-20% of the cells in the pharmaceutical composition are NKX6.1 cells (e.g., as determined by flow cytometry). - / ISL1 + In some embodiments, no more than 50%, no more than 45%, no more than 40%, no more than 35%, no more than 30%, or no more than 25% of the cells in the pharmaceutical composition are NKX6.1 cells (e.g., as determined by flow cytometry). + / ISL1 - In some embodiments, 50% or less of the cells in the pharmaceutical composition are NKX6.1 cells (e.g., as determined by flow cytometry). + / ISL1 - cells (eg, as determined by flow cytometry).

[0133]

[0147] In some embodiments, less than 12% (e.g., about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, or less) of the cells in the population are NKX6.1-negative, ISL1-negative cells. In some embodiments, less than 10%, less than 8%, less than 6%, less than 4%, or between 1% and 11%, 2% and 10%, 2% and 12%, 4% and 12%, 6% and 12%, 8% and 12%, 2% and 8%, 4% and 8%, 3% and 6%, or 3% and 5% of the cells in the population are NKX6.1-negative, ISL1-negative cells. In some embodiments, 2%-12%, 4%-12%, 6%-12%, 8%-12%, 2%-8%, 4%-8%, 3%-6%, or 3%-5% of the cells in the population are NKX6.1-negative, ISL1-negative cells.

[0134]

[0148] In some embodiments, at least 15% (e.g., about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60% or more) of the cells in the population are NKX6.1 negative, ISL1 positive cells. In some embodiments, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, or 15%-60%, 15%-45%, 15%-30%, 30%-60%, 30%-45%, 45%-60% of the cells in the population are NKX6.1 negative, ISL1 positive cells. In some embodiments, 20%-60%, 20%-50%, 20%-40%, 20%-30%, 30%-60%, 30%-50%, 30%-40%, 40%-60%, 40%-50%, or 50%-60% of the cells in the population are NKX6.1 negative, ISL1 positive cells.

[0135]

[0149] In some embodiments, at least 15% (e.g., 20%-60%, 20%-50%, 20%-40%, 20%-30%, 30%-60%, 30%-50%, 30%-40%, 40%-60%, 40%-50%, or 50%-60%) of the cells in the population are NKX6.1-negative, ISL1-positive cells and less than 12% (e.g., 2%-12%, 4%-12%, 6%-12%, 8%-12%, 2%-8%, 4%-8%, 3%-6%, or 3%-5%) of the cells are NKX6.1-negative, ISL1-negative cells.

[0136]

[0150] In some embodiments, at least 60%, at least 65%, at least 70%, at least 73%, at least 74%, at least 75%, at least 80%, at least 85%, at least 90%, about 85-95%, or about 90-95% of the cells in the population are ISL1 positive cells. In some embodiments, 50-90%, 50-85%, 50-80%, 50-75%, 50-70%, 50-60%, 60-90%, 60-85%, 60-80%, 60-75%, 60-70%, 65-90%, 65-85%, 65-80%, 65-75%, 65-70%, 70-90%, 70-85%, 70-80%, 70-75%, 75-90%, 75-85%, 75-80%, 80-90%, 80-85%, or 85-90% of the cells in the population are ISL1 positive cells. In some embodiments, at least 74%, at least 75%, at least 80%, at least 85%, at least 90%, about 85-95%, or about 90-95% of the cells in the population are ISL1 positive cells. In some embodiments, about 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%, or about 99% of the cells in the population are ISL1 positive cells.

[0137]

[0151] In some embodiments, the in vitro differentiated cell populations described herein contain more NKX6.1 negative, ISL1 positive cells than NKX6.1 positive, ISL1 positive cells. In some embodiments, at least 40% of the cells in the population are NKX6.1 negative, ISL1 positive cells. In some embodiments, at least 45%, at least 50%, about 40-50%, about 45-55%, or about 50-55% of the cells in the population are NKX6.1 negative, ISL1 positive cells. In some embodiments, about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, or about 55% of the cells in the population are NKX6.1 negative, ISL1 positive cells.

[0138]

[0152] In some embodiments, at least 20% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 50%, at least 60% or more) of the ISL1 positive cells are NKX6.1 negative. In some embodiments, about 20%-60%, 20%-50%, 20%-40%, 20%-30%, 30%-60%, 30%-50%, 30%-40%, 40%-60%, 40%-50%, or 50%-60% of the ISL1 positive cells are NKX6.1 negative. In some embodiments, about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or more of the ISL1 positive cells are NKX6.1 negative.

[0139]

[0153] In some embodiments, at least 20% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 50%, at least 60% or more) of the cells in the composition are ISL1 positive and NKX6.1 positive. In some embodiments, about 20%-60%, 20%-50%, 20%-40%, 20%-30%, 30%-60%, 30%-50%, 30%-40%, 40%-60%, 40%-50%, or 50%-60% of the cells in the composition are ISL1 positive and NKX6.1 positive. In some embodiments, about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or more of the cells in the composition are ISL1 positive and NKX6.1 positive.

[0140]

[0154] In some embodiments, at least 20% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 50%, at least 60% or more) of the cells in the composition are ISL1 positive and NKX6.1 negative. In some embodiments, about 20%-60%, 20%-50%, 20%-40%, 20%-30%, 30%-60%, 30%-50%, 30%-40%, 40%-60%, 40%-50%, or 50%-60% of the cells in the composition are ISL1 positive and NKX6.1 negative. In some embodiments, about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or more of the cells in the composition are ISL1 positive and NKX6.1 negative.

[0141]

[0155] In some embodiments, the in vitro differentiated cell populations described herein comprise up to 20% (e.g., up to 20%, up to 30%, up to 40%, or up to 50%) of NXK6.1-positive, ISL1-positive cells. In some embodiments, the in vitro differentiated cell populations described herein comprise about 20%-50%, 20%-40%, 20%-30%, 30%-50%, 30%-40%, or 40%-50% of NXK6.1-positive, ISL1-positive cells. In some embodiments, the in vitro differentiated cell populations described herein comprise about 20%-50%, 20%-40%, 20%-30%, 30%-50%, 30%-40%, or 40%-50% of NXK6.1-positive, ISL1-positive cells.

[0142]

[0156] In some embodiments, the NKX6.1 positive, ISL1 positive cells also express PDX1.

[0157] In some embodiments, the disclosure provides a composition comprising a plurality of cells (e.g., a cluster of cells or a composition comprising a plurality of clusters of cells); 30-60%, 30-55%, 30-50%, 30-45%, 30-40%, 30-35%, 35-60%, 35-55%, 35-50%, 35-45%, 35-40%, 40-60%, 40-55%, 40-50%, 40-45%, 45-60%, 45-55%, 45-50%, 50-60%, or 50-55% of the cells in the composition are NKX6.1-positive, ISL1-positive cells; 20-50%, 20-45%, 20-40%, 20-35%, 20-30%, 20-25%, 20-35%, 20-40%, 20-55%, 20-60%, or 20-55% of the cells in the composition are NKX6.1-positive, ISL1-positive cells; 5-50%, 25-45%, 25-40%, 25-35%, 25-30%, 30-50%, 30-45%, 30-40%, 30-35%, 35-50%, 35-35%, 35-40%, 40-50%, 40-45%, or 45-50% of the cells are NKX6.1-negative, ISL1-positive cells; 1-12% of the composition, 1-10%, 1-8%, 1-6%, 1-4%, 3-5%, 1-2%, 2-12%, 2-10%, 2-8%, 2-6%, 2-4%, 4-12%, 4-10%, 4-8%, 4-6%, 6-12%, 6-10%, 6-8%, 8-12%, 8-10%, or 10-12% of the cells are NKX6.1-negative, ISL1-negative cells. In some embodiments, the present disclosure provides a composition comprising a plurality of cells (e.g., a cluster of cells or a composition comprising a cluster of a plurality of cells); 35-50% of the cells in the composition are NKX6.1-positive, ISL1-positive cells; 30-45% of the cells in the composition are NKX6.1-negative, ISL1-positive cells; and 2-12% of the cells in the composition are NKX6.1-negative, ISL1-negative cells. In some embodiments, 3-25%, 3-20%, 3-15%, 3-10%, 3-5%, 5-25%, 5-20%, 5-15%, 5-10%, 10-25%, 10-20%, 10-15%, 15-25%, 15-20% or 20-25% of the cells in the composition are NKX6.1-positive, ISL1-negative cells.

[0143]

[0158] In some embodiments, the disclosure provides a composition comprising a plurality of cells (e.g., a cluster of cells or a composition comprising a plurality of clusters of cells); at least 30% of the cells in the composition are NKX6.1 positive, ISL1 positive cells; at least 25% of the cells in the composition are NKX6.1 negative, ISL1 positive cells; or between 9 and 25% of the cells in the composition are NKX6.1 positive, ISL1 negative cells. In some embodiments, the disclosure provides a composition comprising a plurality of cells (e.g., a cluster of cells or a composition comprising a plurality of clusters of cells); 30-60%, 30-55%, 30-50%, 30-45%, 30-40%, 30-35%, 35-60%, 35-55%, 35-50%, 35-45%, 35-40%, 40-60%, 40-55%, 40-50%, 40-45%, 45-60%, 45-55%, 45-50%, 50-60%, or 50-55% of the cells in the composition are NKX6.1-positive, ISL1-positive cells; 20-50%, 20-45%, 20-40%, 20-35%, 20- 30%, 20-25%, 25-50%, 25-45%, 25-40%, 25-35%, 25-30%, 30-50%, 30-45%, 30-40%, 30-35%, 35-50%, 35-35%, 35-40%, 40-50%, 40-45%, or 45-50% of cells are NKX6.1 negative and ISL1 positive cells; 9-30%, 9-25%, 9-20%, 9-15%, 9-12%, 12-30%, 12-25%, 12-20%, 12-15%, 15-30%, 15-25%, 15-20%, 20-30%, 20-25%, or 25-30% of the cells in the composition are NKX6.1 positive, ISL negative cells. In some embodiments, 1-12%, 1-10%, 1-8%, 1-6%, 1-4%, 3-5%, 1-2%, 2-12%, 2-10%, 2-8%, 2-6%, 2-4%, 4-12%, 4-10%, 4-8%, 4-6%, 6-12%, 6-10%, 6-8%, 8-12%, 8-10%, or 10-12% of the cells in the composition are NKX6.1 negative, ISL1 negative cells.In some embodiments, the disclosure provides a composition comprising a plurality of cells (e.g., a cluster of cells or a composition comprising a plurality of clusters of cells); 35-50% of the cells in the composition are NKX6.1 positive, ISL1 positive cells; 30-45% of the cells in the composition are NKX6.1 negative, ISL1 positive cells; and 9-25% of the cells in the composition are NKX6.1 positive, ISL1 negative cells.

[0144]

[0159] In some embodiments, less than 12% (e.g., about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, or less) of the cells in the composition are NKX6.1-negative, ISL1-negative cells. In some embodiments, less than 10%, less than 8%, less than 6%, less than 4%, 1%-11%, 2%-10%, 2%-12%, 4%-12%, 6%-12%, 8%-12%, 2%-8%, 4%-8%, 3%-6%, or 3%-5% of the cells in the composition are NKX6.1-negative, ISL1-negative cells. In some embodiments, between 2% and 12%, between 4% and 12%, between 6% and 12%, between 8% and 12%, between 2% and 8%, between 4% and 8%, between 3% and 6%, or between 3% and 5% of the cells in the population are NKX6.1-negative, ISL1-negative cells.

[0145]

[0160] In some embodiments, at least 15% (e.g., about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60% or more) of the cells in the composition are NKX6.1 negative, ISL1 positive cells. In some embodiments, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, 15%-60%, 15%-45%, 15%-30%, 30%-60%, 30%-45%, 45%-60% of the cells in the composition are NKX6.1 negative, ISL1 positive cells. In some embodiments, 20%-60%, 20%-50%, 20%-40%, 20%-30%, 30%-60%, 30%-50%, 30%-40%, 40%-60%, 40%-50%, or 50%-60% of the cells in the composition are NKX6.1-negative, ISL1-positive cells.

[0146]

[0161] In some embodiments, at least 15% (e.g., 20%-60%, 20%-50%, 20%-40%, 20%-30%, 30%-60%, 30%-50%, 30%-40%, 40%-60%, 40%-50%, or 50%-60%) of the cells in the composition are NKX6.1-negative, ISL1-positive cells, and less than 12% (e.g., 2%-12%, 4%-12%, 6%-12%, 8%-12%, 2%-8%, 4%-8%, 3%-6%, or 3%-5%) of the cells in the composition are NKX6.1-negative, ISL1-negative cells.

[0147]

[0162] In some embodiments, at least 60%, at least 65%, at least 70%, at least 73%, at least 74%, at least 75%, at least 80%, at least 85%, at least 90%, about 85-95%, or about 90-95% of the cells in the composition are ISL1 positive cells. In some embodiments, 50-90%, 50-85%, 50-80%, 50-75%, 50-70%, 50-60%, 60-90%, 60-85%, 60-80%, 60-75%, 60-70%, 65-90%, 65-85%, 65-80%, 65-75%, 65-70%, 70-90%, 70-85%, 70-80%, 70-75%, 75-90%, 75-85%, 75-80%, 80-90%, 80-85%, or 85-90% of the cells in the composition are ISL1-positive cells. In some embodiments, at least 74%, at least 75%, at least 80%, at least 85%, at least 90%, about 85-95%, or about 90-95% of the cells in the composition are ISL1 positive cells. In some embodiments, about 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%, or about 99% of the cells in the composition are ISL1 positive cells.

[0148]

[0163] In some embodiments, the composition comprises more NKX6.1 positive, ISL1 positive cells than NKX6.1 negative, ISL1 positive cells. In some embodiments, at least 40% of the cells in the composition are NKX6.1 negative, ISL1 positive cells. In some embodiments, at least 45%, at least 50%, about 40-50%, about 45-55%, or about 50-55% of the cells in the composition are NKX6.1 negative, ISL1 positive cells. In some embodiments, about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, or about 55% of the cells in the composition are NKX6.1 negative, ISL1 positive cells.

[0149]

[0164] In some embodiments, at least 20% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 50%, at least 60% or more) of ISL1-positive cells are NKX6.1-negative. In some embodiments, about 20%-60%, 20%-50%, 20%-40%, 20%-30%, 30%-60%, 30%-50%, 30%-40%, 40%-60%, 40%-50%, or 50%-60% of ISL1-positive cells are NKX6.1-negative. In some embodiments, about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or more of ISL1-positive cells are NKX6.1-negative.

[0150]

[0165] In some embodiments, the composition comprises at least 20% (e.g., at least 20%, 30%, 40%, 50% or 60%) of NXK6.1-positive, ISL1-positive cells. In some embodiments, the composition comprises about 20%-50%, 20%-40%, 20%-30%, 30%-50%, 30%-40%, 40%-50%, 40%-60%, or 50%-60% of NXK6.1-positive, ISL1-positive cells. In some embodiments, the composition comprises about 20%-50%, 20%-40%, 20%-30%, 30%-50%, 30%-40%, or 40%-50% of NXK6.1-positive, ISL1-positive cells.

[0151]

[0166] In some embodiments, the composition comprises less than 25% (e.g., less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less) NKX6.1 positive, ISL1 negative cells. In some embodiments, the composition comprises about 2%-25%, 2%-20%, 2%-15%, 2%-10%, 2%-5%, 5%-25%, 5%-20%, 5%-15%, 5%-10%, 10%-25%, 10%-20%, 10%-15%, 15%-25%, 15%-20%, or 20%-25% NKX6.1 positive, ISL1 negative cells. In some embodiments, the composition comprises about 2%-10%, 2%-8%, 2%-6%, 2%-4%, 4%-10%, 4%-8%, 4%-6%, 6%-10%, 6%-8%, or 8%-10% NKX6.1 positive, ISL1 negative cells. In some embodiments, the composition comprises about 2%, 4%, 6%, 8%, or 10% NKX6.1 positive, ISL1 negative cells.

[0152]

[0167] In some embodiments, the composition comprises less than 10% SOX9 positive cells. In some embodiments, the composition comprises less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% SOX9 positive cells. In some embodiments, the composition comprises 0.1-10%, 0.1-7%, 0.1-3%, 0.1-1%, 0.5-10%, 0.5-7%, 0.5-3%, 0.5-1%, 1-10%, 1-5%, 1-3%, 3-10%, 3-5%, or 5-10% SOX9 positive cells.

[0153]

[0168] In some embodiments, the composition comprises less than 5% Ki67 positive cells. In some embodiments, the composition comprises less than 5%, less than 4%, less than 3%, less than 2% or less than 1% Ki67 positive cells. In some embodiments, the composition comprises 0.01-0.1%, 0.1-5%, 0.1-3%, 0.1-1%, 0.5-5%, 0.5-3%, 0.5-1%, 1-5%, 1-3%, or 1-2% Ki67 positive cells.

[0154]

[0169] In some embodiments, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cells in the composition are CHGA positive cells, in some embodiments, 80-100%, 85-100%, 90-100%, 90-99%, 90-98%, 95-99%, or 95-99% of the cells in the composition are CHGA positive cells.

[0155]

[0170] In some embodiments, the percentage of cells expressing the markers provided herein is measured by flow cytometry.Those skilled in the art know the typical method of testing whether a cell or a collection of cells is positive or negative for the expression of a specific gene marker (e.g., NKX6.1, ISL1, INS, GCG, somatostatin, chromogranin A, SOX9, C-peptide, Ki67) by flow cytometry.In some embodiments, a cell is considered to be positive for the expression of a specific gene (e.g., NKX6.1, ISL1, INS, GCG, somatostatin, chromogranin A, SOX9, C-peptide, Ki67) based on median fluorescence intensity (rMFI). As used herein, the term "rMFI" or median relative fluorescence intensity is the ratio of the fluorescence intensity measured using an antibody against a particular target (e.g., NKX6.1, ISL1, INS, GCG, somatostatin, chromogranin A, SOX9, C-peptide, Ki67) to the intensity obtained from a control antibody (isotype control). In some embodiments, anti-(human) NKX6.1, ISL1, INS, GCG, somatostatin, chromogranin A, or SOX9 antibodies are used. Examples of antibodies suitable for use in flow cytometry are any of the antibodies disclosed in Table 1. An example of a suitable flow cytometer is the Accuri 6 flow cytometer. In some embodiments, target-expressing cells (e.g., cells expressing NKX6.1 and / or ISL1) when tested exhibit a target relative median fluorescence intensity (rMFI) of at least 6, 6.5, 7, 8, 9, or 10 measured by flow cytometry. In another embodiment, the rMFI is 6.5-15, 6.5-14, 6.5-13, 6.5-12, or 6.5-10.

[0156] [Table 1]

[0157]

[0171] In some embodiments, the percentage of cells expressing the markers provided herein is measured by qRT-PCR.In some embodiments, the percentage of cells expressing the markers provided herein is measured by single-cell RNA sequencing analysis.Those skilled in the art know how to test whether a cell or a collection of cells is positive for the expression of a specific gene marker (e.g., NKX6.1, ISL1, INS, GCG, ARX, or ghrelin) by single-cell RNA sequencing analysis.

[0158]

[0172] In some embodiments, the in vitro differentiated cell populations described herein comprise less than 25% (e.g., less than 25%, less than 20%, less than 15%, less than 10%, less than 5%) of NKX6.1 positive, ISL1 negative cells. In some embodiments, the in vitro differentiated cell populations described herein comprise about 2%-25%, 2%-20%, 2%-15%, 2%-10%, 2%-5%, 5%-25%, 5%-20%, 5%-15%, 5%-10%, 10%-25%, 10%-20%, 10%-15%, 15%-25%, 15%-20%, or 20%-25% of NKX6.1 positive, ISL1 negative cells. In some embodiments, the in vitro differentiated cell populations described herein comprise about 2%-10%, 2%-8%, 2%-6%, 2%-4%, 4%-10%, 4%-8%, 4%-6%, 6%-10%, 6%-8%, or 8%-10% NKX6.1 positive, ISL1 negative cells. In some embodiments, the in vitro differentiated cell populations described herein comprise about 2%, 4%, 6%, 8%, or 10% NKX6.1 positive, ISL1 negative cells.

[0159]

[0173] In some embodiments, the present disclosure provides a pharmaceutical composition comprising NKX6.1 positive, ISL1 positive cells expressing lower levels of MAFA than NKX6.1 positive, ISL1 positive cells from the pancreas of a healthy adult control subject or a cadaver pancreas. In some embodiments, the pharmaceutical composition comprises NKX6.1 positive, ISL1 positive cells expressing higher levels of MAFB than NKX6.1 positive, ISL1 positive cells from the pancreas of a healthy adult control subject or a cadaver pancreas. In some embodiments, the pharmaceutical composition comprises NKX6.1 positive, ISL1 positive cells expressing higher levels of SIX2, HOPX, IAPP and / or UCN3 than NKX6.1 positive, ISL1 positive cells from the pancreas of a healthy adult control subject or a cadaver pancreas. In some embodiments, the pharmaceutical composition comprises NKX6.1 positive, ISL1 positive cells that do not express MAFA. In some embodiments, the pharmaceutical composition comprises NKX6.1 positive, ISL1 positive cells expressing MAFB. In some embodiments, the pharmaceutical composition comprises genetically modified cells (e.g., using gene editing techniques such as CRISPR). In some embodiments, the pharmaceutical composition comprises NKX6.1-positive, ISL1-positive cells that express lower levels of beta-2 microglobulin, CIITA, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLADR than NKX6.1-positive, ISL1-positive cells from healthy adult control subjects or cadaveric pancreases. In some embodiments, the pharmaceutical composition comprises NKX6.1 positive, ISL1 positive cells that express increased levels of CD47, PDL1, HLA-G, CD46, CD55, CD59 and CTLA compared to NKX6.1 positive, ISL1 positive cells from the pancreas of a healthy adult control subject or a cadaver pancreas. In some embodiments, any of the cell markers disclosed herein (e.g., NKX6.1, PDX1, MAFA, MAFB, SIX2, HOPX, IAPP and / or UCN3) are detected by flow cytometry.

[0160]

[0174] In some embodiments, the pharmaceutical composition is derived from stem cells in vitro. In some embodiments, the stem cells are genetically modified (e.g., using gene editing techniques such as CRISPR). In some embodiments, the stem cells have reduced expression of one or more of beta-2 microglobulin, CIITA, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLADR compared to non-genetically modified stem cells. In some embodiments, the stem cells have increased expression of CD47, PDL1, HLA-G, CD46, CD55, CD59, and CTLA compared to non-genetically modified stem cells. In some embodiments, any of the cell markers disclosed herein (e.g., NKX6.1, PDX1, MAFA, MAFB, SIX2, HOPX, IAPP, and / or UCN3) are detected by flow cytometry.

[0161]

[0175] In some cases, the cell population or cell cluster disclosed herein is an isolated cell population or cell cluster that has not been sorted, e.g., has not undergone a cell sorting process. In some embodiments, the cell cluster disclosed herein can refer to a cell cluster formed by the self-aggregation of cells cultured in a given environment, e.g., 3D suspension culture. Cell sorting as described herein can refer to a process of separating a group of cells from a plurality of cells depending on differences in cell size, shape (morphology), expression of surface proteins, expression of endogenous signal proteins, or combinations thereof. In some cases, cell sorting includes subjecting cells to flow cytometry. Flow cytometry can be a laser or impedance-based biophysical technique. In flow cytometry, cells can be suspended in a liquid stream and passed through an electronic detection device. In a type of flow cytometry, fluorescence-activated cell sorting (FACS), flow cytometry can be used to physically separate and thereby purify cells of interest based on one or more parameters of the optical properties of the cells (e.g., emission wavelength upon laser excitation). As described herein, an unsorted cell cluster may be a cell cluster formed by a plurality of cells that have not undergone an active cell sorting process, such as flow cytometry. In some cases, the flow cytometry discussed herein may be based on one or more signal peptides expressed in the cells. For example, the cell cluster may include cells that express a signal peptide (e.g., a fluorescent protein, such as green fluorescent protein (GFP) or tdTomato). In some cases, the signal peptide is expressed as an indicator of insulin expression in the cells. For example, the cell cluster may include cells that carry an exogenous nucleic acid sequence that encodes GFP under the control of an insulin promoter. The insulin promoter may be an endogenous promoter or an exogenous promoter. In some cases, the expression of GFP in these cells may indicate the expression of insulin in the cells. Thus, the GFP signal may be a marker for pancreatic beta cells.In some cases, the cell sorting described herein involves subjecting the cells to a magnetically activated sorting process, in which magnetic antibodies or other ligands are used to label different types of cells, and differences in magnetic properties can be used for cell sorting.

