Methods to promote the differentiation of stem cells into beta cells

Abstract

This provides an unlimited supply method for human β-cells from stem cells. [Solution] Compositions and methods for promoting the differentiation of stem cells into beta cells using one or more epigenetically modified compounds are disclosed herein. The disclosure also relates to compositions and methods for selecting and enriching differentiated beta cells. The disclosure also relates to compositions and methods for irradiating a cell population to reduce proliferation. In some embodiments, methods are provided herein, comprising the step of contacting a population of pancreatic progenitor cells or their precursors with an epigenetically modified compound, wherein the contact step results in a population of endocrine cells in which the proportion of chromogranin A-positive (CHGA+) cells or C-peptide-positive and NKX6.1-positive (C-PEP+, NKX6.1+) cells is increased compared to a corresponding population of endocrine cells that have not been contacted with the epigenetically modified compound.

Description

[Technical Field]

[0001] cross reference

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 637,923 filed March 2, 2018, which is incorporated herein by reference in its entirety. [Background technology]

[0002]

[0002] Elucidating the molecular mechanisms that govern the regeneration, plasticity, and function of pancreatic islet cells will allow for the improvement and expansion of β-cell replacement strategies for treating diabetes. The production of β-cells induced from stem cells can provide a potentially useful step toward the production of pancreatic islet cells and pancreatic organs. Diabetes is one of the rapidly progressing diseases that can be treated with tissues induced from stem cells. Type 1 diabetes is caused by the autoimmune destruction of β-cells in the pancreatic islets. Type 2 diabetes is caused by insulin resistance in peripheral tissues and dysfunction of β-cells. Patients with diabetes, especially those with type 1 diabetes, may be cured by transplantation of new β-cells. Patients who have received transplantation of cadaveric human islets can become insulin-free for 5 years or more with this strategy, but this approach has limitations due to the rarity and quality of donor islets. If an unlimited supply of human β-cells could be produced from stem cells, this therapy could be extended to millions of new patients, which could be an important test case for translating stem cell biology into clinical practice. Embedding by reference

[0001] All publications, patents, and patent applications described herein are incorporated by reference to the same extent as each individual publication, patent, or patent application is specifically and individually indicated as being incorporated by reference. Unless otherwise indicated, the publications, patents, and patent applications described herein are incorporated by reference as a whole. [Overview of the project]

[0003]

[0002] In some embodiments, the present invention provides a method comprising the step of contacting a population of pancreatic progenitor cells or their precursors with an epigenetic modification compound, wherein the contact step results in a population of endocrine cells in which the proportion of chromogranin A-positive (CHGA+) cells is increased or the proportion of C-peptide-positive and NKX6.1-positive (C-PEP+, NKX6.1+) cells is increased compared to a corresponding population of endocrine cells that have not been contacted with the epigenetic modification compound.

[0004]

[0003] In some embodiments, the present invention provides a method comprising the step of contacting a population of pancreatic progenitor cells or their precursors with an epigenetic modification compound, wherein the contact step results in a population of endocrine cells in which the proportion of cells expressing VMAT or Cdx2 is reduced compared to a corresponding population of endocrine cells that have not been contacted with the epigenetic modification compound.

[0005]

[0004] In some examples, the epigenetic modification compound comprises one or more of the following: DNA methylation inhibitors, histone acetyltransferase inhibitors, histone deacetylase inhibitors, histone methyltransferase inhibitors, or bromodomain inhibitors. In some examples, the epigenetic modification compound comprises a histone methyltransferase inhibitor. In some examples, the histone methyltransferase inhibitor is an EZH2 inhibitor. In some examples, the histone methyltransferase inhibitor is selected from the group consisting of DZNep, GSK126, and EPZ6438. In some examples, the histone methyltransferase inhibitor is DZNep. In some examples, pancreatic progenitor cells or their precursors The concentration of DZNep brought into contact with the population of carcinogens is approximately 0.05 μM to 50 μM, 0.1 μM to 10 μM, 0.5 μM to 5 μM, 0.75 μM to 2.5 μM, or 1 μM to 2 μM. In some examples, the concentration of DZNep is at least 0.5 μM. In some examples, the concentration of DZNep is approximately 1 μM. In some examples, the epigenetic modification compound includes a histone deacetylase (HDAC) inhibitor. In some examples, the HDAC inhibitor is a class I HDAC inhibitor, a class II HDAC inhibitor, or a combination thereof. In some examples, the HDAC inhibitor is selected from the group consisting of KD5170, MC1568, and TMP195. In some examples, the HDAC inhibitor is KD5170. In some examples, the epigenetic modification compound includes an HDAC inhibitor and an EZH2 inhibitor. In some cases, the epigenetic modification compounds include DZNep and KD5170. In some cases, this method is performed in vitro.

[0006]

[0005] In some examples, the method further includes the step of contacting a population of pancreatic progenitor cells or their precursors with a drug selected from the group consisting of (i) SHH pathway inhibitors, (ii) retinoic acid (RA) signaling pathway activators, (iii) γ-secretase inhibitors, (iv) growth factors from the epidermal growth factor (EGF) family, (v) bone morphogenetic protein (BMP) signaling pathway inhibitors, (vi) TGF-β signaling pathway inhibitors, (vii) thyroid hormone signaling pathway activators, (viii) protein kinase inhibitors, and (ix) ROCK inhibitors. In some cases, (A) the SHH pathway inhibitor includes SANT1, (B) the RA signaling pathway activator includes retinoic acid, (C) the γ-secretase inhibitor includes XXI, (D) the growth factor from the EGF family includes beta-cerulin, (E) the BMP signaling pathway inhibitor includes LDN, (F) the TGF-β signaling pathway inhibitor includes Alk5i II, (G) the thyroid hormone signaling pathway activator includes GC-1, (H) the protein kinase inhibitor includes staurosporine, or (I) the ROCK inhibitor includes thiazobinin. In some cases, the method includes the step of contacting a population of pancreatic progenitor cells or their precursors with a drug selected from the group consisting of beta-cerulin, thiazobinin, retinoic acid, SANT1, XXI, Alk5i II, GC-1, LDN, and staurosporine. In some cases, the contact step is for at least 3 days. In some cases, the contact step includes contacting a population of pancreatic progenitor cells or their precursors with an epigenetic modification compound for a period of more than 3 days, and removing an SHH pathway inhibitor, an RA signaling pathway activator, or a growth factor from the EGF family after the contact step with the population of pancreatic progenitor cells or their precursors during the first 3 days of the above period. In some cases, the contact step is at least 5 days. In some cases, the contact step is approximately 7 days. In some cases, at least one cell in the population of pancreatic progenitor cells expresses at least one of PDX1 and NKX6-1. In some cases, at least one cell in the population of pancreatic progenitor cells expresses both PDX1 and NKX6-1.In some cases, at least one cell in a population of endocrine cells expresses CHGA. In some cases, at least one cell in a population of endocrine cells expresses C-peptide and NKX6.1. In some cases, a population of endocrine cells, as measured by flow cytometry, contains at least approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 180%, 200%, 220%, 250%, 280%, 300%, 320%, 350%, 380%, 400%, 420%, 450%, 480%, or 500% higher percentages of CHGA+ cells than the corresponding population of endocrine cells not in contact with the epigenetic modification compound. In some cases, a population of endocrine cells, as measured by flow cytometry, contains at least approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 180%, 200%, 220%, 250%, 280%, 300%, 320%, 350%, 380%, 400%, 420%, 450%, 480%, or 500% higher percentages of C-PEP+, NKX6.1+ cells compared to the corresponding population of endocrine cells not in contact with epigenetic modification compounds. In the example, a population of endocrine cells, as measured by flow cytometry, contains cells expressing VMAT or Cdx2 at a rate at least approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 180%, 200%, 220%, 250%, 280%, 300%, 320%, 350%, 380%, or 400% lower than the corresponding population of endocrine cells not in contact with at least one epigenetic modification compound.

[0007]

[0006] In some embodiments, cells produced by any method provided herein are provided herein. In one aspect, a composition comprising a cell population, wherein the cell population is measured by flow cytometry and comprises (a) at least about 20% of cells that express C-peptide and NKX6.1, (b) at least about 60% of cells that express CHGA, (c) at most about 20% of cells that express Cdx2, or (d) at most about 45% of cells that express VMAT1, is provided herein.

[0008] In one aspect, a composition comprising a cell population that is measured by flow cytometry and comprises at least about 30% ISL1-positive, NKX6.1-positive cells and at most about 20% ISL1-negative, NKX6.1-negative cells is provided herein.

[0009]

[0009] In some examples, the cell population contains at least about 35% ISL1-positive, NKX6.1-positive cells. In some examples, the cell population contains at least about 40% ISL1-positive, NKX6.1-positive cells. In some examples, the cell population contains at most about 15% ISL1-negative, NKX6.1-negative cells. In some examples, the composition, as measured by flow cytometry, contains (a) at least about 20% of cells expressing C-peptide and NKX6.1, (b) at least about 60% of cells expressing CHGA, and (c) at most about 20% of cells expressing Cdx2. In some examples, the composition, as measured by flow cytometry, contains at most about 45% of cells expressing VMAT1. In some examples, the composition further contains an epigenetic modification compound. In some examples, the epigenetic modification compound comprises one or more of the following: a DNA methylation inhibitor, a histone acetyltransferase inhibitor, a histone deacetylase inhibitor, a histone methyltransferase inhibitor, or a bromodomain inhibitor. In some examples, the epigenetic modification compound comprises a histone methyltransferase inhibitor. In some examples, the histone methyltransferase inhibitor is an EZH2 inhibitor. In some examples, the histone methyltransferase inhibitor is selected from the group consisting of DZNep, GSK126, and EPZ6438. In some examples, the histone methyltransferase composition is DZNep. In some examples, the concentration of DZNep to be contacted with a population of pancreatic progenitor cells or their precursors is approximately 0.05 μM to approximately 50 μM, approximately 0.1 μM to approximately 10 μM, approximately 0.5 μM to approximately 5 μM, approximately 0.75 μM to approximately 2.5 μM, or approximately 1 μM to approximately 2 μM. In some examples, the concentration of DZNep is at least approximately 0.5 μM. In some cases, the concentration of DZNep is approximately 1 μM. In some cases, the epigenetic modification compound includes a histone deacetylase (HDAC) inhibitor. In some cases, the HDAC inhibitor is a class I HDAC inhibitor, a class II HDAC inhibitor, or a combination thereof. In some cases, the HDAC inhibitor is selected from the group consisting of KD5170, MC1568, and TMP195. In some cases, the HDAC inhibitor is KD5170.In some examples, the epigenetic modification compound includes an HDAC inhibitor and an EZH2 inhibitor. In some examples, the epigenetic modification compound includes DZNep and KD5170. In some examples, the composition further includes a drug selected from the group consisting of (i) an SHH pathway inhibitor, (ii) a retinoic acid (RA) signaling pathway activator, (iii) a γ-secretase inhibitor, (iv) a growth factor from the epidermal growth factor (EGF) family, (v) a bone morphogenetic protein (BMP) signaling pathway inhibitor, (vi) a TGF-β signaling pathway inhibitor, (vii) a thyroid hormone signaling pathway activator, (vii i) a protein kinase inhibitor, and (ix) a ROCK inhibitor. In some examples of the composition, (A) the SHH pathway inhibitor includes SANT1, (B) the RA signaling pathway activator includes retinoic acid, (C) the γ-secretase inhibitor includes XXI, (D) the growth factor from the EGF family includes betacellulin, (E) the BMP signaling pathway inhibitor includes LDN, (F) the TGF-β signaling pathway inhibitor includes Alk5i II, (G) the thyroid hormone signaling pathway activator includes GC-1, (H) the protein kinase inhibitor includes staurosporine, or (I) the ROCK inhibitor includes thiazovivin. In some examples, the composition includes a drug selected from the group consisting of betacellulin, thiazovivin, retinoic acid, SANT1, XXI, Alk5i II, GC-1, LDN, and staurosporine.

[0010]

[0010] In some embodiments, provided herein is a composition comprising pancreatic progenitor cells and at least one of a histone deacetylase (HDAC) inhibitor or a histone methyltransferase inhibitor.

[0011]

[0011] In some examples, the composition further comprises endocrine cells. In some examples of the composition, the HDAC inhibitor is a class I HDAC inhibitor, a class II HDAC inhibitor, or a combination thereof. In some examples, the HDAC inhibitor is selected from the group consisting of KD5170, MC1568, and TMP195. In some examples, the HDAC inhibitor is KD5170. In some examples, the concentration of KD5170 in the composition is about 0.05 μM to about 50 μM, about 0.1 μM to about 10 μM, about 0.5 μM to about 5 μM, about 0.75 μM to about 2.5 μM, or about 1 μM to about 2 μM. In some examples, the concentration of KD5170 is at least 0.5 μM. In some examples, the concentration of KD5170 is about 1 μM. In some examples, the histone methyltransferase inhibitor is an EZH2 inhibitor. In some cases, the histone methyltransferase inhibitor is selected from the group consisting of DZNep, GSK126, and EPZ6438. In some cases, the histone methyltransferase inhibitor is DZNep. In some cases, the concentration of DZNep in the composition is approximately 0.05 μM to approximately 50 μM, approximately 0.1 μM to approximately 10 μM, approximately 0.5 μM to approximately 5 μM, approximately 0.75 μM to approximately 2.5 μM, or approximately 1 μM to approximately 2 μM. In some cases, the concentration of DZNep is at least 0.5 μM. In some cases, the concentration of DZNep is approximately 1 μM. In some cases, the HDAC inhibitor is KD5170 and the histone methyltransferase inhibitor is DZNep. In some cases, the composition is an in vitro composition. In some examples, the composition further comprises agents selected from the group consisting of (i) SHH pathway inhibitors, (ii) retinoic acid (RA) signaling pathway activators, (iii) γ-secretase inhibitors, (iv) growth factors from the epidermal growth factor (EGF) family, (v) bone morphogenetic protein (BMP) signaling pathway inhibitors, (vi) TGF-β signaling pathway inhibitors, (vii) thyroid hormone signaling pathway activators, (viii) protein kinase inhibitors, and (ix) ROCK inhibitors.In some examples, (A) the SHH pathway inhibitor includes SANT1, (B) the RA signaling pathway activator includes retinoic acid, (C) the γ-secretase inhibitor includes XXI, (D) the growth factor from the EGF family includes beta-cellulin, (E) the BMP signaling pathway inhibitor includes LDN, (F) the TGF-β signaling pathway inhibitor includes Alk5i II, (G) the thyroid hormone signaling pathway activator includes GC-1, (H) the protein kinase inhibitor includes staurosporine, or (I) the ROCK inhibitor includes thiazobinin. In some examples, the composition further includes agents selected from the group consisting of beta-cellulin, thiazobinin, retinoic acid, SANT1, XXI, Alk5i II, GC-1, LDN, and staurosporine.

[0012]

[0012] In some embodiments, the steps include contacting a cell population containing pancreatic progenitor cells or their precursors with a histone methyltransferase inhibitor to produce a cell population containing endocrine cells, and maturing the cell population containing endocrine cells to respond to a glucose challenge in v A method is provided herein that includes the step of obtaining at least one pancreatic β-cell that exhibits an itro-glucose-stimulated insulin secretion response.

[0013]

[0013] In some examples, the method includes contacting a cell population with a drug selected from the group consisting of (i) an SHH pathway inhibitor, (ii) a retinoic acid (RA) signaling pathway activator, (iii) a γ-secretase inhibitor, (iv) a growth factor from the epidermal growth factor (EGF) family, (v) a bone morphogenetic protein (BMP) signaling pathway inhibitor, (vi) a TGF-β signaling pathway inhibitor, (vii) a thyroid hormone signaling pathway activator, (viii) a protein kinase inhibitor, and (ix) a ROCK inhibitor. In some examples, (A) the SHH pathway inhibitor is SANT1, (B) the RA signaling pathway activator is retinoic acid, (C) the γ-secretase inhibitor is XXI, (D) the growth factor from the EGF family is beta-cell phosphate, (E) the BMP signaling pathway inhibitor is LDN, and (F) the TGF-β signaling pathway inhibitor is Alk5i (G) a thyroid hormone signaling pathway activator including II, (H) a protein kinase inhibitor including staurosporine, or (I) a ROCK inhibitor including thiazobinin. In some examples, the method includes the step of contacting a cell population with a drug selected from the group consisting of beta-cerulin, thiazobinin, retinoic acid, SANT1, XXI, Alk5i II, GC-1, LDN, and staurosporine. In some examples, the method further includes the step of contacting a cell population with a histone deacetylase (HDAC) inhibitor. In some examples, the HDAC inhibitor is KD5170. In some examples, the histone methyltransferase inhibitor is selected from the group consisting of DZNep, GSK126, and EPZ6438. In some examples, the histone methyltransferase inhibitor is DZNep. In some cases, the step of contacting with a histone methyltransferase inhibitor results in a population containing endocrine cells with an increased proportion of chromogranin A-positive (CHGA+) cells or an increased proportion of C-peptide-positive and NKX6.1-positive (C-PEP+, NKX6.1+) cells compared to a corresponding population of endocrine cells not contacted with the histone methyltransferase inhibitor. In some cases, the step of contacting with a histone methyltransferase inhibitor results in a population containing endocrine cells with a decreased proportion of cells expressing VMAT or Cdx2 compared to a corresponding population of endocrine cells not contacted with the histone methyltransferase inhibitor. In some cases, at least one cell among the pancreatic progenitor cells or their precursors expresses both Pdx1 and NKX6.1. In some cases, the method further includes the step of differentiating multiple stem cells in vitro to obtain a cell population containing pancreatic progenitor cells or their precursors.

[0014]

[0014] In some embodiments, pancreatic β-cells produced by any method provided herein are provided herein.

[0015] In some embodiments, the present invention provides a method comprising the steps of (a) contacting a population of Pdx1-negative, NKX6.1-negative gastrulatic cells with a bone morphogenetic protein (BMP) signaling pathway inhibitor and a growth factor from the transformation growth factor β (TGF-β) superfamily to produce a cell population including Pdx1-positive, NKX6.1-positive pancreatic progenitor cells, and (b) contacting a cell population including Pdx1-positive, NKX6.1-positive pancreatic progenitor cells with an epigenetic modification compound to produce a cell population including endocrine cells.

[0015]

[0016] In some cases, BMP signaling pathway inhibitors include DMH-1, its derivatives, analogs, or variants. In some cases, the concentration of DMH-1 exposed to a population of Pdx1-negative, NKX6.1-negative gastrulatic cells is approximately 0.01 μM to 10 μM, approximately 0.05 μM to 5 μM, approximately 0.1 μM to 1 μM, or approximately 0.15 μM to 0.5 μM. In some cases, the concentration of DMH-1 exposed to a population of Pdx1-negative, NKX6.1-negative gastrulatic cells is approximately 0.25 μM. In some cases, TGF-β superfluid The growth factor from Lee contains activin A. In some cases, the concentration of activin A applied to a population of Pdx1-negative, NKX6.1-negative gastrula cells is approximately 0.5 ng / mL to 200 ng / mL, 1 ng / mL to 100 ng / mL, 2 ng / mL to 50 ng / mL, or 5 ng / mL to 30 ng / mL. In some cases, the concentration of activin A applied to a population of Pdx1-negative, NKX6.1-negative gastrula cells is at least approximately 5 ng / mL or at least approximately 10 ng / mL. In some cases, the concentration of activin A applied to a population of Pdx1-negative, NKX6.1-negative gastrula cells is approximately 20 ng / mL. In some examples, the step of contacting a population of Pdx1-negative, NKX6.1-negative gastrulatic cells further includes the step of contacting them with a drug selected from the group consisting of growth factors from the FGF family, SHH pathway inhibitors, RA signaling pathway activators, protein kinase C activators, and ROCK inhibitors. In some examples, the epigenetic modification compound includes a compound selected from the group consisting of DNA methylation inhibitors, histone acetyltransferase inhibitors, histone deacetylase inhibitors, histone methyltransferase inhibitors, and bromodomain inhibitors. In some examples, the epigenetic modification compound includes a histone methyltransferase inhibitor. In some examples, the histone methyltransferase inhibitor is an EZH2 inhibitor. In some examples, the histone methyltransferase inhibitor is selected from the group consisting of DZNep, GSK126, and EPZ6438. In some examples, the histone methyltransferase inhibitor is DZNep. In some cases, the concentration of DZNep exposed to a population of pancreatic progenitor cells or their precursors is approximately 0.05 μM to 50 μM, 0.1 μM to 10 μM, 0.5 μM to 5 μM, 0.75 μM to 2.5 μM, or 1 μM to 2 μM. In some cases, the concentration of DZNep is at least 0.5 μM. In some cases, the concentration of DZNep is approximately 1 μM. In some cases, the epigenetic modification compound includes a histone deacetylase (HDAC) inhibitor.In some cases, the HDAC inhibitor is a class I HDAC inhibitor, a class II HDAC inhibitor, or a combination thereof. In some cases, the HDAC inhibitor is selected from the group consisting of KD5170, MC1568, and TMP195. In some cases, the HDAC inhibitor is KD5170. In some cases, the method is performed in vitro. In some cases, the method further includes the step of contacting a population containing Pdx1-positive, NKX6.1-positive pancreatic progenitor cells with a drug selected from the group consisting of (i) SHH pathway inhibitors, (ii) retinoic acid (RA) signaling pathway activators, (iii) γ-secretase inhibitors, (iv) growth factors from the epidermal growth factor (EGF) family, (v) bone morphogenetic protein (BMP) signaling pathway inhibitors, (vi) TGF-β signaling pathway inhibitors, (vii) thyroid hormone signaling pathway activators, (viii) protein kinase inhibitors, and (ix) ROCK inhibitors. In some cases, (A) the SHH pathway inhibitor includes SANT1, (B) the RA signaling pathway activator includes retinoic acid, (C) the γ-secretase inhibitor includes XXI, (D) the growth factor from the EGF family includes beta-cerulin, (E) the BMP signaling pathway inhibitor includes LDN, (F) the TGF-β signaling pathway inhibitor includes Alk5i II, (G) the thyroid hormone signaling pathway activator includes GC-1, (H) the protein kinase inhibitor includes staurosporine, or (I) the ROCK inhibitor includes thiazobinin. In some cases, the method includes the step of exposing a population of Pdx1-positive, NKX6.1-positive pancreatic progenitor cells to a drug selected from the group consisting of beta-cerulin, thiazobinin, retinoic acid, SANT1, XXI, Alk5i II, GC-1, LDN, and staurosporine. In some cases, the exposure step is for at least 3 days.In some cases, the contact step includes contacting a population of Pdx1-positive, NKX6.1-positive pancreatic progenitor cells with an epigenetic modification compound for a period of more than 3 days, and removing an SHH pathway inhibitor, RA signaling pathway activator, or growth factor from the EGF family after the contact step with the population of pancreatic progenitor cells or their precursors during the first 3 days of the above period. In some cases, the contact step is at least 5 days. In some cases, the contact step is about 7 days. The duration is several days. In some cases, cell populations containing Pdx1-positive, NKX6.1-positive pancreatic progenitor cells contain at most about 10% of cells expressing Cdx2 as measured by flow cytometry. In some cases, cell populations containing Pdx1-positive, NKX6.1-positive pancreatic progenitor cells contain a lower proportion of cells expressing Cdx2 compared to the corresponding cell population not in contact with bone morphogenetic protein (BMP) signaling pathway inhibitors and growth factors from the transformation growth factor β (TGF-β) superfamily. In some cases, cell populations containing endocrine cells contain at least about 40% of cells expressing ISL1 and NKX6.1 as measured by flow cytometry. In some cases, cell populations containing endocrine cells contain a higher proportion of cells expressing ISL1 and NKX6.1 compared to the corresponding cell population not in contact with bone morphogenetic protein (BMP) signaling pathway inhibitors and growth factors from the transformation growth factor β (TGF-β) superfamily. In some cases, cell populations containing endocrine cells contain at most about 15% ISL-1-negative, NKX6.1-negative cells, as measured by flow cytometry. In some cases, cell populations containing endocrine cells contain a lower proportion of ISL-1-negative, NKX6.1-negative cells compared to corresponding cell populations not exposed to bone morphogenetic protein (BMP) signaling pathway inhibitors and growth factors from the transformation growth factor β (TGF-β) superfamily. In some cases, the method further includes the step of cryopreserving the cell population containing endocrine cells. In some cases, the cell population containing endocrine cells is a cell cluster, and the method further includes the steps of (a) separating multiple cells from the cell cluster, and (b) culturing the multiple cells from step (a) in reaggregation medium to form a second cell cluster in at least a portion of the multiple cells. In some cases, the separation step does not include the step of subjecting multiple cells to flow cytometry. In some cases, the reaggregation medium does not contain serum. In some cases, the reaggregation medium does not contain exogenous differentiation factors.In some cases, the method further includes the step of maturing endocrine cells in vitro to obtain at least one pancreatic β-cell that exhibits an in vitro glucose-stimulated insulin secretion response in response to a glucose challenge. In some cases, the maturation step is carried out in serum-free medium. In some cases, the maturation step is carried out in xeno-free medium. In some cases, the maturation step is carried out in a medium that does not contain exogenous differentiation factors. In some cases, the maturation step is carried out in the presence of human serum albumin (HSA). In some cases, HSA is present at concentrations of approximately 0.1% to 5%, and approximately 0.5% to 2%. In some cases, HSA is present at a concentration of approximately 1%.

[0016]

[0017] In some examples, the methods provided herein include: (a) differentiating pluripotent stem cells into endoderm cells by contacting pluripotent stem cells in a population with growth factors from the TGF-β superfamily and WNT signaling pathway activators; (b) differentiating at least some endoderm cells into gastrulatic cells by contacting endoderm cells with growth factors from the FGF family; (c) differentiating at least some gastrulatic cells into Pdx1-positive pancreatic progenitor cells by contacting gastrulatic cells with ROCK inhibitors, growth factors from the FGF family, BMP signaling pathway inhibitors, PKC activators, retinoic acid signaling pathway activators, SHH pathway inhibitors, and growth factors from the TGF-β superfamily; and (d) differentiating Pdx1-positive pancreatic progenitor cells with ROCK inhibitors. (e) Differentiating at least some Pdx1-positive pancreatic progenitor cells into Pdx1-positive, NKX6.1-positive pancreatic progenitor cells by contacting them with a CK inhibitor, growth factors from the TGFβ superfamily, growth factors from the FGF family, RA signaling pathway activators, and SHH pathway inhibitors; and (e) differentiating at least some Pdx1-positive, NKX6.1-positive pancreatic progenitor cells into at least one NKX6.1-positive pancreatic progenitor cells by contacting them with a TGF-β signaling pathway inhibitor, growth factors from the EGF family, RA signaling pathway activators, SHH pathway inhibitors, TH signaling pathway activators, γ-secretase inhibitors, protein kinase inhibitors, ROCK inhibitors, BMP signaling pathway inhibitors, and epigenetic modification compounds. The method includes the step of differentiating cells into a population containing cells that are 1+ and C-peptide+.