[0162]

[0176] The percentage of cells expressing one or more specific markers, such as PDX1, NKX6.1, insulin, NGN3, or CHGA, as described herein, can be a percentage value detected using a technique such as flow cytometry assay. In some cases, during flow cytometry assay, the cell population or cell clusters discussed herein are dispersed into a single cell suspension by incubation in a digestive enzyme, such as trypsin or TrypLE™ Express. The dispersed cells can be washed with a suitable buffer, such as PBS, centrifuged, and then resuspended in a fixation buffer, such as 4% PFA. Incubation can then be performed with a primary antibody against the cell marker of interest, followed by incubation with a secondary antibody. After antibody incubation, the cells can be washed and separated by flow cytometry. Techniques other than flow cytometry can also be used to characterize the cells described herein, such as determining the cell percentage. Non-limiting examples of cell characterization methods include gene sequencing, microscopy techniques (fluorescence microscopy, atomic force microscopy), karyotyping, isozyme analysis, DNA characterization, viral susceptibility, and the like.

[0163]

[0177] In some embodiments, any of the cells disclosed herein comprises a genomic disruption in at least one gene sequence, the disruption reducing or eliminating expression of a protein encoded by the gene sequence. In some embodiments, the cell comprises a genomic disruption in at least one gene sequence, the disruption reducing or eliminating expression of a protein encoded by the gene sequence. In some embodiments, the cell comprises a genomic disruption in at least one gene sequence, the disruption reducing or eliminating expression of a protein encoded by the gene sequence. In some embodiments, any of the cells disclosed herein (e.g., any of the SC-β cells or any of the cells of the clusters disclosed herein) comprises a genomic disruption in at least one gene sequence, the disruption reducing or eliminating expression of a protein encoded by the gene sequence. In some embodiments, the at least one gene sequence encodes an MHC-class I gene. In some embodiments, the MHC-class I gene encodes beta-2 microglobulin (B2M), HLA-A, HLA-B, or HLA-C. In some embodiments, the at least one gene sequence encodes CIITA. In some embodiments, the cells comprise a genomic disruption in genes encoding HLA-A and HLAB, but not in genes encoding HLA-C. In some embodiments, the cells comprise a genomic disruption in a natural killer cell activating ligand gene. In some embodiments, the natural killer cell activating ligand gene encodes intercellular adhesion molecule 1 (ICAM1), CD58, CD155, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), cell adhesion molecule 1 (CADM1), MHC class I polypeptide-related sequence A (MICA), or MHC class I polypeptide-related sequence B (MICB). In some embodiments, the cells have reduced expression of one or more of beta-2 microglobulin, CIITA, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLADR, compared to a cell that is not genetically modified.In some embodiments, the cells have increased expression of CD47, PDL1, HLA-G, CD46, CD55, CD59 and CTLA compared to cells that are not genetically modified. In certain embodiments, the pancreatic islet cells (e.g., SC-beta cells) disclosed herein have increased expression of PDL1 compared to endogenous pancreatic islet cells from healthy control subjects. In certain embodiments, the pancreatic islet cells (e.g., SC-beta cells) disclosed herein have increased expression of CD47 compared to endogenous pancreatic islet cells from healthy control subjects. In some embodiments, genome disruption is induced by the use of a gene editing system, for example, CRISPR Cas technology.

[0164]

[0178] In some embodiments, any of the cells disclosed herein comprises a genomic disruption in at least one gene sequence, the disruption reducing or eliminating expression of a protein encoded by the gene sequence (e.g., by knocking out one or both functional copies of the gene in the subject). In some embodiments, the at least one gene sequence is an ABO sequence, such that the disruption results in the cell having blood type O. In some embodiments, the at least one gene sequence encodes an MHC class I gene. In some embodiments, the MHC class I gene encodes beta 2-microglobulin (B2M), HLA-A, HLA-B, or HLA-C. In some embodiments, the at least one gene sequence encodes CIITA. In some embodiments, the cell comprises a genomic disruption in genes encoding HLA-A and HLA-B, but not in genes encoding HLA-C. In some embodiments, the cell comprises a genomic disruption in a gene encoding CXCL10. In some embodiments, the cell comprises a genomic disruption in a gene encoding renalase. In some embodiments, the cell comprises a genomic disruption in a natural killer cell activating ligand gene. In some embodiments, the natural killer cell activating ligand gene encodes intercellular adhesion molecule 1 (ICAM1), CD58, CD155, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), intercellular adhesion molecule 1 (CADM1), MHC class I polypeptide-related sequence A (MICA), or MHC class I polypeptide-related sequence B (MICB). In some embodiments, the cells have reduced expression of one or more of beta2-microglobulin, CIITA, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLADR compared to a stem cell that has not been genetically modified.In some embodiments, the cells have increased expression of CD47, PDL1, HLA-G, CD46, CD55, CD59, CTLA, PDL2, HLA-C, HLA-E, HLA-G, C1-inhibitor, IL-35, DUX4, IDO1, IL10, CCL21, CCL22, CD16, CD52, H2-M3, CD200, FASLG, MFGE8, and / or SERPINB9 compared to cells that are not genetically modified. In certain embodiments, the pancreatic islet cells (e.g., SC-β cells) disclosed herein have increased expression of PDL1 compared to endogenous pancreatic islet cells from a healthy control subject. In certain embodiments, the pancreatic islet cells (e.g., SC-β cells) disclosed herein have increased expression of CD47 compared to endogenous pancreatic islet cells from a healthy control subject. In some embodiments, the genome disruption is induced by the use of a gene editing system, e.g., CRISPR Cas technology. In some embodiments, any of the isolated cells (e.g., stem cells or NKX6.1 positive, ISL1 positive cells) described herein are selected from the group consisting of B2M, CIITA, CXCL10, Renase, HLA-A, HLA-B, HLA-C, RFX-ANK, NFY-A, NLRC5, RFX5, RFX-AP, HLA-G, HLA-E, NFY-B, PD-L1, NFY-C, IRF1, TAPI, GITR, 4-1BB, CD28, B7-1, CD47, B7-2, OX40, CD27, HVEM, SLAM, CD226, ICOS, LAG3, TIGIT, T and / or GFAP.In some embodiments, the disruption of the gene results in at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% decrease in expression compared to expression of the gene in the same type of cell without the disruption. In some embodiments, the gene is disrupted using CRISPR / Cas, piggyBac transposon, TALEN, and / or zinc finger technology.

[0165]

[0179] In some embodiments, the cells described herein (e.g., isolated stem cells, or NKX6.1 positive, ISL1 positive cells) are A antigen negative and B antigen negative. In some embodiments, the cells described herein are A antigen negative. In some embodiments, the cells described herein are B antigen negative. In some embodiments, the cells described herein (e.g., isolated stem cells, or NKX6.1 positive, ISL1 positive cells) are Rh antigen negative. In some embodiments, the cells described herein (e.g., isolated stem cells, or NKX6.1 positive, ISL1 positive cells) are A antigen negative, B antigen negative, Rh antigen negative. As used herein, "A antigen" refers to a histo-blood group antigen produced by 3α-N-acetylgalactosaminyltransferase and expressed as a cell surface antigen. As used herein, "B antigen" refers to a histo-blood group antigen produced by 3α-galactosaminyltransferase and expressed as a cell surface antigen. In some embodiments, the cells include a disruption of the ABO gene. In some embodiments, the cells comprise a disruption of the ABO gene, thereby reducing or eliminating the levels of A and B antigens. In some embodiments, the cells comprise a disruption of the FUT1 gene. In some embodiments, the cells comprise a disruption of the FUT1 gene, thereby reducing or eliminating the expression of galactoside 2-alpha-L-fucosyltransferase 1. As used herein, "Rh antigen" refers to a highly immunogenic antigen encoded by two highly polymorphic genes, RHD and RHCE. Rh antigen proteins are transmembrane proteins. In some embodiments, the cells comprise a disruption of the RHAG gene. In some embodiments, the cells comprise a disruption of the RHAG gene, thereby reducing or eliminating the levels of Rh-associated glycoproteins. In some embodiments, the cells comprise a reduced or eliminated expression of an Rh protein antigen selected from the group consisting of Rh C antigen, Rh E antigen, Kell K antigen (KEL), Duffy (FY) Fya antigen, Duffy Fy3 antigen, Kidd (JK) Jkb antigen, MNS antigen U, and MNS antigen S.

[0166]

[0180] In some embodiments, any of the cells disclosed herein (e.g., any of the SC islet cells disclosed herein) comprises a "safety switch." In some embodiments, the safety switch is a nucleic acid construct that encodes a switch protein that inducibly causes cell death or stops cell proliferation. In some embodiments, the safety switch is inserted into a defined, specific target locus (e.g., a safe harbor locus) in the genome of the engineered cell, typically in both alleles of the target locus. In some embodiments, the target locus is a safe harbor locus such as ActB or CLYBL. In some embodiments, the target locus is a gene targeted for disruption (e.g., B2M or CIITA). In some embodiments, the switch protein is activated by contacting with an effective amount of a clinically tolerated ortholog small molecule. In some embodiments, when activated, the safety switch stops cell proliferation, in some embodiments by activating apoptosis of the cell. In some embodiments, the switch protein comprises herpes-simplex-thymidine kinase. In some embodiments, the switch protein comprises a human caspase protein, such as caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 14, etc. In certain embodiments, the protein is human caspase 9. In some embodiments, the caspase protein is fused to a sequence that provides chemically induced dimerization (CID), where dimerization occurs only in the presence of an orthologous activator. One or more CID domains can be fused to the caspase protein, for example, two different CID domains can be fused to the caspase protein. In some embodiments, the CID domain is a dimerization domain of FKBPl or FRb (FKBP-rapamycin binding) domain, which is activated with a rapamycin analog.In some embodiments, the safety switch is any of the safety switches described in International Publication No. WO2021 / 173449 and Jones et al., 2014, Frontiers in Pharmacology, 5(254):1-8, each of which is incorporated herein in its entirety.

[0167]

[0181] In some embodiments, in the pharmaceutical compositions disclosed herein, at least a portion of the cells in the cell population are present in multiple cell clusters. In some cases, the cell clusters are about 50 μm to about 500 μm, about 50 μm to about 400 μm, about 50 μm to about 300 μm, about 60 μm to about 400 μm, about 60 μm to about 300 μm, about 60 μm to about 250 μm, about 75 μm to about 400 μm, about 75 μm to about 300 μm, about 75 μm to about 250 μm, about 125 μm to about 225 μm, about 130 μm to about 160 μm, about 170 μm to about 225 μm, about 140 μm to about 200 μm, about 140 μm to about 170 μm, about 160 μm to about 220 μm, about 170 μm to about 215 μm, or about 170 μm to about 200 μm in diameter. In some cases, in the pharmaceutical compositions disclosed herein, the cell population is present as a single cell suspension.In some embodiments, in the pharmaceutical compositions disclosed herein, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, or at least 99% of the cells are present in cell clusters.In some embodiments, in the pharmaceutical compositions disclosed herein, substantially all the cells are present in cell clusters, for example, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.9%, at least 99.999%, or at least 99.9999% of the cells are present.

[0168] Pharmaceutically acceptable excipients and / or carriers

[0182] In some embodiments, the pharmaceutical composition disclosed herein comprises a cell population in liquid suspension.The liquid suspension can comprise an aqueous solution comprising pharma- ceutically acceptable excipients and / or carriers.For example, the pharmaceutical composition can further comprise a physiologically compatible solution comprising, for example, phosphate buffered saline.

[0169]

[0183] In some cases, the disclosure provides pharmaceutical compositions that can utilize non-native pancreatic β cell populations and cellular components and products in various methods for treating diseases (e.g., diabetes). Certain cases include pharmaceutical compositions that include live cells (e.g., non-native pancreatic β cells alone or mixed with other cell types). Other cases include pharmaceutical compositions that include components (e.g., cell lysates, soluble cell fractions, conditioned medium, ECM, or any of the foregoing components) or products (e.g., trophic and other biological factors produced by the non-native pancreatic β cells or by genetic modification, conditioned medium from culture of the non-native pancreatic β cells). In any case, the pharmaceutical compositions may further include other active agents, such as anti-inflammatory agents, exogenous small molecule agonists, exogenous small molecule antagonists, anti-apoptotic agents, antioxidants, and / or growth factors known to those of skill in the art.

[0170]

[0184] The pharmaceutical compositions of the present disclosure can include non-native pancreatic beta cells, or components or products thereof, formulated with a pharma- ceutically acceptable carrier (e.g., medium or excipient). The term pharma- ceutically acceptable carrier (or medium or excipient) may be used interchangeably with the term biologically compatible carrier or medium and may refer to reagents, cells, compounds, materials, compositions, and / or dosage forms that are compatible with cells and other agents administered therapeutically, as well as suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other complications. Suitable pharma- ceutically acceptable carriers can include water, salt solutions (e.g., Ringer's solution), alcohol, oils, gelatin, and carbohydrates such as lactose, amylose, or starch, fatty acid esters, hydroxymethylcellulose, and polyvinylpyrrolidone. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, and colorants. Pharmaceutical compositions that do not contain living cells, but contain cellular constituents or products, can be formulated as liquid suspensions.

[0171]

[0185] The pharmaceutical composition may include auxiliary components familiar to those skilled in the art. For example, antioxidants may be included in different ranges depending on the type of antioxidant used. Reasonable ranges of commonly used antioxidants are about 0.01 to about 0.15% by weight to volume of EDTA, about 0.01 to about 2.0% by weight to volume of sodium sulfite, and about 0.01 to about 2.0% by weight to volume of sodium metabisulfite. Those skilled in the art may use a concentration of about 0.1% by weight to volume for each of the above. Other representative compounds include mercaptopropionylglycine, N-acetylcysteine, beta-mercaptoethylamine, glutathione and similar species, although other antioxidants suitable for administration to the kidney may also be employed, such as ascorbic acid and its salts, or sulfite or sodium metabisulfite.

[0172]

[0186] A buffering agent can be used to maintain the pH of the formulation in the range of about 4.0 to about 8.0 to minimize irritation in the target tissue. For direct intraperitoneal injection, the formulation should be at pH 7.2 to 7.5, preferably pH 7.35 to 7.45. The composition may also include an isotonicity agent suitable for administration to the kidney. Of these, sodium chloride is preferred to make the formulation approximately isotonic with blood.

[0173]

[0187] In certain cases, the pharmaceutical composition is formulated with a viscosity enhancer. Exemplary agents are hydroxyethylcellulose, hydroxypropylcellulose, methylcellulose, polyvinylpyrrolidone. The pharmaceutical composition may have a co-solvent added if necessary. Suitable co-solvents may include glycerin, polyethylene glycol (PEG), polysorbate, propylene glycol, polyvinyl alcohol. Preservatives may also be included, such as benzalkonium chloride, benzethonium chloride, chlorobutanol, phenylmercuric acetate or phenylmercuric nitrate, thimerosal, or methyl or propyl paraben.

[0174]

[0188] The pharmaceutical composition comprising the cells, cell components, or cell products may be delivered to the liver of a patient by one or more of several delivery methods known in the art. In some cases, the composition is delivered to the liver. In another embodiment, the composition may be delivered to the liver by intraportal injection.

[0175]

[0189] In some embodiments, any of the cell compositions disclosed herein (e.g., compositions comprising in vitro differentiated pancreatic islet cells) further comprises a liquid solution (or medium). In some embodiments, the liquid solution comprises a sugar. In some embodiments, the sugar is sucrose or glucose. In some embodiments, the liquid solution comprises a sugar at a concentration of about 0.05% to about 1.5%. In some embodiments, the liquid solution is CMRL medium. In some embodiments, the composition comprises HypoThermosol® FRS storage medium. In some cases, the liquid solution is serum-free. In some cases, the liquid solution in which the cells are suspended is protein-free. In some cases, the liquid solution in which the cells are suspended is animal component-free. In some cases, the liquid solution in which the cells are suspended is protein- and animal component-free. In some cases, the liquid solution has a physiological osmolality. In some cases, the liquid solution is pH buffered.

[0176]

[0190] In some cases, pharmaceutical compositions of stem cell-derived islet cells are formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and auxiliary agents that facilitate the processing of active compounds into medicament-usable preparations.Suitable formulations depend on the route of administration selected.Summary of pharmaceutical compositions described herein can be found, for example, in Remington: The Science and Practice of Pharmacy, 19th Edition (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, HA and Lachman, L., eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Edition (Lippincott Williams & Wilkins 1999).

[0177]

[0191] The pharmaceutical compositions are optionally manufactured in a conventional manner, by way of example only, by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compressing processes.

[0178]

[0192] In certain embodiments, the composition may also include one or more pH adjusting or buffering agents, including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and tris-hydroxymethylaminomethane; buffers such as citrate / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in amounts necessary to maintain the pH of the composition in an acceptable range.

[0179]

[0193] In other embodiments, the composition may also include one or more salts in an amount necessary to bring the osmolality of the composition into an acceptable range. Such salts include salts having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions, with suitable salts including sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfate and ammonium sulfate.

[0180]

[0194] The pharmaceutical compositions described herein are administered by any suitable route of administration, including, but not limited to, oral, parenteral (e.g., intravenous, subcutaneous, intramuscular, intracerebral, intraventricular, intraarticular, intraperitoneal, or intracranial), intranasal, buccal, sublingual, or rectal administration. In some cases, the pharmaceutical composition is formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular, intracerebral, intraventricular, intraarticular, intraperitoneal, or intracranial) administration. In some embodiments, any of the pharmaceutical compositions disclosed herein is administered to a subject by injection via the hepatic portal vein.

[0181]

[0195] The pharmaceutical compositions described herein may be formulated into any suitable dosage form, including, but not limited to, aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions, etc. (for oral ingestion by the individual being treated), solid oral dosage forms, aerosols, controlled release formulations, fast dissolving formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, capsules, delayed release formulations, sustained release formulations, pulsatile release formulations, multiparticulate formulations, and immediate mixed release and controlled release formulations. In some embodiments, the pharmaceutical compositions are formulated into capsules. In some embodiments, the pharmaceutical compositions are formulated into solutions (e.g., for IV administration). In some cases, the pharmaceutical compositions are formulated as infusions. In some cases, the pharmaceutical compositions are formulated as injections.

[0182]

[0196] The pharmaceutical solid dosage forms described herein optionally comprise a compound described herein and one or more pharma- ceutically acceptable excipients, such as a compatible carrier, a binder, a filler, a suspending agent, a flavoring agent, a sweetener, a disintegration agent, a dispersing agent, a surfactant, a lubricant, a coloring agent, a diluent, a solubilizing agent, a moisturizing agent, a plasticizer, a stabilizer, a penetration enhancer, a wetting agent, an antifoaming agent, an antioxidant, a preservative, or one or more combinations thereof.

[0183]

[0197] In yet another aspect, a film coating is applied around the composition using standard coating procedures, such as those described in Remington's Pharmaceutical Sciences, 20th Edition (2000).In some embodiments, the composition is formulated into particles (e.g., for administration in capsules), and some or all of the particles are coated.In some embodiments, the composition is formulated into particles (e.g., for administration in capsules), and some or all of the particles are microencapsulated.In some embodiments, the composition is formulated into particles (e.g., for administration in capsules), and some or all of the particles are not microencapsulated and are not coated.

[0184]

[0198] In certain embodiments, the compositions provided herein may contain one or more preservatives to inhibit microbial activity.Suitable preservatives include mercury-containing substances, such as merfen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds, such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.

[0185]

[0199] In some embodiments, the compositions of the present disclosure can include stem cell-derived islet cells in an amount effective for treating or preventing diabetes, for example. Pharmaceutical compositions can include the stem cell-derived islet cells described herein in combination with one or more pharmacologic or physiologically acceptable carriers, diluents or excipients. Such compositions can include buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0186]

[0200] The pharmaceutical composition may include auxiliary compounds, as is well known to those skilled in the art. For example, the pharmaceutical composition may contain antioxidants within a range that varies depending on the type of antioxidant used. Reasonable ranges of commonly used antioxidants are about 0.01% to about 0.15% by weight by volume EDTA, about 0.01% to about 2.0% by weight by volume sodium sulfite, and about 0.01% to about 2.0% by weight by volume sodium metabisulfite. Those skilled in the art may use a concentration of about 0.1% by weight by volume for each of the above. Other preservative compounds include mercaptopropionylglycine, N-acetylcysteine, β-mercaptoethylamine, glutathione and similar species, although other antioxidants suitable for renal administration, such as ascorbic acid and its salts or sulfites or sodium metabisulfite, may also be used.

[0187]

[0201] A buffering agent can be used to maintain the pH of the formulation in the range of about 4.0 to about 8.0 to minimize irritation in the target tissue. For direct intraperitoneal injection, the formulation should be at pH 7.2 to 7.5, preferably pH 7.35 to 7.45. The composition may also include an isotonicity agent suitable for administration to the kidney. Of these, sodium chloride is preferred to make the formulation approximately isotonic with blood.

[0188]

[0202] In certain cases, the pharmaceutical composition is formulated with a viscosity enhancing agent. Exemplary agents are hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, and polyvinylpyrrolidone. The pharmaceutical composition may have a cosolvent added if necessary. Suitable cosolvents may include glycerin, polyethylene glycol (PEG), polysorbate, propylene glycol, and polyvinyl alcohol. Preservatives may also be included, such as benzalkonium chloride, benzethonium chloride, chlorobutanol, phenylmercuric acetate or phenylmercuric nitrate, thimerosal, or methyl or propyl paraben.

[0189]

[0203] The formulations described herein can benefit from antioxidants, metal chelators, thiol-containing compounds and other general stabilizing agents. Examples of such stabilizers include, but are not limited to, (a) about 0.5% to about 2% w / v glycerol, (b) about 0.1% to about 1% w / v methionine, (c) about 0.1 to about 2% w / v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01% to about 2% w / v ascorbic acid, (f) about 0.003% to about 0.02% w / v polysorbate 80, (g) 0.001% to about 0.5% w / v polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrin, (l) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc; or (n) combinations thereof.

[0190]

[0204] "Binders" provide adhesion and include, for example, alginic acid and its salts; cellulose derivatives such as carboxymethylcellulose, methylcellulose (e.g., Methocel®), hydroxypropylmethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose (e.g., Klucel®), ethylcellulose (e.g., Ethocel®), and microcrystalline cellulose (e.g., Avicel®); microcrystalline dextrose; amylose; magnesium aluminum silicate; polysaccharide acids; bentonite; gelatin; polyvinylpyrrolidone / vinyl acetate copolymer; crospovidone; povidone; starch; pregelatinized starch; tragacanth, dextrins, sugars such as sucrose (e.g., Dipac®), glucose, dextrose, molasses, mannitol, sorbitol, xylitol (e.g., Xylitab®), and lactose; natural or synthetic gums such as acacia, tragacanth, ghatti gum, isapol shells, and the like. These include husk mucus, polyvinylpyrrolidone (e.g., Polyvidone® CL, Kollidon® CL, Polyplasdone® XL-10), larch arabogalactan, Veegum®, polyethylene glycol, waxes, sodium alginate, and the like.