[0017]

[0018] In some embodiments, a cell population comprising endocrine cells produced by any method provided herein is provided herein. In some embodiments, a cell population comprising SC-β cells produced by any method provided herein is provided herein.

[0018]

[0019] In some embodiments, the method provided herein includes the step of exposing an in vitro cell population, including endocrine cells, to irradiation at a dose of approximately 100 rad to approximately 100,000 rad for a time of approximately 1 minute to approximately 60 minutes.

[0019]

[0020] In some embodiments, a method for reducing cell proliferation is provided herein, comprising the step of exposing a population of cells including stem cells, endoderm cells of an embryo, gastrulatum cells, pancreatic progenitor cells, or endocrine cells to irradiation, wherein the irradiation results in a population of cells with reduced proliferative capacity compared to the corresponding cell population that has not been subjected to irradiation.

[0020]

[0021] In some embodiments, the cell population is exposed to irradiation of approximately 100 rad to 50,000 rad, approximately 100 rad to 25,000 rad, approximately 100 rad to 10,000 rad, approximately 250 rad to 25,000 rad, approximately 500 rad to 25,000 rad, approximately 1,000 rad to 25,000 rad, approximately 2,500 rad to 25,000 rad, approximately 5,000 rad to 25,000 rad, or approximately 10,000 rad to 15,000 rad. In some examples, the cell population is exposed to irradiation of approximately 10,000 rad. In some cases, the cell population is exposed to irradiation for approximately 1 to 55 minutes, 1 to 50 minutes, 1 to 45 minutes, 1 to 40 minutes, 1 to 35 minutes, 1 to 30 minutes, 1 to 25 minutes, 1 to 20 minutes, 1 to 10 minutes, 1 to 5 minutes, 10 to 55 minutes, 15 to 55 minutes, 20 to 55 minutes, 25 to 55 minutes, 30 to 55 minutes, 20 to 40 minutes, or 25 to 35 minutes. In some cases, the cell population is exposed to irradiation for approximately 30 minutes. In some cases, the irradiation includes ionization irradiation. In some cases, the ionization irradiation includes gamma rays, X-rays, ultraviolet radiation, alpha rays, beta rays, or neutrons. In some cases, irradiation results in a cell population with reduced proliferation compared to the corresponding cell cluster that has not been irradiated. In some cases, the cell population includes a second cell cluster having a diameter of approximately 50 μm to 500 μm, 50 μm to 300 μm, 50 μm to 200 μm, 50 μm to 150 μm, 600 μm to 150 μm, 700 μm to 150 μm, 80 μm to 150 μm, or 60 μm to 100 μm. In some cases, the method further includes (a) the step of separating a plurality of cells from the first cell cluster, and (b) the step of culturing the plurality of cells from step (a) in a re-aggregation medium to cause at least a portion of the plurality of cells to form a second cell cluster. In some cases, the separation step does not include the step of subjecting the plurality of cells to flow cytometry. In some cases, the first cell cluster is obtained by separating the third cell cluster and culturing the cells separated from the third cell cluster to form the first cell cluster.In some cases, cell clusters are cryopreserved before irradiation, and the method further includes the step of thawing the cryopreserved cell clusters before irradiation. In some cases, cell clusters are cryopreserved while being subjected to irradiation. In some cases, the method further includes the step of thawing the cryopreserved cell clusters after irradiation and differentiating at least some endocrine cells. In some cases, the method further includes the step of obtaining a cell population containing endocrine cells by differentiating pancreatic progenitor cells or their precursors in vitro. In some cases, the method further includes the step of differentiating stem cells in vitro to produce a cell population containing endocrine cells. In some cases, the method further includes the step of maturing at least some endocrine cells into pancreatic β cells in vitro to produce a cell population containing. In some cases, the method further includes the step of implanting pancreatic β cells into a subject requiring implantation of pancreatic β cells. In some cases, the implanted pancreatic β. The cells are configured to control blood glucose levels in the subject for at least approximately 50, 60, 70, 80, 90 days, or longer.

[0021]

[0022] In some embodiments, a cell population comprising endocrine cells produced by any irradiation method described herein is provided herein. In some embodiments, a cell population comprising pancreatic β-cells produced by any irradiation method described herein is provided herein.

[0022]

[0023] In some embodiments, a method is provided herein that includes the step of contacting a population of pancreatic progenitor cells or their precursors with a composition comprising at least one epigenetic modification compound, wherein the contact step results in a population of endocrine cells in which the proportion of cells expressing VMAT or Cdx2 is reduced compared to a corresponding population of endocrine cells that have not been contacted with at least one epigenetic modification compound.

[0023]

[0024] In some embodiments, the epigenetic modification compound comprises one or more of the following: a DNA methylation inhibitor, a histone acetyltransferase inhibitor, a histone deacetylase inhibitor, a histone methyltransferase inhibitor, or a bromodomain inhibitor. In some embodiments, the epigenetic modification compound comprises a histone methyltransferase inhibitor. In some embodiments, the histone methyltransferase inhibitor is an EZH2 inhibitor.

[0024]

[0025] In some embodiments, the histone methyltransferase inhibitor is at least one of DZNep, GSK126, and EPZ6438. In some embodiments, the histone methyltransferase inhibitor is DZNep. In some embodiments, the concentration of DZNep in the composition is greater than 0.1 μM. In some embodiments, the concentration of DZNep is at least 0.5 μM. In some embodiments, the concentration of DZNep is about 1 μM. In some embodiments, at least one epigenetic modification compound comprises a histone deacetylase (HDAC) inhibitor. In some embodiments, the HDAC inhibitor is a class I HDAC inhibitor, a class II HDAC inhibitor, or a combination thereof. In some embodiments, the HDAC inhibitor is at least one of KD5170, MC1568, and TMP195. In some embodiments, the HDAC inhibitor is KD5170. In some embodiments, at least one epigenetic modification compound comprises an HDAC inhibitor and an EZH2 inhibitor. In some embodiments, at least one epigenetic modification compound comprises DZNep and KD5170.

[0025]

[0026] In some embodiments, in the method provided herein, at least one of the cells expressing VMAT is INS -In some embodiments, at least some cells in the population of pancreatic progenitor cells differentiate into a population of PH cells. In some embodiments, an increased proportion of cells in the population of endocrine cells differentiate into NKX6.1 cells compared to the corresponding population of endocrine cells that have not come into contact with at least one epigenetic modification compound. - or ChromA + In some embodiments, at least one of the increased proportion of cells is NKX6.1 - and ChromA + In some embodiments, at least some cells from a population of pancreatic progenitor cells differentiate into a population of β cells. In some embodiments, the β cells are β(SC-β) cells derived from stem cells. In some embodiments, the β cells express C-PEP and NKX6-1. In some embodiments, the β cells exhibit an in vitro glucose-stimulated insulin secretion response to a glucose challenge.

[0026]

[0027] In some embodiments, the composition of the method described herein comprises at least one of beta-cellulin, thiazobinin, retinoic acid, SANT1, XXI, Alk5i II, GC-1, LDN, or staurosporine. In some embodiments, the contact step is at least 3 days. In some embodiments, the contact step is less At most, it is 5 days. In some embodiments, the contact step is about 7 days.

[0027]

[0028] In some embodiments, at least one pancreatic progenitor cell in a population of pancreatic progenitor cells expresses at least one of PDX1 and NKX6-1. In some embodiments, at least one endocrine cell in a population of endocrine cells expresses CHGA.

[0028]

[0029] Endocrine cells produced by any of the methods described herein are provided herein.

[0030] Compositions comprising pancreatic progenitor cells, a histone deacetylase (HDAC) inhibitor, a histone methyltransferase inhibitor, and optionally endocrine cells are provided herein. In some embodiments, the HDAC inhibitor is a class I HDAC inhibitor, a class II HDAC inhibitor, or a combination thereof. In some embodiments, the HDAC inhibitor is at least one of KD5170, MC1568, or TMP195. In some embodiments, the HDAC inhibitor is KD5170. In some embodiments, the concentration of KD5170 in the composition is at least 0.1 μM. In some embodiments, the concentration of KD5170 is at least 0.5 μM. In some embodiments, the concentration of KD5170 is about 1 μM. In some embodiments, the histone methyltransferase inhibitor is an EZH2 inhibitor. In some embodiments, the histone methyltransferase inhibitor is at least one of DZNep, GSK126, or EPZ6438. In some embodiments, the histone methyltransferase inhibitor is DZNep. In some embodiments, the concentration of DZNep is at least 0.1 μM. In some embodiments, the concentration of DZNep is at least 0.5 μM. In some embodiments, the concentration of DZNep is approximately 1 μM. In some embodiments, the HDAC inhibitor is KD5170 and the histone methyltransferase inhibitor is DZNep. In some embodiments, the composition is an in vitro composition. In some embodiments, the composition further comprises at least one of beta-cellulin, thiazobinin, retinoic acid, SANT1, XXI, Alk5i II, GC-1, LDN, or staurosporine.

[0029]

[0031] A method is provided herein comprising the step of contacting pancreatic progenitor cells or their precursors with a histone deacetylase (HDAC) inhibitor and a histone methyltransferase inhibitor, wherein the contact step induces differentiation of the pancreatic progenitor cells. In some embodiments, the pancreatic progenitor cells are differentiated into β cells. In some embodiments, the β cells are β (SC-β) cells derived from stem cells. In some embodiments, the β cells express C-PEP and NKX6-1. In some embodiments, the β cells exhibit an in vitro glucose-stimulated insulin secretion response to a glucose challenge. In some embodiments, the HDAC inhibitor is a class I HDAC inhibitor, a class II HDAC inhibitor, or a combination thereof. In some embodiments, the HDAC inhibitor is at least one of KD5170, MC1568, or TMP195. In some embodiments, the HDAC inhibitor is KD5170. In some embodiments, the histone methyltransferase inhibitor is an EZH2 inhibitor. In some embodiments, the histone methyltransferase inhibitor is at least one of DZNep, GSK126, or EPZ6438. In some embodiments, the histone methyltransferase inhibitor is DZNep. In some embodiments, the HDAC inhibitor is KD5170 and the histone methyltransferase inhibitor is DZNep. In some embodiments, the method is performed in vitro.

[0030]

[0032] A method is provided herein that includes the step of contacting pancreatic progenitor cells or their precursors with a sufficient amount of KD5170 to induce cell differentiation. In some embodiments, the method further includes the step of contacting pancreatic progenitor cells with a histone methyltransferase inhibitor. In some embodiments, the histone methyltransferase inhibitor is DZNe The histone methyltransferase inhibitor is at least one of p, GSK126, or EPZ6438. In some embodiments, the histone methyltransferase inhibitor is DZNep. In some embodiments, pancreatic progenitor cells differentiate into endocrine cells. In some embodiments, pancreatic progenitor cells differentiate into β cells. In some embodiments, the β cells are β(SC-β) cells derived from stem cells. In some embodiments, the β cells express C-PEP and NKX6-1. In some embodiments, the β cells exhibit an in vitro glucose-stimulated insulin secretion response to a glucose challenge.

[0031]

[0033] A method is provided herein that comprises the steps of (a) differentiating a plurality of stem cells in vitro to obtain a cell population containing pancreatic progenitor cells or their precursors, (b) contacting the cell population in vitro with a histone deacetylase (HDAC) inhibitor to produce at least one endocrine cell, and (c) maturing the endocrine cell in vitro to obtain at least one SC-β cell. In some embodiments, the stem cells are human pluripotent stem cells. In some embodiments, the method further comprises the step of contacting the cell population with at least one of beta-cellulin, thiazobinin, retinoic acid, SANT1, XXI, Alk5i II, GC-1, LDN, or staurosporine. In some embodiments, the SC-β cells express C-PEP and NKX6-1. In some embodiments, the SC-β cells exhibit an in vitro glucose-stimulated insulin secretion response to a glucose challenge. In some embodiments, the method further comprises the step of contacting the cell population with a histone methyltransferase inhibitor. In some embodiments, the histone methyltransferase inhibitor is at least one of DZNep, GSK126, or EPZ6438. In some embodiments, the histone methyltransferase inhibitor is DZNep. In some embodiments, the HDAC inhibitor is KD5170.

[0032]

[0034] A method is provided herein that includes the steps of contacting a cell population containing pancreatic progenitor cells or their precursors in vitro with a sufficient amount of a histone methyltransferase inhibitor to produce endocrine cells, and maturing the endocrine cells in vitro to obtain at least one SC-β cell that exhibits an in vitro glucose-stimulated insulin secretion response to a glucose challenge. In some embodiments, the method further includes the step of differentiating a plurality of stem cells in vitro to obtain a cell population containing pancreatic progenitor cells or their precursors. In some embodiments, the method further includes the step of contacting the cell population with at least one of beta-cellulin, thiazobinin, retinoic acid, SANT1, XXI, Alk5i II, GC-1, LDN, or staurosporine. In some embodiments, the method further includes the step of contacting the cell population with a histone deacetylase (HDAC) inhibitor. In some embodiments, the HDAC inhibitor is KD5170. In some embodiments, the histone methyltransferase inhibitor is at least one of DZNep, GSK126, or EPZ6438. In some embodiments, the histone methyltransferase inhibitor is DZNep.

[0033]

[0035] A method for selecting target cells from a population of cells is provided herein, comprising the steps of (i) contacting target cells with an irritant compound, wherein the contact step induces and localizes a selection marker for the target cells to the cell surface of the target cells, and (ii) selecting target cells based on the localization of the selection marker on the cell surface. In some embodiments, the selection marker includes PSA-NCAM. In some embodiments, the step of selecting target cells is by cell sorting. In some embodiments, the selection step includes contacting the selection marker for the target cells with an antigen-binding polypeptide when the selection marker is localized to the surface of the target cells. In some embodiments, the antigen-binding polypeptide includes an antibody. In some embodiments, the antigen-binding polypeptide binds to PSA-NCAM. In some embodiments, the method selects a population of cells from which at least The method further includes the step of treating a single cell with a compound that removes a selection marker from its cell surface. In some embodiments, a population of cells is treated with the compound before the step of contacting target cells with the irritating compound. In some embodiments, the compound cleaves the selection marker from the cell surface of at least one cell. In some embodiments, the compound is an enzyme. In some embodiments, the compound is an endosialidase. In some embodiments, the endosialidase is an endoneuraminidase (Endo-N). In some embodiments, the target cells are endocrine cells. In some embodiments, the irritating compound comprises at least one of arginine or glucose. In some embodiments, the endocrine cells are β cells. In some embodiments, the β cells are SC-β cells. In some embodiments, the irritating compound comprises isoproterenol. In some embodiments, the endocrine cells are EC cells. In some embodiments, one or more cells in a population of cells are unable to localize the selection marker to their cell surface when in contact with the irritating compound. In some embodiments, the irritating compound comprises at least one of glucose or arginine, and one or more cells are EC cells. In some embodiments, the irritant compound is isoproterenol, and one or more cells are β cells. In some embodiments, the step of selecting target cells involves isolating target cells from one or more cells in a population of cells.

[0034]

[0036] A method is provided herein that comprises the step of contacting a population of pancreatic progenitor cells or their precursors with a composition comprising at least one epigenetically modified compound, wherein the contact step results in an increased proportion of islet cells compared to a corresponding population of pancreatic progenitor cells that have not been contacted with at least one epigenetically modified compound. In some embodiments, the islet cells comprise at least one β-cell. In some embodiments, the β-cell comprises SC-β-cells. In some embodiments, the SC-β-cells exhibit an in vitro glucose-stimulated insulin secretion response to a glucose challenge. In some embodiments, the islet cells comprise at least one alpha-cell. In some embodiments, the islet cells comprise delta-cells. In some embodiments, the islet cells comprise polyformonal (PH) cells. In some embodiments, the method further comprises the step of differentiating a plurality of stem cells in vitro to obtain a population of pancreatic progenitor cells or their precursors. In some embodiments, the stem cells are human pluripotent stem cells. In some embodiments, the at least one epigenetically modified compound comprises one or more of the following: DNA methylation inhibitors, histone acetyltransferase inhibitors, histone deacetylase inhibitors, histone methyltransferase inhibitors, or bromodomain inhibitors. In some embodiments, at least one epigenetic modification compound comprises a histone methyltransferase inhibitor. In some embodiments, the histone methyltransferase inhibitor is an EZH2 inhibitor. In some embodiments, the histone methyltransferase inhibitor is at least one of DZNep, GSK126, or EPZ6438. In some embodiments, the histone methyltransferase inhibitor is DZNep. In some embodiments, the concentration of DZNep in the composition is greater than 0.1 μM. In some embodiments, the concentration of DZNep is at least 0.5 μM. In some embodiments, the concentration of DZNep is approximately 1 μM. In some embodiments, at least one epigenetic modification compound comprises a histone deacetylase (HDAC) inhibitor. In some embodiments, the HDAC inhibitor is a class I HDAC inhibitor, a class II HDAC inhibitor, or a combination thereof.In some embodiments, the HDAC inhibitor is at least one of KD5170, MC1568, or TMP195. In some embodiments, the HDAC inhibitor is KD5170. In some embodiments, at least one epigenetic modification compound comprises an HDAC inhibitor and an EZH2 inhibitor. In some embodiments, at least one epigenetic modification compound comprises DZNep and KD5170.

[0035]

[0037] The features of this disclosure are described in detail in the appended claims. A more complete understanding of the features and advantages of this disclosure can be found in exemplary embodiments in which the principles of this disclosure are utilized. This can be obtained by referring to the following detailed explanation and the attached drawings. [Brief explanation of the drawing]

[0036] [Figure 1]

[0038] The chemical structures of 3-deazanepranosin A hydrochloride (DZNep), GSK126, and EPZ6438 are shown. [Figure 2]

[0039] The chemical structures of KD5170 and MC1568 are shown. [Figure 3]

[0040] The chemical structure of a methyltransferase inhibitor is shown. [Figure 4]

[0041] The chemical structure of the DZNep analog is shown. [Figure 5]

[0042] This is an overview of the differentiation stages described herein. [Figure 6]

[0043] This is a schematic diagram of differentiation induction from hPSCs to INS+ cells, including the inhibition of stage 5 histone methylation and deacetylation as described herein. DE: Embryonic endoderm; PGT: Gastrulatum; PPT1: Early pancreatic progenitor cells; PPT2: PDX1+ / NKX6.1+ pancreatic progenitor cells; EN: NKX6.1 / C-peptide+ cells; SC-β: Stem cell-derived beta cells. [Figure 7]

[0044] This shows that stage 5 cells express NGN3 and initiate differentiation of stem cells (SCs) into pancreatic islet cells. [Figure 8]

[0045] This study demonstrates that inhibiting EZH2 or HDAC in stage 5 increases endocrine cells and SC-β cells. [Figure 9]

[0046] This study demonstrates that co-inhibition of EZH2 and HDAC in stage 5 significantly increases endocrine cells. [Figure 10]

[0047] This study demonstrates that co-inhibition of EZH2 and HDAC in stage 5 significantly increases endocrine cells. [Figure 11]

[0048] This study demonstrates that combined inhibition of EZH2 and HDAC in stage 5 significantly increases SC β cells. [Figure 12]

[0049] This shows an increase in endocrine cells in stage 5 (n=2). [Figure 13]

[0050] This study demonstrates that co-inhibition of EZH2 and HDAC increases neurogenin 3+ progenitor cells in stage 5. [Figure 14]

[0051] This demonstrates that DZNep is superior to other EZH2 inhibitors. [Figure 15]

[0052] This demonstrates that KD5170 is superior to other HDAC inhibitors. [Figure 16]

[0053] This study demonstrates that co-inhibition of EZH2 and HDAC in stage 5 increases NKX6-1+ progenitor cells. [Figure 17]

[0054] This is an outline of the experiment to test EZH2 and HDAC in Stage 5. [Figure 18]

[0055] This is a schematic diagram of the candidate screening settings. [Figure 19]

[0056] (VMAT1+ INS-) shows a specific decrease in the EC population. [Figure 20]

[0057] This indicates a simultaneous increase in (NKX6.1- ChromA+)PH cells. PH: Polyhormone cells. [Figure 21]

[0058] This indicates that the percentage of the (NKX6.1+ INS+)SC-β population was unaffected. [Figure 22]

[0059] This shows various plates used for cell surface marker detection screening. [Figure 23]

[0060] This shows the screening of MACS markers for stage 5. [Figure 24]

[0061] A schematic diagram of SC-beta cell labeling and MACS marker screening is shown. [Figure 25]

[0062] This study demonstrates that sorting based on PSA-NCAM microbeads enriches on-target cells and reduces SOX9+ cells. [Figure 26]

[0063] This shows that EC cells (VMAT1+) remain after PSA-NCAM sorting. [Figure 27]

[0064] This study demonstrates a significant decrease in PSA-NCAM expression upon endo-N enzyme treatment. Endo-N is an endosialidase that rapidly and specifically degrades linear sialic acid polymers containing α-2,8-links, characterized by a minimum sialic acid residue length of 7-9 residues associated with NCAM. The study also shows cleavage of PSA in NCAM under physiological conditions. [Figure 28]

[0065] This is a schematic diagram showing the removal of EC cells using microbead sorting with PSA-NCAM. [Figure 29]

[0066] This suggests that EC cells can arise from intestinal progenitor cells that have become specialized early in the differentiation process. [Figure 30]

[0067] A low percentage of OCT4 at the completion of Stage 0 is associated with a higher percentage of CDX2 in later stages. A high Oct4% is required for stable differentiation. The variability in Sox17 induction remains similar to that of a high Oct4%. [Figure 31]

[0068] This is a schematic diagram of a compound screening method for identifying inhibitors of EC cell differentiation. [Figure 32]

[0069] This is a schematic diagram of compound screening on stage 5 cells. [Figure 33]

[0070] Proliferating cells show a dose-dependent reduction. [Figure 34]

[0071] This study demonstrates that high-dose gamma-ray irradiation did not have a significant effect on the composition and function of SC-pancreatic islets. [Figure 35]

[0072] This indicates that the cryopreserved SC-pancreatic islets lost their ability to control background grafts 60 days after irradiation. [Figure 36]

[0073] This shows the number of beta cells in the irradiated sample compared to the control sample. [Figure 37]

[0074] This shows the blood glucose control maintained by irradiated transplanted mRA pancreatic islet cells. [Figure 38]

[0075] This shows the blood glucose control in all animals with irradiated SC-pancreatic islets. [Figure 39]

[0076] Figures of two exemplary protocols (v11 and v12) for differentiating human pluripotent stem cells into stem cell-derived pancreatic β-cells according to this disclosure are shown. [Figure 40]

[0077] The v12 protocol, compared to the v11 protocol, produced a higher proportion of cells expressing ISL1 and NKX6.1 at stage 5 (ISL1-positive, NKX6.1-positive), a lower proportion of ISL-negative, NKX6.1-negative cells, and a lower proportion of CDX2-positive cells at stage 4. [Figure 41]

[0078] This shows that cell clusters produced by the v12 protocol had a higher recovery rate after cryopreservation compared to those produced by the v11 protocol. [Figure 42]

[0079] This shows that protocols v11 and v12 produced similar proportions of SC-β cells. [Figure 43]

[0080] This shows that the v11 and v12 protocols produced cell clusters with equivalent GSIS response and insulin content. [Figure 44]

[0081] The insulin-releasing performance and insulin content of exemplary cell populations produced in a bioreactor according to the method provided in this invention are summarized, respectively, in response to low glucose (LG), high glucose (HG), and potassium chloride (KCl) challenges. [Modes for carrying out the invention]

[0037]

[0082] Embodiments of this disclosure will be described in detail by the following description and examples. It should be understood that this disclosure is not limited to the specific embodiments described herein and is therefore subject to change. Those skilled in the art will recognize that numerous variations and modifications exist within the scope of this disclosure.

[0038]

[0083] All terms are intended to be understood as they are understood by those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as they are commonly understood by those skilled in the art to which this disclosure relates.

[0039]

[0084] The headings used in this specification are for organizational purposes only and should not be interpreted as limiting the subjects described.

[0085] Various features of this disclosure can be described in relation to a single embodiment, but features can also be provided individually or in any suitable combination. Conversely, for clarity, this disclosure can be described herein in relation to individual embodiments, but it can also be implemented in a single embodiment.

[0040]

[0086] The following definitions supplement those in this Art and are directed to this application, and do not belong to any related or unrelated examples, such as any widely shared patent or patent application. Any methods and materials similar to or equivalent to those described herein may be used in practices to test this disclosure, but preferred materials and methods are described herein. Accordingly, the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit them. definition

[0087] In this application, the use of the singular form includes the plural form unless otherwise specified. It should be noted that, as used herein, the singular forms "a," "an," and "the" include multiple referents unless the context explicitly indicates otherwise.

[0041]

[0088] In this application, the use of “or” means “and / or” unless otherwise stated. The terms “and / or” and “any combination thereof” as used herein, 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, as well as A, B, and C.” The term “or” may be used conjunctively or disjunctively unless the context specifically implies a disjunctive use.

[0042]

[0089] Furthermore, the use of the term "including," as well as other forms such as "include" and "included," is not limited to these.

[0090] References in the specification to “some embodiments,” “an embodiment,” or “other embodiments” refer to the features described in relation to those embodiments. Certain features, structures, or properties are included in at least some embodiments of this disclosure, but not necessarily in all embodiments.

[0043]

[0091] As used herein and in the 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"), and "including" "Includes" and any other form of "Include," or "containing" (and any other form of "containing," such as "contains" and "contain") is a encapsulation. This disclosure is comprehensive or open-ended and does not exclude additional elements or method steps not mentioned herein. Any embodiment discussed herein may be carried out with respect to any method or composition of this disclosure, and vice versa. Furthermore, the compositions of this disclosure may be used to achieve the methods of this disclosure.

[0044]

[0092] The term "about" used herein in reference to numerical values ​​and their grammatical equivalents may include the numerical value itself and a range of values ​​within plus or minus 10% of that value.

[0093] The terms “approximately” or “nearly” mean a range of acceptable error for a particular value, as determined by those skilled in the art, which is partly due to how the value was measured or determined, for example, the limitations of the measuring system. For example, “approximately” may mean within or exceeding one standard deviation by convention in the art. Or, "Approximately" can mean a range of up to 20%, 10%, 5%, or 1% of a given value. In another example, the quantity "approximately 10" includes 10 and any quantity from 9 through 11. In yet another example, the term "approximately" in relation to a reference number 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 "approximately" can mean within one order of magnitude of a value, preferably within five times, and more preferably within two times. Where a specific value is stated in the application and claims, unless otherwise stated, the term "approximately" should be assumed to mean within an acceptable margin of error for that specific value.

[0045]

[0094] As used herein, the term “diabetes” and its grammatical equivalents may mean a disease characterized by long-term high blood glucose levels. For example, as used herein, the term “diabetes” and its grammatical equivalents may mean all or any type of diabetes, including, but not limited to, type 1 diabetes, type 2 diabetes, cystic fibrosis-associated diabetes, surgical diabetes, gestational diabetes, and mitochondrial diabetes. In some cases, diabetes may be a form of hereditary diabetes.

[0046]

[0095] The term "endocrine cell," unless otherwise specified, may refer to hormone-producing cells present in the pancreas of a living organism, including "islets," "islet cells," "islet equivalents," "islet-like cells," "pancreatic islets," and their grammatical equivalents. In one embodiment, endocrine cells can differentiate from pancreatic progenitor cells or precursors. Islet cells may include, but are not limited to, various types of cells, including pancreatic α cells, pancreatic β cells, pancreatic δ cells, pancreatic F cells, and / or pancreatic ε cells. Islet cells may also refer to groups of cells, cell clusters, and so on.

[0047]

[0096] The terms “progenitor cell” and “precursor” are used interchangeably herein and refer to cells that have a more primitive cellular phenotype (e.g., at an earlier step in the developmental pathway or development than fully differentiated cells) compared to cells that can be produced by differentiation. Progenitor cells may also have significant or extremely high proliferative potential. Depending on the developmental pathway and the environment in which the cell develops and differentiates, progenitor cells can give rise to many different differentiated cell types or a single differentiated cell type.

[0048]

[0097] As a term related to insulin-positive endocrine cells, “its precursor” may mean any cell that can differentiate into insulin-positive endocrine cells when cultured under conditions suitable for differentiation into insulin-positive endocrine cells, including, for example, pluripotent stem cells, endoderm cells, gastrulatal cells, pancreatic progenitor cells, or endocrine progenitor cells.

[0049]

[0098] As used herein, the term “exocrine cell” may mean the cells of an exocrine gland, i.e., a gland that releases its secretions through a duct. In certain embodiments, exocrine cell may mean pancreatic exocrine cell, which is a pancreatic cell capable of producing enzymes secreted into the small intestine. These enzymes can help digest food as it passes through the gastrointestinal tract. Pancreatic exocrine cells are also known as islets of Langerhans, which can secrete two hormones, namely insulin and glucagon. Pancreatic exocrine cells can be one of several cell types, namely α-2 cells (which can produce the hormone glucagon), or β-cells (which can produce the hormone insulin) and α-1 cells (which can produce the regulatory factor somatostatin). Non-insulin-producing exocrine cells may mean α-2 cells or α-1 cells as used herein. The term "exocrine pancreatic cells" encompasses "endocrine pancreatic cells," which can refer to pancreatic cells that produce hormones secreted into the bloodstream (e.g., insulin (produced by β cells), glucagon (produced by α-2 cells), somatostatin (produced by delta cells), and pancreatic polypeptide (produced by F cells).

[0050]

[0099] The terms “stem cell-derived β-cells,” “SC-β-cells,” “functional β-cells,” “functional pancreatic β-cells,” “mature SC-β-cells,” and their grammatical equivalents may mean cells (e.g., non-native pancreatic β-cells) that exhibit at least one marker indicating pancreatic β-cells (e.g., PDX-1 or NKX6.1), secrete insulin, and exhibit a glucose-stimulated insulin secretion (GSIS) response characteristic of endocrine mature β-cells. In some embodiments, the terms “SC-β-cells” and “non-native β-cells” as used herein are interchangeable. In some embodiments, “SC-β-cells” include mature pancreatic cells. It should be understood that SC-β-cells do not necessarily need to be derived from stem cells (e.g., directly). This is because the method of the present disclosure can induce SC-β cells from any insulin-positive endocrine cell or its precursor using any cell as a starting point (the present invention is not intended to limit itself to this form, so for example, 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 original somatic cell from which it was induced), pluripotent cells, totipotent cells, transdifferentiated versions of any of the above cells, and others can be used). In some embodiments, 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 SC-β cells is similar to that of endogenous β cells. In some embodiments, SC-β cells exhibit an in vitro GSIS response similar to that of endogenous β cells. In some embodiments, SC-β cells exhibit an in vivo GSIS response similar to that of endogenous β cells. In some embodiments, SC-β cells exhibit both in vitro and in vivo GSIS responses similar to those of endogenous β cells. The GSIS response of SC-β cells can be observed within two weeks after transplantation of SC-β cells into a host (e.g., human or animal).In some embodiments, SC-β cells encapsulate insulin in secretory granules. In some embodiments, SC-β cells exhibit encapsulated crystalline insulin granules. In some embodiments, SC-β cells exhibit a stimulation index greater than 1. In some embodiments, SC-β cells exhibit a stimulation index greater than 1.1. In some embodiments, SC-β cells exhibit a stimulation index greater than 2. In some embodiments, SC-β cells exhibit cytokine-induced apoptosis in response to cytokines. In some embodiments, insulin secretion from SC-β cells is enhanced in response to known antidiabetic drugs (e.g., secretagogues). In some embodiments, SC-β cells are monoformal. In some embodiments, SC-β cells do not abnormally co-express other hormones, such as glucagon, somatostatin, or pancreatic polypeptides. In some embodiments, SC-β cells exhibit a low replication rate. In some embodiments, SC-β cells increase intracellular Ca2+ in response to glucose.

[0051]

[0100] As used herein, the term “insulin-producing cells” and its grammatical equivalent mean cells that differentiate from pancreatic progenitor cells or their precursors and secrete insulin. Insulin-producing cells may include pancreatic β-cells, as well as pancreatic β-like cells (e.g., insulin-positive endocrine cells) that synthesize (e.g., transcribe the insulin gene, translate proinsulin mRNA, and modify proinsulin mRNA to insulin protein), express (e.g., realize the phenotypic traits carried by the insulin gene), or secrete (release insulin into extracellular space) insulin in a structural or inducible manner. For example, a population of insulin-producing cells produced by differentiating insulin-positive endocrine cells or their precursors into SC-β-cells according to the methods disclosed herein may be pancreatic β-cells or β-like cells (e.g., cells having at least one or at least two characteristics of endogenous β-cells and exhibiting a glucose-stimulated insulin secretion (GSIS) response similar to that of endogenous mature β-cells). For example, a population of insulin-producing cells produced by the methods disclosed herein may include mature pancreatic β-cells or SC-β-cells, and non-insulin-positive endocrine cells. Phosphoric cells (for example, cells that have a cell-like phenotype except that they do not produce or secrete insulin) may also be included.

[0052]

[0101] 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 indicating pancreatic β-cells and also express insulin, but lack the glucose-stimulated insulin secretion (GSIS) response characteristic of endogenous β-cells.

[0053]

[0102] The term "β-cell marker" means, without limitation, proteins, peptides, nucleic acids, protein and nucleic acid polymorphisms, splice variants, protein or nucleic acid fragments, elements, and other analytes that are specifically expressed or present in pancreatic β-cells. Exemplary β-cell markers include, but are not limited to, pancreatic and duodenal homeobox 1 (Pdx1) polypeptides, insulin, c-peptide, amylin, E-cadherin, Hnf3β, PCI / 3, B2, Nkx2.2, GLUT2, PC2, ZnT-8, ISL1, Pax6, Pax4, NeuroD, 1 Inf1b, Hnf-6, Hnf-3 beta, and MafA, as well as those described by Zhang et al., Diabetes. 50(10):2231-6 (2001). This includes: In some embodiments, the β-cell marker is a nuclear 3-cell marker. In some embodiments, the β-cell marker is Pdx1 or PH3.

[0054]

[0103] The term “pancreatic endocrine markers” can mean, without limitation, 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.

[0055]

[0104] The terms “pancreatic progenitor cells,” “pancreatic endocrine progenitor cells,” “pancreatic precursor,” “pancreatic endocrine precursor,” and their grammatical equivalents are used interchangeably herein and may mean stem cells that can become pancreatic hormone-expressing cells capable of forming pancreatic endocrine cells, pancreatic exocrine cells, or pancreatic duct cells. These cells are involved in differentiation toward at least one type of pancreatic cell, e.g., insulin-producing β cells, glucagon-producing α cells, somatostatin-producing δ cells (or D cells), and / or pancreatic polypeptide-producing F cells. Such cells may express at least one of the following markers: NGN3, NKX2.2, NeuroD, ISL-1, Pax4, Pax6, or ARX.

[0056]

[0105] As used herein, the term "Pdx1-positive pancreatic progenitor cells" may refer to pancreatic endodermal (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, Ptf1a, HNF6, or NRx2.2. Pdx1 expression can be evaluated by any method known to those skilled in the art, such as immunochemistry using an anti-Pdx1 antibody or quantitative RT-PCR. In some cases, Pdx1-positive pancreatic progenitor cells lack NKX6.1 expression. In some cases, Pdx1-positive pancreatic progenitor cells may also be referred to as Pdx1-positive, NKX6.1-negative pancreatic progenitor cells because they lack NKX6.1 expression. In some cases, Pdx1-positive pancreatic progenitor cells may also be named "foregut endoderm cells."

[0057]

[0106] As used herein, the term "Pdx1-positive, NKX6-1-positive pancreatic progenitor cells" may refer to pancreatic endodermal (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 the markers Pdx1 and NKX6-1. Other markers include, but are not limited to, Cdcp1, Alternatively, Ptf1a, or HNF6 or NRx2.2 may be included. NKX6-1 expression can be evaluated by any method known to those skilled in the art, such as immunochemistry using an anti-NKX6-1 antibody or quantitative RT-PCR. As used herein, the terms "NKX6.1" and "NKX6-1" are equivalent and interchangeable. In some cases, Pdx1-positive, NKX6-1-positive pancreatic progenitor cells may also be named "pancreatic foregut progenitor cells."

[0058]

[0107] As used herein, the term "Ngn3-positive endocrine progenitor cells" may refer to pancreatic endocrine cell precursors that express the transcription factor neurogenin-3 (Ngn3). Progenitor cells are more differentiated than pluripotent stem cells and can differentiate into only a few types of cells. In particular, Ngn3-positive endocrine progenitor cells have the ability to differentiate into five types of pancreatic endocrine cell types (α, β, δ, ε, and PP). Ngn3 expression can be evaluated by any method known to those skilled in the art, such as immunochemistry using an anti-Ngn3 antibody or quantitative RT-PCR.

[0059]

[0108] The terms "NeuroD" and "NeuroD1" are interchangeable and are used to identify proteins expressed in pancreatic endocrine progenitor cells and the genes that encode them.

[0109] The term “selective marker” means a gene, RNA, or protein that, when expressed, confers a selectable phenotype to cells, such as resistance to cytotoxic or cell growth-inhibiting agents (e.g., antibiotic resistance), prototrophicity, or the expression of a specific protein that can be used as a basis for distinguishing cells expressing that protein from cells that do not. As used herein, the term “selective marker” may mean a gene or a gene expression product, e.g., an encoded protein. In some embodiments, a selective marker confers a growth and / or survival advantage to cells expressing it compared to cells that do not express it or cells that express it at significantly low levels. Such a growth and / or survival advantage typically arises when cells are maintained under certain conditions, i.e., “selective conditions.” To ensure effective selection, a population of cells can be maintained under conditions and for a sufficient time such that cells that do not express the marker do not grow and / or survive, and they are eliminated from the population or reduced to a very small fraction of the population. The process of selecting cells that express a marker conferring a proliferation and / or survival advantage by maintaining a population of cells under selective conditions, and thereby primarily or completely eliminating cells that do not express that marker, is referred to herein as “positive selection,” and the marker is referred to as “useful for positive selection.” Negative selection and markers useful for negative selection are also objectives, where applicable, as described herein. The expression of such a marker confers a disadvantage to proliferation and / or survival to cells expressing the marker compared to cells that do not express the marker or express it at significantly low levels (or, to put it another way, cells that do not express the marker have a proliferation and / or survival advantage compared to cells that express the marker). Thus, cells expressing the marker can be primarily or completely eliminated from a population of cells if they are maintained under selective conditions for a sufficient period of time.

[0060]

[0110] The term "epigenetics" refers to heritable changes in gene function that do not involve changes in DNA sequence. While epigenetics most often refers to chromosomal changes that affect gene activity and expression, it can also be used to describe any heritable phenotypic changes that do not originate from genomic modifications. Such effects on cellular and physiological phenotypic traits may be due to external or environmental factors, or they may be part of a normal developmental program. Epigenetics can also refer to functionally relevant changes in the genome that do not involve changes in nucleotide sequence. Examples of mechanisms that produce such changes include DNA methylation and histone modification, each of which alters the manner in which genes are expressed without altering the underlying DNA sequence. Gene expression can be controlled by the action of repressor proteins bound to the silencer region of DNA. These epigenetics can persist through cell division during the lifespan of a cell, and these Even if it does not involve changes in the DNA sequence that underlies the organism, it can persist for multiple generations. One example of epigenetics in eukaryotic cell biology is the process of cell differentiation. During morphogenesis, totipotent stem cells can become various pluripotent cells, which can then become fully differentiated cells.

[0061]

[0111] The term "epigenetic modification compound" refers to a compound that induces epigenetics in a gene, i.e., alters gene expression without altering the DNA sequence. Epigenetics helps determine whether a gene is turned on or off and can influence protein production in certain cells, such as beta cells. Epigenetic modifications, such as DNA methylation and histone modification, alter the accessibility of DNA and the structure of chromatin, thereby regulating the pattern of gene expression. These processes are crucial for the normal development and differentiation of unique cell lineages in adult organisms. They can be modified by exogenous influences and therefore can contribute to, or may result from, environmental modifications of phenotypic or pathological phenotypes. Importantly, epigenetic modifications play a vital role in regulating pluripotency genes, which are inactivated during differentiation. Examples of non-restrictive epigenetic modification compounds include DNA methylation inhibitors, histone acetyltransferase inhibitors, histone deacetylase inhibitors, histone methyltransferase inhibitors, bromodomain inhibitors, or any combination thereof.

[0062]

[0112] The term “differentiated cell” or its grammatical equivalent means any primary cell that, in its natural form, is not pluripotent as defined herein. In other words, the term “differentiated cell” may mean a cell of a highly specific cell type derived from a cell of a less specific cell type (e.g., stem cells such as induced pluripotent stem cells) in the cell differentiation process. While we do not wish to be limited to theory, in the course of normal ontogeny, pluripotent stem cells can first differentiate into endodermal cells that can form pancreatic cells and other endodermal cell types. Further differentiation of endodermal cells leads to the pancreatic pathway, where about 98% of cells become exocrine, ductal, or matrix cells, and about 2% become endocrine cells. Early endocrine cells are pancreatic islet progenitor cells, which can then further differentiate into insulin-producing cells (e.g., functional endocrine cells) that secrete insulin, glucagon, somatostatin, or pancreatic polypeptides. Endoderm cells can also differentiate into other cells of endodermal origin, such as lung, liver, intestine, thymus, etc.

[0063]

[0113] As used herein, the term “somatic cell” may mean any cell that makes up a living organism, as opposed to germline cells. In mammals, germline cells (also known as “gametes”) are sperm and egg cells that fuse during fertilization to produce a cell called a zygote, from which the entire mammalian embryo develops. Apart from sperm and egg cells (germ cells) and undifferentiated stem cells, from which somatic cells are made, all other cell types in the mammalian body are somatic cells. Internal organs, skin, bone, blood, and connective tissue are all made up of somatic cells. In some embodiments, somatic cells are “non-embryonic somatic cells,” meaning somatic cells that are not present in or obtained from an embryo and are not the result of in vitro proliferation of such cells. In some embodiments, somatic cells are “adult somatic cells,” meaning cells that are present in or obtained from living organisms other than embryos or fetuses, or the result of in vitro proliferation of such cells. Unless otherwise specified, methods for converting at least one insulin-positive endocrine cell or its precursor into an insulin-producing, glucose-responsive cell can be performed both in vivo and in vitro (in vivo is performed if at least one insulin-positive endocrine cell or its precursor is present in the subject, and in vitro is performed using at least one insulin-positive endocrine cell or its precursor that has been isolated and maintained in culture medium).

[0064]

[0114] As used herein, the term “adult cell” may mean a cell found throughout the body after embryonic development.

[0115] As used herein, the term “endodermal cell” may mean a cell from one of the three primary embryonic cell layers in a very early embryo (the other two are the mesoderm and ectoderm). The endoderm is the innermost of the three layers. Endoderm cells differentiate to first give rise to the embryonic intestine, then to the respiratory tract and the lining of the digestive tract (e.g., the intestine), the liver, and the pancreas.

[0065]

[0116] As used herein, the term “endodermal-derived cells” may refer to any cells that develop or differentiate from endodermal cells. For example, endodermal-derived cells include liver, lung, pancreas, thymus, intestine, stomach, and thyroid cells. While we do not wish to be bound by theory, hepatic and pancreatic progenitor cells (also called pancreatic progenitor cells) arise from endodermal cells in the pre-embryonic gut. Immediately after their identification, hepatic and pancreatic progenitor cells rapidly acquire entirely different cellular functions and regenerative capabilities. These changes are triggered by highly conserved inductive signals and gene regulatory factors among vertebrates. Interest in organ development and regeneration has been accelerated by the strong need for hepatocytes and pancreatic β-cells in the treatment of liver failure and type 1 diabetes. Studies in diverse model organisms and in humans have revealed evolutionarily conserved networks of inductive signals and transcription factors that induce hepatocyte and pancreatic cell differentiation and provide guidance on how to promote the differentiation of hepatocytes and β-cells from diverse stem cell and progenitor cell types.

[0066]

[0117] As used herein, the term “embryonic endoderm” may mean cells that differentiate from endodermal cells and can differentiate into SC-β cells (e.g., pancreatic β cells). Embryonic endoderm cells express the marker Sox17. Other markers characteristic of embryonic endoderm cells include, but are not limited to, MIXL2, GATA4, HNF3B, GSC, FGF17, VWF, CALCR, FOXQ1, CXCR4, Cerberus, OTX2, Gusecoid, C-Kit, CD99, CMKOR1, and CRIP1. In particular, the embryonic endoderm cells described herein express Sox17 and, in some embodiments, Sox17 and HNF3B, but do not express significant levels of GATA4, SPARC, APF, or DAB. Embryonic endoderm cells are not positive for the marker Pdx1 (e.g., they are Pdx1 negative). Embryonic endoderm cells have the ability to differentiate into cells including those of the liver, lungs, pancreas, thymus, intestines, stomach, and thyroid. The expression of Sox17 and other endoderm markers can be evaluated by any method known to those skilled in the art, such as immunochemistry using anti-Sox17 antibodies or quantitative RT-PCR.

[0067]

[0118] The term "pancreatic endoderm" can refer to endoderm-derived cells that can differentiate into multiple pancreatic lineages, including pancreatic β-cells, but no longer possess the ability to differentiate into non-pancreatic lineages.

[0119] As used herein, the terms “gastrulatory cells” or “intestinal cells” may mean cells that differentiate from endodermal cells and are capable of differentiating into SC-β cells (e.g., pancreatic β cells). Gastrulatory cells express at least one of the following markers: HNP1-β, HNF3-β, or HNF4-α. Gastrulatory cells have the ability to differentiate into cells including lung, liver, pancreas, stomach, and intestinal cells. The expression of HNF1-β and other gastrulatal markers can be evaluated by any method known to those skilled in the art, such as immunochemistry using an anti-HNF1-β antibody.

[0068]

[0120] As used herein, the term “stem cell” may mean an undifferentiated cell that gives rise to a number of progenitor cells capable of proliferating and producing a large number of mother cells that can subsequently give rise to differentiated or differentiateable daughter cells. The daughter cells themselves may be induced to proliferate and subsequently differentiate into offspring that develop into one or more mature cell types, while retaining one or more cells that have the potential to develop into parents. The term “stem cell” may be used in specific contexts. Below, it may mean a subset of progenitor cells that have the ability or potential to differentiate into more specialized or differentiated phenotypes and, under certain circumstances, retain the ability to proliferate substantially without differentiation. In one embodiment, the term stem cell generally refers to a naturally occurring parent cell whose descendants (progeny) specialize, often in different directions, by differentiation, for example, by acquiring completely distinct characteristics, as occurs in the gradual diversification of embryonic cells and tissues. Cell differentiation is a complex process that typically occurs through many cell divisions. Differentiated cells may be pluripotent, and pluripotent cells are themselves, pluripotent, etc. Each of these pluripotent cells is considered a stem cell, but the range of cell types that each can produce can vary considerably. Some differentiated cells also have the ability to produce cells with greater developmental potential. Such ability may be spontaneous or may be artificially induced by treatment with various factors. In many biological examples, stem cells are also "pluripotent" because they can produce descendants with two or more different cell types, but this is not necessary to be "stem cell-like." "Self-renewal" is another classic part of the definition of stem cells, and it is important as used in this book. Theoretically, self-renewal can occur through one of two main mechanisms. Stem cells may divide asymmetrically, with one daughter retaining the stem cell state and the other expressing several different specific functions and phenotypes. Alternatively, some stem cells in a population may divide symmetrically into two stem cells, thereby maintaining some stem cells in the population as a whole, while the other cells in the population produce only differentiated offspring. Formally, cells that begin as stem cells progress toward a differentiated phenotype, but it is also possible to "reverse" this process and reverse-express the stem cell phenotype. This is often referred to by those skilled in the art as "dedifferentiation," "reprogramming," or "reverse differentiation." As used herein, the term "pluripotent stem cells" includes embryonic stem cells, induced pluripotent stem cells, placental stem cells, etc.

[0069]

[0121] As used herein, the term "pluripotency" may mean cells that have the ability to differentiate into two or more differentiated cell types under various conditions, preferably into cell types characteristic of all three germinal layers. Pluripotent cells are primarily characterized by their ability to differentiate into two or more cell types, preferably into all three germinal layers, for example, using a teratoma formation assay in nude mice. Pluripotency can also be demonstrated by the expression of germinal stem (ES) cell markers, but a preferred test for pluripotency is to demonstrate the ability to differentiate into cells of each of the three germinal layers. It should be noted that simply culturing such cells does not make them pluripotent in itself. Reprogrammed pluripotent cells (e.g., iPS cells as defined herein) are also characterized by having an extended ability to passage without losing growth potential, compared to primary parent cells, which generally have only a limited number of divisions during culture.

[0070]

[0122] As used herein, the terms “iPS cells” and “induced pluripotent stem cells” are interchangeable and may mean pluripotent stem cells artificially induced (e.g., induced or by complete inversion) from non-pluripotent cells, typically from adult somatic cells, by, for example, inducing the forced expression of one or more genes.

[0071]

[0123] The term "phenotype" can refer to all of the one or more biological characteristics that define a cell or organism under a particular set of environmental conditions and factors, regardless of the actual genotype.

[0072]

[0124] The terms “subject,” “patient,” or “individual” are used interchangeably herein and may mean an animal from which cells are obtained and / or to which treatment including prophylactic treatment using the cells described herein is provided, such as a human. For treatment of infections, conditions, or disease states specific to a particular animal, such as a human subject, the term “subject” may mean that particular animal. The terms “non-human animal” and “non-human mammary” are used interchangeably herein. The term "animal" includes mammals such as rats, mice, rabbits, sheep, cats, dogs, cattle, pigs, and non-human primates. The term "subject" also includes any vertebrate, including mammals, reptiles, amphibians, and fish, but is not limited to these. However, advantageously, the subject is a mammal such as a human, or other mammals such as dogs, cats, horses, or other livestock, or other mammals such as cattle, sheep, pigs, or other production mammals. "Patient in need" or "subject in need" in this specification means a patient who has been diagnosed with or is suspected of having a disease or disorder, not limited to diabetes.

[0073]

[0125] As used herein, “administer” may mean providing one or more compositions described herein to a patient or subject. Without limiting to examples, administration of a composition, e.g., by injection, may be carried out 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 by time-based stepwise perfusion. Or, or simultaneously, administration may be by oral route. Furthermore, administration may involve surgically depositing a bolus or pellet of cells, or positioning a medical device. In one embodiment, a composition of the Disclosure may comprise engineered cells 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 for treating or preventing proliferative disorders. A pharmaceutical composition may comprise a population of cells described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline or phosphate-buffered saline, carbohydrates such as glucose, mannose, sucrose, or dextran and mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives.

[0074]

[0126] The terms "treat, treating," "processing," and their grammatical equivalents. When applied to isolated cells, this includes subjecting cells to any kind of process or condition, or performing any kind of operation or procedure on cells. When applied to an object, this term means providing medical or surgical attention, care, or management to an individual. An individual typically has a disease or injury, or is at increased risk of becoming ill compared to the average member of a population, and therefore requires such attention, care, or management.

[0075]

[0127] As used herein, the terms “to treat” and “treatment” may mean administering an effective amount of a composition to a subject so that the subject obtains, for example, a favorable or desired clinical outcome, such as a reduction of at least one symptom of the disease or an improvement of the disease. For the purposes of the present invention, favorable or desired clinical outcomes include, but are not limited to, relief of one or more symptoms, disappearance of the degree of the disease, stabilization of the disease state (e.g., no worsening), delay or slowing of the progression of the disease, improvement or reduction of the disease state, and remission (whether partial or complete), whether detected or not. Treating may also mean extended survival compared to the survival expected if no treatment were received. As such, those skilled in the art will recognize that treatment may improve the disease state but may not be a complete cure of the disease. As used herein, the term “treatment” includes prevention. Alternatively, “treatment” is effective if the progression of the disease is attenuated or stopped. “Treatment” may also mean extended survival compared to the survival expected if no treatment were received. Those requiring treatment include individuals who have already been diagnosed with a heart condition, as well as those whose heart condition may progress due to genetic susceptibility or other factors such as weight, diet, and health.

[0076]

[0128] The terms “therapeutic dose,” “therapeutic dose,” or their grammatical equivalents may mean an effective amount to achieve the desired therapeutic outcome in the required dose and duration. The therapeutic dose may vary depending on factors such as the disease state, the individual’s age, sex, and weight, as well as the ability of the compositions described herein to elicit the desired response in one or more subjects. The exact amount of the compositions disclosed herein to be administered can be determined by a clinician, taking into account individual differences in age, weight, tumor size, extent of infection or metastasis, and the patient’s (subject’s) condition.

[0077]

[0129] Alternatively, the pharmacological and / or physiological effect of administering one or more compositions described herein to a patient or subject may be “preventive,” for example, the effect may completely or partially prevent a disease or its symptoms. “Preventive dose” may mean an effective amount to achieve the desired preventive outcome (e.g., prevention of the onset of a disease) in the required dose and duration.

[0078]

[0001] Some of the numbers disclosed in general are referred to as, for example, "X is at least or at least about 100 or 200 [or any number]." This number includes the number itself and the following i) X is at least 100. ii) X is at least 200, iii) X is at least about 100, and iv) X is at least approximately 200. It includes all of the above.

[0079]

[0002] All of these different combinations are intended by the numerical values ​​disclosed throughout. Unless otherwise specifically indicated to the contrary, all disclosed numerical values, whether relating to the administration of therapeutic drugs, days, months, years, weights, doses, etc., should be interpreted in this way.