[0191]

[0205] "Carrier" or "carrier material" includes any excipient commonly used in medicine, and should be selected based on the compatibility with the compounds disclosed herein, such as the compound of ibrutinib and the anticancer drug, and the release profile characteristics of the desired dosage form. Exemplary carrier materials include, for example, binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, etc. "Pharmaceutically compatible carrier materials" may include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerin, magnesium silicate, polyvinylpyrrolidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan, monoglycerides, diglycerides, pregelatinized starch, and the like. See, e.g., Remington: The Science and Practice of Pharmacy, 19th ed. (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, HA and Lachman, L, eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed. (Lippincot T Williams & Wilkins 1999).

[0192]

[0206] "Dispersing agents" and / or "viscosity modifiers" include materials that control the diffusion and homogeneity of drugs through liquid media or granulation or blending methods. In some embodiments, these agents also promote the effectiveness of coatings or erodible matrices. Exemplary diffusion enhancers / dispersing agents include, for example, hydrophilic polymers, electrolytes, Tween® 60 or 80, PEG, polyvinylpyrrolidone (PVP; commercially known as Plasdone®), and carbohydrate-based dispersing agents, such as hydroxypropylcellulose (e.g., HPC, HPC-SL, and HPC-L), hydroxypropylmethylcellulose (e.g., HPMC K100, HPMC K4M, HPMC K15M, and HPMC K100M), sodium carboxymethylcellulose, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate stearate (HPMCAS), amorphous cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol (PVA), vinylpyrrolidone / vinyl acetate copolymer (S630), 4-(1,1,3,3-tetramethylbutyl)-phenol polymer with ethylene oxide and formaldehyde (also known as tyloxapol), poloxamers (e.g., Pluronics F68®, F88®, and F108®, which are block copolymers of ethylene oxide and propylene oxide); and poloxamines (e.g., Tetronic 908®, also known as Poloxamine 908®, which is a tetrafunctional block copolymer derived from the sequential addition of propylene oxide and ethylene oxide to ethylenediamine (BASF Corporation, Parsippany, NJ).), polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, polyvinylpyrrolidone / vinyl acetate copolymer (S-630), polyethylene glycol, for example, the polyethylene glycol may have a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400, sodium carboxymethylcellulose, methylcellulose, polysorbate-80, sodium alginate, gums, for example, Examples of dispersants include gum lagacant and gum acacia, guar gum, xanthan (including xanthan gum), sugars, cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone, carbomer, polyvinyl alcohol (PVA), alginates, chitosan, and combinations thereof. Plasticizers such as cellulose or triethylcellulose can also be used as dispersants. Dispersants particularly useful in liposomal and self-emulsifying dispersions are dimyristoyl phosphatidylcholine, natural phosphatidylcholine from eggs, natural phosphatidylglycerol from eggs, cholesterol, and isopropyl myristate.

[0193]

[0207] Combinations of one or more erosion facilitators with one or more diffusion facilitators may also be used in the compositions of the present invention.

[0208] The term "diluent" refers to chemical compounds that are used to dilute the compound of interest before delivery. Diluents can also be used to stabilize the compound, as they can provide a more stable environment. Salts dissolved in buffer solutions, including but not limited to phosphate buffered saline solutions, which can also provide pH control or maintenance, are utilized as diluents in the art. In certain embodiments, diluents increase the bulk of the composition to facilitate compression or create sufficient bulk for uniform blending for capsule filling. Such compounds include, for example, lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose such as Avicel®; dibasic calcium phosphate, dicalcium phosphate dihydrate; tricalcium phosphate, calcium phosphate; anhydrous lactose, spray-dried lactose; pregelatinized starch, compressible sugars such as Di-Pac® (Amstar); mannitol, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate stearate, sucrose-based diluents, refined powdered sugar; monocalcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactate trihydrate, dextrates; hydrolyzed cereal solids, amylose; powdered cellulose, calcium carbonate; glycine, kaolin; mannitol, sodium chloride; inositol, bentonite, and the like.

[0194]

[0209] "Bulking agents" include compounds such as lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, and polyethylene glycol.

[0195]

[0210] "Lubricants" and "flow agents" are compounds that prevent, reduce or inhibit adhesion or friction of materials. Exemplary lubricants include, for example, stearic acid, calcium hydroxide, talc, sodium stearyl fumarate, hydrocarbons such as mineral oils or hydrogenated vegetable oils such as hydrogenated soybean oil (Sterotex®), higher fatty acids and their alkali metal and alkaline earth metal salts such as aluminum, calcium, magnesium, zinc, stearic acid, sodium stearate, glycerol, talc, wax, Stearowet®, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycol (e.g., PEG-4000) or methoxypolyethylene glycol such as Carbowax™, sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium or sodium lauryl sulfate, colloidal silica such as Syloid™, Cab-O-Sil®, starch such as corn starch, silicone oil, surfactants, and the like.

[0196]

[0211] "Plasticizers" are compounds used to soften microencapsulation materials or film coatings to make them less brittle. Suitable plasticizers include, for example, polyethylene glycols such as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propylene glycol, oleic acid, triethylcellulose, and triacetin. In some embodiments, plasticizers can also function as dispersants or wetting agents.

[0197]

[0212] Examples of "solubilizing agents" include compounds such as triacetin, triethyl citrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium docusate, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cyclodextrin, ethanol, n-butanol, isopropyl alcohol, cholesterol, bile salts, polyethylene glycol 200-600, glycofurol, transcutol, propylene glycol, and dimethyl isosorbide.

[0198]

[0213] "Stabilizers" include compounds such as any antioxidant, buffer, acid, preservative, etc.

[0214] Examples of the "suspending agent" include polyvinylpyrrolidone, such as polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinylpyrrolidone / vinyl acetate copolymer (S630), polyethylene glycol (for example, the polyethylene glycol can have a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400), sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, hydroxymethylcellulose acetate stearyl alcohol, and the like. Examples of suitable cellulose acetate compositions include cellulose acetate, cellulose acetate copolymers ...

[0199]

[0215] "Surfactants" include, for example, compounds such as sodium lauryl sulfate, sodium docusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate, ethylene oxide and propylene oxide, such as Pluronic® (BASF). Some other surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, such as polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkyl ethers and alkyl phenyl ethers, such as octoxynol 10, octoxynol 40. In some embodiments, surfactants may be included to enhance physical stability or for other purposes.

[0200]

[0216] "Viscosity enhancing agents" include, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, hydroxypropylmethylcellulose phthalate, carbomer, polyvinyl alcohol, alginate, acacia, chitosan, and combinations thereof.

[0201]

[0217] "Wetting agents" include, for example, compounds such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate, sodium docusate, triacetin, Tween 80, Vitamin E TPGS, ammonium salts, and the like.

[0202]

[0218] In some embodiments, any of the compositions disclosed herein is encapsulated in any of the devices disclosed herein. Stem Cells and Reprogramming

[0219] Provided herein is the use of stem cells to generate SC-β cells (e.g., mature pancreatic β cells or β-like cells) or precursors thereof. In one embodiment, embryonic cells can be used to provide at least one SC-β cell, either in place of or in conjunction with stem cells, using similar protocols described in US Patent Application Publication Nos. 2015 / 0240212 and 2015 / 0218522, each of which is incorporated herein by reference in its entirety. Suitable embryonic cells can be prepared, for example, from primordial embryonic cells present in human fetal material harvested about 8-11 weeks after the last menstrual cycle. Illustrative methods for preparing embryonic cells are described, for example, in Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998 and U.S. Patent No. 6,090,622.

[0203]

[0220] Provided herein are compositions and methods for generating SC-islet cells (eg, SC-β cells), as well as SC-α cells, and / or SC-δ cells.

[0221] Generally, the at least one SC-pancreatic islet cell or its precursor, eg, pancreatic precursor, produced according to the methods disclosed herein can consist of a mixture or combination of different cells.

[0204]

[0222] The at least one pancreatic alpha, beta and / or delta cell or precursor thereof can be generated according to any suitable culture protocol that differentiates stem cells or pluripotent cells to a desired differentiation stage. In some embodiments, the at least one pancreatic alpha, beta and / or delta cell or precursor thereof is generated by culturing at least one pluripotent cell for a time and under conditions suitable for differentiating the at least one pluripotent cell into at least one pancreatic alpha, beta and / or delta cell or precursor thereof.

[0205]

[0223] In some embodiments, at least one pancreatic alpha, beta and / or delta cell or precursor thereof is a substantially pure population of pancreatic alpha, beta and / or delta cells or precursor thereof. In some embodiments, the population of pancreatic alpha, beta and / or delta cells or precursor thereof comprises a mixture of pluripotent or differentiated cells. In some embodiments, the population of pancreatic alpha, beta and / or delta cells or precursor thereof is substantially free or free of embryonic stem cells or pluripotent cells, iPS cells.

[0206]

[0224] In some embodiments, somatic cells, e.g., fibroblasts, can be isolated from a subject, e.g., as a tissue biopsy, e.g., a skin biopsy, and can be reprogrammed into induced pluripotent stem cells for further differentiation to generate at least one pancreatic alpha, beta, and / or delta cell, or a precursor thereof, for use in the compositions and methods described herein. In some embodiments, the somatic cells, e.g., fibroblasts, are maintained in culture by methods known to those of skill in the art, and in some embodiments, expanded before being converted into pancreatic alpha, beta, and / or delta cells by the methods disclosed herein.

[0207]

[0225] In some embodiments, at least one pancreatic alpha, beta and / or delta cell or precursor thereof is maintained in culture by methods known to those of skill in the art, and in some embodiments expanded before being converted to a pancreatic alpha, beta and / or delta cell by methods described herein.

[0208]

[0226] Furthermore, at least one pancreatic alpha, beta and / or delta cell or its precursor, e.g., pancreatic precursor, can be from any mammalian species, non-limiting examples include murine, bovine, simian, porcine, equine, ovine or human cells. For clarity and simplicity, the method description herein refers to at least one pancreatic alpha, beta and / or delta cell or its precursor from a mammal, but it should be understood that all methods described herein can be easily applied to other cell types of at least one pancreatic alpha, beta and / or delta cell or its precursor. In some embodiments, at least one pancreatic alpha, beta and / or delta cell or its precursor is derived from a human individual.

[0209] stem cells

[0227] The embodiments of the present disclosure relate to the use of stem cells for the production of pancreatic alpha, beta and / or delta cells or their precursors. As used herein, the term "stem cell" may refer to a cell (e.g., a vertebrate stem cell) that has the ability to self-renew and produce differentiated cell types (Morrison et al., (1997) Cell 88:287-298). In the context of cytogenetics, the adjectives "differentiated" or "differentiating" are relative terms. A "differentiated cell" may be a cell that has progressed further down the developmental pathway than the cell to which it is being compared. Thus, pluripotent stem cells can differentiate into lineage-restricted progenitor cells (e.g., definitive endoderm cells), which in turn can differentiate into more restricted cells (e.g., pancreatic progenitor cells), which can play characteristic roles in certain tissue types, and end-stage cells (e.g., terminally differentiated cells, e.g., beta cells, etc.) that may or may not retain the ability to proliferate further. Stem cells can be characterized by the presence of certain markers (e.g., proteins, RNA, etc.) and the absence of certain markers. Stem cells can also be identified by both in vitro and in vivo functional assays, particularly assays related to the ability of stem cells to give rise to multiple differentiated progeny. In one embodiment, the host cells are adult stem cells, somatic stem cells, non-embryonic stem cells, embryonic stem cells, hematopoietic stem cells, including pluripotent stem cells, and trophoblast stem cells.

[0210]

[0228] Stem cells of interest, such as stem cells that can be used in the methods provided herein, can include pluripotent stem cells (PSCs). As used herein, the term "pluripotent stem cells" or "PSCs" refers to stem cells that can generate all cell types of an organism. Thus, PSCs can give rise to cells of all germ layers of an organism (e.g., endoderm, mesoderm, and ectoderm of vertebrates). Pluripotent cells can form teratomas and contribute to ectoderm, mesoderm, or endoderm tissues of an organism. Plant pluripotent stem cells can give rise to all cell types of a plant (e.g., roots, stems, leaves, and other cells).

[0211]

[0229] Embodiments of the present disclosure relate to the use of PSCs for the generation of pancreatic alpha, beta and / or delta cells or their precursors. PSCs in animals can be derived in several different ways. For example, embryonic stem cells (ESCs) can be derived from the inner cell mass of an embryo (Thomson et al., Science. 1998 Nov. 6; 282(5391):1145-7), 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., Nat Protoc. 2007; 2(12):3081-9; Yu et al., Science. 2007 Dec. 21; 318(5858):1917-20. Epub 2007 Nov. 20). The term PSC can refer to pluripotent stem cells regardless of their origin, and therefore can encompass the terms ESC and iPSC, as well as the term fetal embryonic stem cells (EGSC), which is another example of a PSC. PSCs can be in the form of established cell lines, which can be obtained directly from primary embryonic tissue or can be derived from somatic cells.

[0212]

[0230] Embodiments of the present disclosure relate to the use of ESCs for the production of pancreatic β cells or their precursors. "Embryonic stem cells (ESCs)" may refer to PSCs isolated from an embryo, typically from the inner cell mass of a blastocyst. ESC lines include, for example, hESBGN-01, hESBGN-02, hESBGN-03, hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3, HES-4, HES-5, HES-6 (ES Cell International); Miz-hES1 (MizMedi Hospital-Seoul National University); HSF-1, HSF-6 (University of California at San Francisco); and H1, H7, H9, H13, H14 (NIH Human Embryonic Stem Cell Line from Wisconsin Alumni Research Foundation (WiCell Research Institute)). Stem cells of interest are listed in the Registry. Stem cells of interest also include embryonic stem cells from other primates, such as rhesus monkey stem cells and marmoset stem cells. Stem cells can be obtained from any mammalian species, such as humans, horses, cows, pigs, dogs, cats, rodents, such as mice, rats, hamsters, primates, 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 nuclear-cytoplasmic ratios, clear borders, and prominent nuclei. 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 for producing and characterizing ESCs can be found, for example, in U.S. Patent Nos. 7,029,913, 5,843,780, and 6,200,806, each of which is incorporated herein in its entirety.Methods for expanding undifferentiated forms of hESCs are described in WO 99 / 20741, WO 01 / 51616, and WO 03 / 020920, each of which is incorporated herein in its entirety.

[0213]

[0231] "Fetal embryonic stem cells (EGSCs) or fetal embryonic cells" or "EG cells" may refer to embryonic cells and / or embryonic cell precursors, such as PSCs derived from embryonic cells that can become primordial embryonic cells, such as sperm and eggs. Fetal embryonic cells (EG cells) are believed to have similar properties to the fetal stem cells described above. Examples of methods for producing and characterizing EG cells can be found, for example, in U.S. Patent No. 7,153,684; Matsui, Y. et al., (1992) Cell 70:841; Shamblott, M. et al., (2001) Proc. Natl. Acad. Sci. USA 98: 113; Shamblott, M. et al., (1998) Proc. Natl. Acad. Sci. USA, 95:13726; and Koshimizu, U. et al., (1996) Development, 122:1235, each of which is incorporated herein in its entirety.

[0214]

[0232] An embodiment of the present disclosure relates to the use of iPSCs for the production of pancreatic alpha, beta and / or delta cells or their precursors. "Induced pluripotent stem cells" or "iPSCs" may refer to PSCs derived from non-PSC cells (e.g., from cells differentiated to PSCs). iPSCs can be derived from a variety of different cell types, including terminally differentiated cells. iPSCs have an ES cell-like morphology and can grow as flat colonies with large nuclear-cytoplasmic ratios, clear borders, and prominent nuclei. In addition, iPSCs can express one or more important pluripotency markers known to those of skill in the art, including, but not limited to, alkaline phosphatase, SSEA3, SSEA4, Sox2, Oct3 / 4, Nanog, TRA160, TRA181, TDGF1, Dnmt3b, FoxD3, GDF3, Cyp26a1, TERT, and zfp42. Examples of methods for generating and characterizing iPSCs can be found, for example, in U.S. Patent Publication Nos. 2009 / 0047263, 2009 / 0068742, 2009 / 0191159, 2009 / 0227032, 2009 / 0246875, and 2009 / 0304646, each of which is incorporated herein in its entirety. Generally, to generate iPSCs, somatic cells are provided with reprogramming factors known in the art (e.g., Oct4, SOX2, KLF4, MYC, Nanog, Lin28, etc.) that reprogram somatic cells to pluripotent stem cells.

[0215]

[0233] In some embodiments, the cell population is derived from stem cells in vitro. In some embodiments, the stem cells are genetically modified. In some embodiments, the stem cells have reduced expression of one or more of beta-2 microglobulin, CIITA, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR compared to non-genetically modified stem cells. In some embodiments, the stem cells have increased expression of one or more of CD47, PDL1, HLA-G, CD46, CD55, CD59, and / or CTLA compared to non-genetically modified stem cells.

[0216]

[0234] An embodiment of the present disclosure relates to the use of somatic cells for the production of pancreatic α, β and / or δ cells or their precursors. "Somatic cells" may refer to any cell in an organism that does not normally give rise to all types of cells in the organism in the absence of experimental manipulation. In other words, somatic cells may be cells that are fully differentiated and therefore do not naturally give rise to cells of all three germ layers of an organism, such as cells of the ectoderm, mesoderm, and endoderm. For example, somatic cells include neurons and neuronal progenitor cells, which can naturally give rise to all or some cell types of the central nervous system, but cannot give rise to cells of the mesodermal or endodermal lineages.

[0217]

[0235] In certain instances, the stem cells may be undifferentiated (e.g., cells not committed to a particular lineage) prior to exposure to at least one differentiation factor or composition according to the methods disclosed herein, while in other instances, it may be desirable to differentiate the stem cells into one or more intermediate cell types prior to exposure to at least one differentiation factor or composition described herein. For example, stem cells may exhibit morphological, biological, or physical characteristics of undifferentiated cells, which may be used to distinguish them from differentiated cells of fetal or adult origin. In some instances, undifferentiated cells may appear in microscopic two dimensions in colonies of cells with high nuclear / cytoplasmic ratios and prominent nuclei. The stem cells may be used by themselves (e.g., substantially free of any undifferentiated cells) or in the presence of differentiated cells. In certain instances, the stem cells may be cultured in the presence of suitable nutrients and optionally other cells to grow and optionally differentiate. For example, fetal fibroblasts or fibroblast-like cells may be present in the culture to support the growth of the stem cells. Fibroblasts may be present during one stage of stem cell development, but not necessarily at all stages. For example, fibroblasts may be added to a stem cell culture at an initial culture step and not be added to the stem cell culture at one or more subsequent culture steps.

[0218]

[0236] The stem cells used in all aspects of the disclosure may be any cells derived from any type of tissue (e.g., embryonic tissue, such as fetal or pre-fetal tissue, or adult tissue), and these stem cells may be characterized by their ability to generate progeny consisting of various cell types under appropriate conditions, such as derivatives of all or at least one of the three germ layers (endoderm, mesoderm, and ectoderm). These cell types may be provided in the form of established cell lines, or may be obtained directly from primary embryonic tissue and immediately used for differentiation. Cells listed in the NIH Human Embryonic Stem Cell Registry include hESBGN-01, hESBGN-02, hESBGN-03, hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3, HES-4, HES-5, HES-6 (ES Cell International); Miz-hES1 (MizMedi Hospital-Seoul National University); HSF-1, FISF-6 (University of California at San Francisco); and H1, 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 include a step of 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 did not include a step of destroying a human embryo.

[0219]

[0237] In another example, stem cells can be isolated from tissues including solid tissues.In some embodiments, the tissue is skin, adipose tissue (e.g., adipose tissue), muscle tissue, heart or heart tissue.In other embodiments, the tissue is, for example but not limited to, umbilical cord blood, placenta, bone marrow, or cartilage.

[0220]

[0238] Stem cells that can be used in the methods provided herein can also include various types of embryonic cells exemplified by human embryonic stem (hES) cells described by Thomson et al. (1998) Science 282:1145, embryonic stem cells from other primates such as rhesus monkey stem cells (Thomson et al., (1995) Proc. Natl. Acad. Sci. USA 92:7844), marmoset stem cells (Thomson et al., (1996) Biol. Reprod. 55:254), and human fetal embryo (hEG) cells (Shambloft et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). The methods provided herein are also applicable to stem cells committed to lineages such as mesodermal stem cells and other early cardiac developmental cells (see, e.g., Reyes et al., (2001) Blood 98:2615-2625; Eisenberg and Bader, (1996) Circ Res. 78(2):205-16). Stem cells can be obtained from any mammalian species, e.g., human, horse, cow, pig, dog, cat, rodent, e.g., mouse, rat, hamster, primate, etc. In some embodiments, human embryos have not been destroyed for a source of pluripotent cells for use in the methods and compositions disclosed herein. In some embodiments, human embryos have not been destroyed for a source of pluripotent cells for use in the methods and compositions disclosed herein.

[0221]

[0239] A mixture of cells from a suitable source of endothelial, muscle, and / or neuronal stem cells can be harvested from a mammalian donor for the purpose of this disclosure. A suitable source is a hematopoietic microenvironment. For example, circulating peripheral blood, preferably fixed (e.g., mobilized), can be removed from a subject. In one embodiment, the stem cells can be reprogrammed stem cells, such as stem cells derived from somatic cells or differentiated cells. In such an embodiment, the dedifferentiated stem cells can be induced and reprogrammed cells, such as, but not limited to, neoplastic cells, tumor cells, and cancer cells, or induced pluripotent stem cells or iPS cells.

[0222]

[0240] In some embodiments, the pancreatic alpha, beta and / or delta cells described herein are selected from the group consisting of hair cells, keratinocytes, gonadotropes, corticotropes, thyrotropes, somatotropes, lactotropes, chromaffin cells, parafollicular cells, glomus cells, melanocytes, nevus cells, Merkel cells, odontoblasts, cementoblasts, corneal keratinocytes, retinal muller cells, retinal pigment epithelial cells, neurons, glial cells (e.g., oligodendrocytes, astrocytes), ependymal cells, pineal cells, lung cells (e.g., type I pneumocytes and type II pneumocytes), Clara cells, goblet cells, G cells, D cells, ECL cells, gastric chief cells, parietal cells, 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), gallbladder cells, atrial cells, pancreatic stellate cells, pancreatic α cells, pancreatic β cells, pancreatic δ cells, pancreatic F cells (e.g. PP cells), pancreatic ε cells, thyroid (e.g. follicular cells), parathyroid (e.g. parathyroid chief cells), eosinophilic cells, urothelial cells, osteoblasts, osteocytes, chondroblasts, chondrocytes, fibroblasts, fibrocytes, myoblasts, myocytes, myosatellite cells, tendon cells, cardiac myocytes, lipoblasts, adipocytes, interstitial cells of Cajal, angioblasts, endothelial cells, mesangial cells (e.g. intraglomerular and extraglomerular mesangial cells), juxtaglomerular cells, macula densa cells, interstitial cells The cells may be derived from one or more of: epithelial cells, stromal cells, telocyte simple epithelial cells, podocytes, renal proximal tubule brush border cells, Sertoli cells, Leydig cells, granulosa cells, Pegg cells, germ layer cells, sperm, eggs, lymphocytes, myeloid cells, endothelial progenitor cells, endothelial stem cells, hemangioblasts, mesoangioblasts, perithelial mural cells, splenocytes (e.g., T lymphocytes, B lymphocytes, dendritic cells, microphages, leukocytes), trophoblast stem cells, or any combination thereof.

[0223] Reprogramming

[0241] The term "reprogramming" as used herein may refer to a process of modifying or reversing the differentiation state of a somatic cell. The cell may be partially or terminally differentiated prior to reprogramming. Reprogramming may include a complete reversal of the differentiation state of a somatic cell to a pluripotent cell. Such a complete reversal of differentiation may generate an induced pluripotent (iPS) cell. As used herein, reprogramming may also include a partial reversal of the differentiation state of a cell, e.g., to a pluripotent state cell or a somatic cell, which is a cell that is neither pluripotent nor pluripotent but has lost one or more specific characteristics of the differentiated cell from which it originated, e.g., direct reprogramming of differentiated cells to various somatic cell types. Reprogramming may include modification, e.g., reversal, of at least some of the heritable patterns of nucleic acid modifications (e.g., methylation), chromatin condensation, epigenetics, genomic imprinting, etc., that occur during the differentiation of a cell as the zygote develops into an adult.