[0080]

[0003] The range disclosed in whole may be referred to, for example, as "X is administered on day 1-2 or approximately day 1-2, or on day 2-3 or approximately day 2-3 [or within any numerical range]." This range includes the number itself (e.g., the end of the range) and the following i) X is administered between day 1 and day 2. ii) X is administered between the second and third day. iii) X is administered between approximately day 1 and day 2. iv) X is administered between approximately the second and third day. v) X is administered between day 1 and approximately day 2. vi) X is administered between day 2 and approximately day 3. vii)X is administered between approximately day 1 and day 2, and viii)X is administered between approximately the second and third day. It includes all of the above.

[0081]

[0130] All these different combinations are intended by the scope of the disclosure as a whole. Unless otherwise specifically indicated to the contrary, all disclosures, whether concerning the administration of therapeutic drugs, days, months, years, weights, doses, etc., should be interpreted in this way. I. Abstract

[0131] In some embodiments, this disclosure provides compositions and methods for differentiating pancreatic progenitor cells. The compositions and methods provided herein may propose pancreatic β-cells, cell populations, or cell clusters having high purity, high insulin content, and excellent glucose-dependent insulin secretion response that may be similar to native pancreatic β-cells or native pancreatic islets.

[0082]

[0132] In some embodiments, a method is provided herein that includes contacting a population of pancreatic progenitor cells or their precursors with an epigenetically modified compound, wherein the contact step results in a population of endocrine cells in which the proportion of chromogranin A-positive (CHGA+) cells is increased or the proportion of C-peptide-positive and NKX6.1-positive (C-PEP+, NKX6.1+) cells is increased compared to a corresponding population of endocrine cells that have not been contacted with the epigenetically modified compound.

[0083]

[0133] In some embodiments, the present disclosure provides a method comprising the step of contacting a population of pancreatic progenitor cells or their precursors with an epigenetic modification compound, wherein the contact step results in a population of endocrine cells in which the proportion of cells expressing VMAT or Cdx2 is reduced compared to a corresponding population of endocrine cells that have not been contacted with the epigenetic modification compound.

[0084]

[0134] In some embodiments, the Disclosure provides a composition comprising a cell population, wherein the cell population, as measured by flow cytometry, comprises (a) at least about 20% of cells expressing C-peptide and NKX6.1, (b) at least about 60% of cells expressing CHGA, (c) at most about 20% of cells expressing Cdx2, or (d) at most about 45% of cells expressing VMAT1. In some examples, the composition, as measured by flow cytometry, comprises (a) at least about 20% of cells expressing C-peptide and NKX6.1, (b) at least about 60% of cells expressing CHGA, and (c) at most about 20% of cells expressing Cdx2. In some examples, the composition also comprises at most about 45% of cells expressing VMAT1, as measured by flow cytometry. In some examples, the composition further comprises an epigenetic modification compound.

[0085]

[0135] In some embodiments, the Disclosure provides a composition comprising a cell population comprising, as measured by flow cytometry, at least about 30% ISL1-positive, NKX6.1-positive cells and at most about 20% ISL1-negative, NKX6.1-negative cells. The composition of claim 34, the cell population comprising at least about 35% ISL1-positive, NKX6.1-positive cells. In some examples, the cell population comprising at least about 40% ISL1-positive, NKX6.1-positive cells. In some examples, the cell population comprising at most about 15% ISL1-negative, NKX6.1-negative cells. In some examples, the composition further comprises an epigenetic modification compound.

[0086]

[0136] In some embodiments, the disclosure provides a composition comprising pancreatic progenitor cells and at least one of a histone deacetylase (HDAC) inhibitor or a histone methyltransferase inhibitor. In some embodiments, the disclosure provides a method comprising the steps of contacting a cell population comprising pancreatic progenitor cells or their precursors with a histone methyltransferase inhibitor to produce a cell population comprising endocrine cells, and maturing the cell population comprising endocrine cells to obtain at least one pancreatic β-cell that exhibits an in vitro glucose-stimulated insulin secretion response to a glucose challenge. In some examples, the maturation step is performed after in vivo implantation of the cell population comprising endocrine cells. In some examples, the maturation step is performed in vitro.

[0087]

[0137] In some embodiments, the Disclosure provides a method comprising (a) contacting a population of Pdx-1-negative, NKX6.1-negative gastrulatic cells with a bone morphogenetic protein (BMP) signaling pathway inhibitor and a growth factor from the transformation growth factor β (TGF-β) superfamily to produce a cell population including Pdx1-positive, NKX6.1-positive pancreatic progenitor cells, and (b) contacting a cell population including Pdx1-positive, NKX6.1-positive pancreatic progenitor cells with an epigenetic modification compound to produce a cell population including endocrine cells.

[0088]

[0138] In some examples, the methods provided herein include: (a) differentiating pluripotent stem cells into endoderm cells by contacting pluripotent stem cells in a population with growth factors from the TGF-β superfamily and WNT signaling pathway activators; (b) differentiating at least some endoderm cells into gastrula cells by contacting endoderm cells with growth factors from the FGF family; (c) differentiating at least some gastrula cells into Pdx1-positive pancreatic progenitor cells by contacting gastrula cells with ROCK inhibitors, growth factors from the FGF family, BMP signaling pathway inhibitors, PKC activators, retinoic acid signaling pathway activators, SHH pathway inhibitors, and growth factors from the TGF-β superfamily; and (d) differentiating Pdx1-positive pancreatic progenitor cells with ROCK inhibitors, TGF-β superfamily The method comprises (e) differentiating at least some Pdx1-positive pancreatic progenitor cells into Pdx1-positive, NKX6.1-positive pancreatic progenitor cells by contacting them with growth factors from Lee, growth factors from the FGF family, RA signaling pathway activators, and SHH pathway inhibitors; and (e) differentiating at least some Pdx1-positive, NKX6.1-positive pancreatic progenitor cells into a cell population containing at least one NKX6.1+ and C-peptide+ cell by contacting them with TGF-β signaling pathway inhibitors, growth factors from the EGF family, RA signaling pathway activators, SHH pathway inhibitors, TH signaling pathway activators, γ-secretase inhibitors, protein kinase inhibitors, ROCK inhibitors, BMP signaling pathway inhibitors, and epigenetic modification compounds.

[0089]

[0139] In some embodiments, the disclosure provides a method for irradiating cells to reduce their proliferation. In some examples, the method includes the step of exposing an in vitro cell population, including endocrine cells, to irradiation at a dose of approximately 100 rad to approximately 100,000 rad for a time of approximately 1 minute to approximately 60 minutes.

[0090]

[0140] In some cases, a method for reducing cell proliferation includes the step of exposing a population of cells, including stem cells, endoderm cells, gastrulatal cells, pancreatic progenitor cells, or endocrine cells, to irradiation, resulting in a population of cells with reduced proliferative capacity compared to the corresponding cell population that has not been irradiated. II. Methods for producing endocrine cells

[0141] In aspects, this disclosure relates to compositions and methods for producing endocrine cells from pancreatic progenitor cells or precursors. A particular exemplary detailed protocol for producing endocrine cells from stem cells to provide at least one SC-β cell is described in U.S. Patent Application Publications 2015 / 0240212 and 2015 / 0218522, which are incorporated herein by reference in their entirety.

[0091]

[0142] In some cases, a method for producing a first population of endocrine cells involves contacting a population of pancreatic progenitor cells or their precursors with a first composition containing at least one epigenetic modification compound to produce a first population of endocrine cells, wherein a reduced proportion of cells in the first population of endocrine cells are VMAT compared to a second population of endocrine cells produced using a second composition lacking at least one epigenetic modification compound. + or Cdx2 + The process includes the step of expressing [the specified expression]. In some embodiments, the epigenetic modification compound is added in stage 5 (Figures 5-6), which can induce changes in the proportion of endocrine cells such as: (1) reducing the population of endocrine cells marked by the VMAT1 marker (Figure 19) or Cdx2, (2) increasing the proportion of cells destined to become islet cells, which are alpha cells and other non-beta cells (Figure 20), and (3) increasing the proportion of beta cells in the composition (Figure 8).

[0092]

[0143] In some embodiments, the first population of endocrine cells is VMAT+ and INS-. In some embodiments, the population of pancreatic progenitor cells differentiates into the population of PH cells. In some embodiments, an increased proportion of cells in the first population of endocrine cells compared to a second population of endocrine cells produced using a second composition lacking an epigenetic modification compound are NKX6.1 - or ChromA + In some embodiments, the increased proportion of cells are NKX6.1 - and ChromA + In some embodiments, the first population of pancreatic progenitor cells differentiates into the population of β cells. In some embodiments, the β cells are β(SC-β) cells derived from stem cells. In some embodiments, the β cells express C-PEP and NKX6-1. In some embodiments, the β cells exhibit an in vitro glucose-stimulated insulin secretion response to a glucose challenge. In some embodiments, the methods described herein are performed in vitro.

[0093]

[0144] In some examples, the first composition and the second composition comprise at least one of: i) a SHH pathway inhibitor, ii) a RA signaling pathway activator, iii) a γ-secretase inhibitor, iv) at least one growth factor from the epithelial cell growth factor (EGF) family, v) a protein kinase inhibitor, vi) a BMP signaling pathway inhibitor, vii) a TGF-β signaling pathway inhibitor, viii) a thyroid hormone signaling pathway agonist, or ix) a ROCK inhibitor. In some embodiments, the first composition and the second composition comprise at least one of betacellulin, thiazovivin, retinoic acid, SANT1, XXI, Alk5i II, GC-1, LDN, staurosporine, or any combination thereof. In some examples, the first composition and the second composition both comprise betacellulin, thiazovivin, retinoic acid, SANT1, XXI, Alk5i II, GC-1, LDN, and staurosporine.

[0094]

[0145] A method for producing endocrine cells is provided herein, comprising the step of contacting pancreatic progenitor cells or their precursors with a histone methyltransferase inhibitor, wherein the contact step induces the pancreatic progenitor cells to differentiate into endocrine cells. In some examples, the histone methyltransferase inhibitor includes DZNep. In some examples, the method further comprises the step of contacting pancreatic progenitor cells or their precursors with at least one of the following: i) an SHH pathway inhibitor, ii) an RA signaling pathway activator, iii) a γ-secretase inhibitor, iv) at least one growth factor from the epidermal growth factor (EGF) family, v) a protein kinase inhibitor, vi) a BMP signaling pathway inhibitor, vii) a TGF-β signaling pathway inhibitor, viii) a thyroid hormone signaling pathway agonist, or ix) a ROCK inhibitor.

[0095]

[0146] A method for producing endocrine cells is provided herein, comprising the step of contacting pancreatic progenitor cells or their precursors with a histone deacetylase (HDAC) inhibitor and a histone methyltransferase inhibitor, wherein the contact step induces the pancreatic progenitor cells to differentiate into endocrine cells. In some examples, the method further comprises the step of contacting pancreatic progenitor cells or their precursors with at least one of the following: i) an SHH pathway inhibitor, ii) an RA signaling pathway activator, iii) a γ-secretase inhibitor, iv) at least one growth factor from the epidermal growth factor (EGF) family, v) a protein kinase inhibitor, vi) a BMP signaling pathway inhibitor, vii) a TGF-β signaling pathway inhibitor, viii) a thyroid hormone signaling pathway agonist, or ix) a ROCK inhibitor.

[0096]

[0147] In some embodiments, endocrine cells differentiate into β cells. In some embodiments, the β cells are β(SC-β) cells derived from stem cells. In some embodiments, the β cells express C-PEP and NKX6-1. In some embodiments, the β cells exhibit an in vitro glucose-stimulated insulin secretion response to glucose challenge. In some embodiments, the HDAC inhibitor is a class I HDAC inhibitor, a class II HDAC inhibitor. It is a drug, or a combination thereof.

[0097]

[0148] A method for producing endocrine cells is provided herein, comprising the step of contacting pancreatic progenitor cells or their precursors with a histone deacetylase (HDAC) inhibitor, wherein the HDAC inhibitor is KD5170. In some embodiments, the method further comprises the step of contacting pancreatic progenitor cells with a histone methyltransferase inhibitor. In some embodiments, the endocrine cells express CHGA. In some embodiments, the endocrine cells differentiate into β cells. In some embodiments, the β cells are β (SC-β) cells derived from stem cells. In some embodiments, the β cells express C-PEP and NKX6-1. In some embodiments, the β cells exhibit an in vitro glucose-stimulated insulin secretion response to a glucose challenge.

[0098]

[0149] Compositions comprising a population of cells and a culture medium are also provided herein, wherein the cells comprise pancreatic progenitor cells and the culture medium comprises a histone deacetylase (HDAC) inhibitor and a histone methyltransferase inhibitor. In some embodiments, pancreatic progenitor cells are induced to differentiate into endocrine cells upon contact with the culture medium.

[0099]

[0150] Examples of non-restrictive epigenetic modification compounds include DNA methylation inhibitors, histone acetyltransferase inhibitors, histone deacetylase inhibitors, histone methyltransferase inhibitors, bromodomain inhibitors, or any combination thereof.

[0100]

[0151] In one embodiment, a histone methyltransferase inhibitor is an inhibitor of the enhancer of zest homolog 2 (EZH2). EZH2 is a histone-lysine N-methyltransferase enzyme. Non-limiting examples of EZH2 inhibitors include 3-deazanepranosine A (DZNep), EPZ6438, EPZ005687 (a competitive S-adenosylmethionine (SAM) inhibitor), EI1, GSK126, and UNC1999. DZNep inhibits the hydrolysis of S-adenosyl-L-homocysteine ​​(SAH), a product inhibitor of all protein methyltransferases, leading to an increase in the intracellular concentration of SAH, which in turn inhibits EZH2. DZNep is not specific to EZH2 and also inhibits other DNA methyltransferases. GSK126 is a SAM-competitive EZH2 inhibitor with 150 times the selectivity of EZH1. UNC1999 is an analog of GSK126, but has lower selectivity than its counterpart, GSK126.

[0101]

[0152] In one embodiment, the histone methyltransferase inhibitor is DZNep. In one embodiment, the HDAC inhibitor is a class I HDAC inhibitor, a class II HDAC inhibitor, or a combination thereof. In one embodiment, the HDAC inhibitor is KD5170 (mercaptoketone HDAC inhibitor), MC1568 (class IIa HDAC inhibitor), TMP195 (class IIa HDAC inhibitor), or any combination thereof. In some embodiments, the HDAC inhibitor is vorinostat, romidepsin (Istodax), thidamide, panobinostat (Faridac), bellinostat (PXD101), panobinostat (LBH589), valproic acid, mosetinostat (MGCD0103), avexinostat (PCI-24781), entinostat (MS-275), SB939, Resmino These include Stat (4SC-201), Gibinostat (ITF2357), Xynostat (JNJ-26481585), HBI-8000 (Benzamide HDI), Kebetrin, CUDC-101, AR-42, CHR-2845, CHR-3996, 4SC-202, CG200745, ACY-1215, ME-344, Sulforaphane, or any variant thereof.

[0102]

[0153] In some cases, histone methyltransferase inhibitors (e.g., DZNep) The concentration of may be 0.01–10 μM or approximately 0.01–10 μM. For example, the concentration of a histone methyltransferase inhibitor (e.g., DZNep) may be approximately 0.01–1, 0.1–1, 0.25–1, 0.5–1, 1–5, or 1–10 μM. The concentration of a histone methyltransferase inhibitor (e.g., DZNep) may be approximately 5, 4, 3, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, or less than 0.01 μM.

[0103]

[0154] In some embodiments, the method includes the step of contacting pancreatic progenitor cells or precursors with the first or second composition for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days. In some embodiments, the method includes the step of contacting pancreatic progenitor cells or precursors with the first or second composition for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, or about 20 days.

[0104]

[0155] In some embodiments, pancreatic progenitor cells express at least one of PDX1 and NKX6.1. In some embodiments, pancreatic progenitor cells express both PDX1 and NKX6.1. In some embodiments, endocrine cells express CHGA. Epigenetic modification

[0156] Epigenetics can refer to heritable modifications that do not involve changes in DNA sequence. Rather, epigenetic modifications such as DNA methylation and histone modification alter the accessibility of DNA and the structure of chromatin, thereby regulating the pattern of gene expression. These processes may be important for the normal development and differentiation of unique cell lineages in adult organisms. They can be modified by exogenous influences and therefore may contribute to, or be a result of, environmental modifications of phenotypic or pathological phenotypes. Importantly, epigenetic programming can play a crucial role in regulating pluripotency genes, which are inactivated during differentiation.

[0105]

[0157] Chromatin is a complex of chromosomal DNA associated with nuclear proteins. The DNA in chromatin is packed around histone proteins within units called nucleosomes. A nucleosome can contain 147 base pairs of DNA associated with an octameric core of histone proteins consisting of two H3-H4 histone dimers surrounded by two H2A-H2B dimers. The N-terminal histone tail can protrude from the nucleosome into the nuclear lumen. H1 histones can associate with linker DNA located between nucleosomes. The spacing of nucleosomes determines the structure of chromatin, which can be broadly divided into heterochromatin and euchromatin. The structure of chromatin and the accessibility of genes to the transcriptional machinery can be regulated by modifications to the DNA and histone tails.

[0106]

[0158] In differentiated mammalian cells, the major epigenetic modification found in DNA may be the covalent addition of a methyl group to the C5 position of cytosine residues in CpG dinucleotide sequences (DNA methylation). In undifferentiated stem cells, cytosines other than those in CpG can also be methylated, and methylation of non-CpG cytosines may be particularly important for gene regulation in embryonic stem cells. However, CpG methylation is an important mechanism for ensuring the repression of repeat elements and transposons, and may also play an important role in imprinting and X chromosome inactivation. Silencing of transcriptional genes by CpG methylation is also... By suppressing tissue-specific genes in non-expressing cells, the expression of several tissue-specific genes during development and differentiation can be restricted.

[0107]

[0159] CpG methylation can repress transcription through several mechanisms. The presence of methyl groups at specific CpGs can directly block the recognition and binding of DNA by some transcription factors. Alternatively, other factors may preferentially bind to methylated DNA, blocking the access of transcription factors. For example, MeCP2 and other family members can contribute to transcriptional repression by binding to methyl CpGs and recruiting histone-modifying proteins such as histone deacetylases (HDACs). Subsequently, histone deacetylation promotes chromatin condensation, further repressing transcription. This sequence of events explains how both DNA methylation and certain histone modifications contribute to the on / off state of transcription in genes targeted by epigenetic modifications.

[0108]

[0160] The DNA methyltransferase (DNMT) family is involved in de novo DNA methylation and its maintenance. During embryonic development, de novo methylation can be carried out by DNMT3A and DNMT3B. Universally expressed DNMT1 may be primarily involved in maintaining intracellular levels of CpG methylation. DNMT1 can recognize semi-methylated DNA and function in the complex to add methyl groups to unmethylated daughter strands formed during replication. CpG base pairing allows for the mutual maintenance of methylation during subsequent replication cycles. In this way, non-genetic traits (DNA methylation) can be transmitted from cell to cell, along with contextual effects on gene expression. Thus, methylation can be considered a long-term, relatively stable epigenetic trait, and its effects can contribute to maintaining the cell's phenotype.

[0109]

[0161] DNA methylation can promote the persistence of certain histone states, such as deacetylation, thereby providing a mechanism for maintaining posttranslational histone modifications. Histones are modified posttranslation and can reconfigure chromatin in many ways, including phosphorylation, ubiquitous distribution, acetylation, and methylation. Among these histone modifications, histone acetylation at the ε-amino group of lysine residues in the tails of H3 and H4 has been highly consistently associated with transcriptional enhancement. The structure of chromatin opened by acetylation also allows access for transcriptional repressors. For example, several bromodomain-containing factors, such as BRG1 and Brd4, target acetylated histones, and these factors can intervene here in the formation of repressor (or activator) complexes. Acetylation can target chromatin regions through the recognition and binding of DNA sequence-specific transcription factors that recruit CREB-binding proteins (CBPs) and one of the growing families of histone acetyltransferase (HAT) cofactors, such as p300, MYST, and GNAT.

[0110]

[0162] Histone deacetylation may correlate with CpG methylation and chromatin inactivity. There are four classes of histone deacetylase enzymes (HDACs), and their members can deacetylate histones and / or other protein targets. These regulatory proteins are themselves subject to regulation by acetylation, phosphorylation, and smoylation, which can affect their function, intracellular distribution, and protein-protein association. Interactions with sequence-specific DNA-binding proteins and corepressor complexes may allow certain HDAC proteins to target histones in a gene-specific manner.

[0111]

[0163] Most histone lysine methyltransferases are SET homologues. The HDAC family can have a broad family of proteins that can be grouped into seven subfamilies based on their structural similarities. Members of the SET1 family can specifically promote active chromatin by methylating H3K4. Other histone lysine methyltransferase families can methylate several histone targets. Furthermore, some of these methyltransferases may have additional domains that specifically bind to methylated DNA or other proteins such as CBP39. In humans, HDAC proteins can comprise a family of 18 members, which are divided into four classes based on their size, intracellular localization, number of catalytic active sites, and homology to yeast HDAC proteins. Class I includes HDAC1, HDAC2, HDAC3, and HDAC8. Class II consists of six HDAC proteins, which are further divided into two subclasses. Class IIa includes HDAC4, HDAC5, HDAC7, and HDAC9, each containing a single catalytic active site. Class IIb includes HDAC6 and HDAC10, both of which contain two active sites, but only HDAC6 has two active sites that are effective as catalysts. HDAC11 is the only member of Class IV based on phylogenetic analysis. Class I, II, and IV HDAC proteins can act by metal ion-dependent mechanisms, as shown by crystallographic analysis. In contrast, Class III HDAC proteins, referred to as sirtuins (SIRT1-7), can act by NAD+-dependent mechanisms independent of other HDAC proteins. In one embodiment, HDAC inhibitors of HDAC proteins induce cell differentiation. In another embodiment, HDAC inhibitors upregulate important genes related to cell differentiation. Epigenetic Modified Compounds

[0164] The term "epigenetic modification compound" can refer to a compound that induces epigenetics in a gene, i.e., alters gene expression without altering the DNA sequence. Epigenetics helps determine whether a gene is turned on or off and can influence protein production in certain cells, such as β-cells. Epigenetic modifications, such as DNA methylation and histone modification, alter DNA accessibility and chromatin structure, thereby regulating the pattern of gene expression. These processes can be crucial for the normal development and differentiation of unique cell lineages in adult organisms. They can be modified by exogenous influences and therefore can contribute to, or be a result of, environmental modifications of phenotype or pathological phenotype. Importantly, epigenetic modifications can play a significant role in regulating pluripotency genes, which are inactivated during differentiation. Examples of non-restrictive epigenetic modification compounds include DNA methylation inhibitors, histone acetyltransferase inhibitors, histone deacetylase inhibitors, histone methyltransferase inhibitors, bromodomain inhibitors, or any combination thereof.

[0112]

[0165] In one embodiment, a histone methyltransferase inhibitor is an inhibitor of the enhancer of zest homolog 2 (EZH2). EZH2 is a histone-lysine N-methyltransferase enzyme. Non-limiting examples of EZH2 inhibitors that can be used in the methods provided herein include 3-deazanepranosine A (DZNep), EPZ6438, EPZ005687 (a competitive S-adenosylmethionine (SAM) inhibitor), EI1, GSK126, and UNC1999. DZNep inhibits the hydrolysis of S-adenosyl-L-homocysteine ​​(SAH), a product system inhibitor of all protein methyltransferases, leading to an increase in the intracellular concentration of SAH, which can then inhibit EZH2. DZNep is not specific to EZH2 and can also inhibit other DNA methyltransferases. GSK126 is a SAM competitive EZH2 inhibitor with 150 times the selectivity of EZH1. UNC1999 is an analog of GSK126, but has lower selectivity than its counterpart, GSK126.

[0113]

[0166] In one embodiment, the histone methyltransferase inhibitor is DZNep. In one embodiment, the HDAC inhibitor is a class I HDAC inhibitor, a class II HDAC inhibitor, or a combination thereof. In one embodiment, the HDAC inhibitor is KD5170 (mercaptoketone HDAC inhibitor), MC1568 (class IIa HDAC inhibitor), TMP195 (class IIa HDAC inhibitor), or any combination thereof. In some embodiments, the HDAC inhibitor is vorinostat, romidepsin (Istodax), thidamide, panobinostat (Faridac), bellinostat (PXD101), panobinostat (LBH589), valproic acid, mosetinostat (MGCD0103), avexinostat (PCI-24781), entinostat (MS-275), SB939, Resmino These include Stat (4SC-201), Gibinostat (ITF2357), Xynostat (JNJ-26481585), HBI-8000 (Benzamide HDI), Kebetrin, CUDC-101, AR-42, CHR-2845, CHR-3996, 4SC-202, CG200745, ACY-1215, ME-344, Sulforaphane, or any variant thereof. III. Methods for producing pancreatic progenitor cells

[0167] In some embodiments, the disclosure relates to compositions and methods for differentiating gastrulatic cells into Pdx1-positive pancreatic β-cells. In some examples, the method includes contacting gastrulatic cells with a composition comprising a bone morphogenetic protein (BMP) signaling pathway inhibitor and growth factors from the transformation growth factor β (TGF-β) superfamily. In some examples, the composition further comprises one or more additional differentiation factors, but is not limited to, growth factors from the fibroblast growth factor (FGF) family, sonic hedgehog (SHH) pathway inhibitors, retinoic acid (RA) signaling pathway activators, protein kinase C (PKC) activators, and Rho-related protein kinase (ROCK) inhibitors.

[0114]

[0168] In some cases, the method provided herein includes the step of producing a population or cell cluster of cells, including Pdx1-positive pancreatic progenitor cells, by contacting a population of cells including gastrullous cells with a first composition comprising a BMP signaling pathway inhibitor and growth factors from the TGF-β superfamily, wherein the gastrullous cells differentiate into Pdx1-positive, NKX6.1-positive pancreatic progenitor cells. In some cases, the contact step takes approximately 1, 2, or 3 days. In some cases, the contact step takes approximately 1 day. In some cases, the gastrullous cells differentiate into Pdx1-positive, NKX6.1-negative pancreatic progenitor cells by contacting the composition comprising a BMP signaling pathway inhibitor and growth factors from the TGF-β superfamily. In some cases, the production step further includes differentiating Pdx1-positive, NKX6.1-positive pancreatic progenitor cells into Pdx1-positive, NKX6.1-positive pancreatic progenitor cells by contacting Pdx1-positive, NKX6.1-negative pancreatic progenitor cells with a second composition comprising one or more differentiation factors, but not limited to, growth factors from the TGF-β superfamily, growth factors from the FGF family, SHH pathway inhibitors, RA signaling pathway activators, and ROCK inhibitors. In some cases, the second composition does not contain BMP signaling pathway inhibitors.

[0115]

[0169] In some cases, by differentiating a population of cells including gastrullary cells into a population or cell cluster of cells including Pdx1-positive, NKX6.1-positive pancreatic progenitor cells, a population or cell cluster containing at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, or at most about 1% CHGA-positive cells can be obtained. In some cases, by differentiating a population of cells including gastrullary cells into a population or cell cluster of cells including Pdx1-positive, NKX6.1-positive pancreatic progenitor cells, a population or cell cluster containing at most about 25%, at most about 20%, at most about 15%, or at most about 10% CDX2-positive cells can be obtained, as measured by flow cytometry. In some examples, by differentiating a population of cells including gastrullous cells into a population or cell cluster of cells including Pdx1-positive, NKX6.1-positive pancreatic progenitor cells, a population or cell cluster containing at most about 30% CHGA-positive cells and at most about 30% CDX2-positive cells can be obtained using the method provided herein. In some examples, by differentiating a population of cells including gastrullous cells into a population or cell cluster of cells including Pdx1-positive, NKX6.1-positive pancreatic progenitor cells, a population or cell cluster containing at most about 20% CHGA-positive cells and at most about 5% CDX2-positive cells can be obtained using the method provided herein. In some examples, by differentiating a population of cells including gastrullous cells into a population or cell cluster of cells including Pdx1-positive, NKX6.1-positive pancreatic progenitor cells, a population or cell cluster containing at most about 15% CHGA-positive cells and at most about 3% CDX2-positive cells can be obtained using the method provided herein.

[0116]

[0170] In some examples, the BMP signaling pathway inhibitors provided herein include DMH-1, its derivatives, analogs, or variants. In some embodiments, the BMP signaling pathway provided herein includes DMH-1. In some embodiments, the method includes contacting gastrulatous cells with about 0.01 μM to about 10 μM, about 0.05 μM to about 5 μM, about 0.1 μM to about 1 μM, or about 0.15 μM to about 0.5 μM of DMH-1. In some embodiments, this method involves reducing gastrulation cells to approximately 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0. The method includes a step of contacting with 31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.42, 0.45, 0.48, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1, 1.2, 1.4, 1.5, 1.6, 1.7, 1.8, 2.0, 4.0, 6.0, 8.0, or 10 μM. In some embodiments, the method includes a step of contacting gastrullous cells with approximately 0.25 μM. In some examples, the BMP signaling pathway inhibitor used for gastrullous cell differentiation does not include LDN193189 (also referred to herein as "LDN").

[0117]

[0171] In some examples, the methods provided herein include the step of producing a population or cell cluster of cells containing Pdx1-positive, NKX6.1-positive pancreatic progenitor cells by contacting a population of cells containing gastrullary cells with DMH-1, or its derivatives, analogs, or variants.

[0118]

[0172] While we do not wish to be bound by any particular theory, in some embodiments of the methods disclosed herein, inhibition of the BMP signaling pathway during the differentiation of gastrullous cells into Pdx1-positive pancreatic progenitor cells may contribute to reducing the production of untargeted cells, such as intestinal cells or CDX2 gene-positive cells. Conversely, in some cases, activation of the type II receptor-mediated TGF-β signaling pathway during the differentiation of gastrullous cells into Pdx1-positive pancreatic progenitor cells or Pdx1-positive, NKX6.1-positive pancreatic progenitor cells may contribute to reducing the early induction of neurogenin 3 (Ngn3) or chromogranin A (CHGA). These early inductions, in some cases, lead to the production of polyhormonal cells rather than mature SC-β cells. Mature SC-β cells are, in some cases, monohormonal, secreting only insulin, for example, but not other pancreatic hormones such as somatostatin or glucagon. Crosstalk between the BMP signaling pathway and the TGF-β signaling pathway may exist. In some cases, BMP signaling pathway inhibitors may have side effects, such as blocking the type II receptor-mediated TGF-β signaling pathway. For example, LDN193189, a BMP signaling pathway inhibitor with relatively low selectivity... Inhibition of the type II receptor-mediated TGF-β signaling pathway can lead to the early induction of NGN3 / CHGA, as shown in Figure 15, thereby producing polyhormone cells. While we do not wish to be bound by any particular theory, in some cases, the inhibitory effect on the type II receptor-mediated TGF-β signaling pathway can be reduced by using highly selective BMP signaling pathway inhibitors, such as DMH-1 or its derivatives, analogs, or variants. In other cases, while we do not wish to be bound by any particular theory, co-incubating growth factors from the TGF-β superfamily with BMP signaling pathway inhibitors can selectively inhibit the BMP signaling pathway while maintaining a relatively high level of activation of the type II receptor-mediated TGF-β signaling pathway. In some cases, co-incubating growth factors from the TGF-β superfamily with BMP signaling pathway inhibitors can reduce the production of untargeted cells, such as CDX2-positive cells, and reduce the production of polyhormone cells as a result of the early induction of NGN3 or CHGA, for example, in cells differentiated from gastrullous cells.

[0119]

[0173] In some embodiments, the disclosure provides a method for generating NKX6-1 positive pancreatic progenitor cells from Pdx1 positive pancreatic progenitor cells, comprising the step of exposing a population of cells containing Pdx1 positive pancreatic progenitor cells or precursors to at least two β-cell maturation factors, including a) at least one growth factor from the fibroblast growth factor (FGF) family, b) a sonic hedgehog pathway inhibitor, and optionally c) a low concentration of a retinoic acid (RA) signaling pathway activator, for at least 5 days under conditions that promote cell clustering, thereby inducing differentiation of at least one Pdx positive pancreatic progenitor cell in the population into an NKX6-1 positive pancreatic progenitor cell, wherein the NKX6-1 positive pancreatic progenitor cell expresses NKX6-1.

[0120]

[0174] In some embodiments, at least 10% of Pdx1-positive pancreatic progenitor cells in a population are induced to differentiate into NKX6-1-positive pancreatic progenitor cells. In some embodiments, at least 95% of Pdx1-positive pancreatic progenitor cells in a population are induced to differentiate into NKX6-1-positive pancreatic progenitor cells. In some embodiments, NKX6-1-positive pancreatic progenitor cells express Pdx1, NKX6-1, and FoxA2. In some embodiments, Pdx1-positive pancreatic progenitor cells are generated from a population of pluripotent stem cells selected from the group consisting of embryonic stem cells and induced pluripotent stem cells. IV. Stem Cells and Reprogramming

[0175] The use of stem cells to generate SC-β cells (e.g., mature pancreatic β cells or β-like cells) or their precursors is provided herein. In one embodiment, embryonic cells may be used instead of or in conjunction with stem cells to provide at least one SC-β cell, using protocols similar to those described herein in whole by reference in U.S. Patent Application Publications 2015 / 0240212 and 2015 / 0218522. Suitable embryonic cells can be prepared, for example, from primordial embryonic cells present in human fetal material collected approximately 8 to 11 weeks after the last menstrual cycle. Descriptive methods for preparing embryonic cells are described, for example, by Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998 and U.S. Patent No. 6,090,622.

[0121]

[0176] Compositions and methods for producing SC-β cells (e.g., pancreatic β cells) are provided herein. Generally, at least one SC-β cell or its precursor, for example, pancreatic progenitor cells produced by the methods disclosed herein, may include a mixture or combination of various cells, such as gastrullary cells, Pdx1-positive pancreatic progenitor cells, Pdx1-positive, NKX6-1-positive pancreatic progenitor cells, Ngn3-positive endocrine progenitor cells, insulin-positive endocrine cells (e.g., β-like cells), and / or other pluripotent cells or stem cell mixtures.

[0122]

[0177] At least one SC-β cell or its precursor can be generated according to any suitable culture protocol for differentiating stem cells or pluripotent cells to a desired differentiation stage. In some embodiments, at least one SC-β cell or its precursor is produced by culturing at least one pluripotent cell under conditions and for a time suitable for differentiating at least one SC-β cell or its precursor.

[0123]

[0178] In some embodiments, at least one SC-β cell or its precursor is a population of substantially pure SC-β cells or their precursors. In some embodiments, the population of SC-β cells or its precursors includes a mixture of pluripotent cells or differentiated cells. In some embodiments, the population of SC-β cells or its precursors substantially does not contain or lacks embryonic stem cells, pluripotent cells, or iPS cells.

[0124]

[0179] In some embodiments, somatic cells, such as fibroblasts, can be isolated from a subject, for example, as a tissue biopsy, such as a skin biopsy, and can be reprogrammed into induced pluripotent stem cells for further differentiation to produce at least one SC-β cell or its precursor for use in the compositions and methods described herein. In some embodiments, somatic cells, such as fibroblasts, are maintained in a culture medium by a method known to those skilled in the art, and in some embodiments, are increased before being converted to SC-β cells by a method disclosed herein.

[0125]

[0180] In some embodiments, at least one SC-β cell or its precursor is maintained in a culture medium by a method known to those skilled in the art, and in some embodiments, is increased before being converted to SC-β cells by the method described herein.

[0126]

[0181] Furthermore, at least one SC-β cell or its precursor, such as a pancreatic progenitor cell, may be from any mammalian species, non-limiting examples including cells from murids, bovines, primates, pigs, equids, sheep, or humans. For clarity and simplicity, the method descriptions herein refer to at least one mammalian SC-β cell or its precursor, but it should be understood that all methods described herein are readily applicable to other cell types of at least one SC-β cell or its precursor. In some embodiments, at least one SC-β cell or its precursor is derived from a human individual. stem cells

[0182] Embodiments of this disclosure may relate to the use of stem cells for the production of pancreatic β-cells or their precursors. As used herein, the term “stem cells” may mean cells that have the ability to self-renew and produce differentiated cell types (e.g., plant stem cells, vertebrate stem cells) (Morrison et al., (1997) Cell 88:287-298). In the context of cell development, the adjectives “differentiated” or “in the process of differentiation” are relative terms. A “differentiated cell” may be a cell that has progressed further downstream in the developmental pathway than the cell being compared. Thus, pluripotent stem cells can differentiate into lineage-restricted progenitor cells (e.g., mesodermal stem cells), which can then differentiate into further restricted cells (e.g., neuronal progenitor cells), which can then differentiate into terminal cells (e.g., terminally differentiated cells, e.g., neurons, cardiomyocytes, etc.). Terminal cells may or may not retain the ability to proliferate further, and they may play characteristic roles in certain tissue types. Stem cells can be characterized by the presence or absence of certain markers (e.g., proteins, RNA, etc.). Stem cells can also be identified by functional assays both in vitro and in vivo, particularly those related to the ability of stem cells to produce multiple differentiated offspring. In one embodiment, host cells include adult stem cells, somatic stem cells, non-embryonic stem cells, embryonic stem cells, hematopoietic stem cells, pluripotent stem cells, and trophoblast stem cells.

[0127]

[0183] The target stem cells, for example, the stem cells that can be used in the methods provided herein, may include pluripotent stem cells (PSCs). The terms “pluripotent stem cells” or “PSC” as used herein are Pluripotent stem cells (PSCs) are capable of generating all cell types of an organism. Therefore, PSCs can give rise to cells of all germ layers of an organism (e.g., endoderm, mesoderm, and ectoderm in vertebrates). Pluripotent cells can form teratomas and contribute to the ectoderm, mesoderm, or endoderm tissues of an organism. Plant pluripotent stem cells can give rise to all cell types of the plant (e.g., roots, stems, leaves, and other cells).

[0128]

[0184] Embodiments of this disclosure may relate to the use of PSCs for the production of pancreatic β-cells or their precursors. Animal PSCs can be induced in several different ways. For example, embryonic stem cells (ESCs) can be induced from the inner cell cluster of an embryo (Thomson et al., Science. 1998 Nov. 6; 282(5391):1145-7). On the other hand, induced pluripotent stem cells (iPSCs) can be induced from somatic cells. It can be guided (Takahashi et al., Cell. 2007 Nov. 30; 131(5):861-72; Takahashi et al.) Nat Protoc. 2007; 2(12):3081-9; Yu et al., Science. 2007 Dec. 21; 318(5858):1917-20. Epub 2007 Nov. 20). The term PSC refers to pluripotent stem cells regardless of their origin. Therefore, the term PSC can also encompass the terms ESC and iPSC, as well as another example of PSC, the term fetal embryonic stem cell (EGSC). PSCs may be in the form of established cell lines, which can be obtained directly from primary embryonic tissue or induced from somatic cells.

[0129]

[0185] Embodiments of this disclosure may relate to the use of ESCs for the production of pancreatic β-cells or their precursors. “Embryo stem cells (ESCs)” may mean PSCs isolated from an embryo, typically from the inner cell cluster of a blastocyst. ESC strains include, for example, hESBGN-01, hESBGN-02, hESBGN-03, hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3, HES-4, HES-5, HES-6 (ES Cell International); Miz-hES1 (MizMedi Hospital-Seoul National University); HSF-1, HSF-6 (University of California at San Francisco); and H1, H7, H9, H13, H14 (Wisconsin Alumni The Research Foundation (WiCell Research Institute) is listed in the NIH Human Embryonic Stem Cell Registry. Target stem cells 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, cattle, pigs, dogs, cats, rodents (e.g., 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, ES ESCs can grow as flat colonies with a large nucleus-to-cytoplasmic ratio, clear boundaries, and prominent nuclei. Furthermore, ESCs can express SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase, but not SSEA-1. Examples of methods for producing and characterizing ESCs can be found, for example, in U.S. Patents 7,029,913, 5,843,780, and 6,200,806, each of which is incorporated herein by reference as a whole. Methods for growing undifferentiated morphological hESCs are described in WO 99 / 20741, WO 01 / 51616, and WO 03 / 020920, each of which is incorporated herein by reference as a whole.

[0130]

[0186] "Fetal embryonic stem cells (EGSCs) or fetal embryonic cells" or "EG cells" may mean embryonic cells and / or embryonic cell progenitor cells, e.g., primordial embryonic cells, e.g., PSCs derived from embryonic cells that can become sperm and oocytes. Fetal embryonic cells (EG cells) are thought to have similar properties to the fetal stem cells described above. Examples of methods for producing and characterizing EG cells are, for example, U.S. Patent No. 7,153,684, each incorporated herein as a whole. It can be found in: 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.

[0131]

[0187] Embodiments of this disclosure may relate to the use of iPSCs for the production of pancreatic β-cells or their precursors. "Induced pluripotent stem cells" or "iPSC" may mean PSCs induced from non-PSC cells (e.g., from cells differentiated to PSCs). iPSCs can be induced from a wide variety of different cell types, including ultimately differentiated cells. iPSCs have ES cell-like morphology and can grow as flat colonies with a large nucleo-cytoplasmic ratio, well-defined boundaries, and prominent nuclei. Furthermore, iPSCs can express one or more important pluripotency markers known to those skilled in the art, including, but not limited to, alkaline phosphatase, SSEA3, SSEA4, Sox2, Oct3 / 4, Nanog, TRA160, TRA181, TDGF1, Dnmt3b, FoxD3, GDF3, Cyp26a1, TERT, and zfp42. Examples of methods for producing and characterizing iPSCs can be found, for example, in U.S. Patent Publications 2009 / 0047263, 2009 / 0068742, 2009 / 0191159, 2009 / 0227032, 2009 / 0246875, and 2009 / 0304646, each of which is incorporated herein by reference. Generally, to produce iPSCs, somatic cells are provided with reprogramming factors known in this technology (e.g., Oct4, SOX2, KLF4, MYC, Nanog, Lin28, etc.) that reprogram somatic cells into pluripotent stem cells.

[0132]

[0188] Embodiments of this disclosure may relate to the use of somatic cells for the production of pancreatic β-cells or their precursors. “Somatic cells” may mean any cells in a living organism that do not normally produce all types of cells in the organism in the absence of experimental manipulation. In other words, somatic cells may be fully differentiated cells that do not naturally produce cells of all three germ layers of the organism, such as ectoderm, mesoderm, and endoderm. For example, somatic cells include neurons and neuronal progenitor cells, which can naturally produce all or some cell types of the central nervous system but cannot produce cells of the mesoderm or endoderm lineages.

[0133]

[0189] In certain cases, stem cells may be undifferentiated (e.g., cells not belonging to a particular lineage) before exposure to at least one differentiation factor or composition as disclosed herein, whereas in other cases, it may be desirable to differentiate stem cells into one or more intermediate cell types before exposure to at least one differentiation factor or composition described herein. For example, stem cells may exhibit morphological, biological, or physical characteristics of undifferentiated cells, which can be used to distinguish these cells from differentiated cells of fetal or adult origin. In some cases, undifferentiated cells may appear in a microscopic two-dimensional manner in colonies of cells with a high nucleus / cytoplasmic ratio and prominent nuclei. Stem cells may be on their own (e.g., substantially free of undifferentiated cells) or used in the presence of differentiated cells. In certain cases, stem cells may be cultured in the presence of suitable nutrients and optionally other cells so that they may grow and optionally differentiate. For example, fetal fibroblasts or fibroblast-like cells may be present in culture to aid in the growth of stem cells. Fibroblasts may be present during one stage of stem cell growth, but do not necessarily have to be present at all stages. For example, fibroblasts may be added to the stem cell culture in the initial culture stage, but may not be added to the stem cell culture in one or more subsequent culture stages.

[0134]

[0190] The stem cells used in all aspects of the present invention 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 of various cell types, for example, three germ layers (endoderm, mesoderm) under appropriate conditions. These cell types may be characterized by their ability to produce offspring consisting of all or at least one derivative of the ectoderm (and ectoderm). These cell types can be provided in the form of established cell lines, or they can be obtained directly from primary embryonic tissue and used immediately for differentiation. Cells listed in the NIH Human Embryonic Stem Cell Registry 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, 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 the step of destroying a human embryo.

[0135]

[0191] In another example, stem cells can be isolated from tissues including solid tissue. In some embodiments, the tissue is skin, adipose tissue (e.g., agitated tissue), muscle tissue, heart, or cardiac tissue. In other embodiments, the tissue is, for example, but not limited to, umbilical cord blood, placenta, bone marrow, or cartilage.

[0136]

[0192] The stem cells that may be used in the methods provided herein may also include various types of embryonic cells, as exemplified by human germinal 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 involved in lineages such as mesodermal stem cells and other early cardiac development cells (Reyes et al., (2001) Blood 98:2615-2625; Eisenberg and Bader, (1996) Circ Res. 78(2):205-16, etc.). (See [reference]). Stem cells can be obtained from any mammalian species, such as humans, horses, cattle, pigs, dogs, cats, rodents, such as mice, rats, hamsters, primates, etc. In some embodiments, human embryos were not destroyed for the source of pluripotent cells used in the methods and compositions disclosed herein. In some embodiments, human embryos were not destroyed for the source of pluripotent cells used in the methods and compositions disclosed herein.

[0137]

[0193] A mixture of cells from suitable sources of endothelial, muscle, and / or neuronal stem cells can be harvested from a mammalian donor for the purposes of this disclosure. A suitable source is the hematopoietic microenvironment. For example, preferably immobilized (e.g., mobilized) circulating peripheral blood can be taken from the subject. In one embodiment, the stem cells may be reprogrammed stem cells, such as stem cells induced from somatic cells or differentiated cells. In such embodiments, the dedifferentiated stem cells may be induced and reprogrammed cells such as neoplastic cells, tumor cells, and cancer cells, or induced pluripotent stem cells or iPS cells, but not limited to these.

[0138]

[0194] In some embodiments, the pancreatic β-cells described herein include hair cells, keratinocytes, gonadotropin-producing cells, adrenocorticotropin-producing cells, thyroid-stimulating hormone-producing cells, somatotropin-producing cells, mammary gland-stimulating hormone-producing cells, chromaffin cells, parafollicular cells, glomus cells, melanin-forming cells, nevus cells, Merkel cells, odontoblasts, and cementoblasts. Cells, corneal keratinocytes, retinal Muller cells, retinal pigment epithelial cells, neurons, glial cells (e.g., oligodendritic cells, astrocytes), ependymal cells, pineal cells, lung cells (e.g., type I and type II lung cells), Clara cells, goblet cells, G cells, D cells, ECL cells, gastric chief cells, parietal cells, pitted cells, K cells, D cells, I cells, goblet cells, Panes cells, intestinal cells, microfold cells, hepatocytes, hepatic stellate cells (e.g., Kupffer cells from mesoderm), gallbladder cells, atrial heart cells, pancreatic stellate cells, pancreatic alpha cells, pancreatic beta cells Cells, pancreatic delta cells, pancreatic f cells (e.g., PP cells), pancreatic epsilon cells, thyroid (e.g., follicular cells), parathyroid (e.g., chief parathyroid cells), eosinophilic cells, urothelial cells, osteoblasts, osteocytes, chondrocytes, chondrocytes, fibroblasts, fibrocytes, myoblasts, myocytes, muscle satellite cells, tendinocytes, cardiomyocytes, lipoblasts, adipocytes, Caffar interstitial cells, angioblasts, endothelial cells, mesangial cells (e.g., intraglomerular mesangial cells and extraglomerular mesangial cells), juxtaglomerular cells, macula densa cells, stromal cells, interstitial cells, telocytic simple epithelial cells, They can be induced from one or more of the following: podocytes, renal proximal tubule brush margin cells, Sertoli cells, Leydig cells, granulosa cells, Peg cells, germ layer cells, sperm, oocytes, lymphocytes, myeloid cells, endothelial progenitor cells, endothelial stem cells, angioblasts, mesovascular angioblasts, pericutaneous cells, splenic cells (e.g., T lymphocytes, B lymphocytes, dendritic cells, microphages, leukocytes), trophoblast stem cells, or any combination thereof. Reprogramming

[0195] As used herein, the term “reprogramming” may mean the process of altering or reversing the differentiated state of a somatic cell. Cells can be partially or fully differentiated before reprogramming. Reprogramming may include the complete reversal of the differentiated state of a somatic cell to a pluripotent cell. Such a complete reversal of differentiation may produce induced pluripotent (iPS) cells. As used herein, reprogramming may also include the partial reversal of the differentiated state of a cell to, for example, a pluripotent cell or somatic cell, which is a cell that is neither pluripotent nor pluripotent but has lost one or more specific characteristics of the original differentiated cell from which it originated, or to, for example, a direct reprogramming of a differentiated cell to various somatic cell types. Reprogramming may include, for example, the reversal of at least some alterations of heritable patterns that occur during cell differentiation as a zygote develops into an adult, such as nucleic acid modifications (e.g., methylation), chromatin condensation, epigenetics, and genomic imprinting.

[0139]

[0196] As used herein, the term “reprogramming factor” may mean molecules associated with the “reprogramming” of cells, i.e., differentiation, and / or dedifferentiation, and / or transdifferentiation, thereby transforming cells into different cell types or phenotypes. Reprogramming factors generally affect the expression of genes associated with cell differentiation, dedifferentiation, and / or transdifferentiation. Transcription factors are an example of reprogramming factors.

[0140]

[0197] As used herein, the terms “differentiation” and its grammatical equivalent mean the process by which less specific cells (e.g., more primitive cells with higher cellular capabilities) become more specific cell types (e.g., less primitive cells with lower cellular capabilities); the term “dedifferentiation” means the process by which more specific cells become less specific cell types (e.g., more primitive cells with higher cellular capabilities); and the term “transdifferentiation” may mean the process by which cells of a particular cell type are converted to another cell type without a significant change in their “cellular capabilities” or “primitiveness.” While we do not wish to be bound by theory, cells are considered to have “transdifferentiated” if they are converted from one lineage-related cell type or ultimately differentiated cell type to another lineage-related cell type or ultimately differentiated cell type without a significant change in their “cellular capabilities” or “primitiveness.”

[0141]

[0198] As used herein, the term “cell differentiation ability” should be understood to mean the ability of a cell to differentiate into different cell lineages. For example, pluripotent cells (e.g., stem cells) have three germ layers: endoderm (internal lining of the stomach, gastrointestinal tract, lungs) and mesoderm (muscle, bone, blood, urogenitalia). Cells have the potential to differentiate into either the organelle or ectoderm (epithelial tissue and nervous system), and therefore possess high cell differentiation potential. Pluripotent cells (e.g., stem cells or certain types of induced stem cells) have the ability to generate cells from a large but limited number of lineages (e.g., hematopoietic stem cells, cardiac stem cells, or neural stem cells) and have relatively lower cell differentiation potential than pluripotent cells. Cells involved in or ultimately differentiated from a particular lineage may have even lower cell differentiation potential. Specific examples of transdifferentiation known in this technique include, for example, the conversion of fibroblast beta cells or pancreatic exocrine cells to beta cells.

[0142]

[0199] Therefore, cells can be differentiated into more primitive cells (for example, the finally differentiated cells can differentiate into pluripotent or pluripotent cells), or cells can be dedifferentiated into less primitive cells (for example, pluripotent or pluripotent cells can differentiate into lineage-related cells or the finally differentiated cells). However, in one embodiment, cells can be transformed or transdifferentiated from one cell type (or phenotype) to another cell type (or phenotype), for example, at a similar level of cellular capability. Therefore, in one embodiment of the present disclosure, the steps induced by the present disclosure can reprogram the cells of the present disclosure to differentiate, dedifferentiate, and / or transdifferentiate.

[0143]

[0200] Methods for reprogramming or inducing a particular type of cell into another type of cell, for example by differentiation, dedifferentiation, and / or transdifferentiation, using one or more exogenous polynucleotides or polypeptide reprogramming factors, are known to those skilled in the art. Such methods may rely on the introduction of genetic material encoding one or more transcription factors or other polypeptides related to cell reprogramming. For example, Pdx1, Ngn3, and MafA, or their functional fragments, are all known to encode peptides that can induce cell differentiation, dedifferentiation, and / or transdifferentiation of the cells of this disclosure. In some methods known to those skilled in the art, an exogenous polypeptide (e.g., a recombinant polypeptide) encoded by a reprogramming gene (e.g., the genes mentioned above) comes into contact with a cell to induce, for example, the cells of this disclosure. Those skilled in the art will recognize that other genes are also related to cell reprogramming, and that the exogenous molecules (or their functional fragments) encoding such genes and the encoded polypeptides are also considered to be polynucleotide or polypeptide reprogramming factors (e.g., polynucleotides or polypeptides that subsequently affect the expression level of another gene related to cell reprogramming). For example, it has been shown that introducing exogenous polynucleotide or polypeptide epigenetic gene silencers that reduce p53 inactivation increases the efficiency of inducing induced pluripotent stem cells (iPSCs). Therefore, exogenous polynucleotides or polypeptides encoding epigenetic silencers, as well as other genes or proteins that appear to be directly or indirectly involved in cell reprogramming or increased cell programming efficiency, are considered to constitute exogenous polynucleotide or polypeptide reprogramming factors. Those skilled in the art will recognize that other methods exist that affect cell reprogramming, such as the introduction of RNAi molecules (or genetic material encoding RNAi molecules) that can knock down the expression of genes involved in inhibiting cell reprogramming.Therefore, any exogenous polynucleotide or polypeptide molecule that is associated with or promotes cellular reprogramming should be understood as an exogenous polynucleotide or polypeptide reprogramming factor as described herein.