[0224]

[0242] As used herein, the term "reprogramming factor" may refer to a molecule associated with the "reprogramming" of a cell, i.e., differentiation, and / or dedifferentiation, and / or transdifferentiation, whereby a cell is converted to a different cell type or phenotype. Reprogramming factors generally affect the expression of genes associated with differentiation, dedifferentiation, and / or transdifferentiation of a cell. Transcription factors are examples of reprogramming factors.

[0225]

[0243] As used herein, the term "differentiation" and its grammatical equivalents refer to the process by which a less specialized cell (e.g., a more primitive cell with a higher cellular potential) becomes a more specialized cell type (e.g., a less primitive cell with a lower cellular potential), the term "dedifferentiation" refers to the process by which a more specialized cell becomes a less specialized cell type (e.g., a more primitive cell with a higher cellular potential), and the term "transdifferentiation" can refer to the process by which a cell of a particular cell type is converted to another cell type without significantly changing its level of "cellular potential" or "primitiveness." Without wishing to be bound by theory, a cell is considered to "transdifferentiate" if it is converted from one lineage-committed or terminally differentiated cell type to another lineage-committed or terminally differentiated cell type without significantly changing its level of "cellular potential" or "primitiveness."

[0226]

[0244] As used herein, the term "cell differentiation potential" is understood to mean the ability of a cell to differentiate into cells of different lineages. For example, a pluripotent cell (e.g., stem cell) has the potential to differentiate into any of the three germ layers, i.e., endoderm (internal lining of the stomach, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, urogenital tract), or ectoderm (epithelial tissue and nervous system), and thus has high cell differentiation potential. A pluripotent cell (e.g., stem cell or a type of induced stem cell) has the ability to generate cells from a large but limited number of lineages (e.g., hematopoietic stem cell, cardiac stem cell, or neural stem cell, etc.), and has a relatively lower cell differentiation potential than a pluripotent cell. Cells committed to a specific lineage or terminally differentiated may have even lower cell differentiation potential. Specific examples of transdifferentiation known in the art include, for example, the conversion of exocrine pancreatic cells to beta cells, etc.

[0227]

[0245] Thus, cells can be differentiated into more primitive cells (e.g., terminally differentiated cells can be differentiated to pluripotent or multipotent), or cells can be dedifferentiated into less primitive cells (e.g., pluripotent or multipotent cells can be differentiated into lineage committed or terminally differentiated cells). However, in one embodiment, cells can be converted or transdifferentiated from one cell type (or phenotype) to another cell type (or phenotype), e.g., at a similar cell potential level. Thus, in one embodiment of the present disclosure, the inducing step of the present disclosure can reprogram the cells of the present disclosure to differentiate, dedifferentiate, and / or transdifferentiate. In one embodiment of the present disclosure, the inducing step of the present disclosure can reprogram the cells to transdifferentiate.

[0228]

[0246] Those skilled in the art are aware of methods for reprogramming or inducing a particular type of cell to become another type of cell, for example, by differentiation, dedifferentiation, and / or transdifferentiation, using one or more exogenous polynucleotide or polypeptide reprogramming factors. Such methods may rely on the introduction of genetic material encoding one or more transcription factors or other polypeptides 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, dedifferentiation, and / or transdifferentiation of cells of the present disclosure. In some methods known to those skilled in the art, an exogenous polypeptide (e.g., a recombinant polypeptide) encoded by a reprogramming gene (such as the genes listed above) is contacted with a cell to, for example, induce a cell of the present disclosure. Those skilled in the art will recognize that other genes are also associated with cell reprogramming, and that exogenous molecules encoding such genes (or functional fragments thereof) and the encoded polypeptides are also considered to be polynucleotide or polypeptide reprogramming factors (e.g., polynucleotides or polypeptides that subsequently affect the expression levels of other genes associated with cell reprogramming). For example, it has been shown that the introduction of exogenous polynucleotide or polypeptide epigenetic gene silencer that reduces the inactivation of p53 increases the efficiency of inducing induced pluripotent stem cells (iPSCs).Therefore, exogenous polynucleotide or polypeptide encoding epigenetic silencer, as well as other genes or proteins that may directly or indirectly be involved in cell reprogramming or increase cell programming efficiency, are considered to constitute exogenous polynucleotide or polypeptide reprogramming factors.Those skilled in the art will recognize that there are other methods to affect cell reprogramming, such as the introduction of RNAi molecules (or genetic material that codes for RNAi molecules) that can knock down the expression of genes involved in inhibiting cell reprogramming.Thus, any exogenous polynucleotide or polypeptide molecule that is associated with or promotes the reprogramming of a cell should be understood to be an exogenous polynucleotide or polypeptide reprogramming factor as described herein.

[0229]

[0247] Thus, in one embodiment, the present disclosure does not include culturing cells with one or more exogenous polynucleotide or polypeptide reprogramming factors. Thus, in one embodiment, the method of the present disclosure does not include the introduction of one or more exogenous polynucleotide or polypeptide reprogramming factors, for example by introducing a transposon, a viral transgenic vector (such as a retroviral vector), a plasmid, an mRNA, an miRNA, a peptide, or a fragment of any of these molecules, that are involved in producing induced alpha, beta and / or delta cells or otherwise inducing the cells of the present disclosure to differentiate, dedifferentiate and / or transdifferentiate.

[0230]

[0248] That is, in one embodiment, the method is performed in the absence of one or more exogenous polynucleotide or polypeptide reprogramming factors (e.g., activin A). It should therefore be understood that in one embodiment, the disclosed method utilizes small molecules to reprogram cells without the addition of polypeptide transcription factors, other polypeptide factors specifically associated with inducing differentiation, dedifferentiation, and / or transdifferentiation, polynucleotide sequences encoding polypeptide transcription factors, polynucleotide sequences encoding other polypeptide factors specifically associated with inducing differentiation, dedifferentiation, and / or transdifferentiation, mRNA, interfering RNA, microRNA, and fragments thereof.

[0231] Methods for Producing Stem Cell-Derived Pancreatic Islet Cells - Patent application

[0249] Provided herein is a method for producing SC-islet cells (e.g., non-natural pancreatic β cells).Detailed examples of protocols for generating endocrine cells from stem cells to provide at least one SC-islet cell (e.g., SC-β cells) are described in US Patent Application Publication No. 20150240212, US Patent Application Publication No. 20150218522, US Patent Application Publication No. 20210198632, and US Patent Application Publication No. 20220090020, PCT Publication No. WO2022 / 147056 and WO2022 / 192300, and US Patent No. 11,466,256, each of which is incorporated herein by reference in its entirety.

[0232]

[0250] Endoderm can give rise to the digestive and respiratory tracts, thyroid, liver, and pancreas. A representative disease of the endodermal lineage is type 1 diabetes, which results from the destruction of insulin-producing beta cells. The production of functional beta cells from human pluripotent stem cells (hPSCs) in vitro could be a practical and renewable cell source for replacement cell therapy of type 1 diabetes. Embryonic stem (ES) cells, produced from the inner cell mass of blast stage embryos, represent a promising cell source for transplantation of any damaged cells or cell-based therapy. They can be maintained in culture, renewed by themselves, and propagated indefinitely as undifferentiated ES cells. ES cells can differentiate into all cell types of the organism as ectodermal, mesodermal, and endodermal lineage cells or tissues. The main advantage of ES cells is the possibility of stable self-renewal and differentiation in culture.

[0233]

[0251] Definitive endoderm is produced in vivo from the inner cell mass by the process of gastrulation during embryonic development, in which epiblast cells are directed to form the three germ layers. Definitive endoderm can give rise to a variety of cells and tissues that contribute to vital organs, such as pancreatic beta cells, hepatocytes in the liver, alveolar cells in the lungs, thyroid gland, thymus, and the epithelial lining of the nutrient and respiratory tracts. This is distinct from the primitive endoderm of extraembryonic tissues, which can give rise to visceral and parietal endoderm. Definitive endoderm derived from ES cells can theoretically become any endodermal derivative, and directing ES cells to an endodermal lineage is a prerequisite for generating therapeutic endodermal derivatives.

[0234]

[0252] Precise patterning of the anterior-posterior axis of the definitive endoderm can ultimately lead to the formation of the primitive gut tube. The primitive gut tube derived by the definitive endoderm gives rise to the pharynx, esophagus, stomach, duodenum, small intestine, and large intestine along the anterior-posterior axis, as well as associated organs such as the pancreas, lung, thyroid gland, thymus, parathyroid gland, and liver. The anterior part of the foregut of the primitive gut tube gives rise to the lung, thyroid gland, esophagus, and stomach. The pancreas, liver, and duodenum originate from the posterior part of the foregut. The midgut and hindgut of the primitive gut tube give rise to the small intestine and large intestine. The anterior foregut expresses developmental markers, NK2 homeobox (NKX2-1) and SRY (sex determining region Y)-box 2 (SOX2), the posterior foregut expresses hematopoietically expressed homeobox (HHEX), pancreatic and duodenal homeobox 1 (PDX1), onecut homeobox 1 (ONECUT1, also known as HNF6), and hepatocyte nuclear factor 4 alpha (HNF4A), and the midgut / hindgut expresses caudal homeobox 1 (CDX1), caudal homeobox 2 (CDX2), and motor neuron and pancreatic homeobox 1 (MNX1) (3, 19, 20).

[0235]

[0253] Successful differentiation of pancreatic α, β, and / or δ cells can entail that the differentiated cells synthesize and secrete physiologically relevant amounts of insulin. An exemplary stepwise protocol directing hPSC cell differentiation has been developed, involving a differentiation process that recapitulates the major stages of normal pancreatic endocrine development. Differentiation of hPSC cells into hormone-expressing pancreatic endocrine cells is achieved by transitioning hPSC cells through the major stages of embryonic development; differentiation into mesoderm and definitive endoderm, establishment of archenteron endoderm, patterning of the posterior foregut, and specification and maturation of pancreatic endoderm and endocrine precursor cells. Through these stages, hPSC cells can acquire a pancreatic endocrine phenotype and the capacity for glucose-responsive insulin secretion in vitro.

[0236]

[0254] Generally, at least one pancreatic alpha, beta and / or delta cell or precursor thereof, e.g., pancreatic progenitor, produced according to the methods disclosed herein may comprise a mixture or combination of different cells, e.g., a mixture of cells such as PDX1-positive pancreatic progenitor cells, pancreatic progenitor cells co-expressing PDX1 and NKX6-1, Ngn3-positive endocrine precursor cells, insulin-positive endocrine cells (e.g., NKX6.1-positive cells, ISL1-positive cells, or beta-like cells), and / or other pluripotent or stem cells.

[0237]

[0255] The at least one pancreatic alpha, beta and / or delta cell or precursor thereof can be produced according to any suitable culture protocol for differentiating stem cells or pluripotent cells to a desired differentiation stage. In some embodiments, the at least one pancreatic alpha, beta and / or delta cell or precursor thereof is produced by culturing at least one pluripotent cell for a period of time and under conditions suitable for the at least one pluripotent cell to differentiate into at least one pancreatic alpha, beta and / or delta cell or precursor thereof.

[0238]

[0256] In some embodiments, the at least one pancreatic alpha, beta and / or delta cell or precursor thereof is a substantially pure population of pancreatic alpha, beta and / or delta cells or precursors thereof. In some embodiments, the population of pancreatic alpha, beta and / or delta cells or precursors thereof comprises a mixture of pluripotent cells or differentiated cells. In some embodiments, the population of pancreatic alpha, beta and / or delta cells or precursors thereof is substantially free of or devoid of embryonic stem cells or pluripotent cells or iPS cells.

[0239]

[0257] In some embodiments, somatic cells, e.g., fibroblasts, can be isolated from a subject, e.g., as a tissue biopsy, e.g., a skin biopsy, and reprogrammed into induced pluripotent stem cells for further differentiation to generate at least one SC-β cell or a precursor thereof for use in the compositions and methods described herein. In some embodiments, the somatic cells, e.g., fibroblasts, are maintained in culture by methods known to those of skill in the art, and in some embodiments, expanded before being converted into pancreatic α, β and / or δ cells by the methods disclosed herein.

[0240]

[0258] In some embodiments, at least one pancreatic alpha, beta and / or delta cell or precursor thereof is maintained in culture by methods known to those of skill in the art, and in some embodiments expanded before being converted to a pancreatic alpha, beta and / or delta cell by the methods disclosed herein.

[0241]

[0259] Furthermore, at least one pancreatic α, β and / or δ cell or its precursor, e.g., pancreatic precursor, may be from any mammalian species, non-limiting examples include murine, bovine, simian, porcine, equine, ovine, or human cells. For clarity and simplicity, the method description herein refers to at least one pancreatic α, β and / or δ cell or its precursor from a mammal, but it should be understood that all of the methods described herein can be easily applied to other cell types of at least one pancreatic α, β and / or δ cell or its precursor. In some embodiments, at least one SC-β cell or its precursor is derived from a human individual.

[0242] Differentiation stage

[0260] In some embodiments, the pancreatic differentiation disclosed herein is performed in a stepwise manner. In the stepwise progression, "Stage 1" or "S1" refers to the first stage of the differentiation process, i.e., the differentiation of pluripotent stem cells into cells expressing markers characteristic of definitive endoderm cells ("DE", "Stage 1 cells" or "S1 cells"). "Stage 2" refers to the second stage, where cells expressing markers characteristic of definitive endoderm cells are differentiated into cells expressing markers characteristic of intestinal cells ("GT", "Stage 2 cells" or "S2 cells"). "Stage 3" refers to the third stage, where cells expressing markers characteristic of intestinal cells are differentiated into cells expressing markers characteristic of pancreatic progenitor 1 cells ("PP1", "Stage 3 cells" or "S3 cells"). "Stage 4" refers to the fourth stage, where cells expressing markers characteristic of pancreatic progenitor 1 cells are differentiated into cells expressing markers characteristic of pancreatic progenitor 2 cells ("PP2", "Stage 4 cells" or "S4 cells"). "Stage 5" refers to the fifth stage, in which cells express markers characteristic of pancreatic progenitor 2 cells (e.g., PDX.1 + , NKX6.1 +"Stage 6" refers to the differentiation of cells expressing markers characteristic of pancreatic endoderm cells and / or pancreatic endocrine precursor cells (e.g., insulin+) ("EN", "Stage 5 cells" or "S5 cells"). "Stage 6" refers to the differentiation of cells expressing markers characteristic of pancreatic endocrine precursor cells (e.g., insulin) to cells expressing markers characteristic of pancreatic endocrine beta cells ("SC-beta cells") or pancreatic endocrine alpha cells ("SC-alpha cells"). However, it should be understood that not all cells within a particular population progress through these stages at the same rate, i.e., a cell may progress less or more along the differentiation pathway than the majority of cells present in the population. For example, in some embodiments, SC-beta cells may be identified during stage 5, at the end of stage 5, at the beginning of stage 6, etc. Examples of methods for producing any one of stages 1 to 6 cells are provided in, for example, U.S. Patent No. 10,030,229; U.S. Patent No. 10,443,042; U.S. Patent No. 11,466,256, U.S. Patent Application Publication No. 20200332262, U.S. Patent Application Publication No. 20150240212, U.S. Patent Application Publication No. 20150218522, U.S. Patent Application Publication No. 20210198632, and U.S. Patent Application Publication No. 20220090020; and PCT Publication Nos. WO2022 / 147056 and WO2022192300, each of which is incorporated by reference in its entirety.

[0243] Definitive endoderm cells

[0261] The embodiments of the present disclosure include definitive endoderm cells.Definitive endoderm cells used herein can be derived from any source or produced according to any suitable protocol.In some embodiments, pluripotent stem cells, such as iPSCs or hESCs, are differentiated into endoderm cells.In some embodiments, endoderm cells (stage 1) are further differentiated into, for example, primitive gut tube cells (stage 2), PDX1 positive, NKX6.1 negative pancreatic progenitor cells (stage 3), NKX6.1 positive pancreatic progenitor cells (stage 4), or Ngn3 positive endocrine progenitor cells or insulin positive endocrine cells (stage 5), and then induced or matured into SC-β cells (stage 6).

[0244]

[0262] In some examples, definitive endoderm cells can be obtained by differentiating at least some of the pluripotent cells in the population into definitive endoderm cells, for example by contacting the population of pluripotent cells with i) at least one growth factor from the TGF-β superfamily and ii) a WNT signaling pathway activator to induce differentiation of at least some of the pluripotent cells into definitive endoderm cells, where the definitive endoderm cells express at least one marker characteristic of definitive endoderm.

[0245]

[0263] Any growth factor from the TGF-β superfamily that can induce pluripotent stem cells (e.g., alone or in combination with a WNT signaling pathway activator) to differentiate into definitive endoderm cells can be used in the methods provided herein. In some examples, the growth factor from the TGF-β superfamily comprises activin A. In some examples, the growth factor from the TGF-β superfamily comprises growth differentiation factor 8 (GDF8). Any WNT signaling pathway activator that can induce pluripotent stem cells (e.g., alone or in combination with a growth factor from the TGF-β superfamily) to differentiate into definitive endoderm cells can be used in the methods provided herein. In some examples, the WNT signaling pathway activator comprises CHIR99021, 3F8, A 1070722, AR-A 014418, BIO, BIO-acetoxime, FRATide, 10Z-hymenialdisine, indirubin-3'oxime, kenpaullone, L803, L803-mts, lithium carbonate, NSC 693868, SB216763, SB 415286, TC-G 24, TCS 2002, TCS 21311, or TWS 119. In some embodiments, the WNT signaling pathway activator comprises CHIR99021. In some examples, the WNT signaling pathway activator comprises Wnt3a recombinant protein.

[0246]

[0264] In some examples, differentiating at least some of the pluripotent cells in the population into definitive endoderm cells is accomplished by contacting the population of pluripotent cells with i) activin A, and ii) CHIR99021 for a suitable period of time, e.g., about 2 days, about 3 days, about 4 days, or about 5 days, to induce differentiation of at least some of the pluripotent cells in the population into definitive endoderm cells, the definitive endoderm cells expressing at least one marker characteristic of definitive endoderm. In some embodiments, the population of pluripotent cells is contacted with a growth factor from the TGF-β superfamily (e.g., activin A) and a WNT signaling pathway activator (e.g., CHIR99021) for 1, 2, or 3 days (e.g., 1 day), and the population of pluripotent cells is further contacted with a TGF-β superfamily (e.g., activin A) in the absence of the WNT signaling pathway activator (e.g., CHIR99021) for 1, 2, 3, or 4 days (e.g., 2 days).

[0247]

[0265] In some examples, the method includes differentiating the pluripotent cells into definitive endoderm cells by contacting the population of pluripotent cells with a suitable concentration, for example, about 10 ng / mL, about 20 ng / mL, about 50 ng / mL, about 75 ng / mL, about 80 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 110 ng / mL, about 120 ng / mL, about 130 ng / mL, about 140 ng / mL, about 150 ng / mL, about 175 ng / mL, about 180 ng / mL, about 200 ng / mL, about 250 ng / mL, or about 300 ng / mL of a growth factor from the TGF-β superfamily (e.g., activin A). In some examples, the method includes the use of about 100 ng / mL of activin A for the differentiation of the pluripotent cells into definitive endoderm cells. In some examples, the methods include the use of about 200 ng / mL of activin A for the differentiation of pluripotent cells to definitive endoderm cells.

[0248]

[0266] In some examples, the method includes differentiating the pluripotent cells into definitive endoderm cells by contacting the population of pluripotent cells with a suitable concentration, for example, about 0.01 μM, about 0.05 μM, about 0.1 μM, about 0.2 μM, about 0.5 μM, about 0.8 μM, about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM, about 5 μM, about 8 μM, about 10 μM, about 12 μM, about 15 μM, about 20 μM, about 30 μM, about 50 μM, about 100 μM, or about 200 μM of a WNT signaling pathway activator (e.g., CHIR99021). In some examples, the method includes using about 2 μM of CHIR99021 for the differentiation of the pluripotent cells into definitive endoderm cells. In some examples, the methods include the use of about 5 μM CHIR99021 for the differentiation of pluripotent cells to definitive endoderm cells.

[0249]

[0267] In some examples, the definitive endoderm cells generated by the methods disclosed herein express at least one marker selected from the group consisting of Nodal, Tmprss2, Tmem30b, St14, Spink3, Sh3gl2, Ripk4, Rab1S, Npnt, Clic6, Cldn5, Cacna1b, Bnip1, Anxa4, Emb, FoxA1, Sox17, and Rbm35a, and the expression of at least one marker is upregulated in a statistically significant amount in the definitive endoderm cells relative to the pluripotent stem cells from which it is derived.In some examples, the definitive endoderm cells generated by the methods disclosed herein do not express at least one marker selected from the group consisting of Gata4, SPARC, AFP, and Dab2 in a statistically significant amount relative to the pluripotent stem cells from which it is derived. In some examples, the definitive endoderm cells generated by the methods disclosed herein do not express at least one marker selected from the group consisting of Zic1, Pax6, Flk1, and CD31 in a statistically significant amount relative to the pluripotent stem cells from which they are derived. In some examples, the definitive endoderm cells generated by the methods disclosed herein have a statistically significant higher level of phosphorylation of Smad2 relative to the pluripotent stem cells from which they are derived. In some examples, the definitive endoderm cells generated by the methods disclosed herein have the ability to form a gut tube in vivo. In some examples, the definitive endoderm cells generated by the methods disclosed herein can be differentiated into cells with morphology characteristic of gut cells, and the cells with morphology characteristic of gut cells express FoxA2 and / or Claudin6. In some examples, the definitive endoderm cells generated by the methods disclosed herein can be further differentiated into cells of endodermal origin.

[0250]

[0268] In some examples, the population of pluripotent stem cells is cultured in the presence of at least one β cell differentiation factor before any differentiation or during the first stage of differentiation. Any pluripotent stem cells can be used, such as human pluripotent stem cells or human iPS cells, or any pluripotent stem cells discussed herein or other suitable pluripotent stem cells. In some examples, the β cell differentiation factor described herein can be present in the culture medium of the population of pluripotent stem cells, or can be added once or periodically during the growth (e.g., replication or expansion) of the population of pluripotent stem cells. In certain examples, the population of pluripotent stem cells can be exposed to at least one β cell differentiation factor before any differentiation. In other examples, the population of pluripotent stem cells can be exposed to at least one β cell differentiation factor during the first stage of differentiation.

[0251] gastrula cells

[0269] The embodiments of the present disclosure include primary gut cells.The primary gut cells used herein can be derived from any source or produced according to any suitable protocol.In some embodiments, definitive endoderm cells are differentiated into primary gut cells.In some embodiments, primary gut cells are further differentiated into, for example, PDX1 positive, NKX6.1 negative pancreatic progenitor cells, NKX6.1 positive pancreatic progenitor cells, Ngn3 positive endocrine progenitor cells, insulin positive endocrine cells, and then induced or matured into SC-β cells.

[0252]

[0270] In some examples, primitive gut tube cells can be obtained by differentiating at least some of the definitive endoderm cells in the population into primitive gut tube cells, for example, by contacting the definitive endoderm cells with at least one growth factor from the fibroblast growth factor (FGF) family to induce differentiation of at least some of the definitive endoderm cells into primitive gut tube cells, where the primitive gut tube cells express at least one marker characteristic of primitive gut tube cells.

[0253]

[0271] Any growth factor from the FGF family that can induce definitive endoderm cells (e.g., alone or in combination with other factors) to differentiate into primitive gut cells can be used in the methods provided herein. In some examples, at least one growth factor from the FGF family comprises keratinocyte growth factor (KGF). In some examples, at least one growth factor from the FGF family comprises FGF2. In some examples, at least one growth factor from the FGF family comprises FGF8B. In some examples, at least one growth factor from the FGF family comprises FGF10. In some examples, at least one growth factor from the FGF family comprises FGF21.

[0254]

[0272] In some examples, primitive gut tube cells can be obtained by differentiating at least some of the definitive endoderm cells in the population into primitive gut tube cells, for example, by contacting the definitive endoderm cells with KGF for a period of time, for example, about 1 day, about 2 days, about 3 days, about 4 days, or about 5 days, to induce differentiation of at least some of the definitive endoderm cells into primitive gut tube cells.

[0255]

[0273] In some examples, the method includes differentiating definitive endoderm cells into primitive gut cells by contacting the definitive endoderm cells with a suitable concentration of, for example, about 10ng / mL, about 20ng / mL, about 50ng / mL, about 75ng / mL, about 80ng / mL, about 90ng / mL, about 95ng / mL, about 100ng / mL, about 110ng / mL, about 120ng / mL, about 130ng / mL, about 140ng / mL, about 150ng / mL, about 175ng / mL, about 180ng / mL, about 200ng / mL, about 250ng / mL, or about 300ng / mL of a growth factor from the FGF family (e.g., KGF). In some examples, the method includes the use of about 50ng / mL of KGF for the differentiation of definitive endoderm cells into primitive gut cells. In some examples, the method includes the use of about 100ng / mL of KGF for the differentiation of definitive endoderm cells into primitive gut cells.

[0256] PDX1-positive pancreatic progenitor cells

[0274] The embodiments of the present disclosure include PDX1 positive, NKX6.1 negative pancreatic progenitor cells.The PDX1 positive, NKX6.1 negative pancreatic progenitor cells used herein can be derived from any source or produced according to any suitable protocol.In some embodiments, primary gut cells are differentiated into PDX1 positive, NKX6.1 negative pancreatic progenitor cells.In some embodiments, the PDX1 positive, NKX6.1 negative pancreatic progenitor cells are further differentiated into, for example, NKX6.1 positive pancreatic progenitor cells, Ngn3 positive endocrine progenitor cells, insulin positive endocrine cells, and then induced or matured into pancreatic alpha, beta and / or delta cells.

[0257]

[0275] In some embodiments, the PDX1-positive, NKX6.1-negative pancreatic progenitor cells can be obtained by differentiating at least a portion of the primary gut cells in the population into PDX1-positive, NKX6.1-negative pancreatic progenitor cells, e.g., by contacting the primary gut cells with i) at least one BMP signaling pathway inhibitor, ii) a growth factor from the TGF-β superfamily, iii) at least one growth factor from the FGF family, iv) at least one SHH pathway inhibitor, v) at least one retinoic acid (RA) signaling pathway activator, vi) at least one protein kinase C activator, and vii) a ROCK inhibitor, to induce differentiation of at least a portion of the primary gut cells into PDX1-positive, NKX6.1-negative pancreatic progenitor cells, wherein the PDX1-positive, NKX6.1-negative pancreatic progenitor cells express PDX1.

[0258]

[0276] In some embodiments, PDX1-positive, NKX6.1-negative pancreatic progenitor cells can be obtained by differentiating at least a portion of the primary gut cells in the population into PDX1-positive, NKX6.1-negative pancreatic progenitor cells, for example, by contacting the primary gut cells with i) at least one BMP signaling pathway inhibitor, ii) a growth factor from the TGF-β superfamily, iii) at least one growth factor from the FGF family, iv) at least one SHH pathway inhibitor, v) at least one retinoic acid (RA) signaling pathway activator, and vi) at least one protein kinase C activator to induce differentiation of at least a portion of the primary gut cells into PDX1-positive, NKX6.1-negative pancreatic progenitor cells.

[0259]

[0277] In some cases, the PDX1-positive, NKX6.1-negative pancreatic progenitor cells can be obtained by differentiating at least a portion of the primitive duct cells in the population into PDX1-positive, NKX6.1-negative pancreatic progenitor cells, e.g., by contacting the primitive duct cells with i) at least one BMP signaling pathway inhibitor, ii) at least one growth factor from the FGF family, iii) at least one SHH pathway inhibitor, iv) at least one retinoic acid (RA) signaling pathway activator, and v) at least one protein kinase C activator to induce differentiation of at least a portion of the primitive duct cells into PDX1-positive, NKX6.1-negative pancreatic progenitor cells, wherein the PDX1-positive, NKX6.1-negative pancreatic progenitor cells express PDX1.

[0260]

[0278] In some examples, the PDX1-positive, NKX6.1-negative pancreatic progenitor cells can be obtained by differentiating at least a portion of the primitive gut cells in the population into PDX1-positive, NKX6.1-negative pancreatic progenitor cells, e.g., by contacting the primitive gut cells with i) at least one SHH pathway inhibitor, ii) at least one retinoic acid (RA) signaling pathway activator; and iii) at least one protein kinase C activator, wherein the PDX1-positive, NKX6.1-negative pancreatic progenitor cells express PDX1.

[0261]

[0279] In some examples, PDX1-positive, NKX6.1-negative pancreatic progenitor cells can be obtained by differentiating at least a portion of the primary gut cells in the population into PDX1-positive, NKX6.1-negative pancreatic progenitor cells, for example, by contacting the primary gut cells with i) at least one growth factor from the FGF family, and ii) at least one activator of the retinoic acid (RA) signaling pathway to induce differentiation of at least a portion of the primary gut cells into PDX1-positive, NKX6.1-negative pancreatic progenitor cells, wherein the PDX1-positive, NKX6.1-negative pancreatic progenitor cells express PDX1.

[0262]

[0280] Any BMP signaling pathway inhibitor that can induce primary gut cells to differentiate into PDX1-positive, NKX6.1-negative pancreatic progenitor cells (e.g., alone or in combination with a growth factor from the TGF-β superfamily, at least one growth factor from the FGF family, at least one SHH pathway inhibitor, at least one retinoic acid signaling pathway activator, at least one protein kinase C activator, and a ROCK inhibitor) can be used in the methods provided herein. In some examples, the BMP signaling pathway inhibitor comprises LDN193189 or DMH-1. In some examples, the method includes contacting the gut cells with a BMP signaling pathway inhibitor (e.g., LDN193189) at a concentration of, for example, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 110 nM, about 120 nM, about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about 200 nM, about 210 nM, about 220 nM, about 230 nM, about 240 nM, about 250 nM, about 280 nM, about 300 nM, about 400 nM, about 500 nM, or about 1 μM. In some examples, the method includes contacting the gut cells with a BMP signaling pathway inhibitor (e.g., DMH-1) at a concentration of, for example, about 0.01 μM, about 0.02 μM, about 0.05 μM, about 0.1 μM, about 0.2 μM, about 0.5 μM, about 0.8 μM, about 1 μM, about 1.2 μM, about 1.5 μM, about 1.75 μM, about 2 μM, about 2.2 μM, about 2.5 μM, about 2.75 μM, about 3 μM, about 3.25 μM, about 3.5 μM, about 3.75 μM, about 4 μM, about 4.5 μM, about 5 μM, about 8 μM, about 10 μM, about 15 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, or about 100 μM.

[0263]

[0281] Any growth factor from the TGF-β superfamily that can induce the primitive gut cells to differentiate into PDX1-positive, NKX6.1-negative pancreatic progenitor cells (e.g., alone or in combination with at least one BMP signaling pathway inhibitor, a growth factor from the FGF family, at least one SHH pathway inhibitor, at least one retinoic acid signaling pathway activator, at least one protein kinase C activator, and a ROCK inhibitor) can be used. In some examples, the growth factor from the TGF-β family includes activin A. In some examples, the growth factor from the TGF-β family includes activin A or GDF8. In some examples, the method includes contacting the primary gut cells with a growth factor from the TGF-β superfamily (e.g., activin A) at a concentration of, for example, about 5 ng / mL, about 7.5 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 11 ng / mL, about 12 ng / mL, about 13 ng / mL, about 14 ng / mL, about 15 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, about 19 ng / mL, about 20 ng / mL, about 21 ng / mL, about 22 ng / mL, about 23 ng / mL, about 24 ng / mL, about 25 ng / mL, about 26 ng / mL, about 27 ng / mL, about 28 ng / mL, about 29 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, or about 100 ng / mL.

[0264]

[0282] Any growth factor from the FGF family that can induce the primitive gut cells to differentiate into PDX1-positive, NKX6.1-negative pancreatic progenitor cells (for example, alone or in combination with at least one BMP signaling pathway inhibitor, a growth factor from the TGF-β superfamily, at least one SHH pathway inhibitor, at least one retinoic acid signaling pathway activator, at least one protein kinase C activator, and a ROCK inhibitor) can be used. In some examples, the at least one growth factor from the FGF family includes keratinocyte growth factor (KGF). In some examples, the at least one growth factor from the FGF family is selected from the group consisting of FGF2, FGF8B, FGF10, and FGF21. In some examples, the method includes contacting the primary gut cells with a growth factor from the FGF family (e.g., KGF) at a concentration of, for example, about 10 ng / mL, about 20 ng / mL, about 50 ng / mL, about 75 ng / mL, about 80 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 110 ng / mL, about 120 ng / mL, about 130 ng / mL, about 140 ng / mL, about 150 ng / mL, about 175 ng / mL, about 180 ng / mL, about 200 ng / mL, about 250 ng / mL, or about 300 nM / mL. In some examples, the method includes contacting the primary gut cells with a growth factor from the FGF family (e.g., KGF) at a concentration of 10-200 ng / mL, 10-150 ng / mL, 10-100 ng / mL, 25-75 ng / mL, 40-60 ng / mL, or 45-55 ng / mL, etc.

[0265]

[0283] Any SHH pathway inhibitor that can induce primary gut cells to differentiate into PDX1-positive, NKX6.1-negative pancreatic progenitor cells (e.g., alone or in combination with at least one BMP signaling pathway inhibitor, at least one growth factor from the FGF family, a growth factor from the TGF-β superfamily, at least one retinoic acid signaling pathway activator, at least one protein kinase C activator, and a ROCK inhibitor) can be used. In some examples, the SHH pathway inhibitor comprises Sant1. In some examples, the method comprises administering to the primitive gut cells at about 0.001 μM, about 0.002 μM, about 0.005 μM, about 0.01 μM, about 0.02 μM, about 0.03 μM, about 0.05 μM, about 0.08 μM, about 0.1 μM, about 0.12 μM, about 0.13 μM, about 0.14 μM, about 0.15 μM, about 0.16 μM, about 0.17 μM, about 0.18 μM, about 0.19 μM, about 0.2 μM, about 0.21 μM, about 0.22 μM, about 0.23 μM, about 0.24 μM, about 0.25 μM, about 0.26 μM, about 0.27 μM, about 0.28 μM, about 0.29 μM, about 0.30 μM, about 0.31 μM, about 0.32 μM, about 0.33 μM, about 0.34 μM, about 0.35 μM, about 0.36 μM, about 0.37 μM, about 0.38 μM, about 0.39 μM, about 0.40 μM, about 0.41 μM, about 0.42 μM, about 0.43 μM, about 0.44 μM, about 0.45 μM, about 0.46 μM, about 0.47 μM, about 0.48 μM, about 0.49 μM, about 0.50 μM, about 0.51 μM, about 0.52 μM, about 0.53 μM, about 0.54 μM, about 0.55 μM, about 0.56 μM, about 0.57 μM, The method includes contacting the cell with an SHH pathway inhibitor (e.g., Sant1) at a concentration of about 0.24 μM, about 0.25 μM, about 0.26 μM, about 0.27 μM, about 0.28 μM, about 0.29 μM, about 0.3 μM, about 0.31 μM, about 0.32 μM, about 0.33 μM, about 0.34 μM, about 0.35 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about 0.6 μM, about 0.8 μM, about 1 μM, about 2 μM, or about 5 μM. In some examples, the method includes contacting the gut cells with an SHH pathway inhibitor (e.g., Sant1) at a concentration of 50-1000 nM, 50-500 nM, 50-300 nM, 100-300 nM, 200-300 nM, 200-500 nM, or 225-275 nM.

[0266]

[0284] Any RA signaling pathway activator that can induce primitive gut cells to differentiate into PDX1-positive, NKX6.1-negative pancreatic progenitor cells (e.g., alone or in combination with at least one BMP signaling pathway inhibitor, at least one growth factor from the FGF family, at least one SHH pathway inhibitor, at least one protein kinase C activator, and a ROCK inhibitor) can be used. In some examples, the RA signaling pathway activator comprises retinoic acid. In some examples, the method comprises administering to the primary intestine cells at about 0.02 μM, about 0.1 μM, about 0.2 μM, about 0.25 μM, about 0.3 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about 0.55 μM, about 0.6 μM, about 0.65 μM, about 0.7 μM, about 0.75 μM, about 0.8 μM, about 0.85 μM, about 0.9 μM, about 1 μM, about 1.1 μM, about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM, about 1.8 μM, about 1.9 μM, about 2 μM, about 2.1 μM, about 2.2 μM, about 2.3 μM, about 2.4 μM, about 2.5 μM , about 2.6 μM, about 2.7 μM, about 2.8 μM, about 3 μM, about 3.2 μM, about 3.4 μM, about 3.6 μM, about 3.8 μM, about 4 μM, about 4.2 μM, about 4.4 μM, about 4.6 μM, about 4.8 μM, about 5 μM, about 5.5 μM, about 6 μM, about 6.5 μM, about 7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM, about 10 μM, about 12 μM, about 14 μM, about 15 μM, about 16 μM, about 18 μM, about 20 μM, about 50 μM, or about 100 μM of a RA signaling pathway activator (e.g., retinoic acid). In some examples, the method includes contacting the gut cells with an RA signaling pathway activator (e.g., retinoic acid) at a concentration of 0.2-5 μM, 0.8-3 μM, 0.8-2.5 μM, 1-2.5 μM, 1.5-2.5 μM, 1.8-2.2 μM, or 1.9-2.1 μM, etc.

[0267]

[0285] Any PKC activator that can induce primary gut cells to differentiate into PDX1-positive, NKX6.1-negative pancreatic progenitor cells (e.g., alone or in combination with at least one BMP signaling pathway inhibitor, at least one growth factor from the FGF family, at least one SHH pathway inhibitor, at least one RA signaling pathway activator, and a ROCK inhibitor) can be used. In some examples, the PKC activator comprises PdBU. In some examples, the PKC activator comprises TPPB. In some examples, the method includes administering to the primitive gut cells at a concentration of, for example, about 10 nM, 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM, 1 μM, 10 μM, about 20 μM, about 50 μM, about 75 μM, about 80 μM, about 100 μM, about 120 μM, about 140 μM, about 150 μM, about 175 μM, about 180 μM, about 200 μM, about 210 μM, about 220 μM, about 240 μM, about 250 μM, about 26 The method includes contacting the PKC activator (e.g., PdBU or TPPB) at a concentration of about 0 μM, about 280 μM, about 300 μM, about 320 μM, about 340 μM, about 360 μM, about 380 μM, about 400 μM, about 420 μM, about 440 μM, about 460 μM, about 480 μM, about 500 μM, about 520 μM, about 540 μM, about 560 μM, about 580 μM, about 600 μM, about 620 μM, about 640 μM, about 660 μM, about 680 μM, about 700 μM, about 750 μM, about 800 μM, about 850 μM, about 900 μM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, or about 5 mM. In some embodiments, the method includes contacting the primary gut cells with a PKC activator (e.g., PdBU or TPPB) at a concentration of, for example, 10 nM to 1 mM, 10 nM to 500 μM, 10 nM to 1 μM, 10 to 800 nM, 100 to 900 nM, 300 to 800 nM, 300 to 600 nM, 400 to 600 nM, 450 to 550 nM, or about 500 nM. In some embodiments, the primary gut cells are not treated with a PKC activator (e.g., PDBU).

[0268]

[0286] Any ROCK inhibitor that can induce primitive gut cells to differentiate into PDX1 positive, NKX6.1 negative pancreatic progenitor cells (for example, alone or in combination with at least one BMP signaling pathway inhibitor, at least one growth factor from FGF family, at least one SHH pathway inhibitor, PKC activator, and at least one RA signaling pathway activator) can be used. In some examples, the ROCK inhibitor comprises Thiazovivin, Y-27632, Fasudil / HA1077, or H-1152. In some examples, the ROCK inhibitor comprises Y-27632. In some examples, the ROCK inhibitor comprises Thiazovivin. In some examples, the method further comprises administering to the gut cells at about 0.2 μM, about 0.5 μM, about 0.75 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 7.5 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, The method includes contacting the primary intestinal cells with a ROCK inhibitor (e.g., Y-27632 or Thiazovivin) at a concentration of about 19 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 35 μM, about 40 μM, about 50 μM, or about 100 μM. In some examples, the method includes contacting the primary intestinal cells with a ROCK inhibitor (e.g., Y-27632 or Thiazovivin) at a concentration of 0.2 to 5 μM, 0.8 to 3 μM, 1 to 4 μM, 1.5 to 4 μM, 1.8 to 3.5 μM, 2 to 3 μM, 2.4 to 2.6 μM, etc.

[0269]

[0287] In some examples, PDX1-positive, NKX6.1-negative pancreatic progenitor cells can be obtained by differentiating at least some of the primary gut cells in the population into PDX1-positive, NKX6.1-negative pancreatic progenitor cells, for example, by contacting the primary gut cells with retinoic acid, KGF, Sant1, DMH-1, PdBU, thiazovivin, and activin A for a suitable period of time, for example, about 1 day, about 2 days, about 3 days, or about 4 days. In some examples, PDX1-positive, NKX6.1-negative pancreatic progenitor cells can be obtained by differentiating at least some of the primary gut cells in the population into PDX1-positive, NKX6.1-negative pancreatic progenitor cells, for example, by contacting the primary gut cells with retinoic acid, KGF, Sant1, DMH-1, PdBU, thiazovivin, and activin A for about 2 days.

[0270]

[0288] In some embodiments, PDX1 positive, NKX6.1 negative pancreatic progenitor cells are differentiated by differentiating at least a portion of the primary gut cells in a population into PDX1 positive, NKX6.1 negative pancreatic progenitor cells, e.g., by treating the primary gut cells with i) at least one BMP signaling pathway inhibitor (e.g., DMH-1), ii) a growth factor from the TGF-β superfamily (e.g., activin A), iii) at least one growth factor from the FGF family (e.g., KGF), iv) at least one SHH pathway inhibitor (e.g., Sant1), v) at least one retinoic acid (RA) signaling pathway activator (e.g., retinoic acid), vi) at least one protein kinase C activator (e.g., PDBU), and vii) at least one ROCK inhibitor. (e.g., thiazovivin) for 1, 2, 3, 4, or 4 days (e.g., 1 day), and then contacting with i) a growth factor from the TGF-β superfamily (e.g., activin A), ii) at least one growth factor from the FGF family (e.g., KGF), iii) at least one SHH pathway inhibitor (e.g., Sant1), iv) at least one retinoic acid (RA) signaling pathway activator (e.g., retinoic acid), v) at least one protein kinase C activator (e.g., PDBU), and vi) at least one ROCK inhibitor (e.g., thiazovivin) for 1, 2, 3, 4, or 5 days (e.g., 1 day) in the absence of a BMP signaling pathway inhibitor (e.g., DMH-1).

[0271] NKX6.1-positive pancreatic progenitor cells

[0289] The embodiments of the present disclosure include PDX1 positive, NKX6.1 positive pancreatic progenitor cells.The PDX1 positive, NKX6.1 positive pancreatic progenitor cells used herein can be derived from any source or produced according to any suitable protocol.In some embodiments, the PDX1 positive, NKX6.1 negative pancreatic progenitor cells are differentiated into PDX1 positive, NKX6.1 positive pancreatic progenitor cells.In some embodiments, the PDX1 positive, NKX6.1 positive pancreatic progenitor cells are further differentiated into, for example, Ngn3 positive endocrine progenitor cells or insulin positive endocrine cells, and then induced or matured into SC-β cells.

[0272]

[0290] In some embodiments, a method for generating PDX1-positive, NKX6.1-positive pancreatic progenitor cells from PDX1-positive, NKX6.1-negative pancreatic progenitor cells includes contacting a cell population comprising PDX1-positive, NKX6.1-negative pancreatic progenitor cells (e.g., under conditions that promote cell clustering and / or promote cell survival) with at least two beta cell differentiation factors comprising a) at least one growth factor from the fibroblast growth factor (FGF) family, b) a sonic hedgehog pathway inhibitor, and optionally c) a retinoic acid (RA) signaling pathway activator to induce differentiation of at least one PDX1-positive, NKX6.1-negative pancreatic progenitor cell in the population into a PDX1-positive, NKX6.1-positive pancreatic progenitor cell, wherein the PDX1-positive, NKX6.1-positive pancreatic progenitor cell expresses NKX6.1.

[0273]

[0291] In some cases, the PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive, NKX6.1-negative pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) an RA signaling pathway activator to induce differentiation of at least some of the PDX1-positive, NKX6.1-positive pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells, wherein the PDX1-positive, NKX6.1-positive pancreatic progenitor cells express PDX1 and NKX6.1.

[0274]

[0292] In some cases, the PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive, NKX6.1-negative pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) an RA signaling pathway activator, iv) a ROCK inhibitor, v) at least one growth factor from the TGF-β superfamily to induce differentiation of at least some of the PDX1-positive, NKX6.1-negative pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells. In some embodiments, after 3, 4, or 5 days of contact, PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive, NKX6.1-negative pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) RA signaling pathway activator, iv) ROCK inhibitor, and v) at least one growth factor from the TGF-β superfamily; the cells are then contacted with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) RA signaling pathway activator, iv) ROCK inhibitor, and v) at least one growth factor from the TGF-β superfamily, and vi) a PKC activator and optionally a gamma-secretase inhibitor. In some cases, PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive, NKX6.1-negative pancreatic progenitor cells with at least one growth factor from the FGF family. In some cases, the growth factor from the FGF family is KGF.

[0275]

[0293] Any growth factor from the FGF family that can induce PDX1 positive, NKX6.1 negative pancreatic progenitor cells to differentiate into PDX1 positive, NKX6.1 positive pancreatic progenitor cells (for example, alone or in combination with at least one SHH pathway inhibitor, ROCK inhibitor, growth factor from TGF-β superfamily, and at least one retinoic acid signaling pathway activator) can be used in the methods provided herein. In some examples, the at least one growth factor from the FGF family comprises keratinocyte growth factor (KGF). In some examples, the at least one growth factor from the FGF family is selected from the group consisting of FGF8B, FGF10, and FGF21. In some examples, the method includes contacting PDX1 positive, NKX6.1 negative pancreatic progenitor cells with a growth factor from the FGF family (e.g., KGF) at a concentration of, for example, about 10 ng / mL, about 20 ng / mL, about 50 ng / mL, about 75 ng / mL, about 80 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 110 ng / mL, about 120 ng / mL, about 130 ng / mL, about 140 ng / mL, about 150 ng / mL, about 175 ng / mL, about 180 ng / mL, about 200 ng / mL, about 250 ng / mL, or about 300 ng / mL. In some examples, the method includes contacting PDX1-positive, NKX6.1-negative pancreatic progenitor cells with a growth factor from the FGF family (e.g., KGF) at a concentration of 10-200 ng / mL, 10-150 ng / mL, 10-100 ng / mL, 25-75 ng / mL, 40-60 ng / mL, or 45-55 ng / mL, etc.