[0144]

[0201] In some embodiments of this disclosure, the method does not involve the use of non-small molecule reprogramming factors. However, the method does not involve the use of culture media, serum, serum substitutes, adjuvants, antibiotics, etc., such as RPMI, renal epithelial basal medium (REBM), Dulbecco's modified Eagle medium (DMEM), MCDB131 medium, CMRL1066 medium, F12, fetal bovine serum (FCS). It is recognized that “routine” tissue culture components such as fetal bovine serum (FBS), bovine serum albumin (BSA), D-glucose, L-glutamine, GlutaMAX.TM-1 (dipeptide, L-alanine-L-glutamine), B27, heparin, progesterone, putrescine, laminin, nicotinamide, insulin, transferrin, sodium selenite, selenium, ethanolamine, human epidermal growth factor (hEGF), basic fibroblast growth factor (bFGF), hydrocortisone, epinephrine, normaline, penicillin, streptomycin, gentamicin, and amphotericin are available. These typical tissue culture components (and other similar tissue culture components routinely used in tissue culture) are not small molecule reprogramming molecules for the purposes of this disclosure. These components are neither small molecules nor reprogramming factors as defined herein.

[0145]

[0202] Accordingly, in one embodiment, the disclosure does not involve the step of culturing cells with one or more exogenous polynucleotide or polypeptide reprogramming factors. Accordingly, in one embodiment, the method of the disclosure does not involve the introduction of one or more exogenous polynucleotide or polypeptide reprogramming factors, for example, by introducing transposons, viral transgenic vectors (such as retroviral vectors), plasmids, mRNA, miRNA, peptides, or fragments of any of these molecules, which are involved in producing induced β cells or otherwise inducing differentiation, dedifferentiation, and / or transdifferentiation of the cells of the disclosure.

[0146]

[0203] That is, in one embodiment, the method is carried out in the absence of one or more exogenous polynucleotide or polypeptide reprogramming factors. Therefore, in one embodiment, it should be understood that the method of the disclosure reprograms cells using small molecules (e.g., HDAC inhibitors) without adding polypeptide transcription factors, other polypeptide factors specifically associated with the induction of differentiation, dedifferentiation, and / or transdifferentiation, polynucleotide sequences encoding polypeptide transcription factors, polynucleotide sequences encoding other polypeptide factors specifically associated with the induction of differentiation, dedifferentiation, and / or transdifferentiation, mRNA, interfering RNA, microRNA, and fragments thereof. V. Xenofree Medium

[0204] In some embodiments, the present disclosure relates to a method for producing pancreatic β-cells, such as SC-β-cells, comprising the step of differentiating progenitor cells (e.g., stem cells such as iPSC cells, endoderm cells, gastrulatal cells, Pdx1-positive pancreatic progenitor cells, NKX6.1-positive pancreatic progenitor cells, or insulin-positive endocrine cells) in a xeno-free medium. A xeno-free medium for culturing animal-derived cells and / or cell clusters does not need to contain products from other animals. In some examples, a xeno-free medium for culturing human cells and / or cell clusters does not need to contain any products from non-human animals. For example, a xeno-free medium for culturing human cells and / or cell clusters may contain human serum albumin (HSA) or human platelet lysate (PLT) instead of fetal bovine serum (FBS) or bovine serum albumin (BSA).

[0147]

[0205] In some embodiments, the methods provided herein include the step of producing pancreatic β-cells, such as SC-β-cells, by differentiating progenitor cells (e.g., stem cells such as iPSC cells, endoderm cells, gastrulatinous cells, Pdx1-positive pancreatic progenitor cells, NKX6.1-positive pancreatic progenitor cells, or insulin-positive endocrine cells) in a serum albumin-deficient medium. In some examples, a population or cluster of cells containing pancreatic β-cells produced by the methods provided herein without the use of serum albumin or HSA in the medium may have a significant improvement compared to a population or cluster of cells containing pancreatic β-cells produced by other equivalent methods using BSA instead. Improvements include a higher percentage of pancreatic β-cells in the final cell population, a higher GSIS response (e.g., more insulin release in response to glucose challenge), a higher GSIS stimulation index, and a higher cell cluster. This may include greater uniformity in the distribution of pancreatic β-cells in the raster, or any combination thereof.

[0148]

[0206] In some embodiments, the method provided herein includes the step of differentiating a population of cells, including stem cells, such as hES cells or iPS cells, in a culture medium containing human serum albumin (HSA). In some examples, the stem cells are differentiated into endoderm cells. In some embodiments, the method provided herein includes the step of differentiating a population of cells, including endoderm cells, in a culture medium containing human serum albumin (HSA). In some examples, the endoderm cells are differentiated into gastrula cells. In some embodiments, the method provided herein includes the step of differentiating a population of cells, including gastrula cells, in a culture medium containing human serum albumin (HSA). In some examples, the gastrula cells are differentiated into Pdx1-positive pancreatic progenitor cells (e.g., Pdx1-positive, NKX6.1-negative pancreatic progenitor cells or Pdx1-positive, NKX6.1-positive pancreatic progenitor cells). In some embodiments, the method provided herein includes the step of differentiating a population of cells, including Pdx1-positive, NKX6.1-negative pancreatic progenitor cells, in a culture medium containing human serum albumin (HSA). In some cases, Pdx1-positive, NKX6.1-negative pancreatic progenitor cells are differentiated into Pdx1-positive, NKX6.1-positive pancreatic progenitor cells. In some embodiments, the method provided herein includes the step of differentiating a population of cells, including Pdx1-positive, NKX6.1-positive pancreatic progenitor cells, in a medium containing human serum albumin (HSA). In some embodiments, Pdx1-positive, NKX6.1-positive pancreatic progenitor cells are differentiated into insulin-positive endocrine cells. In some embodiments, the method provided herein includes the step of differentiating a population of cells, including insulin-positive endocrine cells, in a medium containing human serum albumin (HSA). In some embodiments, insulin-positive endocrine cells are differentiated into pancreatic β-cells, such as SC-β-cells.

[0149]

[0207] In some embodiments, the methods provided herein involve the use of a culture medium containing HSA in concentrations of approximately 0.001% to approximately 5% (w / v), approximately 0.005% to approximately 4% (w / v), approximately 0.01% to approximately 3% (w / v), approximately 0.02% to approximately 2.5% (w / v), approximately 0.03% to approximately 2% (w / v), approximately 0.04% to approximately 1% (w / v), approximately 0.045% to approximately 0.5% (w / v), or approximately 0.05% to approximately 0.1% (w / v). In some embodiments, the methods provided herein are approximately 0.001%, 0.002%, 0.0025%, 0.005%, 0.0075%, 0.01%, 0.0125%, 0.015%, 0.0175%, 0.02%, 0.0225%, 0.025%, 0.0275%, 0.03%, 0.0325%, 0.035%, 0.0375%, 0.04%, 0.0425%, 0.045%, 0.0475%, 0.05%, 0.0525%, 0.055% This includes the use of media containing HSA at concentrations of %, 0.575%, 0.06%, 0.0625%, 0.065%, 0.0675%, 0.07%, 0.0725%, 0.075%, 0.0775%, 0.08%, 0.085%, 0.09%, 0.1%, 0.12%, 0.15%, 0.2%, 0.25%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, or 4%, 5% (w / v). The term "w / v" is an abbreviation for weight / volume or percentage of weight per volume. For example, 1 mg of HSA in 100 mL of medium has a concentration of 1% (w / v).

[0150]

[0208] In some cases, by differentiating a population of cells including gastrullary cells into a population or cell cluster of cells including Pdx1-positive, NKX6.1-negative pancreatic progenitor cells, a population or cell cluster containing at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% Pdx1-positive cells can be obtained by the method provided herein. By differentiating a population of cells into a population or cluster of cells containing Pdx1-positive, NKX6.1-negative pancreatic progenitor cells, a population or cluster of cells containing at most approximately 60%, at most approximately 50%, at most approximately 40%, at most approximately 35%, at most approximately 30%, at most approximately 25%, at most approximately 22%, at most approximately 20%, at most approximately 18%, at most approximately 15%, at most approximately 14%, at most approximately 13%, at most approximately 11%, at most approximately 12%, at most approximately 10%, or at most approximately 5% CDX2-positive cells can be obtained. VI. Method for producing beta cells derived from stem cells

[0209] Methods for producing SC-β cells (e.g., pancreatic β cells) are provided herein. Detailed protocols for producing endocrine cells from stem cells to provide at least one SC-β cell are described in U.S. Patent Application Publications 2015 / 0240212 and 2015 / 0218522, respectively, which are incorporated herein by reference as a whole.

[0151]

[0210] The endoderm can give rise to the gastrointestinal and respiratory tracts, the thyroid gland, the liver, and the pancreas. A representative disease of the endodermal lineage is type 1 diabetes, which is caused by the destruction of insulin-producing β cells. The production of functional β cells from human pluripotent stem cells (hPSCs) in vitro could be a practical and renewable cell source for replacement cell therapy for type 1 diabetes. Embryonic stem (ES) cells, produced from the inner cell clusters of blast-stage embryos, represent a promising cell source for transplantation of any damaged cells or for cell-based therapies. They can be maintained in culture medium, self-renewed, and proliferate indefinitely as undifferentiated ES cells. ES cells can differentiate into all cell types of the organism, as ectoderm, mesoderm, and endoderm lineage cells or tissues. The main advantage of ES cells is their potential for stable self-renewal and differentiation in culture medium.

[0152]

[0211] Embryonic endoderm is produced in vivo from the inner cell cluster during the gastrulation process of embryonic development, in which the cells of the blastocyst epidermis are instructed to form the three germ layers. Embryonic endoderm can give rise to a variety of cells and tissues that contribute to living organs, such as pancreatic β-cells, liver hepatocytes, lung alveolar cells, thyroid gland, thymus, and epithelial lining of the nutrient and respiratory tracts. This is different from the primitive endoderm of extraembryonic tissue, which can give rise to visceral and parietal endoderm. Embryonic endoderm induced from ES cells can theoretically become any endodermal derivative, and guiding ES cells into an endodermal lineage is a prerequisite for producing therapeutic endodermal derivatives.

[0153]

[0212] Precise patterning of the anterior-posterior axis of the endoderm can ultimately lead to the formation of the gastrula. The gastrula, induced by the endoderm, gives rise to the pharynx, esophagus, stomach, duodenum, small intestine, and large intestine along the anterior-posterior axis, as well as related organs such as the pancreas, lungs, thyroid gland, thymus, parathyroid gland, and liver. The anterior part of the foregut of the gastrula becomes the lungs, thyroid gland, esophagus, and stomach. The pancreas, liver, and duodenum originate from the posterior part of the foregut. The midgut and hindgut of the gastrula give rise to the small intestine and large intestine. The anterior part of the foregut expresses developmental markers, NK2 homeobox (NKX2-1), and SRY (sex-determining region Y)-box 2 (SOX2); the posterior part of the foregut expresses hematopoietic homeobox (HHEX), pancreatic and duodenal homeobox 1 (PDX1), onecut homeobox 1 (ONECUT1, known as HNF6), and hepatocyte nuclear factor 4 alpha (HNF4A); and the midgut / hindgut expresses tail-type homeobox 1 (CDX1), tail-type homeobox 2 (CDX2), and motor neuron and pancreatic homeobox 1 (MNX1) (3, 19, 20).

[0154]

[0213] Successful differentiation of pancreatic β-cells requires that the differentiated cells synthesize and secrete physiologically appropriate amounts of insulin. Exemplary stepwise protocols have been developed to guide the differentiation of hPSC cells, involving a differentiation process that replicates the main stages of normal pancreatic endocrine development (Figure 5). Differentiation of hPSC cells into hormone-expressing pancreatic endocrine cells involves the development of the hPSC cells, differentiation into mesoendoderm and endoderm, establishment of the gastrulate, and patterning of the posterior foregut. This is carried out by passing the cells through key stages consisting of the identification and maturation of the pancreatic endoderm and endocrine precursors. Through these stages, hPSC cells can acquire the pancreatic endocrine phenotype and the ability to secrete glucose-responsive insulin in vitro.

[0155]

[0214] In general, at least one SC-β cell or its precursor, for example, pancreatic progenitor cells produced by the methods disclosed herein, may include a mixture or combination of various cells, such as Pdx1-positive pancreatic progenitor cells, pancreatic progenitor cells co-expressing Pdx1 and NKX6-1, Ngn3-positive endocrine progenitor cells, insulin-positive endocrine cells (e.g., β-like cells), and insulin-positive endocrine cells, as well as / or other pluripotent cells or stem cells.

[0156]

[0215] At least one SC-β cell or its precursor can be generated according to any suitable culture protocol for differentiating stem cells or pluripotent cells to a desired differentiation stage. In some embodiments, at least one SC-β cell or its precursor is generated by culturing at least one pluripotent cell for a time and under conditions suitable for differentiating at least one SC-β cell or its precursor.

[0157]

[0216] In some embodiments, at least one SC-β cell or its precursor is a population of substantially pure SC-β cells or their precursors. In some embodiments, the population of SC-β cells or their precursors includes a mixture of pluripotent cells or differentiated cells. In some embodiments, the population of SC-β cells or their precursors substantially does not contain or lacks embryonic stem cells, pluripotent cells, or iPS cells.

[0158]

[0217] In some embodiments, somatic cells, such as fibroblasts, can be isolated from a subject, for example, as a tissue biopsy, such as a skin biopsy, and can be reprogrammed into induced pluripotent stem cells for further differentiation to produce at least one SC-β cell or its precursor for use in the compositions and methods described herein. In some embodiments, somatic cells, such as fibroblasts, are maintained in a culture medium by a method known to those skilled in the art, and in some embodiments, are increased before being converted to SC-β cells by a method disclosed herein.

[0159]

[0218] In some embodiments, at least one SC-β cell or its precursor is maintained in a culture medium by a method known to those skilled in the art, and in some embodiments, is increased before being converted to SC-β cells by the method disclosed herein.

[0160]

[0219] Furthermore, at least one SC-β cell or its precursor, such as a pancreatic progenitor cell, may be from any mammalian species, non-limiting examples including cells from murids, bovines, primates, pigs, equids, sheep, or humans. For clarity and simplicity, the method descriptions herein refer to at least one mammalian SC-β cell or its precursor, but it should be understood that all methods described herein are readily applicable to other cell types of at least one SC-β cell or its precursor. In some embodiments, at least one SC-β cell or its precursor is derived from a human individual.

[0161]

[0220] A method for producing stem cell-derived β(SC-β) cells is provided herein, comprising the step of contacting a cell population containing pancreatic progenitor cells or their precursors with a histone deacetylase (HDAC) inhibitor to produce SC-β cells, wherein the cell population is induced in vitro from stem cells. In some embodiments, the stem cells are human pluripotent stem cells. In some embodiments, the method further comprises the step of contacting the cell population with at least one of beta-cell phosphate, thiazobinin, retinoic acid, SANT1, XXI, Alk5i II, GC-1, LDN, staurosporine, or any combination thereof. In some embodiments, SC-β cells express C-PEP and NKX6-1. In some embodiments, SC-β cells exhibit a glucose-stimulated insulin secretion response in vitro to a glucose challenge. In some embodiments, the method further includes the step of contacting a cell population with a histone methyltransferase inhibitor.

[0162]

[0221] A method for producing stem cell-derived β(SC-β) cells is provided herein, comprising the step of contacting a cell population containing pancreatic progenitor cells or their precursors with a histone methyltransferase inhibitor to produce SC-β cells, wherein the cell population is induced in vitro from stem cells, and the SC-β cells exhibit a glucose-stimulated insulin secretion response in vitro to a glucose challenge. In some embodiments, the stem cells are human pluripotent stem cells. In some embodiments, the method further comprises the step of contacting the cell population with at least one of beta-cellulin, thiazobinin, retinoic acid, SANT1, XXI, Alk5i II, GC-1, LDN, staurosporine, or any combination thereof. In some embodiments, the method further comprises the step of contacting the cell population with a histone deacetylase (HDAC) inhibitor.

[0163]

[0222] Non-limiting and exemplary epigenetic modification compounds include DNA methylation inhibitors, histone acetyltransferase inhibitors, histone deacetylase inhibitors, histone methyltransferase inhibitors, bromodomain inhibitors, or any combination thereof.

[0164]

[0223] In one embodiment, a histone methyltransferase inhibitor is an inhibitor of the enhancer of zest homolog 2 (EZH2). EZH2 is a histone-lysine N-methyltransferase enzyme. Non-limiting examples of EZH2 inhibitors include 3-deazanepranosine A (DZNep), EPZ6438, EPZ005687 (a competitive S-adenosylmethionine (SAM) inhibitor), EI1, GSK126, and UNC1999. DZNep inhibits the hydrolysis of S-adenosyl-L-homocysteine ​​(SAH), a product inhibitor of all protein methyltransferases, leading to an increase in the intracellular concentration of SAH, which in turn inhibits EZH2. DZNep is not specific to EZH2 and also inhibits other DNA methyltransferases. GSK126 is a SAM-competitive EZH2 inhibitor with 150 times the selectivity of EZH1. UNC1999 is an analog of GSK126, but has lower selectivity than its counterpart, GSK126.

[0165]

[0224] In one embodiment, the histone methyltransferase inhibitor is DZNep. In one embodiment, the HDAC inhibitor is a class I HDAC inhibitor, a class II HDAC inhibitor, or a combination thereof. In one embodiment, the histone methyltransferase inhibitor is KD5170 (mercaptoketone HDAC inhibitor), MC1568 (class IIa HDAC inhibitor), TMP195 (class IIa HDAC inhibitor), or any combination thereof. In some embodiments, the HDAC inhibitor is vorinostat, romidepsin (Istodax), thidamide, panobinostat (Faridac), bellinostat (PXD101), panobinostat (LBH589), valproic acid, mosetinostat (MGCD0103), avexinostat (PCI-24781), entinostat (MS-275), SB939, Resmino These include Stat (4SC-201), Gibinostat (ITF2357), Xynostat (JNJ-26481585), HBI-8000 (Benzamide HDI), Kebetrin, CUDC-101, AR-42, CHR-2845, CHR-3996, 4SC-202, CG200745, ACY-1215, ME-344, Sulforaphane, or any variant thereof.

[0166]

[0225] In some cases, the concentration of a histone methyltransferase inhibitor (e.g., DZNep) may be 0.01–10 μM or approximately 0.01–10 μM. For example, The concentration of the histone methyltransferase inhibitor (e.g., DZNep) may be approximately 0.01–1, 0.1–1, 0.25–1, 0.5–1, 1–5, or 1–10 μM. The concentration of the histone methyltransferase inhibitor (e.g., DZNep) may be approximately 5, 4, 3, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, or less than 0.01 μM.

[0167]

[0226] Embodiments of this disclosure include endoderm cells. Endoderm cells used herein may be derived from any source or produced according to any preferred 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, gastrulatum cells (stage 2), Pdx1-positive pancreatic progenitor cells (stage 3), NKX6.1-positive pancreatic progenitor cells (stage 4), or Ngn3-positive endocrine progenitor cells or insulin-positive endocrine cells (stage 5), and subsequently induced into or mature into SC-β cells (stage 6).

[0168]

[0227] In some cases, endoderm cells can be obtained by differentiating at least some pluripotent cells in a population into endoderm cells, for example by contacting a population of pluripotent cells with i) at least one growth factor from the TGF-β superfamily and ii) a WNT signaling pathway activator to induce differentiation of at least some pluripotent cells into endoderm cells, and these endoderm cells express at least one marker characteristic of endoderm.

[0169]

[0228] Any growth factor from the TGF-β superfamily that can induce pluripotent stem cells (for example, alone or in combination with a WNT signaling pathway activator) to differentiate into embryonic endoderm cells can be used in the methods provided herein. In some examples, the growth factor from the TGF-β superfamily includes activin A. In some examples, the growth factor from the TGF-β superfamily includes growth differentiation factor 8 (GDF8). Any WNT signaling pathway activator that can induce pluripotent stem cells (for example, alone or in combination with a growth factor from the TGF-β superfamily) to differentiate into embryonic endoderm cells can be used in the methods provided herein. In some examples, the WNT signaling pathway activator includes CHIR99Q21. In some examples, the WNT signaling pathway activator includes Wnt3a recombinant protein.

[0170]

[0229] In some cases, the step of differentiating at least some pluripotent cells in a population into endoderm cells is achieved by a process in which the population of pluripotent cells is exposed to i) activin A and ii) CHIR99021 for a suitable period, e.g., about 2, 3, 4, or 5 days, to induce differentiation of at least some pluripotent cells in the population into endoderm cells, and the endoderm cells express at least one marker characteristic of endoderm.

[0171]

[0230] In some cases, the method includes the step of differentiating pluripotent cells into endoderm cells by contacting a population of pluripotent cells with a growth factor from the TGF-β superfamily (e.g., activin A) at a suitable concentration, for example, about 10 ng / mL, about 20 ng / mL, about 50 ng / mL, about 75 ng / mL, about 80 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 110 ng / mL, about 120 ng / mL, about 130 ng / mL, about 140 ng / mL, about 150 ng / mL, about 175 ng / mL, about 180 ng / mL, about 200 ng / mL, about 250 ng / mL, or about 300 ng / mL. In some cases, the method includes the use of about 100 ng / mL of activin A for differentiation of pluripotent cells into endoderm cells. In some cases, the method includes the differentiation of pluripotent cells into endoderm cells. This includes the use of approximately 200 ng / mL of activin A for differentiation.

[0172]

[0231] In some cases, the method includes the step of differentiating pluripotent cells into endoderm cells by contacting a 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 cases, the method includes the use of about 2 μM of CHIR99021 for the differentiation of pluripotent cells into endoderm cells. In some cases, this method involves the use of approximately 5 μM of CHIR99021 for the differentiation of pluripotent cells into endoderm cells of the embryo.

[0173]

[0232] In some cases, 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 statistically upregulated in endoderm cells compared to the original pluripotent stem cells from which it was induced. In some cases, 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 statistically significant amounts compared to the original pluripotent stem cells from which it was induced. In some cases, 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 statistically significant amounts compared to the original pluripotent stem cells from which they were induced. In some cases, endoderm cells generated by the methods disclosed herein have statistically significant levels of Smad2 phosphorylation compared to the original pluripotent stem cells from which they were induced. In some cases, endoderm cells generated by the methods disclosed herein have the ability to form the intestinal tract in vivo. In some cases, endoderm cells generated by the methods disclosed herein can differentiate into cells with a morphology characteristic of intestinal cells, and these cells with a morphology characteristic of intestinal cells express FoxA2 and / or Claudin6. In some cases, endoderm cells generated by the methods disclosed herein can further differentiate into cells of endodermal origin.

[0174]

[0233] In some cases, a 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, such as human pluripotent stem cells or human iPS cells, or any pluripotent stem cells or other suitable pluripotent stem cells discussed herein can be used. In some cases, the β-cell differentiation factors described herein may be present in the culture medium of the population of pluripotent stem cells, or may be added once or periodically during the growth (e.g., replication or increase) of the population of pluripotent stem cells. In certain cases, the population of pluripotent stem cells may be exposed to at least one β-cell differentiation factor before any differentiation. In other cases, the population of pluripotent stem cells may be exposed to at least one β-cell differentiation factor during the first stage of differentiation.

[0175]

[0234] Aspects of this disclosure include gastrulatic cells. The gastrulatic cells used herein may be derived from any source or produced according to any preferred protocol. In some aspects, endoderm cells of the embryo are differentiated into gastrulatic cells. In some aspects, the gastrulatic cells are further differentiated into, for example, Pdx1-positive pancreatic progenitor cells, NKX6.1-positive pancreatic progenitor cells, Ngn3-positive endocrine progenitor cells, insulin-positive endocrine cells, and subsequently induced into or mature into SC-β cells.

[0176]

[0235] In some cases, gastrulatic cells can be obtained by differentiating at least a number of endoderm cells in a population into gastrulatic cells, for example, by contacting endoderm cells with at least one growth factor from the fibroblast growth factor (FGF) family to induce differentiation of at least a number of endoderm cells into gastrulatic cells, and gastrulatic cells express at least one marker characteristic of gastrulatic cells.

[0177]

[0236] Any growth factor from the FGF family that can induce (for example, alone or in combination with other factors) the differentiation of endoderm cells into gastrullous cells can be used in the methods provided herein. In some examples, at least one growth factor from the FGF family is keratinocyte growth factor (KGF). In some examples, at least one growth factor from the FGF family is FGF2. In some examples, at least one growth factor from the FGF family is FGF8B. In some examples, at least one growth factor from the FGF family is FGF10. In some examples, at least one growth factor from the FGF family is FGF21.

[0178]

[0237] In some cases, gastrullous cells can be obtained by differentiating at least some endoderm cells in a population into gastrullous cells, for example, by exposing endoderm cells to KGF for a period of time, e.g., about 1 day, 2 days, 3 days, or 4 days, to induce differentiation of at least some endoderm cells into gastrullous cells.

[0179]

[0238] In some cases, the method includes the step of differentiating endoderm cells into gastrullary cells by contacting them with a growth factor from the FGF family (e.g., KGF) at a suitable concentration of approximately 10 ng / mL, 20 ng / mL, 50 ng / mL, 75 ng / mL, 80 ng / mL, 90 ng / mL, 95 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL, 175 ng / mL, 180 ng / mL, 200 ng / mL, 250 ng / mL, or 300 ng / mL. In some cases, the method includes the use of approximately 50 ng / mL of KGF for the differentiation of endoderm cells into gastrullary cells. In some cases, the method includes the use of approximately 100 ng / mL of KGF for the differentiation of endoderm cells into gastrullary cells.

[0180]

[0239] Aspects of this disclosure include Pdx1-positive pancreatic progenitor cells. Pdx1-positive pancreatic progenitor cells used herein may be induced from any source or produced according to any preferred protocol. In some aspects, gastrulatic cells are differentiated into Pdx1-positive pancreatic progenitor cells. In some aspects, Pdx1-positive 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 subsequently induced into or mature into SC-β cells.

[0181]

[0240] In some embodiments, Pdx1-positive pancreatic progenitor cells can be obtained by differentiating at least some gastrulatic cells in a population into Pdx1-positive pancreatic progenitor cells, for example by contacting gastrulatic 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 gastrulatic cells into at least some Pdx1-positive pancreatic progenitor cells, which express Pdx1.

[0182]

[0241] In some embodiments, Pdx1-positive pancreatic progenitor cells differentiate at least some gastrulatic cells in a population into Pdx1-positive pancreatic progenitor cells, for example, by i) reducing the number of gastrulatic cells. It can be obtained by inducing the differentiation of gastrulatic cells into at least several Pdx1-positive pancreatic progenitor cells by contacting them with 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, the Pdx1-positive pancreatic progenitor cells expressing Pdx1.

[0183]

[0242] In some cases, Pdx1-positive pancreatic progenitor cells can be obtained by differentiating at least some gastrulatic cells in a population into Pdx1-positive pancreatic progenitor cells, for example by contacting gastrulatic 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 gastrulatic cells into at least some Pdx1-positive pancreatic progenitor cells, which express Pdx1.

[0184]

[0243] In some cases, Pdx1-positive pancreatic progenitor cells can be obtained by differentiating at least a number of gastrulatic cells in a population into Pdx1-positive pancreatic progenitor cells, for example by contacting gastrulatic 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, and Pdx1-positive pancreatic progenitor cells express Pdx1.