[0276]

[0294] Any SHH pathway inhibitor that can induce PDX1 positive, NKX6.1 negative pancreatic progenitor cells to differentiate into PDX1 positive, NKX6.1 positive pancreatic progenitor cells (for example, alone or in combination with at least one growth factor from FGF family, at least one retinoic acid signaling pathway activator, ROCK inhibitor, and at least one growth factor from TGF-β superfamily) can be used in the methods provided herein. In some examples, the SHH pathway inhibitor comprises Sant1. In some examples, the method comprises administering to the patient PDX1-positive, NKX6.1-negative pancreatic progenitor cells at a concentration of, for example, about 0.001 μM, about 0.002 μM, about 0.005 μM, about 0.01 μM, about 0.02 μM, about 0.03 μM, about 0.05 μM, about 0.08 μM, about 0.1 μM, about 0.12 μM, about 0.13 μM, about 0.14 μM, about 0.15 μM, about 0.16 μM, about 0.17 μM, about 0.18 μM, about 0.19 μM, about 0.2 μM, about 0.21 μM, about 0.22 μM, about 0.26 μM, about 0.28 μM, about 0.29 μM, about 0.30 μM, about 0.31 μM, about 0.32 μM, about 0.33 μM, about 0.34 μM, about 0.35 μM, about 0.36 μM, about 0.37 μM, about 0.38 μM, about 0.39 μM, about 0.40 μM, about 0.41 μM, about 0.42 μM, about 0.43 μM, about 0.44 μM, about 0.45 μM, about 0.46 μM, about 0.47 μM, about 0.48 μM, about 0.49 μM, about 0.50 μM, about 0.51 μM, about 0.52 μM, about 0.53 μM, about 0.54 μM, about 0.55 μM, about 0.56 μM, about 0.57 μM, about 0.58 μ The method includes contacting the cell with an SHH pathway inhibitor (e.g., Sant1) at a concentration of about 0.23 μM, about 0.24 μM, about 0.25 μM, about 0.26 μM, about 0.27 μM, about 0.28 μM, about 0.29 μM, about 0.3 μM, about 0.31 μM, about 0.32 μM, about 0.33 μM, about 0.34 μM, about 0.35 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about 0.6 μM, about 0.8 μM, about 1 μM, about 2 μM, or about 5 μM. In some examples, the method includes contacting PDX1-positive, NKX6.1-negative pancreatic progenitor cells with a SHH pathway inhibitor (e.g., Sant1) at a concentration of 50-1000 nM, 50-500 nM, 50-300 nM, 100-300 nM, 200-300 nM, 200-500 nM, or 225-275 nM, etc.

[0277]

[0295] Any RA signaling pathway activator that can induce PDX1 positive, NKX6.1 negative pancreatic progenitor cells to differentiate into PDX1 positive, NKX6.1 positive pancreatic progenitor cells (e.g., alone or in combination with at least one growth factor from the FGF family, at least one SHH pathway inhibitor, a ROCK inhibitor, and at least one growth factor from the TGF-β superfamily) can be used. In some examples, the RA signaling pathway activator comprises retinoic acid. In some examples, the method comprises administering to the patient a PDX1 positive, NKX6.1 negative pancreatic progenitor cell at a concentration of, for example, about 0.02 μM, about 0.1 μM, about 0.2 μM, about 0.25 μM, about 0.3 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about 0.55 μM, about 0.6 μM, about 0.65 μM, about 0.7 μM, about 0.75 μM, about 0.8 μM, about 0.85 μM, about 0.9 μM, about 1 μM, about 1.1 μM, about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM, about 1.8 μM, about 1.9 μM, about 2 μM, about 2.1 μM, about 2.2 μM, about 2.3 μM, about 2.4 μM, about 2.5 μM, about 2.6 μM, about 2.7 μM, about 2.8 μM, about 2.9 μM, about 3.0 μM, about 3.1 μM, about 3.2 μM, about 3.3 μM, about 3.4 μM, about 3.5 μM, about 3.6 μM, about 3.7 μM, about 3.8 μM, about 3.9 μM, about 4.0 μM, about 4.1 μM, about 4.2 μM, about 4.3 μM, about 4.4 μM, about 4.5 μM, about 4.6 μM, about 4.7 μM, about 4.8 μM, about 4.9 μM, about 5.0 μM, about 5.1 μM, about 5 M, about 2.5 μM, about 2.6 μM, about 2.7 μM, about 2.8 μM, about 3 μM, about 3.2 μM, about 3.4 μM, about 3.6 μM, about 3.8 μM, about 4 μM, about 4.2 μM, about 4.4 μM, about 4.6 μM, about 4.8 μM, about 5 μM, about 5.5 μM, about 6 μM, about 6.5 μM, about 7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM, about 10 μM, about 12 μM, about 14 μM, about 15 μM, about 16 μM, about 18 μM, about 20 μM, about 50 μM, or about 100 μM of a RA signaling pathway activator (e.g., retinoic acid). In some examples, the method includes contacting PDX1-positive, NKX6.1-negative pancreatic progenitor cells with a RA signaling pathway activator (e.g., retinoic acid) at a concentration of 1-500 nM, 50-400 nM, 50-250 nM, 50-150 nM, 80-200 nM, 75-125 nM, or 90-110 nM, etc.

[0278]

[0296] Any ROCK inhibitor that can induce PDX1 positive, NKX6.1 negative pancreatic progenitor cells to differentiate into PDX1 positive, NKX6.1 positive pancreatic progenitor cells (for example, alone or in combination with at least one growth factor from FGF family, at least one SHH pathway inhibitor, RA signaling pathway activator, and at least one growth factor from TGF-β superfamily) can be used. In some examples, the ROCK inhibitor comprises Thiazovivin, Y-27632, Fasudil / HA1077, or 14-1152. In some examples, the method comprises administering to the patient PDX1-positive, NKX6.1-negative pancreatic progenitor cells at a concentration of, for example, about 0.2 μM, about 0.5 μM, about 0.75 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 7.5 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 31 μM, about 32 μM, about 33 μM, about 34 μM, about 35 μM, about 36 μM, about 37 μM, about 38 μM, about 39 μM, about 40 μM, about 41 μM, about 42 μM, about 43 μM, about 44 μM, about 45 μM, about 46 μM, about 47 μM, about 48 μM, about 49 μM, about 50 μM, about 51 μM, about 52 μM, about 53 μM, about 54 μM, about 55 μM, about 56 μM, about 57 μM, about 58 μM, about 59 μM, about 60 μM, about 61 μM, about 62 μM, about 63 μM, about 64 μM, about M, about 18 μM, about 19 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 35 μM, about 40 μM, about 50 μM, or about 100 μM of a ROCK inhibitor (e.g., Y-27632 or Thiazovivin). In some examples, the method includes contacting the PDX1-positive, NKX6.1-negative pancreatic progenitor cells with a ROCK inhibitor (e.g., Y-27632 or Thiazovivin) at a concentration of 0.2-5 μM, 0.8-3 μM, 1-4 μM, 1.5-4 μM, 1.8-3.5 μM, 2-3 μM, 2.4-2.6 μM, or the like.

[0279]

[0297] Any activator from the TGF-β superfamily can be used that can induce PDX1 positive, NKX6.1 negative pancreatic progenitor cells to differentiate into PDX1 positive, NKX6.1 positive pancreatic progenitor cells (for example, alone or in combination with at least one growth factor from the FGF family, at least one SHH pathway inhibitor, RA signaling pathway activator, and ROCK inhibitor).In some examples, the activator from the TGF-β superfamily includes activin A or GDF8. In some examples, the method involves treating PDX1 positive, NKX6.1 negative pancreatic progenitor cells with a concentration of a growth factor from the TGF-β superfamily (e.g., activin A), such as about 0.1 ng / mL, about 0.2 ng / mL, about 0.3 ng / mL, about 0.4 ng / mL, about 0.5 ng / mL, about 0.6 ng / mL, about 0.7 ng / mL, about 0.8 ng / mL, about 1 ng / mL, about 1.2 ng / mL, about 1.4 ng / mL, about 1.6 ng / mL, about 1.8 ng / mL, about 2 ng / mL, about 2.2 ng / mL, about 2.4 ng / mL, about 2.6 ng / mL, about 2.8 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 11 ng / mL, about 12 ng / mL, about 13 ng / mL, about 14 ng / mL, about 15 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, about 19 ng / mL, about 22 ng / mL, about 23 ng / mL, about 24 ng / mL, about 25 ng / mL, about 26 ng / mL, about 27 ng / mL, about 28 ng / mL, about 29 ng / mL, about 30 ng / mL, about 31 ng / mL, about 32 ng / mL, about 33 ng / mL, about 34 ng / mL, about 35 ng / mL, about 36 ng / The method includes contacting the antibody with about 3.2 ng / mL, about 3.4 ng / mL, about 3.6 ng / mL, about 3.8 ng / mL, about 4 ng / mL, about 4.2 ng / mL, about 4.4 ng / mL, about 4.6 ng / mL, about 4.8 ng / mL, about 5 ng / mL, about 5.2 ng / mL, about 5.4 ng / mL, about 5.6 ng / mL, about 5.8 ng / mL, about 6 ng / mL, about 6.2 ng / mL, about 6.4 ng / mL, about 6.6 ng / mL, about 6.8 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, or about 50 ng / mL. In some examples, the method includes contacting PDX1-positive, NKX6.1-negative pancreatic progenitor cells with a concentration of a growth factor from the TGF-β superfamily (e.g., activin A), e.g., about 5 ng / mL. In some embodiments, the concentration of the growth factor from the TGF-β superfamily (e.g., activin A) is 1-15 ng / mL, 3-12 ng / mL, 5-12 ng / mL, 5-20 ng / mL, 8-20 ng / mL, 8-15 ng / mL, 9-11 ng / mL, or 8-12 ng / mL.

[0280]

[0298] In some cases, the PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive, NKX6.1-negative pancreatic progenitor cells with KGF, Sant1, and RA for 5 or 6 days. In some cases, the PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive, NKX6.1-negative pancreatic progenitor cells with KGF, Sant1, RA, thiazovivin, and activin A for 5 or 6 days. In some cases, the PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive, NKX6.1-negative pancreatic progenitor cells with KGF for 5 days. In some cases, the PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting the PDX1-positive, NKX6.1-negative pancreatic progenitor cells with i) at least one growth factor from the FGF family (e.g., KGF), ii) at least one SHH pathway inhibitor (e.g., Sant1), iii) at least one RA signaling pathway activator (e.g., retinoic acid), iv) at least one ROCK inhibitor (e.g., thiazovivin), and v) at least one growth factor from the TGF-β superfamily (e.g., activin A) for 1, 2, 3, 4, 5, 6, 7, 8, or 9 days (e.g., 5 or 6 days).

[0281] Insulin-positive endocrine cells

[0299] The embodiments of the present disclosure include insulin-positive endocrine cells (e.g., NKX6.1 positive, ISL1 positive, or β-like cells).The insulin-positive endocrine cells used herein can be derived from any source or produced according to any suitable protocol.In some embodiments, PDX1 positive, NKX6.1 positive pancreatic progenitor cells are differentiated into insulin-positive endocrine cells (e.g., NKX6.1 positive, ISL1 positive, or β-like cells).In some embodiments, the insulin-positive endocrine cells are further differentiated, for example, by induction or maturation into SC-β cells.

[0282]

[0300] In some embodiments, a method for producing insulin-positive endocrine cells from PDX1-positive, NKX6.1-positive pancreatic progenitor cells includes contacting a cell population comprising PDX1-positive, NKX6-1-positive pancreatic progenitor cells (e.g., under conditions that promote cell clustering) with a) a TGF-β signaling pathway inhibitor, b) a thyroid hormone signaling pathway activator, c) a gamma-secretase inhibitor, and d) a protein kinase inhibitor to induce differentiation of at least one PDX1-positive, NKX6.1-positive pancreatic progenitor cell in the population into an insulin-positive endocrine cell, wherein the insulin-positive endocrine cell expresses insulin. In some examples, the insulin-positive endocrine cell expresses PDX1, NKX6.1, ISL1, NKX2.2, Mafb, glis3, Sur1, Kir6.2, Znt8, SLC2A1, SLC2A3, and / or insulin.

[0283]

[0301] Any TGF-β signaling pathway inhibitor that can induce the differentiation of PDX1 positive, NKX6.1 positive pancreatic progenitor cells (e.g., alone or in combination with other β cell differentiation factors, e.g., thyroid hormone signaling pathway activators) into insulin positive endocrine cells can be used. In some examples, the TGF-β signaling pathway comprises TGF-β type I receptor kinase signaling. In some examples, the TGF-β signaling pathway inhibitor comprises Alk5 inhibitor II.

[0284]

[0302] Any thyroid hormone signaling pathway activator that can induce differentiation of PDX1-positive, NKX6.1-positive pancreatic progenitor cells (e.g., alone or in combination with other β-cell differentiation factors, such as TGF-β sig...

Claims

1. 1. A method for treating a subject in need of treatment, comprising administering to the subject by injection a first pharmaceutical composition comprising a cell population in a liquid suspension, wherein the cell population is greater than or equal to 1×10 ８ ~10x10 ８ , 3×10 ８ ~8.5 x 10 ８ , 4 × 10 ８ ~8.5 x 10 ８ , or 5 × 10 ８ ~8.5 x 10 ８ The method comprises the step of: providing a cell population comprising a non-native cell that expresses C-peptide and ISL1;

2. The stimulated blood C-peptide levels of the subject are determined by: a) at least 450 pmol / L, 460 pmol / L, 470 pmol / L, 480 pmol / L, 490 pmol / L, 500 pmol / L, 510 pmol / L, 520 pmol / L, 530 pmol / L, 540 pmol / L, 550 pmol / L, 700 pmol / L, 800 pmol / L, 900 pmol / L, 1000 pmol / L, 1400 pmol / L, and / or b) 150 to 350 pmol / L, 250 to 400 pmol / L, 400 to 1400 pmol / L, 400 to 1200 pmol / L, 400 to 1000 pmol / L, 400 to 800 pmol / L, 400 to 600 pmol / L, 600 to 800 pmol / L, 600 to 1400 pmol / L, or 900 to 1200 pmol / L; 10. The method of claim 1, wherein the increased cholesterol level is measured using a mixed meal tolerance test at least about 1, 2, 3, 4, 5, or 6 months after administration.

3. The stimulated blood C-peptide levels of the subject a) at least 450 pmol / L, 460 pmol / L, 470 pmol / L, 480 pmol / L, 490 pmol / L, 500 pmol / L, 510 pmol / L, 520 pmol / L, 530 pmol / L, 540 pmol / L, 550 pmol / L, 700 pmol / L, 800 pmol / L, 900 pmol / L, 1000 pmol / L, 1400 pmol / L, and / or b) 150 to 350 pmol / L, 250 to 400 pmol / L, 400 to 1400 pmol / L, 400 to 1200 pmol / L, 400 to 1000 pmol / L, 400 to 800 pmol / L, 400 to 600 pmol / L, 600 to 800 pmol / L, 600 to 1400 pmol / L, or 900 to 1200 pmol / L; 2. The method of claim 1, wherein c) the increased cholesterol level is increased as measured using a mixed meal tolerance test at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after administration.

4. The stimulated blood C-peptide levels of the subject are determined by: a) at least 200 pmol / L, 300 pmol / L, 450 pmol / L, 460 pmol / L, 470 pmol / L, 480 pmol / L, 490 pmol / L, 500 pmol / L, 510 pmol / L, 520 pmol / L, 530 pmol / L, 540 pmol / L, 550 pmol / L, 600 pmol / L, or 650 pmol / L, and / or b) 150 to 350 pmol / L, 200 to 700 pmol / L, 150 to 700 pmol / L, 300 to 650 pmol / L, or 500 to 700; c) increased as measured using a mixed meal tolerance test about 3 months after administration.

5. The stimulated blood C-peptide levels of the subject are determined by: a) at least 450 pmol / L, 460 pmol / L, 470 pmol / L, 480 pmol / L, 490 pmol / L, 500 pmol / L, 510 pmol / L, 520 pmol / L, 530 pmol l / L, 540 pmol / L, 550 pmol / L, 560 pmol / L, 700 pmol / L, 800 pmol / L, 900 pmol / L, 1000 pmol / L, 1400 pmol / L, and / or b) 150 to 350 pmol / L, 250 to 400 pmol / L, 400 to 1400 pmol / L, 400 to 1200 pmol / L, 400 to 1000 pmol / L, 400 to 800 pmol / L, 400 to 600 pmol / L, 600 to 800 pmol / L, 600 to 1400 pmol / L, or 900 to 1200 pmol / L; 2. The method of claim 1, wherein c) the level of erythrocyte proliferation is increased as measured using a mixed meal tolerance test about 6 months after administration.

6. 6. The method of any one of claims 1 to 5, which increases the subject's blood C-peptide level to at least 100 pmol / L, 120 pmol / L, 130 pmol / L, 150 pmol / L, 160 pmol / L, 180 pmol / L, 200 pmol / L, 210 pmol / L, 220 pmol / L, 230 pmol / L, 240 pmol / L, 250 pmol / L, 260 pmol / L, 270 pmol / L, or 280 pmol / L under fasting conditions when measured at least about 1, 2, 3, 4, 5, or 6 months after administration.

7. 6. The method of any one of claims 1 to 5, wherein the method increases the subject's blood C-peptide level under fasting conditions to at least 100 pmol / L, 120 pmol / L, 130 pmol / L, 150 pmol / L, 160 pmol / L, 180 pmol / L, 200 pmol / L, 210 pmol / L, 220 pmol / L, 230 pmol / L, 240 pmol / L, 250 pmol / L, 260 pmol / L, 270 pmol / L, or 280 pmol / L when measured about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after administration.

8. 6. The method of any one of claims 1 to 5, which increases the subject's blood C-peptide concentration under fasting conditions to at least 100 pmol / L, 120 pmol / L, 130 pmol / L, 150 pmol / L, 160 pmol / L, 180 pmol / L, 200 pmol / L, 210 pmol / L, 220 pmol / L, 230 pmol / L, 240 pmol / L, 250 pmol / L, 260 pmol / L, 270 pmol / L, or 280 pmol / L when measured about 3 months after administration.

9. 6. The method of any one of claims 1 to 5, wherein the method increases the subject's blood C-peptide level to about 200 pmol / L, 220 pmol / L, 240 pmol / L, 260 pmol / L, or 280 pmol / L under fasting conditions when measured about three months after administration.

10. 10. The method of any one of claims 1 to 9, wherein prior to administration, the subject has a stimulated blood C-peptide level of less than 100 pmol / L, less than 80 pmol / L, less than 60 pmol / L, less than 40 pmol / L, less than 30 pmol / L, less than 20 pmol / L, or less than 10 pmol / L as measured using a mixed meal tolerance test.

11. 10. The method of any one of claims 1 to 9, wherein prior to administration, the subject has undetectable stimulated blood C-peptide levels as measured using a mixed meal tolerance test.

12. 10. The method of any one of claims 1 to 9, wherein prior to administration, the subject has undetectable stimulated blood C-peptide levels when measured under fasting conditions.

13. The subject's hemoglobin A1c level (Hb1Ac) level is determined by: a) less than 8%, less than 7.5%, less than 7.0%, less than 6.5%, less than 6.0%, or less than 5.5%, and / or b) 5-8%, 5-7.5%, 5-7%, 5-6.5%, 5-5.5%, 5.5-8%, 5.5-7%, 5.5-6.5%, 6-8%, 6-7%, or 6-6.5%, 13. The method of any one of claims 1 to 12, wherein the method results in a reduction as measured using an HbA1c test at least about 1, 2, 3, 4, 5, or 6 months after administration.

14. The subject's Hb1Ac level is determined by: a) less than 8%, less than 7.5%, less than 7.0%, less than 6.5%, less than 6.0%, less than 5.5%, or less than 5%, and / or b) 5-8%, 5-7.5%, 5-7%, 5-6.5%, 5-5.5%, 5.5-8%, 5.5-7%, 5.5-6.5%, 6-8%, 6-7%, or 6-6.5%, 13. The method of any one of claims 1 to 12, wherein the method results in a reduction as measured by an HbA1c test about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after administration.

15. The subject's Hb1Ac level is determined by: a) less than 8%, less than 7.5%, less than 7.0%, or less than 6.75%, and / or b) 5-8.0%, 6.5-8.0%, 6.5-7.5%, 6.5-7.0%, or 7-8%, 13. The method of claim 1, wherein the method reduces as measured about 3 months after administration.

16. The subject's Hb1Ac level is determined by: a) less than 8%, less than 7.5%, less than 7.0%, less than 6.5%, less than 6.0%, less than 5.5%, or less than 5%, and / or b) 4.5-8%, 5-8%, 5-7.5%, 5-7%, 5-6.5%, 5-5.5%, 5.5-8%, 5.5-7%, 5.5-6.5%, 6-8%, 6-7%, or 6-6.5%, 13. The method of claim 1, wherein the method reduces as measured about 9 months after administration.

17. 17. The method of any one of claims 1 to 16, wherein prior to administration, the subject has a baseline Hb1Ac value of 7.0%, 7.3%, 7.8%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.8%, or 9.0% or greater.

18. 18. The method of any one of claims 1 to 17, wherein prior to administration, the subject receives an infusion of insulin at a level of at least about 20 U / day, 22 U / day, 24 U / day, 26 U / day, 28 U / day, 30 U / day, 32 U / day, or 34 U / day.

19. 19. The method of any one of claims 1 to 18, wherein prior to administration, the subject has a history of a severe hypoglycemic event.

20. 20. The method of any one of claims 1 to 19, wherein prior to administration, the subject has a history of impaired hypoglycemic awareness.

21. 21. The method of any one of claims 1 to 20, wherein prior to administration, the subject demonstrates no residual endogenous pancreatic islet cell function.

22. 22. The method of claim 21, wherein the absence of residual endogenous pancreatic islet cell function is indicated by undetectable fasting C-peptide and by undetectable stimulated C-peptide in a mixed meal tolerance test.

23. 23. The method of claim 21 or 22, wherein the absence of residual endogenous pancreatic islet cell function is indicated by sustained stimulated glucose levels of greater than 350, 400, 450, 475, or 500 mg / dL in a mixed meal tolerance test.

24. 24. The method of any one of claims 1 to 23, wherein prior to administration, the subject had an HbA1c value of greater than 7.7%, 8%, 8.2%, or 8.5%.

25. 25. The method of any one of claims 1 to 24, wherein prior to administration, the subject was receiving more than 10, 15, 20, 25, 30 or 35 units of insulin per day.

26. 26. The method of any one of claims 1 to 25, wherein the subject has a disease characterized by high blood glucose levels over an extended period of time.

27. 27. The method of claim 26, wherein the disease is diabetes.

28. 27. The method of claim 26, wherein the disease is type 1 diabetes.

29. The cell population is at most about 10×10 ８ , 8 x 10 ８ , 7 x 10 ８ , 6.5×10 ８ , 6 × 10 ８ , 5.5×10 ８ , 5×10 ８ , 4.5×10 ８ , 4 × 10 ８ , 3.5×10 ８ , 3×10 ８ , 2.5×10 ８ , or 2 × 10 ８ 29. The method of any one of claims 1 to 28, comprising cells.

30. The cell population is approximately 1×10 ８ ~Approx. 7×10 ８ , about 1×10 ８ ~Approx. 6×10 ８ , about 1×10 ８ ~Approx. 5×10 ８ , about 1×10 ８ ~About 4×10 ８ , about 1×10 ８ ~Approx. 3×10 ８ , about 1×10 ８ ~Approx. 2×10 ８ , about 1.5×10 ８ ~Approx. 6.5×10 ８ , about 2×10 ８ ~Approx. 7×10 ８ , about 2×10 ８ ~Approx. 6×10 ８ , about 2×10 ８ ~Approx. 5×10 ８ , about 2×10 ８ ~About 4×10 ８ , about 2×10 ８ ~Approx. 3×10 ８ , about 2.5×10 ８ ~Approx. 5.5×10 ８ , about 3×10 ８ ~Approx. 7×10 ８ , about 3×10 ８ ~Approx. 6×10 ８ , about 3×10 ８ ~Approx. 5×10 ８ , about 3×10 ８ ~About 4×10 ８ , about 3.5×10 ８ ~Approx. 4.5×10 ８ , or about 3.8×10 ８ ~Approx. 4.2×10 ８ 29. The method of any one of claims 1 to 28, comprising cells.

31. The cell population was approximately 3.5×10 ８ ~Approx. 4.5×10 ８ 29. The method of any one of claims 1 to 28, comprising cells.

32. The cell population was approximately 3.5×10 ８ ~Approx. 8.5×10 ８ 32. The method of any one of claims 1 to 31, comprising cells.

33. The subject is administered by injection a second pharmaceutical composition comprising the cell population in liquid suspension, the cell population being at least 1×10 ８ ~10x10 ８ , 3×10 ８ ~8.5 x 10 ８ , 4 × 10 ８ ~8.5 x 10 ８ , or 5 × 10 ８ ~8.5 x 10 ８ 33. The method of any one of claims 1 to 32, wherein the cell population comprises non-native cells that express C-peptide and ISL1, and wherein the second pharmaceutical composition is administered to the subject at a later time than the first pharmaceutical composition.

34. 34. The method of claim 33, wherein the second pharmaceutical composition is administered to the subject at least 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, or 3-12 months, 3-10 months, 3-9 months, 3-7 months, 3-5 months, 5-12 months, 8-10 months, 7-12 months, 9-15 months, or 9-12 months after the subject was administered the first pharmaceutical composition.

35. The first pharmaceutical composition is 3.5×10 ８ ~Approx. 8.5×10 ８ cells, and the second pharmaceutical composition comprises 3.5×10 ８ ~Approx. 8.5×10 ８ 35. The method of claim 33 or 34, comprising cells.

36. The first pharmaceutical composition is 3.5×10 ８ ~about 8.5~10 ８ cells, and the second pharmaceutical composition comprises 3.5×10 ８ ~Approx. 4.5×10 ８ 35. The method of claim 33 or 34, comprising cells.

37. 37. The method of any one of claims 1 to 36, wherein the subject is also administered at least one immunosuppressant agent.

38. 38. The method of claim 37, wherein the at least one immunosuppressant comprises one selected from the group consisting of thymoglobulin, etanercept, basiliximab, tacrolimus, sirolimus, and mycophenolate mofetil.

39. 37. The method of any one of claims 1 to 36, wherein the subject is also administered thymoglobulin and etanercept.

40. 37. The method of any one of claims 1 to 36, wherein the subject is also administered thymoglobulin and basiliximab.

41. 37. The method of any one of claims 1 to 36, wherein the subject is also administered tacrolimus and sirolimus.

42. 37. The method of any one of claims 1 to 36, wherein the subject is also administered tacrolimus and basiliximab.

43. 37. The method of any one of claims 1 to 36, wherein the subject is also administered sirolimus and mycophenolate.

44. 44. The method of any one of claims 1 to 43, wherein the first pharmaceutical composition comprises a sugar.

45. 45. The method of claim 44, wherein the sugar is sucrose or glucose.

46. 46. ​​The method of claim 44 or 45, wherein the liquid suspension comprises sugar at a concentration of about 0.05% to about 1.5%.

47. 47. The method of any one of claims 1 to 46, wherein the first pharmaceutical composition comprises CMRL medium.

48. 47. The method of any one of claims 1 to 46, wherein the first pharmaceutical composition comprises HypoThermosol® FRS storage medium.

49. 49. The method of any one of claims 1 to 48, wherein the cell population in the first pharmaceutical composition further comprises non-naturally occurring cells that express glucagon but do not express somatostatin, and non-naturally occurring cells that express somatostatin but do not express glucagon.

50. (a) 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 30-40%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-90%, 50-80%, 50-70%, 50-60%, 60-90%, 60-80%, 60-70%, 70-90%, 70-80%, or 80-90% of the cells in the cell population in the first pharmaceutical composition express C-peptide and ISL1 but do not express VMAT1; (b) 3-40%, 3-35%, 3-30%, 3-25%, 3-20%, 3-15%, 3-10%, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-40%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15% in the cell population in the first pharmaceutical composition %, 15-40%, 15-35%, 15-30%, 15-25%, 15-20%, 20-40%, 20-35%, 20-30%, 20-25%, 25-40%, 25-35%, 25-30%, 30-40%, 30-35% or 35-40% of the cells express glucagon but do not express somatostatin; and / or (c) 1-20%, 1-15%, 1-12%, 1-10%, 1-8%, 1-5%, 2-20%, 2-15%, 2-12%, 2-10%, 2-8%, 2-5%, 3-20%, 3-15%, 3-12%, 3-10%, 3-8%, 3-5%, 4-20%, 4-15%, 4-12%, 4-10%, 4-8%, 4-5%, 5-20%, 5-15%, 50. The method of claim 49, wherein 5-12%, 5-10%, 5-8%, 7-20%, 7-15%, 7-12%, 7-10%, 9-20%, 9-15%, 9-12%, 8-10%, 8-12%, 8-15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15% or 15-20% of the cells express somatostatin but do not express glucagon.

51. (a) 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 30-40%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-90%, 50-80%, 50-70%, 50-60%, 60-90%, 60-80%, 60-70%, 70-90%, 70-80%, or 80-90% of the cells in a population of cells express C-peptide and ISL1 but do not express VMAT1; (b) 3-40%, 3-35%, 3-30%, 3-25%, 3-20%, 3-15%, 3-10%, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-40%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 1 5-40%, 15-35%, 15-30%, 15-25%, 15-20%, 20-40%, 20-35%, 20-30%, 20-25%, 25-40%, 25-35%, 25-30%, 30-40%, 30-35%, or 35-40% of the cells express glucagon but do not express somatostatin; and (c) 1-20%, 1-15%, 1-12%, 1-10%, 1-8%, 1-5%, 2-20%, 2-15%, 2-12%, 2-10%, 2-8%, 2-5%, 3-20%, 3-15%, 3-12%, 3-10%, 3-8%, 3-5%, 4-20%, 4-15%, 4-12%, 4-10%, 4-8%, 4-5%, 5-20%, 5-15%, 5-12% of the cell population 50. The method of claim 49, wherein 5-10%, 5-8%, 7-20%, 7-15%, 7-12%, 7-10%, 9-20%, 9-15%, 9-12%, 8-10%, 8-12%, 8-15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15% or 15-20% of the cells express somatostatin but do not express glucagon.

52. (a) 35-60% of cells in the cell population express C-peptide and ISL1, but do not express VMAT1; (b) 4-25% of the cells in the cell population express glucagon but do not express somatostatin; and 50. The method of claim 49, wherein (c) 1-10% of the cells in the cell population express somatostatin but do not express glucagon.

53. (a) 40-60% of cells in the cell population express C-peptide and ISL1, but do not express VMAT1; (b) 10-25% of the cells in the cell population express glucagon but do not express somatostatin; and 50. The method of claim 49, wherein (c) 4-10% of the cells in the cell population express somatostatin but do not express glucagon.

54. 54. The method of any one of claims 1 to 53, wherein less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 18%, less than 15%, less than 12%, or less than 10% of the cells in the cell population express VMAT1 but do not express C-peptide.

55. 50%, 40%, 30%, or 20% or more of the cells in the first pharmaceutical composition are NKX6.1 as determined by flow cytometry. ＋ / ISL1 ＋ 55. The method of any one of claims 1 to 54, which is a cell.

56. 20% or more of the cells in the first pharmaceutical composition express NKX6.1 as determined by flow cytometry. ＋ / ISL1 ＋ 56. The method of claim 55, which is a cell.

57. 40%, 35%, 30%, 26%, 25%, or 20% or more of the cells in the first pharmaceutical composition are NKX6.1 as determined by flow cytometry. － / ISL1 ＋ 57. The method of any one of claims 1 to 56, which is a cell.

58. 26% or more of the cells in the first pharmaceutical composition express NKX6.1 as determined by flow cytometry. － / ISL1 ＋ 58. The method of claim 57, which is a cell.

59. 5-25%, 5-40%, 5-35%, or 8-20% of the cells in the first pharmaceutical composition are NKX6.1 as determined by flow cytometry. － / ISL1 ＋ 59. The method of any one of claims 1 to 58, which is a cell.

60. no more than 50%, 45%, 40%, 35%, 30%, or 25% of the cells in the first pharmaceutical composition are NKX6.1 as determined by flow cytometry. ＋ / ISL1 － 60. The method of any one of claims 1 to 59, which is a cell.

61. 50% or less of the cells in the first pharmaceutical composition express NKX6.1 as determined by flow cytometry. ＋ / ISL1 － 61. The method of claim 60, which is a cell.

62. 20-60%, 20-50%, 20-45%, 20-40%, 20-35%, 20-30%, 20-25%, 25-50%, 25-40%, 25-35%, 30-60%, 30-50%, 30-40%, 30-35%, 35-50%, 40-50% of the cells in the pharmaceutical composition are NKX6.1 as determined by flow cytometry. ＋ / ISL1 ＋ 62. The method of any one of claims 1 to 61, which is a cell.

63. 20-60%, 20-50%, 20-45%, 20-40%, 20-35%, 20-30%, 20-25%, 25-50%, 25-40%, 25-35%, 30-60%, 30-50%, 30-40%, 30-35%, 35-50%, or 40-50% of the cells in the pharmaceutical composition are NKX6.1 as determined by flow cytometry. － / ISL1 ＋ 63. The method of any one of claims 1 to 62, which is a cell.

64. 20-50%, 20-45%, 20-40%, 20-35%, 20-30%, 20-25%, 25-50%, 25-40%, 25-35%, 30-60%, 30-50%, 30-40%, 30-35%, 35-50%, 40-50%, 10-20%, or 10-25% of the cells in the pharmaceutical composition are NKX6.1 as determined by flow cytometry. ＋ / ISL1 － 63. The method of any one of claims 1 to 62, which is a cell.

65. a) at least 30% of the cells in the composition are NKX6.1 positive, ISL1 positive cells; b) at least 25% of the cells in the composition are NKX6.1 negative, ISL1 positive cells; c) less than 12% of the cells in the composition are NKX6.1-negative, ISL1-negative cells; and / or d) A method according to any one of claims 1 to 64, wherein 9-25% of the cells in the composition are NKX6.1 positive, ISL1 negative cells.

66. Compositions that exhibit GSIS in vitro NKX6.1 ＋ / ISL1 ＋ 66. The method of any one of claims 1 to 65, comprising a cell.

67. The composition exhibits GSIS in vivo NKX6.1 ＋ / ISL1 ＋ 67. The method of any one of claims 1 to 66, comprising a cell.

68. 68. The method of any one of claims 1 to 67, wherein the cell population is generated in vitro from stem cells.

69. 69. The method of any one of claims 1 to 68, wherein the non-native cells in the cell population that express C-peptide and ISL1, but do not express VMAT1, exhibit a glucose-stimulated insulin secretion response in vitro.

70. 70. The method of claim 69, wherein in response to a glucose challenge, the secretion of insulin by the non-native cell that expresses C-peptide and ISL1, but does not express VMAT1, is proportional to the glucose concentration of the glucose challenge.

71. The method of claim 69 or 70, wherein the secretion of insulin by a non-native cell that expresses C-peptide and ISL1 but does not express VMAT1 secretes insulin in response to a first glucose challenge, a second glucose challenge, and a third glucose challenge, wherein the first glucose challenge, the second glucose challenge, and the third glucose challenge are applied sequentially.

72. 72. The method of any one of claims 1 to 71, wherein at least some of the cells in the cell population are present in multiple cell clusters.

73. 73. The method of claim 72, wherein the cell clusters are about 50 μm to about 500 μm, about 50 μm to about 400 μm, about 50 μm to about 300 μm, about 60 μm to about 400 μm, about 60 μm to about 300 μm, about 60 μm to about 250 μm, about 75 μm to about 400 μm, about 75 μm to about 300 μm, about 75 μm to about 250 μm, about 125 μm to about 225 μm, about 130 μm to about 160 μm, about 170 μm to about 225 μm, about 140 μm to about 200 μm, about 140 μm to about 170 μm, about 160 μm to about 220 μm, about 170 μm to about 215 μm, or about 170 μm to about 200 μm in diameter.

74. 72. The method of any one of claims 1 to 71, wherein the cell population is present in the first pharmaceutical composition as a single cell suspension.

75. 75. The method of any one of claims 1 to 74, wherein in the first pharmaceutical composition, the cell population is suspended in a serum-free solution.

76. 76. The method of any one of claims 1 to 75, further comprising the step of administering an immune response modulator to the subject.

77. 77. The method of claim 76, wherein the immune response modulator is administered simultaneously with the first pharmaceutical composition.

78. 77. The method of claim 76, wherein the immune response modulator is administered before or after the first pharmaceutical composition.

79. 79. The method of any one of claims 76 to 78, wherein the immune response modulator is not a steroid.

80. Immune response modulators include azathioprine, mycophenolate, leflunomide, teriflunomide, methotrexate, tacrolimus, cyclosporine, pimecrolimus, avetimus, gusperimus, lenalidomide, pomalidomide, thalidomide, PDE4 inhibitors, apremilast, anakinra, sirolimus, everolimus, ridaforolimus, temsirolimus, umirolimus, zotarolimus, antithymocyte globulin antibodies, antilymphocyte globulins, Antibody, CTLA-4, abatacept, belatacept, etanercept, pegsunercept, aflibercept, alefacept, rilonacept, eculizumab, adalimumab, afelimomab, certolizumab pegol, golimumab, infliximab, nerelimomab, mepolizumab, omalizumab, faralimomab, ersilimomab, lebrikizumab, ustekinumab, secukinumab, muromonab-CD3, otelixizumab, teplizumab, Bisilizumab, Clenoliximab, Keliximab, Zanolimumab, Efalizumab, Erlizumab, Obinutuzumab, Rituximab, Ocrelizumab, Pascolizumab, Gomiliximab, Rumiliximab, Teneliximab, Toralizumab, Acelizumab, Galiximab, Gavilimomab, Ruplizumab, Belimumab, Brisibimod, Ipilimumab, Tremelimumab, Bertilimumab, Lerdelimumab, Metelimumab, Natalizumab, Tocilizumab 80. The method of any one of claims 76 to 79, comprising one or more of the following: , odurimomab, basiliximab, daclizumab, inolimomab, atrolimumab, cedelizumab, fontolizumab, maslimomab, morolimumab, pexelizumab, reslizumab, rovelizumab, siplizumab, talizumab, terimomab alitox, bapaliximab, beparimomab, alemtuzumab, mycophenolate mofetil, FTY720, or any combination thereof.

81. 80. The method of any one of claims 76 to 79, wherein the immune response modulator comprises tacrolimus, cyclosporine, mycophenolate mofetil, azathioprine, everolimus, sirolimus, abatacept, belatacept, antithymocyte globulin, alemtuzumab, rituximab, basiliximab, daclizumab, muromonab-CD3, efalizumab, FTY720, or any combination thereof.

82. 79. The method of any one of claims 76 to 78, wherein the immune response modulator comprises a corticosteroid.

83. 83. The method of any one of claims 1 to 82, wherein the first pharmaceutical composition is injected into the subject's portal vein.

84. 84. The method of any one of claims 1 to 83, wherein the subject receives a single dose of the first pharmaceutical composition.

85. 85. The method of any one of claims 1 to 84, wherein the non-native cells expressing C-peptide and ISL1 cells are obtained from a culture of cells comprising PDX1 positive, NKX6.1 positive, insulin positive cells that have been treated with i) a TGF-β signaling pathway inhibitor (e.g., Alk5i), ii) a thyroid hormone signaling pathway activator (e.g., GC-1), iii) a BMP pathway inhibitor (e.g., LDN193189), iv) a ROCK inhibitor (e.g., thiazovivin), v) a protein kinase inhibitor (e.g., staurosporine), and / or vi) an epigenetic modifier (e.g., DZNEP).

86. 86. The method of any one of claims 1 to 85, wherein the non-native cells expressing C-peptide and ISL1 cells are obtained from a culture of cells comprising PDX1 positive, NKX6.1 positive, insulin positive cells that have been treated with i) a TGF-β signaling pathway inhibitor (e.g., Alk5i), ii) a thyroid hormone signaling pathway activator (e.g., GC-1), iii) a BMP pathway inhibitor (e.g., LDN193189), iv) a ROCK inhibitor (e.g., thiazovivin), v) a protein kinase inhibitor (e.g., staurosporine), and / or vi) an epigenetic modifier (e.g., DZNEP) for at least three days.

87. The method of claim 85 or 86, wherein the PDX1 positive, NKX6.1 positive, insulin positive cells are also treated with human serum albumin.

88. 88. The method of any one of claims 85-87, wherein the PDX1 positive, NKX6.1 positive, insulin positive cells were also treated with human serum albumin for at least 6 days.

89. 88. The method of any one of claims 85-87, wherein the PDX1 positive, NKX6.1 positive, insulin positive cells are obtained from a cell culture comprising PDX1 positive, NKX6.1 positive, insulin negative cells that have been treated with i) a TGF-β signaling pathway inhibitor (e.g., Alk5i), ii) a TH signaling pathway activator (e.g., GC-1), iii) at least one SHH pathway inhibitor (e.g., Sant1), iv) an RA signaling pathway activator (e.g., retinoic acid), v) a gamma secretase inhibitor (e.g., XXI), vi) at least one growth factor from the epidermal growth factor (EGF) family (e.g., betacellulin), vii) a BMP pathway inhibitor (e.g., LDN193189), viii) a ROCK inhibitor (e.g., thiazovivin), ix) a protein kinase inhibitor (e.g., staurosporine), and / or x) an epigenetic modifier (e.g., DZNEP).

90. PDX1 positive, NKX6.1 positive, insulin positive cells are treated with i) a TGF-β signaling pathway inhibitor (e.g., Alk5i), ii) a TH signaling pathway activator (e.g., GC-1), iii) at least one SHH pathway inhibitor (e.g., Sant1), iv) an RA signaling pathway activator (e.g., retinoic acid), v) a γ-secretase inhibitor (e.g., XXI), vi) at least one growth factor from the epidermal growth factor (EGF) family (e.g., betacellulin), vii) a BMP pathway inhibitor (e.g., LDN193189), viii) a ROCK inhibitor (e.g., thiazovivin), ix) a protein kinase inhibitor (e.g., staurosporine), and / or x) an epigenetic inhibitor (e.g., β-lactamase inhibitor), 90. The method of claim 89, wherein the PDX1 positive, NKX6.1 positive, insulin negative cell culture is obtained from a culture of cells comprising PDX1 positive, NKX6.1 positive, insulin negative cells that have been treated with an epigenetic modifier (e.g., DZNEP) for 2 or 3 days and subsequently contacting the PDX1 positive, NKX6.1 positive pancreatic progenitor cells with i) a TGF-β signaling pathway inhibitor (e.g., Alk5i), ii) a TH signaling pathway activator (e.g., GC-1), iii) a gamma secretase inhibitor (e.g., XXI), iv) a BMP pathway inhibitor (e.g., LDN193189), v) a ROCK inhibitor (e.g., thiazovivin), vi) a protein kinase inhibitor (e.g., staurosporine), and vii) an epigenetic modifier (e.g., DZNEP) for 4 or 5 days.

91. PDX1-positive, NKX6.1-positive, insulin-positive cells are treated with i) a TGF-β signaling pathway inhibitor (e.g., Alk5i), ii) a TH signaling pathway activator (e.g., GC-1), iii) at least one SHH pathway inhibitor (e.g., Sant1), iv) an RA signaling pathway activator (e.g., retinoic acid), v) a γ-secretase inhibitor (e.g., XXI), vi) at least one growth factor from the epidermal growth factor (EGF) family (e.g., betacellulin), vii) a BMP pathway inhibitor (e.g., LDN193189), viii) a ROCK inhibitor (e.g., thiazovivin), ix) a protein kinase inhibitor (e.g., staurosporine), and / or x) an epigenetic modifier (e.g., DZNEP) for 2 or 3 days, followed by the generation of PDX1-positive, NKX6.1-positive pancreatic tumor cells.

91. The method of claim 90, wherein the progenitor cells are obtained from a culture of cells comprising PDX1 positive, NKX6.1 positive, insulin negative cells, in which the progenitor cells have been contacted with i) a TGF-β signaling pathway inhibitor (e.g., Alk5i), ii) a TH signaling pathway activator (e.g., GC-1), iii) a gamma secretase inhibitor (e.g., XXI), iv) a BMP pathway inhibitor (e.g., LDN193189), v) a ROCK inhibitor (e.g., thiazovivin), vi) a protein kinase inhibitor (e.g., staurosporine), and vii) an epigenetic modifier (e.g., DZNEP) for 4 or 5 days in the absence of i) an SHH pathway inhibitor (e.g., Sant1), ii) an RA signaling pathway activator (e.g., retinoic acid), and iii) a growth factor from the epidermal growth factor (EGF) family (e.g., betacellulin).

92. The method of any one of claims 89 to 91, wherein the PDX1 positive, NKX6.1 positive, insulin negative cells are obtained from a cell culture comprising PDX1 positive, NKX6.1 negative cells treated with i) at least one growth factor from the FGF family (e.g., KGF), ii) at least one SHH pathway inhibitor (e.g., Sant1), iii) an RA signaling pathway activator (e.g., retinoic acid), iv) a ROCK inhibitor (e.g., thiazovivin), and v) at least one factor from the TGFβ superfamily (e.g., activin A).

93. The method of claim 92, wherein the PDX1 positive, NKX6.1 positive, insulin negative cells are obtained from a cell culture comprising PDX1 positive, NKX6.1 negative cells that have been treated for 5 days with i) at least one growth factor from the FGF family (e.g., KGF), ii) at least one SHH pathway inhibitor (e.g., Sant1), iii) an RA signaling pathway activator (e.g., retinoic acid), iv) a ROCK inhibitor (e.g., thiazovivin), and v) at least one factor from the TGFβ superfamily (e.g., activin A).

94. The method of claim 92 or 93, wherein the PDX1 positive, NKX6.1 negative cells are obtained from a culture of cells comprising PDX1 negative gastrula cells treated with i) a retinoic acid signaling pathway activator, ii) at least one factor from the FGF family (e.g., KGF), iii) an SHH pathway inhibitor (e.g., Sant1), iv) a PKC activator (e.g., PDBU), v) a ROCK inhibitor (e.g., thiazovivin), and vi) a BMP pathway inhibitor (e.g., DMH-1).

95. 95. The method of claim 94, wherein the PDX1 positive, NKX6.1 negative cells are obtained from a culture of cells comprising PDX1 negative gastrula cells, in which the PDX1 positive, NKX6.1 negative cells are treated with i) a retinoic acid signaling pathway activator, ii) at least one factor from the FGF family (e.g., KGF), iii) an SHH pathway inhibitor (e.g., Sant1), iv) a PKC activator (e.g., PDBU), v) a ROCK inhibitor (e.g., thiazovivin), and vi) a BMP pathway inhibitor (e.g., DMH-1) for one day, followed by contacting the cells with i) a retinoic acid signaling pathway activator, ii) at least one factor from the FGF family (e.g., KGF), iii) an SHH pathway inhibitor (e.g., Sant1), iv) a PKC activator (e.g., PDBU), v) a ROCK inhibitor (e.g., thiazovivin) for an additional day.

96. 96. The method of claim 95, wherein the PDX1 positive, NKX6.1 negative cells are obtained from a culture of cells comprising PDX1 negative gastrula cells, wherein the PDX1 positive, NKX6.1 negative cells are treated with i) a retinoic acid signaling pathway activator, ii) at least one factor from the FGF family (e.g., KGF), iii) an SHH pathway inhibitor (e.g., Sant1), iv) a PKC activator (e.g., PDBU), v) a ROCK inhibitor (e.g., thiazovivin), and vi) a BMP pathway inhibitor (e.g., DMH-1) for one day, and subsequently contacting the cells with i) a retinoic acid signaling pathway activator, ii) at least one factor from the FGF family (e.g., KGF), iii) an SHH pathway inhibitor (e.g., Sant1), iv) a PKC activator (e.g., PDBU), v) a ROCK inhibitor (e.g., thiazovivin) for an additional day in the absence of the BMP pathway inhibitor.