[0185]

[0244] In some cases, Pdx1-positive pancreatic progenitor cells can be obtained by differentiating at least some gastrulatic cells in a population into Pdx1-positive pancreatic progenitor cells, for example by contacting gastrulatic cells with i) at least one growth factor from the FGF family and ii) at least one retinoic acid (RA) signaling pathway activator to induce differentiation of at least some gastrulatic cells into Pdx1-positive pancreatic progenitor cells, which express Pdx1.

[0186]

[0245] Any BMP signaling pathway inhibitor capable of inducing gastrulation cells to differentiate into Pdx1-positive pancreatic progenitor cells (for example, alone or in combination with a growth factor from the TGF-β superfamily, at least one growth factor from the FGF family, at least one SHH pathway inhibitor, at least one retinoic acid signaling pathway activator, at least one protein kinase C activator, and a ROCK inhibitor) can be used in the methods provided herein. In some examples, the BMP signaling pathway inhibitor includes LDN193189 or DMH-1. In some cases, the method involves contacting gastrulatous cells with a BMP signaling pathway inhibitor (e.g., LDN193189) at a concentration of, for example, approximately 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, 200 nM, 210 nM, 220 nM, 230 nM, 240 nM, 250 nM, 280 nM, 300 nM, 400 nM, 500 nM, or approximately 1 μM. In some cases, the method includes contacting gastrulatous cells with a BMP signaling pathway inhibitor (e.g., DMH-1) at concentrations of approximately 0.01 μM, 0.02 μM, 0.05 μM, 0.1 μM, 0.2 μM, 0.5 μM, 0.8 μM, 1 μM, 1.2 μM, 1.5 μM, 1.75 μM, 2 μM, 2.2 μM, 2.5 μM, 2.75 μM, 3 μM, 3.25 μM, 3.5 μM, 3.75 μM, 4 μM, 4.5 μM, 5 μM, 8 μM, 10 μM, 15 μM, 20 μM, 30 μM, 40 μM, 50 μM, or 100 μM.

[0187]

[0246] Any growth factor from the TGF-β superfamily can be used to induce gastrulation cells to differentiate into Pdx1-positive pancreatic progenitor cells (for example, 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). In some cases, the growth factor from the TGF-β family contains activin A. In some cases, the growth factor from the TGF-β family contains either activin A or GDF8. In some cases, the method includes the step of contacting gastrulation 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.

[0188]

[0247] Any growth factor from the FGF family can be used to induce gastrulation cells to differentiate into Pdx1-positive 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). In some cases, at least one growth factor from the FGF family includes keratinocyte growth factor (KGF). In some cases, at least one growth factor from the FGF family is selected from the group consisting of FGF2, FGF8B, FGF10, and FGF21. In some cases, the method includes the step of exposing gastrulatous cells to 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.

[0189]

[0248] Any SHH pathway inhibitor can be used to induce gastrulation cells to differentiate into Pdx1-positive pancreatic progenitor cells (for example, alone or in combination with at least one BMP signaling pathway inhibitor, at least one growth factor from the FGF family, 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). In some cases, the SHH pathway inhibitors include Sant1. In some cases, this method involves reducing gastrulatal cells to approximately 0.001 μM, 0.002 μM, 0.005 μM, 0.01 μM, 0.02 μM, 0.03 μM, 0.05 μM, 0.08 μM, 0.1 μM, 0.12 μM, 0.13 μM, 0.14 μM, 0.15 μM, 0.16 μM, 0.17 μM, 0.18 μM, 0.19 μM, 0.2 μM, 0.21 μM, 0.22 μM, and 0.23 μM. The process includes contacting the material with an SHH pathway inhibitor (e.g., Sant1) at concentrations of approximately μM, 0.24 μM, 0.25 μM, 0.26 μM, 0.27 μM, 0.28 μM, 0.29 μM, 0.3 μM, 0.31 μM, 0.32 μM, 0.33 μM, 0.34 μM, 0.35 μM, 0.4 μM, 0.45 μM, 0.5 μM, 0.6 μM, 0.8 μM, 1 μM, 2 μM, or 5 μM.

[0190]

[0249] Any RA signaling pathway activator capable of inducing gastrulation cells to differentiate into Pdx1-positive pancreatic progenitor cells (e.g., alone or in combination with at least one BMP signaling pathway inhibitor, at least one growth factor from the FGF family, at least one SHH pathway inhibitor, at least one protein kinase C activator, and a ROCK inhibitor) can be used. In some cases, the RA signaling pathway activator includes retinoic acid. In some cases, this method involves gastrulatum cells with concentrations of approximately 0.02 μM, 0.1 μM, 0.2 μM, 0.25 μM, 0.3 μM, 0.4 μM, 0.45 μM, 0.5 μM, 0.55 μM, 0.6 μM, 0.65 μM, 0.7 μM, 0.75 μM, 0.8 μM, 0.85 μM, 0.9 μM, 1 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, 2 μM, 2.1 μM, 2.2 μM, 2.3 μM, 2.4 μM, and 2.5 μM. The process includes contacting the material with an RA signaling pathway activator (e.g., retinoic acid) at concentrations of approximately 2.6 μM, 2.7 μM, 2.8 μM, 3 μM, 3.2 μM, 3.4 μM, 3.6 μM, 3.8 μM, 4 μM, 4.2 μM, 4.4 μM, 4.6 μM, 4.8 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, 8.5 μM, 9 μM, 9.5 μM, 10 μM, 12 μM, 14 μM, 15 μM, 16 μM, 18 μM, 20 μM, 50 μM, or 100 μM.

[0191]

[0250] Any PKC activator capable of inducing gastrulation cells to differentiate into Pdx1-positive pancreatic progenitor cells (e.g., alone or in combination with at least one BMP signaling pathway inhibitor, at least one growth factor from the FGF family, at least one SHH pathway inhibitor, at least one RA signaling pathway activator, and a ROCK inhibitor) can be used. In some cases, the PKC activator contains PdBU. In some cases, the PKC activator contains TPB. In some cases, this method involves preparing gastrulatal cells with concentrations of approximately 10 μM, 20 μM, 50 μM, 75 μM, 80 μM, 100 μM, 120 μM, 140 μM, 150 μM, 175 μM, 180 μM, 200 μM, 210 μM, 220 μM, 240 μM, 250 μM, 260 μM, 280 μM, 300 μM, 320 μM, 340 μM, 360 μM, 380 μM, 400 μM, and 420 μM. The process includes contacting the material with a PKC activator (e.g., PdBU) at concentrations of approximately M, 440 μM, 460 μM, 480 μM, 500 μM, 520 μM, 540 μM, 560 μM, 580 μM, 600 μM, 620 μM, 640 μM, 660 μM, 680 μM, 700 μM, 750 μM, 800 μM, 850 μM, 900 μM, 1 mM, 2 mM, 3 mM, 4 mM, or 5 mM.

[0192]

[0251] Any ROCK inhibitor capable of inducing gastrulation cells to differentiate into Pdx1-positive pancreatic progenitor cells (e.g., alone or in combination with at least one BMP signaling pathway inhibitor, at least one growth factor from the FGF family, at least one SHH pathway inhibitor, a PKC activator, and at least one RA signaling pathway activator) can be used. In some cases, the ROCK inhibitor includes thiazovivin, Y-27632, fasudil / HA1077, or H-1152. In some cases, the ROCK inhibitor includes Y-27632. In some cases, the ROCK inhibitor includes thiazovivin. In some cases, this method involves gastrulatal cells in concentrations of approximately 0.2 μM, 0.5 μM, 0.75 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 7.5 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, and 1 ROCK inhibitors (e.g., Y-2) at concentrations of 6 μM, approximately 17 μM, approximately 18 μM, approximately 19 μM, approximately 20 μM, approximately 21 μM, approximately 22 μM, approximately 23 μM, approximately 24 μM, approximately 25 μM, approximately 26 μM, approximately 27 μM, approximately 28 μM, approximately 29 μM, approximately 30 μM, approximately 35 μM, approximately 40 μM, approximately 50 μM, or approximately 100 μM. The process includes the step of bringing the substance into contact with 7632 or Thiazovivin.

[0193]

[0252] In some cases, Pdx1-positive pancreatic progenitor cells can be obtained by differentiating at least some gastrulatic cells in a population into Pdx1-positive pancreatic progenitor cells, for example by contacting the gastrulatic cells with retinoic acid, KGF, Sant1, LDN193189, PdBU, Y-27632, and activin A for a suitable period, e.g., about 1 day, 2 days, 3 days, or 4 days. In some cases, Pdx1-positive pancreatic progenitor cells can be obtained by differentiating at least some gastrulatic cells in a population into Pdx1-positive pancreatic progenitor cells, for example by contacting the gastrulatic cells with retinoic acid, KGF, Sant1, LDN193189, PdBU, Y-27632, and activin A for about 2 days. In some cases, Pdx1-positive pancreatic progenitor cells can be obtained by differentiating at least some gastrulatic cells in S3 medium.

[0194]

[0253] Aspects of this disclosure include NKX6.1-positive pancreatic progenitor cells. NKX6.1-positive pancreatic progenitor cells used herein may be induced from any source or produced according to any preferred protocol. In some aspects, Pdx1-positive pancreatic progenitor cells are differentiated into NKX6.1-positive pancreatic progenitor cells. In some aspects, NKX6.1-positive pancreatic progenitor cells are further differentiated into, for example, Ngn3-positive endocrine progenitor cells or insulin-positive endocrine cells, and subsequently induced into or mature into SC-β cells.

[0195]

[0254] In some embodiments, a method for generating NKX6.1-positive pancreatic progenitor cells from Pdx1-positive pancreatic progenitor cells comprises the step of contacting a population of cells containing Pdx1-positive pancreatic progenitor cells with at least two β-cell differentiation factors, including a) at least one growth factor from the fibroblast growth factor (FGF) family, b) a Sonic Hedgehog pathway inhibitor, and optionally c) a low concentration of a retinoic acid (RA) signaling pathway activator (under conditions that promote cell clustering and / or cell survival, for example), to induce differentiation of at least one Pdx-positive pancreatic progenitor cell in the population into an NKX6.1-positive pancreatic progenitor cell, the NKX6.1-positive pancreatic progenitor cell expressing NKX6.1.

[0196]

[0255] In some examples, Pdx1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by a step of inducing differentiation of Pdx1-positive pancreatic progenitor cells into at least some Pdx1-positive, NKX6.1-positive pancreatic progenitor cells by contacting the Pdx1-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) a low concentration of an RA signaling pathway activator, and the Pdx1-positive, NKX6.1-positive pancreatic progenitor cells express Pdx1 and NKX6.1.

[0197]

[0256] In some examples, Pdx1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting the Pdx1-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) a low concentration of an RA signaling pathway activator, iv) a ROCK inhibitor, and v) at least one growth factor from the TGF-β superfamily to induce differentiation of the Pdx1-positive pancreatic progenitor cells into at least some Pdx1-positive, NKX6.1-positive pancreatic progenitor cells. In some examples, Pdx1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting the Pdx1-positive pancreatic progenitor cells with at least one growth factor from the FGF family under conditions that promote cell clustering.

[0198]

[0257] In some examples, Pdx1-positive pancreatic progenitor cells are generated from a population of pluripotent cells. In some examples, Pdx1-positive pancreatic progenitor cells are generated from a population of iPS cells. In some examples, Pdx1-positive pancreatic progenitor cells are generated from a population of ESC cells. In some examples, Pdx1-positive pancreatic progenitor cells are generated from a population of definitive endoderm cells. In some examples, Pdx1-positive pan creatic progenitor cells are generated from a population of primitive streak cells.

[0199]

[0258] Any growth factor from the FGF family that can induce Pdx1-positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (for example, alone or in combination with at least one SHH pathway inhibitor, a ROCK inhibitor, a growth factor from the TGF-β superfamily, and at least one retinoic acid signaling pathway activator) can be used in the methods provided herein. In some examples, at least one growth factor from the FGF family includes keratinocyte growth factor (KGF). In some examples, at least one growth factor from the FGF family is selected from the group consisting of FGF2, FGF8B, FGF10, and FGF21. In some cases, the method includes contacting Pdx1-positive pancreatic progenitor cells with a growth factor from the FGF family (e.g., KGF) at concentrations of approximately 10 ng / mL, 20 ng / mL, 50 ng / mL, 75 ng / mL, 80 ng / mL, 90 ng / mL, 95 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL, 175 ng / mL, 180 ng / mL, 200 ng / mL, 250 ng / mL, or 30 ng / mL.

[0200]

[0259] Any SHH pathway inhibitor capable of inducing differentiation of Pdx1-positive pancreatic progenitor cells into NKX6.1-positive pancreatic progenitor cells (e.g., alone or in combination with at least one growth factor from the FGF family, at least one retinoic acid signaling pathway activator, a ROCK inhibitor, and at least one growth factor from the TGF-β superfamily) can be used in the methods provided herein. In some examples, the SHH pathway inhibitor includes Sant1. In some cases, this method involves Pdx1-positive pancreatic progenitor cells in quantities of approximately 0.001 μM, 0.002 μM, 0.005 μM, 0.01 μM, 0.02 μM, 0.03 μM, 0.05 μM, 0.08 μM, 0.1 μM, 0.12 μM, 0.13 μM, 0.14 μM, 0.15 μM, 0.16 μM, 0.17 μM, 0.18 μM, 0.19 μM, 0.2 μM, 0.21 μM, 0.22 μM, and 0. The process includes contacting the material with an SHH pathway inhibitor (e.g., Sant1) at concentrations of 0.23 μM, approximately 0.24 μM, approximately 0.25 μM, approximately 0.26 μM, approximately 0.27 μM, approximately 0.28 μM, approximately 0.29 μM, approximately 0.3 μM, approximately 0.31 μM, approximately 0.32 μM, approximately 0.33 μM, approximately 0.34 μM, approximately 0.35 μM, approximately 0.4 μM, approximately 0.45 μM, approximately 0.5 μM, approximately 0.6 μM, approximately 0.8 μM, approximately 1 μM, approximately 2 μM, or approximately 5 μM.

[0201]

[0260] Any RA signaling pathway activator can be used to induce differentiation of Pdx1-positive pancreatic progenitor cells into 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, a ROCK inhibitor, and at least one growth factor from the TGF-β superfamily). In some cases, the RA signaling pathway activator includes retinoic acid. In some cases, this method involves Pdx1-positive pancreatic progenitor cells in concentrations of approximately 0.02 μM, 0.1 μM, 0.2 μM, 0.25 μM, 0.3 μM, 0.4 μM, 0.45 μM, 0.5 μM, 0.55 μM, 0.6 μM, 0.65 μM, 0.7 μM, 0.75 μM, 0.8 μM, 0.85 μM, 0.9 μM, 1 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, 2 μM, 2.1 μM, 2.2 μM, and 2.3 μM. RA signaling pathway activity at concentrations of approximately μM, 2.4 μM, 2.5 μM, 2.6 μM, 2.7 μM, 2.8 μM, 3 μM, 3.2 μM, 3.4 μM, 3.6 μM, 3.8 μM, 4 μM, 4.2 μM, 4.4 μM, 4.6 μM, 4.8 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, 8.5 μM, 9 μM, 9.5 μM, 10 μM, 12 μM, 14 μM, 15 μM, 16 μM, 18 μM, 20 μM, 50 μM, or 100 μM. The process includes the step of contacting the material with a retinoic acid (e.g., retinoic acid).

[0202]

[0261] Any ROCK inhibitor capable of inducing differentiation of Pdx1-positive pancreatic progenitor cells into NKX6.1-positive pancreatic progenitor cells (e.g., alone or in combination with at least one growth factor from the FGF family, at least one SHH pathway inhibitor, an RA signaling pathway activator, and at least one growth factor from the TGF-β superfamily) can be used. In some examples, ROCK inhibitors include thiazovivin, Y-27632, fasudil / HA1077, or 14-1152. In some cases, this method is used to sample Pdx1-positive pancreatic progenitor cells in concentrations of approximately 0.2 μM, 0.5 μM, 0.75 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 7.5 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, and 18 μM. The process includes the step of contacting the ROCK inhibitor (e.g., Y-27632 or Thiazovivin) at concentrations of approximately μM, 19 μM, 20 μM, 21 μM, 22 μM, 23 μM, 24 μM, 25 μM, 26 μM, 27 μM, 28 μM, 29 μM, 30 μM, 35 μM, 40 μM, 50 μM, or 100 μM.

[0203]

[0262] Any activator from the TGF-β superfamily can be used to induce differentiation of Pdx1-positive pancreatic progenitor cells into NKX6.1-positive pancreatic progenitor cells (for example, alone or in combination with at least one growth factor from the FGF family, at least one SHH pathway inhibitor, an RA signaling pathway activator, and a ROCK inhibitor). In some cases, the activators from the TGF-β superfamily include activin A or GDF8. In some cases, this method was used to process Pdx1-positive pancreatic progenitor cells at concentrations of approximately 0.1 ng / mL, 0.2 ng / mL, 0.3 ng / mL, 0.4 ng / mL, 0.5 ng / mL, 0.6 ng / mL, 0.7 ng / mL, 0.8 ng / mL, 1 ng / mL, 1.2 ng / mL, 1.4 ng / mL, 1.6 ng / mL, 1.8 ng / mL, 2 ng / mL, 2.2 ng / mL, 2.4 ng / mL, 2.6 ng / mL, 2.8 ng / mL, 3 ng / mL, 3.2 ng / mL, 3.4 ng / mL, 3.6 ng / mL, 3.8 ng / mL, and 4 ng / mL. The method includes contacting cells with a growth factor from the TGF-β superfamily (e.g., activin A) at concentrations of approximately ng / mL, 4.2 ng / mL, 4.4 ng / mL, 4.6 ng / mL, 4.8 ng / mL, 5 ng / mL, 5.2 ng / mL, 5.4 ng / mL, 5.6 ng / mL, 5.8 ng / mL, 6 ng / mL, 6.2 ng / mL, 6.4 ng / mL, 6.6 ng / mL, 6.8 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, or 50 ng / mL. In some examples, the method includes contacting Pdx1-positive pancreatic progenitor cells with a growth factor from the TGF-β superfamily (e.g., activin A) at a concentration of approximately 5 ng / mL.

[0204]

[0263] In some cases, Pdx1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting Pdx1-positive pancreatic progenitor cells with KGF, Sant1, and RA for 5 days under conditions that promote cell clustering. In some cases, Pdx1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting Pdx1-positive pancreatic progenitor cells with KGF, Sant1, RA, Y27632, and activin A for 5 days under conditions that promote cell clustering. In some cases, Pdx1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting Pdx1-positive pancreatic progenitor cells with KGF for 5 days under conditions that promote cell clustering. In some cases, Pdx1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting Pdx1-positive pancreatic progenitor cells in S3 medium.

[0205]

[0264] Aspects of this disclosure include insulin-positive endocrine cells. Insulin-positive endocrine cells used herein may be induced from any source or produced according to any preferred protocol. In some aspects, NKX6.1-positive pancreatic progenitor cells are differentiated into insulin-positive endocrine cells. In some aspects, insulin-positive endocrine cells are further differentiated, for example, by induction or maturation into SC-β cells.

[0206]

[0265] In some embodiments, a method for generating insulin-positive endocrine cells from NKX6.1-positive pancreatic progenitor cells comprises the step of contacting a population of cells containing NKX6.1-positive pancreatic progenitor cells with a) a TGF-β signaling pathway inhibitor and b) a thyroid hormone signaling pathway activator (under conditions that promote cell clustering, for example) to induce differentiation of at least one NKX6.1-positive pancreatic progenitor cell in the population into an insulin-positive endocrine cell, the insulin-positive endocrine cell expressing insulin. In some examples, the insulin-positive endocrine cells express Pdx1, NKX6.1, NKX2.2, Mafb, glis3, Sur1, Kir6.2, Znt8, SLC2A1, SLC2A3, and / or insulin.

[0207]

[0266] Any TGF-β signaling pathway inhibitor can be used to induce differentiation of 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. In some cases, the TGF-β signaling pathway includes TGF-β type I receptor kinase signaling. In some cases, the TGF-β signaling pathway inhibitor includes Alk5 inhibitor II.

[0208]

[0267] Any thyroid hormone signaling pathway activator capable of inducing differentiation of NKX6.1-positive pancreatic progenitor cells (e.g., alone or in combination with other β-cell differentiation factors, e.g., TGF-β signaling pathway inhibitors) into insulin-positive endocrine cells can be used. In some cases, the thyroid hormone signaling pathway activator includes triiodothyronine (T3). In some cases, the thyroid hormone signaling pathway activator includes GC-1.

[0209]

[0268] In some cases, the method involves contacting a population of cells (e.g., NKX6.1-positive pancreatic progenitor cells) with at least one additional factor. In some cases, the method involves contacting Pdx1-positive, NKX6.1-positive pancreatic progenitor cells with at least one of the following: i) an SHH pathway inhibitor, ii) an RA signaling pathway activator, iii) a γ-secretase inhibitor, iv) at least one growth factor from the epidermal growth factor (EGF) family, v) a protein kinase inhibitor, vi) a TGF-β signaling pathway inhibitor, or vii) a thyroid hormone signaling pathway activator.

[0210]

[0269] In some cases, the method includes the step of contacting Pdx1-positive, NKX6.1-positive pancreatic progenitor cells with at least one of the following: i) an SHH pathway inhibitor, ii) an RA signaling pathway activator, iii) a γ-secretase inhibitor, iv) at least one growth factor from the epidermal growth factor (EGF) family, v) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, vi) a TGF-β signaling pathway inhibitor, vii) a thyroid hormone signaling pathway activator, viiii) a protein kinase inhibitor, or ix) a ROCK inhibitor.

[0211]

[0270] In some cases, this method involves Pdx1-positive, NKX6.1-positive pancreatic progenitor cells being treated with i) SHH pathway inhibitors, ii) RA signaling pathway activators, iii) γ-secretase inhibitors, iv) at least one growth factor from the epidermal growth factor (EGF) family, v) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, and vi) T The method includes the step of contacting at least one of the following: vii) a GF-β signaling pathway inhibitor, vii) a thyroid hormone signaling pathway activator, viiii) an epigenetic modification compound, ix) a protein kinase inhibitor, or x) a ROCK inhibitor.

[0212]

[0271] In some embodiments, in a method for generating insulin-positive endocrine cells from Pdx1-positive, NKX6.1-positive pancreatic progenitor cells, some differentiation factors are present only during the differentiation step, for the first 1, 2, 3, 4, or 5 days. In some examples, some differentiation factors, such as SHH pathway inhibitors, RA signaling pathway activators, and at least one growth factor from the EGF family, are removed from the culture medium after the first 3 days of incubation.

[0213]

[0272] Any γ-secretase inhibitor capable of inducing differentiation of NKX6.1-positive pancreatic progenitor cells into insulin-positive endocrine cells in a population (e.g., alone or in combination with any TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator) can be used. In some cases, the γ-secretase inhibitor includes XXI. In some cases, the γ-secretase inhibitor includes DAPT. In some cases, this method was used to extract NKX6.1-positive pancreatic progenitor cells in quantities of approximately 0.01 μM, 0.02 μM, 0.05 μM, 0.075 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, 2 μM, 2.1 μM, 2.2 μM, 2.3 μM, 2.4 μM, 2.5 μM, and 2. The process includes contacting the material with a γ-secretase inhibitor (e.g., XXI) at concentrations of 0.6 μM, approximately 2.7 μM, approximately 2.8 μM, approximately 2.9 μM, approximately 3 μM, approximately 3.2 μM, approximately 3.4 μM, approximately 3.6 μM, approximately 3.8 μM, approximately 4 μM, approximately 4.2 μM, approximately 4.4 μM, approximately 4.6 μM, approximately 4.8 μM, approximately 5 μM, approximately 5.2 μM, approximately 5.4 μM, approximately 5.6 μM, approximately 5.8 μM, approximately 6 μM, approximately 6.2 μM, approximately 6.4 μM, approximately 6.6 μM, approximately 6.8 μM, approximately 7 μM, approximately 8 μM, approximately 9 μM, approximately 10 μM, approximately 20 μM, approximately 30 μM, or approximately 50 μM.

[0214]

[0273] Any growth factor from the EGF family can be used to induce differentiation of NKX6.1-positive pancreatic progenitor cells into insulin-positive endocrine cells in a population (e.g., alone or in combination with any of the TGF-β signaling pathway inhibitors and / or thyroid hormone signaling pathway activators). In some cases, at least one growth factor from the EGF family contains beta-cellulin. In some cases, at least one growth factor from the EGF family contains EGF. In some cases, the method includes the step of contacting NKX6.1-positive pancreatic progenitor cells with a growth factor from the EGF family (e.g., beta-cellin) at concentrations of approximately 1 ng / mL, 2 ng / mL, 4 ng / mL, 6 ng / mL, 8 ng / mL, 10 ng / mL, 12 ng / mL, 14 ng / mL, 16 ng / mL, 18 ng / mL, 20 ng / mL, 22 ng / mL, 24 ng / mL, 26 ng / mL, 28 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 75 ng / mL, 80 ng / mL, 90 ng / mL, 95 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL, 250 ng / mL, or 300 ng / mL.

[0215]

[0274] Any RA signaling pathway activator can be used to induce differentiation of NKX6.1-positive pancreatic progenitor cells into insulin-positive endocrine cells (e.g., alone or in combination with any TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway activator). In some cases, the RA signaling pathway activator contains RA. In some cases, this method can be performed by introducing NKX6.1-positive pancreatic progenitor cells in concentrations of approximately 0.02 μM, 0.1 μM, 0.2 μM, 0.25 μM, 0.3 μM, and so on. 0.4μM, approximately 0.45μM, approximately 0.5μM, approximately 0.55μM, approximately 0.6μM, approximately 0.65μM, approximately 0.7μM, approximately 0.75μM, approximately 0.8μM, approximately 0.85μM, approximately 0.9μM, approximately 1μM, approximately 1.1μM, approximately 1.2μM, approximately 1.3μM, approximately 1 .4μM, about 1.5μM, about 1.6μM, about 1.7μM, about 1.8μM, about 1.9μM, about 2μM, about 2.1μM, about 2.2μM, about 2.3μM, about 2.4μM, about 2.5μM, about 2.6μM, about 2.7μM, about 2.8μM, about 3μM, about 3. The process includes contacting the material with an RA signaling pathway activator (e.g., retinoic acid) at concentrations of 2 μM, approximately 3.4 μM, approximately 3.6 μM, approximately 3.8 μM, approximately 4 μM, approximately 4.2 μM, approximately 4.4 μM, approximately 4.6 μM, approximately 4.8 μM, approximately 5 μM, approximately 5.5 μM, approximately 6 μM, approximately 6.5 μM, approximately 7 μM, approximately 7.5 μM, approximately 8 μM, approximately 8.5 μM, approximately 9 μM, approximately 9.5 μM, approximately 10 μM, approximately 12 μM, approximately 14 μM, approximately 15 μM, approximately 16 μM, approximately 18 μM, approximately 20 μM, approximately 50 μM, or approximately 100 μM.