97. 97. The method of any one of claims 94-96, wherein the PDX1-negative archenterocytes are obtained from a culture comprising definitive endoderm cells that have been treated with a factor from the FGF family (e.g., KGF).

98. 98. The method of claim 97, wherein the PDX1-negative archenterocytes are obtained from a culture comprising definitive endoderm cells that have been treated with a factor from the FGF family (e.g., KGF) for three days.

99. The method of claim 97 or 98, wherein the definitive endoderm cells are obtained from a culture comprising pluripotent stem cells treated with a factor from the TGFβ superfamily (e.g., activin A) and a WNT signaling pathway activator (e.g., CHIR99021).

100. The method of claim 99, wherein the definitive endoderm cells are obtained from a culture comprising pluripotent stem cells that have been treated with a factor from the TGFβ superfamily (e.g., activin A) and a WNT signaling pathway activator (e.g., CHIR99021) for one day, followed by contacting the cells with a factor from the TGFβ superfamily (e.g., activin A) for an additional two days.

101. 1. A pharmaceutical composition formulated for injection, comprising a population of cells in liquid suspension, the population of cells being approximately 1×10 ８ ~About 10×10 ８ , 3×10 ８ ~8.5 x 10 ８ , 4 × 10 ８ ~8.5 x 10 ８ , or 5 × 10 ８ ~8.5 x 10 ８ The pharmaceutical composition comprises a non-native cell population comprising a cell, the cell population expressing C-peptide and ISL1.

102. The cell population is at most about 8×10 ８ , 7 x 10 ８ , 6.5×10 ８ , 6 × 10 ８ , 5.5×10 ８ , 5×10 ８ , 4.5×10 ８ , 4 × 10 ８ , 3.5×10 ８ , 3×10 ８ , 2.5×10 ８ , or 2 × 10 ８ The pharmaceutical composition of claim 101, comprising cells.

103. The cell population is approximately 1×10 ８ ~Approx. 7×10 ８ , about 1×10 ８ ~Approx. 6×10 ８ , about 1×10 ８ ~Approx. 5×10 ８ , about 1×10 ８ ~About 4×10 ８ , about 1×10 ８ ~Approx. 3×10 ８ , about 1×10 ８ ~Approx. 2×10 ８ , about 1.5×10 ８ ~Approx. 6.5×10 ８ , about 2×10 ８ ~Approx. 7×10 ８ , about 2×10 ８ ~Approx. 6×10 ８ , about 2×10 ８ ~Approx. 5×10 ８ , about 2×10 ８ ~About 4×10 ８ , about 2×10 ８ ~Approx. 3×10 ８ , about 2.5×10 ８ ~Approx. 5.5×10 ８ , about 3×10 ８ ~Approx. 7×10 ８ , about 3×10 ８ ~Approx. 6×10 ８ , about 3×10 ８ ~Approx. 5×10 ８ , about 3×10 ８ ~About 4×10 ８ , about 3.5×10 ８ ~Approx. 4.5×10 ８ , or about 3.8×10 ８ ~Approx. 4.2×10 ８ The pharmaceutical composition of claim 101, comprising cells.

104. The cell population was approximately 3.5×10 ８ ~Approx. 4.5×10 ８ The pharmaceutical composition of claim 101, comprising cells.

105. The cell population was approximately 3.5×10 ８ ~Approx. 8.5×10 ８ The pharmaceutical composition of claim 101, comprising cells.

106. 106. The pharmaceutical composition of any one of claims 101 to 105, wherein the cell population further comprises non-naturally occurring cells that express glucagon but do not express somatostatin, and non-naturally occurring cells that express somatostatin but do not express glucagon.

107. (a) 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 30-40%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-90%, 50-80%, 50-70%, 50-60%, 60-90%, 60-80%, 60-70%, 70-90%, 70-80%, or 80-90% of the cells in a population of cells express C-peptide and ISL1 but do not express VMAT1; (b) 3-40%, 3-35%, 3-30%, 3-25%, 3-20%, 3-15%, 3-10%, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-40%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15- 40%, 15-35%, 15-30%, 15-25%, 15-20%, 20-40%, 20-35%, 20-30%, 20-25%, 25-40%, 25-35%, 25-30%, 30-40%, 30-35% or 35-40% of the cells express glucagon but do not express somatostatin; and / or (c) 1-20%, 1-15%, 1-12%, 1-10%, 1-8%, 1-5%, 2-20%, 2-15%, 2-12%, 2-10%, 2-8%, 2-5%, 3-20%, 3-15%, 3-12%, 3-10%, 3-8%, 3-5%, 4-20%, 4-15%, 4-12%, 4-10%, 4-8%, 4-5%, 5-20%, 5-15%, 5-12%, 5% of the cell population 107. The pharmaceutical composition of claim 106, wherein up to 10%, 5-8%, 7-20%, 7-15%, 7-12%, 7-10%, 9-20%, 9-15%, 9-12%, 8-10%, 8-12%, 8-15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15% or 15-20% of the cells express somatostatin but do not express glucagon.

108. (a) 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 30-40%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-90%, 50-80%, 50-70%, 50-60%, 60-90%, 60-80%, 60-70%, 70-90%, 70-80%, or 80-90% of the cells in a population of cells express C-peptide and ISL1 but do not express VMAT1; (b) 3-40%, 3-35%, 3-30%, 3-25%, 3-20%, 3-15%, 3-10%, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-40%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 1 5-40%, 15-35%, 15-30%, 15-25%, 15-20%, 20-40%, 20-35%, 20-30%, 20-25%, 25-40%, 25-35%, 25-30%, 30-40%, 30-35%, or 35-40% of the cells express glucagon but do not express somatostatin; and (c) 1-20%, 1-15%, 1-12%, 1-10%, 1-8%, 1-5%, 2-20%, 2-15%, 2-12%, 2-10%, 2-8%, 2-5%, 3-20%, 3-15%, 3-12%, 3-10%, 3-8%, 3-5%, 4-20%, 4-15%, 4-12%, 4-10%, 4-8%, 4-5%, 5-20%, 5-15%, 5-12%, 5% of the cell population 107. The pharmaceutical composition of claim 106, wherein up to 10%, 5-8%, 7-20%, 7-15%, 7-12%, 7-10%, 9-20%, 9-15%, 9-12%, 8-10%, 8-12%, 8-15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15% or 15-20% of the cells express somatostatin but do not express glucagon.

109. (a) 35-60% of cells in the cell population express C-peptide and ISL1, but do not express VMAT1; (b) 4-25% of the cells in the cell population express glucagon but do not express somatostatin; and The pharmaceutical composition of claim 106, wherein (c) 1-10% of the cells in the cell population express somatostatin but do not express glucagon.

110. (a) 40-60% of cells in the cell population express C-peptide and ISL1, but do not express VMAT1; (b) 10-25% of the cells in the cell population express glucagon but do not express somatostatin; and The pharmaceutical composition of claim 106, wherein (c) 4-10% of the cells in the cell population express somatostatin but do not express glucagon.

111. A pharmaceutical composition according to any one of claims 101 to 110, wherein less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 18%, less than 15%, less than 12%, or less than 10% of the cells in the cell population express VMAT1 but do not express C-peptide.

112. 50%, 40%, 30%, or 20% or more of the cells in the pharmaceutical composition are NKX6.1 as determined by flow cytometry. ＋ / ISL1 ＋ 112. The pharmaceutical composition of any one of claims 101 to 111, which is a cell.

113. 20% or more of the cells in the pharmaceutical composition are NKX6.1 as determined by flow cytometry. ＋ / ISL1 ＋ The pharmaceutical composition of claim 112, which is a cell.

114. 40%, 35%, 30%, 26%, 25%, or 20% or more of the cells in the pharmaceutical composition are NKX6.1 as determined by flow cytometry. － / ISL1 ＋ 114. The pharmaceutical composition of any one of claims 101 to 113, which is a cell.

115. 26% or more of the cells in the pharmaceutical composition are NKX6.1 as determined by flow cytometry. － / ISL1 ＋ The pharmaceutical composition of claim 114, which is a cell.

116. 5-25%, 5-40%, 5-35%, or 8-20% of the cells in the pharmaceutical composition are NKX6.1 as determined by flow cytometry. － / ISL1 ＋ 116. The pharmaceutical composition of any one of claims 101 to 115, which is a cell.

117. NKX6.1 or less, as determined by flow cytometry, ＋ / ISL1 － 117. The pharmaceutical composition of any one of claims 101 to 116, which is a cell.

118. As determined by flow cytometry, a) at least 30% of the cells in the composition are NKX6.1 positive, ISL1 positive cells; b) at least 25% of the cells in the composition are NKX6.1 negative, ISL1 positive cells; c) less than 12% of the cells in the composition are NKX6.1-negative, ISL1-negative cells; and / or d) The pharmaceutical composition of claim 117, wherein 9-25% of the cells in the composition are NKX6.1-positive, ISL1-negative cells.

119. 119. The pharmaceutical composition of any one of claims 101 to 118, wherein the cell population is generated in vitro from stem cells.

120. 120. The pharmaceutical composition of any one of claims 101 to 119, wherein the non-native cell expressing C-peptide and ISL1, but not VMAT1, exhibits a glucose-stimulated insulin secretion response in vitro.

121. 121. The pharmaceutical composition of claim 120, wherein in response to a glucose challenge, the secretion of insulin by the non-native cell that expresses C-peptide and ISL1, but does not express VMAT1, is proportional to the glucose concentration of the glucose challenge.

122. The pharmaceutical composition of claim 120 or 121, wherein the secretion of insulin by a non-native cell that expresses C-peptide and ISL1 but does not express VMAT1 secretes insulin in response to a first glucose challenge, a second glucose challenge, and a third glucose challenge, wherein the first glucose challenge, the second glucose challenge, and the third glucose challenge are applied sequentially.

123. 123. The pharmaceutical composition of any one of claims 101 to 122, wherein at least some of the cells in the cell population are present in multiple cell clusters.

124. 124. The pharmaceutical composition of claim 123, wherein the cell clusters are about 50 μm to about 500 μm, about 50 μm to about 400 μm, about 50 μm to about 300 μm, about 60 μm to about 400 μm, about 60 μm to about 300 μm, about 60 μm to about 250 μm, about 75 μm to about 400 μm, about 75 μm to about 300 μm, about 75 μm to about 250 μm, about 125 μm to about 225 μm, about 130 μm to about 160 μm, about 170 μm to about 225 μm, about 140 μm to about 200 μm, about 140 μm to about 170 μm, about 160 μm to about 220 μm, about 170 μm to about 215 μm, or about 170 μm to about 200 μm in diameter.

125. 123. The pharmaceutical composition of any one of claims 101 to 122, wherein the cell population is present in the pharmaceutical composition as a single cell suspension.

126. 126. The pharmaceutical composition of any one of claims 101 to 125, wherein the cell population is suspended in a serum-free solution.

127. (a) a pharmaceutical composition according to any one of claims 101 to 126, and (b) instructions for administering the pharmaceutical composition to a subject in need thereof. Kit including:

128. (a) A pharmaceutical composition formulated for injection, the pharmaceutical composition comprising a population of cells in suspension, the population of cells being at least about 1×10 ８ a pharmaceutical composition comprising a non-native cell, the cell population expressing C-peptide and ISL1; and (b) instructions for administering the pharmaceutical composition to a subject in need thereof. Kit including:

129. 129. The kit of claim 127 or 128, further comprising an immune response modulator.

130. The kit of claim 129, wherein the immune response modulator is not a steroid.

131. Immune response modulators include azathioprine, mycophenolate, leflunomide, teriflunomide, methotrexate, tacrolimus, cyclosporine, pimecrolimus, avetimus, gusperimus, lenalidomide, pomalidomide, thalidomide, PDE4 inhibitors, apremilast, anakinra, sirolimus, everolimus, ridaforolimus, temsirolimus, umirolimus, zotarolimus, antithymocyte globulin antibodies, antilymph ... Brin antibody, CTLA-4, abatacept, belatacept, etanercept, pegsunercept, aflibercept, alefacept, rilonacept, eculizumab, adalimumab, afelimomab, certolizumab pegol, golimumab, infliximab, nerelimomab, mepolizumab, omalizumab, faralimomab, ersilimomab, lebrikizumab, ustekinumab, secukinumab, muromonab-CD3, otelixizumab, tepli Zumab, bisilizumab, clenoliximab, keliximab, zanolimumab, efalizumab, erlizumab, obinutuzumab, rituximab, ocrelizumab, pascolizumab, gomiliximab, rumiliximab, teneliximab, toralizumab, acelizumab, galiximab, gavilimomab, ruplizumab, belimumab, brisibimod, ipilimumab, tremelimumab, bertilimumab, lerdelimumab, metelimumab, natalizumab , tocilizumab, odurimomab, basiliximab, daclizumab, inolimomab, atrolizumab, cedelizumab, fontolizumab, maslimomab, morolimumab, pexelizumab, reslizumab, rovelizumab, siplizumab, talizumab, terimomab alitox, bapaliximab, beparimomab, alemtuzumab, mycophenolate mofetil, FTY720, or any combination thereof.

132. 130. The kit of claim 129, wherein the immune response modulator comprises tacrolimus, cyclosporine, mycophenolate mofetil, azathioprine, everolimus, sirolimus, abatacept, belatacept, antithymocyte globulin, alemtuzumab, rituximab, basiliximab, daclizumab, muromonab-CD3, efalizumab, FTY720, or any combination thereof.

133. 130. The kit of claim 129, wherein the immune response modulator comprises a corticosteroid.

134. 134. The kit of any one of claims 127 to 133, wherein the kit comprises instructions for administering the pharmaceutical composition to a subject with a history of severe hypoglycemic events.

135. 135. The kit of any one of claims 127 to 134, wherein the kit comprises instructions for administering the pharmaceutical composition to a subject having a history of impaired hypoglycemic awareness.

136. 136. The kit of any one of claims 127-135, wherein the kit comprises instructions for administering the pharmaceutical composition to a subject with no residual endogenous pancreatic islet cell function.

137. The kit of claim 136, wherein the absence of residual endogenous pancreatic islet cell function is indicated by undetectable fasting C-peptide and by undetectable stimulated C-peptide in a mixed meal tolerance test.

138. 138. The kit of claim 136 or 137, wherein the absence of residual endogenous islet cell function is indicated by sustained stimulated glucose levels of greater than 350, 400, 450, 475, or 500 mg / dL in a mixed meal tolerance test.

139. 139. The kit of any one of claims 127 to 138, comprising instructions for administering the pharmaceutical composition to a subject having an HbA1c value greater than 7.7%, 8%, 8.2%, or 8.5%.

140. 140. The kit of any one of claims 127 to 139, comprising instructions for administering the pharmaceutical composition to a subject receiving more than 10, 15, 20, 25, 30 or 35 units of insulin per day.

141. 141. The kit of any one of claims 127 to 140, wherein the kit comprises instructions for administering the pharmaceutical composition to a subject having a disease characterized by high blood glucose levels over an extended period of time.

142. 142. The kit of claim 141, wherein the disease is diabetes.

143. 142. The kit of claim 141, wherein the disease is type 1 diabetes.

144. 40 x 10 ６ cells ~ 100 x 10 ６ cells, 50 x 10 ６ cells ~ 90 x 10 ６ cells, 60 x 10 ６ cells ~ 80 x 10 ６ cells, or 70 x 10 ６ cells ~ 80 x 10 ６ An encapsulated composition comprising SC islet cells.

145. 10x10 in device ６ ~60 x 10 ６ , 10×10 ６ ~50 x 10 ６ , 10×10 ６ ~40 x 10 ６ , 10×10 ６ ~30 x 10 ６ , 25×10 ６ ~60 x 10 ６ , or 25 x 10 ６ ~40 x 10 ６ The encapsulated composition of claim 144, wherein the cells are NKX6.1 positive, ISL1 positive cells.

146. 146. The encapsulated composition of claim 144 or 145, wherein the encapsulated composition is a degradable material particularly suitable for sustained release formulations.

147. The encapsulation composition of claim 146, wherein the degradable material comprises a biocompatible polymer, such as poly(lactic acid), poly(lactic-co-glycolic acid), methylcellulose, hyaluronic acid, or collagen.

148. 146. The encapsulated composition of claim 144 or 145, wherein the encapsulated composition is a device.

149. The encapsulated composition of claim 148, wherein the device comprises one or more semipermeable membranes.

150. 150. The encapsulation composition of claim 149, wherein the one or more semipermeable membranes comprise one or more made of poly(lactide) (PLA), poly(glycolic acid) (PGA), poly(lactide-co-glycolide) (PLGA), other polyhydroxy acids, poly(caprolactone), polycarbonate, polyamides, polyanhydrides, polyphosphazenes, polyamino acids, polyorthoesters, polyacetals, polycyanoacrylates, biodegradable polyurethanes, albumin, collagen, fibrin, polyamino acids, polyamines, alginates, agarose, agarose including gelatin, dextran, polyacrylates, ethylene vinyl acetate polymers and other acyl substituted cellulose acetates and derivatives thereof, polyurethanes, polystyrene, polyvinyl chloride, polyvinyl fluoride, poly(vinylimidazole), chlorosulfonated polyolefins, polyethylene oxide, or any combination thereof.

151. 151. The encapsulated composition of claim 149 or 150, wherein the semipermeable membrane is hydrophilic or comprises a hydrophilic coating.

152. The device comprises a plurality of pores, the pores having: a) an average pore size of about 1 nm, 5 nm, 10 nm, 15 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 200 nm, 300 nm or more, and / or any other suitable size range; and / or a pore size of about 2500 nm, 2000 nm, 1700 nm, 1500 nm, 1400 nm, 2500 nm, 2600 nm, 2700 nm, 2800 nm, 2900 nm, 3000 nm, 3100 nm, 3200 nm, 3300 nm, 3400 nm, 3500 nm, 3600 nm, 3700 nm, 3800 nm, 3900 nm, 4000 nm, 4100 nm, 4200 nm, 4300 nm, 4400 nm, 4500 nm, 4600 nm, 4700 nm, 4800 nm, 4900 nm, 5000 nm, 5100 nm, 5200 nm, 5300 nm, 5400 nm, 5500 nm, 5600 nm, 5700 nm, 5800 nm, 5900 nm, 6000 nm, 6100 nm, 6200 nm, 6300 nm, 6400 nm, 6500 nm, 6600 nm, 6700 nm, 6800 nm, 6900 nm, 7000 nm, 7100 nm, 7200 nm, 7300 nm, 7400 nm, 7500 152. The encapsulated composition of any one of claims 148-151, comprising an average pore size that is less than or equal to 0 nm, 1300 nm, 1200 nm, 1100 nm, 1000 nm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm.

153. the device comprises a porous polymeric membrane comprising a plurality of open pores, the open pores having an average pore size of 1 μm or less, said membrane having an average thickness of 10 μm to 150 μm, said membrane having a tensile strength of at least 1 MPa; said membrane configured to exhibit a Dfirst / Dsecond ratio of 2 to 50, Dfirst being a first diffusion coefficient for a first molecule having a first molecular weight of 50 Da to 10 kDa, inclusive; 152. The encapsulated composition of any one of claims 148-151, wherein Dsecond is a second diffusion coefficient for a second molecule having a second molecular weight between 50 kDa and 500 kDa (inclusive), and the ratio of the second molecular weight to the first molecular weight is 10 or greater.

154. 153. The encapsulation composition of any one of claims 148-152, wherein the device comprises: a first membrane having a first surface and a second surface opposite the first surface; and a second membrane opposing and attached to the second surface of the first membrane, the first membrane and the second membrane forming an enclosed compartment configured to house a cell population, and one or both of the first membrane and the second membrane are sintered.

155. 155. The encapsulated composition of any one of claims 144 to 154, wherein the composition comprises DMEM.

156. 156. The encapsulated composition of any one of claims 144 to 155, wherein the composition comprises human serum albumin.

157. 157. A method for treating a subject having type I diabetes comprising administering to the subject one or more of the devices described in any one of claims 144 to 156.

158. One or more devices are 50x10 ６ cells ~ 120 x 10 ６ cells, 50 x 10 ６ cells ~ 500 x 10 ６ cells, 50 x 10 ６ cells ~ 300 x 10 ６ cells, 100 x 10 ６ cells ~ 900 x 10 ６ cells, 100 x 10 ６ cells ~ 600 x 10 ６ cells, 100 x 10 ６ cells ~ 500 x 10 ６ cells, 250 x 10 ６ cells ~ 900 x 10 ６ cells, 250 x 10 ６ cells ~ 500 x 10 ６ cells, 350 x 10 ６ cells ~ 900 x 10 ６ cells, 350 x 10 ６ cells ~ 500 x 10 ６ cells, 400 x 10 ６ cells ~ 500 x 10 ６ cells, 150 x 10 ６ cells ~ 700 x 10 ６ cells, 130 x 10 ６ cells ~ 470 x 10 ６ cells, 450 x 10 ６ cells ~ 900 x 10 ６ cells, or 450 x 10 ６ cells ~ 600 x 10 ６ The method of claim 157, comprising cells.

159. 50x10 on one or more devices ６ ~800 x 10 ６ , 50×10 ６ ~400 x 10 ６ , 50×10 ６ ~300 x 10 ６ , 50×10 ６ ~800 x 10 ６ , 50×10 ６ ~200 x 10 ６ , 150×10 ６ ~800 x 10 ６ , 150×10 ６ ~500 x 10 ６ , 50×10 ６ ~250 x 10 ６ , 200×10 ６ ~800 x 10 ６ , 200×10 ６ ~500 x 10 ６ , 200×10 ６ ~300 x 10 ６ , 300×10 ６ ~800 x 10 ６ , or 500 x 10 ６ ~800 x 10 ６ The method of claim 157 or 158, wherein the cells are NKX6.1 positive, ISL1 positive cells.

160. The method of any one of claims 157 and 159, wherein the patient is administered 1-8, 2-6, or 4-6 devices.

161. The method of any one of claims 157 and 159, wherein the patient is administered 1, 2, 3, 4, 5, 6, 7 or 8 devices.

162. 160. The method of any one of claims 157 to 159, wherein the one or more devices are administered extrahepatically to the subject.

163. 161. The method of any one of claims 157 to 160, wherein the one or more devices are administered subcutaneously to the subject.

164. 164. The method of any one of claims 157 to 163, wherein the one or more devices are administered preperitoneally to the subject.

165. 165. The method of any one of claims 157 to 164, wherein the subject is not administered an immunosuppressant selected from the group consisting of thymoglobulin, etanercept, basiliximab, tacrolimus, sirolimus, and mycophenolate mofetil.

166. 101. The method of any one of claims 1 to 100, wherein: 35-55% of the cells in the composition are NKX6.1 positive, ISL1 positive cells; 25-45% of the cells in the composition are NKX6.1 negative, ISL1 positive cells; and 1-12% of the cells in the plurality of cells are NKX6.1 negative, ISL1 negative cells.

167. The composition of claims 101 to 126, wherein 35-55% of the cells in the composition are NKX6.1 positive, ISL1 positive cells; 25-45% of the cells in the composition are NKX6.1 negative, ISL1 positive cells; and 1-12% of the cells in the plurality of cells are NKX6.1 negative, ISL1 negative cells.

168. 157. The encapsulated composition of claims 144-156, wherein 35-55% of the cells in the encapsulated composition are NKX6.1 positive, ISL1 positive cells; 25-45% of the cells in the encapsulated composition are NKX6.1 negative, ISL1 positive cells; and 1-12% of the cells in the plurality of cells are NKX6.1 negative, ISL1 negative cells.

169. 166. The method of claims 157 to 165, wherein 35-55% of the cells in the encapsulated composition are NKX6.1 positive, ISL1 positive cells; 25-45% of the cells in the encapsulated composition are NKX6.1 negative, ISL1 positive cells; and 1-12% of the cells in the plurality of cells are NKX6.1 negative, ISL1 negative cells.

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