[0216]

[0275] Any SHH pathway inhibitor capable of inducing differentiation of NKX6.1-positive pancreatic progenitor cells (for example, alone or in combination with any TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator) into insulin-positive endocrine cells can be used in the methods provided herein. In some examples, the SHH pathway inhibitor includes Sant1. In some cases, this method involves NKX6.1-positive pancreatic progenitor cells in quantities of approximately 0.001 μM, 0.002 μM, 0.005 μM, 0.01 μM, 0.02 μM, 0.03 μM, 0.05 μM, 0.08 μM, 0.1 μM, 0.12 μM, 0.13 μM, 0.14 μM, 0.15 μM, 0.16 μM, 0.17 μM, 0.18 μM, 0.19 μM, 0.2 μM, 0.21 μM, 0.22 μM, and approximately The process includes contacting the material with an SHH pathway inhibitor (e.g., Sant1) at concentrations of 0.23 μM, approximately 0.24 μM, approximately 0.25 μM, approximately 0.26 μM, approximately 0.27 μM, approximately 0.28 μM, approximately 0.29 μM, approximately 0.3 μM, approximately 0.31 μM, approximately 0.32 μM, approximately 0.33 μM, approximately 0.34 μM, approximately 0.35 μM, approximately 0.4 μM, approximately 0.45 μM, approximately 0.5 μM, approximately 0.6 μM, approximately 0.8 μM, approximately 1 μM, approximately 2 μM, or approximately 5 μM.

[0217]

[0276] Any BMP signaling pathway inhibitor that can induce the differentiation of NKX6.1-positive pancreatic progenitor cells (e.g., alone or in combination with any TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway activator) into insulin-positive endocrine cells can be used. In some examples, the BMP signaling pathway inhibitor includes LDN193189 or DMH-1. In some examples, the method includes contacting NKX6.1-positive pancreatic progenitor cells with a BMP signaling pathway inhibitor (e.g., LDN1931189) at a concentration of, for example, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 110 nM, about 120 nM, about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about 200 nM, about 210 nM, about 220 nM, about 230 nM, about 240 nM, about 250 nM, about 280 nM, about 300 nM, about 400 nM, about 500 nM, or about 1 μM.

[0218]

[0277] Any ROCK inhibitor that can induce the differentiation of NKX6.1-positive pancreatic progenitor cells in the population into insulin-positive endocrine cells (e.g., alone or in combination with any TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway activator) can be used. In some examples, the ROCK inhibitor includes Thiazovivin, Y-27632, Fasudil / HA1077, or H-1152. In some examples, the ROCK inhibitor includes Y-27632. In some examples, the ROCK inhibitor includes Thiazovivin. In some examples, the method includes Pdx1-positive, NKX6.1-positive pancreatic progenitor cells at, 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 1 The process includes contacting the material with a ROCK inhibitor (e.g., Y-27632 or Thiazovivin) at concentrations of 5 μM, approximately 16 μM, approximately 17 μM, approximately 18 μM, approximately 19 μM, approximately 20 μM, approximately 21 μM, approximately 22 μM, approximately 23 μM, approximately 24 μM, approximately 25 μM, approximately 26 μM, approximately 27 μM, approximately 28 μM, approximately 29 μM, approximately 30 μM, approximately 35 μM, approximately 40 μM, approximately 50 μM, or approximately 100 μM.

[0219]

[0278] Any epigenetic modification compound that can induce differentiation of NKX6.1-positive pancreatic progenitor cells in a population into insulin-positive endocrine cells (e.g., alone or in combination with any TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway activator) can be used. In some cases, the epigenetic modification compound includes a histone methyltransferase inhibitor or an HDAC inhibitor. In some cases, the epigenetic modification compound includes a histone methyltransferase inhibitor, e.g., DZNep. In some cases, the epigenetic modification compound includes an HDAC inhibitor, e.g., KD5170. In some cases, the method involves contacting Pdx1-positive, NKX6.1-positive pancreatic progenitor cells with an epigenetic modification compound (e.g., DZNep or KD5170) at concentrations of approximately 0.01 μM, 0.025 μM, 0.05 μM, 0.075 μM, 0.1 μM, 0.15 μM, 0.2 μM, 0.5 μM, 0.75 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 7.5 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 50 μM, or 100 μM.

[0220]

[0279] In some cases, the cell population is optionally exposed to a protein kinase inhibitor. In some cases, the cell population is not exposed to a protein kinase inhibitor. In some cases, the cell population is exposed to a protein kinase inhibitor. Any protein kinase inhibitor capable of inducing differentiation of NKX6.1-positive pancreatic progenitor cells in the population into insulin-positive endocrine cells (e.g., alone or in combination with any TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator) can be used. In some cases, the protein kinase inhibitor includes staurosporine.

[0221]

[0280] In some cases, the method includes a step of contacting a population of cells (e.g., NKX6.1-positive pancreatic progenitor cells) with XXI, Alk5i, T3 or GC-1, RA, Sant1, and beta-cell phosphate for 7 days to induce differentiation of at least one NKX6.1-positive pancreatic progenitor cell in the population into an insulin-positive endocrine cell, which expresses insulin. In some embodiments, one or more differentiation factors are added during a portion of Stage 5, for example, only on the first 1, 2, 3, 4, 5, or 6 days of the Stage 5 period, or on the last 1, 2, 3, 4, 5, or 6 days of the Stage 5 period. In one example, cells are exposed to an SHH signaling pathway inhibitor only during the first 2, 3, 4, or 5 days of Stage 5, after which the SHH signaling pathway inhibitor is removed from the culture medium. In another example, cells are exposed to a BMP signaling pathway inhibitor only during the first 1, 2, or 3 days of Stage 5, after which the BMP signaling pathway inhibitor is removed from the culture medium.

[0222]

[0281] In some cases, this method involves culturing a population of cells (e.g., NKX6.1-positive pancreatic progenitor cells) in BE5 medium and then insulin-stimulating at least one NKX6.1-positive pancreatic progenitor cell in the population. The process includes a step to induce differentiation into insulin-positive endocrine cells, which then express insulin.

[0223]

[0282] Aspects of this disclosure include the step of producing pancreatic β-cells (e.g., non-native pancreatic β-cells). Non-native pancreatic β-cells are, in some cases, similar in morphology and function to endogenous mature β-cells, but are nevertheless distinct from native β-cells.

[0224]

[0283] In some cases, insulin-positive pancreatic endocrine cells produced using the methods provided herein can form cell clusters, either alone or with other types of cells, such as their precursors, such as stem cells, endoderm cells, gastrulatal cells, Pdx1-positive pancreatic progenitor cells, or NKX6.1-positive pancreatic progenitor cells.

[0225]

[0284] In some cases, cell populations including insulin-positive endocrine cells can be directly induced to mature into SC-β cells without the addition of any exogenous differentiation factors (e.g., TGF-β signaling pathway inhibitors, thyroid hormone signaling pathway activators, PKC activators, growth factors from the TGF-β superfamily, FGF family, or EGF family, SHH signaling pathway inhibitors, γ-secretase inhibitors, ROCK inhibitors, or BMP signaling pathway inhibitors).

[0226]

[0285] In some cases, insulin-positive endocrine cells can be brought into contact with differentiation factors to directly induce cell populations containing insulin-positive endocrine cells and mature into SC-β cells. Differentiation factors may include at least one inhibitor of the TGF-β signaling pathway and thyroid hormone signaling pathway activators as described herein. In some cases, SC-β cells can be obtained by bringing a cell population containing insulin-positive endocrine cells into contact with Alk5i and T3 or GC-1.

[0227]

[0286] In some cases, insulin-positive endocrine cells can be matured in NS-GFs medium, MCDB131 medium, DMEM medium, or CMRL medium. In some cases, insulin-positive endocrine cells can be matured in CMRL medium supplemented with 10% FBS. In some cases, insulin-positive endocrine cells can be matured in DMEM medium supplemented with 1% HSA. In other cases, SC-β cells can be obtained by culturing a population of cells, including insulin-positive endocrine cells, in MCDB131 medium, which may be supplemented with 2% BSA. In some cases, MCDB131 medium supplemented with 2% BSA for maturing insulin-positive endocrine cells into SC-β cells does not need to contain the small molecule factors described herein. In some cases, MCDB131 medium supplemented with 2% BSA for maturing insulin-positive endocrine cells into SC-β cells does not need to contain serum (e.g., FBS).

[0228]

[0287] In some embodiments, the present disclosure provides a method for producing SC-β cells from pluripotent cells, the method comprising: a) differentiating pluripotent stem cells in a population into endoderm cells by contacting the pluripotent stem cells with at least one factor from the TGF-β superfamily and a WNT signaling pathway activator for 3 days; b) differentiating at least some endoderm cells into gastrula cells by contacting the endoderm cells with at least one factor from the FGF family for 3 days; c) differentiating at least some gastrula cells into Pdx1-positive pancreatic progenitor cells by contacting the gastrula cells with i) a retinoic acid signaling pathway activator, ii) at least one factor from the FGF family, iii) an SHH pathway inhibitor, iv) a BMP signaling pathway inhibitor (e.g., DMH-1 or LDN193189), v) a PKC activator, and vi) a ROCK inhibitor; and d) differentiating Pdx1-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family and ii) at least one SHH under conditions that promote cell clustering. A step of differentiating at least some Pdx1-positive pancreatic progenitor cells into Pdx1-positive, NKX6.1-positive pancreatic progenitor cells by a process of contacting them every other day for 5 days with a pathway inhibitor, and optionally iii) an RA signaling pathway activator, and optionally iv) a ROCK inhibitor, and v) at least one factor from the TGF-β superfamily, wherein the NKX6.1-positive pancreatic progenitor cells express Pdx1 and NKX6.1, e) Pdx1-positive, NKX6.1-positive pancreatic progenitor cells with i) a TGF-β signaling pathway inhibitor, ii) a TH signaling pathway activator, iii) at least one SHH pathway inhibitor, iv) an RA signaling pathway activator, v) a gamma-secretase inhibitor, and optionally vi) The process involves differentiating at least some Pdx1-positive, NKX6.1-positive pancreatic progenitor cells into Pdx1-positive, NKX6.1-positive, insulin-positive endocrine cells by exposing them to at least one growth factor from the epidermal growth factor (EGF) family and, optionally, vii) a BMP signaling pathway inhibitor every other day for 5 to 7 days, and f) culturing the Pdx1-positive, NKX6.1-positive, insulin-positive endocrine cells in a medium (e.g., NS-GFs medium, MCDB medium supplemented with BSA, MCDB131 medium, or DMEM / F12 medium) every other day for 7 to 14 days without exogenous differentiation factors to induct at least some Pdx1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells. A step comprising differentiating at least some Pdx1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells by a process that induces in vitro maturation, wherein the SC-β cells exhibit a GSIS response in vitro and / or in vivo. In some cases, the GSIS response is similar to that of endogenous mature β cells.

[0229]

[0288] In some embodiments, the present disclosure provides a method for producing SC-β cells from pluripotent cells, the method comprising: a) differentiating pluripotent stem cells in a population into endoderm cells by contacting the pluripotent stem cells with at least one factor from the TGFβ superfamily and a WNT signaling pathway activator for three days; b) differentiating at least some endoderm cells into gastrulatic cells by contacting the endoderm cells with at least one factor from the FGF family for three days; and c) differentiating the gastrulatic cells into i) retinoic acid signaling pathway A step of differentiating at least some gastrullary cells into Pdx1-positive pancreatic progenitor cells by a process of contacting them for 2 days with an activator, ii) at least one factor from the FGF family, iii) an SHH pathway inhibitor, iv) a BMP signaling pathway inhibitor, v) a PKC activator, vi) a ROCK inhibitor, and vii) a growth factor from the TGFβ superfamily, d) Pdx1-positive pancreatic progenitor cells under conditions that promote cell clustering with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and A step of differentiating at least some Pdx1-positive pancreatic progenitor cells into Pdx1-positive, NKX6.1-positive pancreatic progenitor cells by a process of exposing them every other day for 5 days to iii) an RA signaling pathway activator, and optionally iv) a ROCK inhibitor, and v) at least one factor from the TGFβ superfamily, wherein the NKX6.1-positive pancreatic progenitor cells express Pdx1 and NKX6.1; e) exposing the Pdx1-positive, NKX6.1-positive pancreatic progenitor cells to i) a TGF-β signaling pathway inhibitor, and ii) a TH signaling pathway The process involves differentiating at least some Pdx1-positive, NKX6.1-positive pancreatic progenitor cells into Pdx1-positive, NKX6.1-positive, insulin-positive endocrine cells by exposing them every other day for 5 to 7 days to a signaling pathway activator, iii) at least one SHH pathway inhibitor, iv) RA signaling pathway activator, v) γ-secretase inhibitor, optionally vi) at least one growth factor from the epidermal growth factor (EGF) family, and optionally vii) BMP signaling pathway inhibitor, as well as f) Pdx1-positive, NKX6.1-positive, insulin-positive cells.Endocrine cells positive for Pdx1 and insulin were cultured every other day for 7 to 14 days without exogenous differentiation factors in a culture medium (e.g., NS-GFs medium, MCDB medium supplemented with BSA, MCDB131 medium, or DMEM / F12 medium) to produce a small number of Pdx1-positive, NKX6.1-positive, and insulin-positive endocrine cells. A step comprising differentiating at least some Pdx1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells by a process that induces in vitro maturation into at least some SC-β cells, wherein the SC-β cells exhibit a GSIS response in vitro and / or in vivo. In some cases, the GSIS response is similar to that of endogenously matured β cells.

[0230]

[0289] In some embodiments, the present disclosure provides a method for producing SC-β cells from pluripotent cells, the method comprising: a) differentiating pluripotent stem cells in a population into endoderm cells by contacting the pluripotent stem cells with at least one factor from the TGFβ superfamily and a WNT signaling pathway activator for three days; b) differentiating at least some endoderm cells into gastrula cells by contacting the endoderm cells with at least one factor from the FGF family for three days; c) differentiating at least some gastrula cells into Pdx1-positive pancreatic progenitor cells by contacting the gastrula cells with i) a retinoic acid signaling pathway activator, ii) at least one factor from the FGF family, iii) an SHH pathway inhibitor, iv) a PKC activator, and v) a ROCK inhibitor; and d) contacting the Pdx1-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) RA-positive growth factor under conditions that promote cell clustering. A step of differentiating at least some Pdx1-positive pancreatic progenitor cells into Pdx1-positive, NKX6.1-positive pancreatic progenitor cells by a process of contacting them every other day for 5 days with a Gunal signaling pathway activator, and optionally iv) a ROCK inhibitor, and v) at least one factor from the TGFβ superfamily, wherein the NKX6.1-positive pancreatic progenitor cells express Pdx1 and NKX6.1, e) Pdx1-positive, NKX6.1-positive pancreatic progenitor cells i) a TGF-β signaling pathway inhibitor, ii) T A step of differentiating at least some Pdx1-positive, NKX6.1-positive pancreatic progenitor cells into Pdx1-positive, NKX6.1-positive, insulin-positive endocrine cells by a process of contacting them every other day for 5 to 7 days with an H signaling pathway activator, iii) at least one SHH pathway inhibitor, iv) an RA signaling pathway activator, v) a γ-secretase inhibitor, and optionally vi) at least one growth factor from the epidermal growth factor (EGF) family, as well as f) Pdx1-positive, NKX6.1-positive, insulin-positive endocrine cells, andA step to differentiate at least some Pdx1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells by culturing Pdx1-positive, insulin-positive endocrine cells in a culture medium (e.g., NS-GFs medium, MCDB medium supplemented with BSA, MCDB131 medium, or DMEM / F12 medium) every other day for 7 to 14 days without exogenous differentiation factors, thereby inducing in vitro maturation of at least some Pdx1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells, wherein the SC-β cells are in... The procedure includes steps that elicit a GSIS response in vitro and / or in vivo. In some cases, the GSIS response is similar to that of endogenous mature β-cells.

[0231]

[0290] In some embodiments, the present disclosure provides a method for producing SC-β cells from pluripotent cells, the method comprising: a) differentiating pluripotent stem cells in a population into endoderm cells by contacting the pluripotent stem cells with at least one factor from the TGFβ superfamily and a WNT signaling pathway activator for three days; b) differentiating at least some endoderm cells into gastrula cells by contacting the endoderm cells with at least one factor from the FGF family for three days; c) differentiating at least some gastrula cells into Pdx1-positive pancreatic progenitor cells by contacting the gastrula cells with i) a retinoic acid signaling pathway activator, ii) at least one factor from the FGF family, iii) an SHH pathway inhibitor, iv) a BMP signaling pathway inhibitor (e.g., DMH-1 or LDN193189), v) a PKC activator, and vi) a ROCK inhibitor; and d) differentiating Pdx1-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family and ii) at least one SHH pathway inhibitor under conditions that promote cell clustering. A step of differentiating at least some Pdx1-positive pancreatic progenitor cells into Pdx1-positive, NKX6.1-positive pancreatic progenitor cells by a process of contacting them every other day for 5 or 6 days with a pathway inhibitor, and optionally iii) an RA signaling pathway activator, and optionally iv) a ROCK inhibitor, and v) at least one factor from the TGFβ superfamily, wherein the NKX6.1-positive pancreatic progenitor cells express Pdx1 and NKX6.1; e) Pdx1-positive, NKX6.1-positive pancreatic progenitor cells with i) an SHH pathway inhibitor, ii) an RA signaling pathway activator, iii) a γ-secretase inhibitor, iv) at least one growth factor from the epidermal growth factor (EGF) family, v) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, and vi) a TGF-β signaling pathway The process involves differentiating at least some Pdx1-positive, NKX6.1-positive pancreatic progenitor cells into Pdx1-positive, NKX6.1-positive, insulin-positive endocrine cells by exposing them every other day for 5 to 7 days to an inhibitor, vii) a thyroid hormone signaling pathway activator, viiii) an epigenetic modification compound (e.g., DZNep or KD5170), ix) a protein kinase inhibitor, and x) a ROCK inhibitor; and f) culturing the Pdx1-positive, NKX6.1-positive, insulin-positive endocrine cells every other day for 7 to 14 days without exogenous differentiation factors to differentiate at least some Pdx1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells. A step comprising differentiating at least some Pdx1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells by a process that induces in vitro maturation, wherein the SC-β cells exhibit a GSIS response in vitro and / or in vivo. In some cases, the GSIS response is similar to that of endogenous mature β cells.

[0232]

[0291] In some embodiments, the present disclosure provides a method for producing SC-β cells from pluripotent cells, the method comprising: a) differentiating pluripotent stem cells in a population into endoderm cells by contacting the pluripotent stem cells with at least one factor from the TGFβ superfamily and a WNT signaling pathway activator for 3 days; b) differentiating at least some endoderm cells into gastrulation cells by contacting the endoderm cells with at least one factor from the FGF family for 3 days; and c) activating the gastrulation cells i) retinoic acid signaling pathway. A step of differentiating at least some gastrulatic cells into Pdx1-positive pancreatic progenitor cells by a process of contacting the Pdx1-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, iv) BMP signaling pathway inhibitor (e.g., DMH-1 or LDN193189), v) PKC activator, and vi) ROCK inhibitor; d) Pdx1-positive pancreatic progenitor cells under conditions that promote cell clustering with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) RA sigma A step of differentiating at least some Pdx1-positive pancreatic progenitor cells into Pdx1-positive, NKX6.1-positive pancreatic progenitor cells by a process of contacting them every other day for 5 or 6 days with a NAL signaling pathway activator, and optionally iv) a ROCK inhibitor, and v) at least one factor from the TGFβ superfamily, wherein the NKX6.1-positive pancreatic progenitor cells express Pdx1 and NKX6.1, and e) Pdx1-positive, NKX6.1-positive pancreatic progenitor cells with i) a γ-secretase inhibitor, ii) at least one bone morphogenetic protein (BMP) sigma iii) TGF-β signaling pathway inhibitors, iv) thyroid hormone signaling pathway activators, v) epigenetic modification compounds (e.g., DZNep or KD5170), vi) protein kinase inhibitors, and vii) ROCK inhibitors were exposed to Pdx1-positive, NKX6.1-positive pancreatic progenitor cells every other day for 5 to 7 days, and during the first 3 days of the 5 to 7 days, Pdx1-positive, NKX6.1-positive pancreatic progenitor cells were exposed to SHH pathway inhibitors, RA signaling pathway inhibitors, and at least one growth factor from the EGF family, and then they were exposed to Pdx1-positive, NKX6.1-positive cells.The process of removing Pdx1-positive and NKX6.1-positive pancreatic progenitor cells results in Pdx1-positive and NKX6.1-positive pancreatic progenitor cells. The process comprises the steps of differentiating at least some progenitor cells into Pdx1-positive, NKX6.1-positive, insulin-positive endocrine cells, and f) differentiating at least some Pdx1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells by a process of inducing in vitro maturation of at least some Pdx1-positive, NKX6.1-positive, insulin-positive endocrine cells into SC-β cells by culturing the Pdx1-positive, NKX6.1-positive, insulin-positive endocrine cells in a medium (e.g., NS-GFs medium, MCDB medium supplemented with BSA, MCDB131 medium, or DMEM / F12 medium) every other day for 7 to 14 days without exogenous differentiation factors, wherein the SC-β cells exhibit a GSIS response in vitro and / or in vivo. In some cases, the GSIS response is similar to the GSIS response of endogenously matured β cells.

[0233]

[0292] The culture medium used to culture cells isolated from the first cell cluster may be xeno-free. Xeno-free media for culturing cells and / or cell clusters of animal origin cannot contain products of other animal origin. In some cases, xeno-free media for culturing human cells and / or cell clusters cannot contain any products of non-human animal origin. For example, xeno-free media for culturing human cells and / or cell clusters may contain human platelet lysates (PLT) instead of fetal bovine serum (FBS). For example, the medium may contain approximately 1% to 20%, 5% to 15%, 8% to 12%, and 9% to 11% serum. In some cases, the medium may contain approximately 10% serum. In some cases, the medium may not contain small molecules and / or FBS. For example, the medium may contain MCDB131 basal medium supplemented with 2% BSA. In some cases, the medium may not contain serum. In some cases, the culture medium contains exogenous small molecules or signaling pathway agonists or antagonists, such as growth factors from the fibroblast growth factor family (FGF, e.g., FGF2, FGF8B, FGF10, or FGF21), sonic hedgehog antagonists (e.g., Sant1, Sant2, Sant4, Sant4, Cur61414, forskolin, tomatidine, AY9944, tripanol, cyclopamine, or derivatives thereof), and retinoic acid signaling pathways. Transduction agonists (e.g., retinoic acid, CD1530, AM580, TTHPB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, or CD2314), inhibitors of Rho-related coiled-coil protein kinase (ROCK) (e.g., thiazovibin, Y-27632, fasudil / HA1077, or 14-1152), activators of protein kinase C (PKC) (e.g., phorbol 12,13-Dibutyrate (PDBU), TPB, phorbol 12-myristate 13-acetate, briostatin 1, or derivatives thereof), TGFβ superfamily antagonists (e.g., Alk5 inhibitor II (CAS) 446859-33-2), A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB-525334, SD-208, SB-505124, or derivatives thereof), inhibitors of bone morphogenetic protein (BMP) type 1 receptor (e.g., LDN193189 or derivatives thereof), thyroid hormone signaling pathway activators (e.g., T3, GC-1, or derivatives thereof), gamma secretase inhibitors (e.g., XXI, DAPT, or derivatives thereof), activators of the TGF-β signaling pathway (e.g., WNT3a or activin A) Growth factors from the epidermal growth factor (EGF) family (e.g., beta-cell phosphate or EGF), broad-spectrum kinases (e.g., staurosporine or its derivatives), non-essential amino acids, vitamins or antioxidants (e.g., cyclopamine, vitamin D, vitamin C, vitamin A, or their derivatives), or other additives such as N-acetylcysteine, zinc sulfate, or heparin may not be included. In some cases, the reaggregation medium may not contain exogenous extracellular matrix molecules. In some cases, the reaggregation medium may not contain Matrigel®. In some cases, the reaggregation medium may not contain other extracellular matrix molecules or, The material does not contain, for example, collagen, gelatin, poly-L-lysine, poly-D-lysine, vitronectin, laminin, fibronectin, PLO-laminin, fibrin, thrombin, and RetroNectin, or mixtures thereof, or, for example, lysed cell membrane preparations.

[0234]

[0293] Those skilled in the art will recognize that the concentration of serum albumin supplemented in the culture medium can vary. For example, a medium (e.g., MCDB131) may contain approximately 0.01%, 0.05%, 0.1%, 1%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 10%, or approximately 15% BSA. In other cases, the medium may contain approximately 0.01%, 0.05%, 0.1%, 1%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 10%, or approximately 15% HSA. The medium used (e.g., MCDB131 medium) may contain components not found in traditional basal media, such as trace elements, putrescine, adenine, thymidine, and high levels of certain amino acids and vitamins. These additives can enable the supplementation of mediums with very low levels of serum or specified components. The culture medium may not contain proteins and / or growth factors, and may be supplemented with EGF, hydrocortisone, and / or glutamine. The culture medium may contain one or more extracellular matrix molecules (e.g., extracellular proteins). Non-limiting exemplary extracellular matrix molecules used in the culture medium include collagen, placental matrix, fibronectin, laminin, merosine, tenascin, heparin, heparin sulfate, chondroitin sulfate, dermatan sulfate, aggrecan, biglycan, thrombospongin, vitronectin, and decorin. In some cases, the culture medium contains laminin, e.g., LN-332. In some cases, the culture medium contains heparin.

[0235]

[0294] The culture medium can be periodically replaced during culture, for example, to provide the cells in the medium with an optimal environment. When culturing cells separated from the first cell cluster for re-aggregation, the medium can be replaced at least every 4 hours, 12 hours, 24 hours, 48 ​​hours, every 3 days, or every 4 days. For example, the medium can be replaced approximately every 48 hours.

[0236]

[0295] In some cases, cells can be cultured under dynamic conditions (e.g., under conditions where cells are subjected to constant movement or agitation while in suspension culture). To culture cells dynamically, cells can be cultured in a container (e.g., a non-adhesive container such as a spinner flask (e.g., 200 ml to 3000 ml, e.g., 250 ml; 100 ml; or 125 ml in an Erlenmeyer)) that can be connected to a control unit and thus provide a controlled culture system. In some cases, cells can be cultured under non-dynamic conditions (e.g., static culture) while maintaining their proliferative capacity. To culture cells non-dynamically, cells can be cultured in an adhesive culture vessel. The adhesive culture vessel may be coated with any cell adhesion substrate, such as an extracellular matrix (ECM), to improve the adhesion of the vessel surface to cells. The cell adhesion substrate can be any material intended to attach stem cells or feeder cells (if used). Examples of substrates for cell adhesion include collagen, gelatin, poly-L-lysine, poly-D-lysine, vitronectin, laminin, fibronectin, PLO-laminin, fibrin, thrombin, and RetroNectin, as well as mixtures thereof, such as Matrigel®, and lysed cell membrane preparations.

[0237]

[0296] The culture medium in a dynamic cell culture vessel (e.g., a spinner flask) may be agitated (e.g., by a stirrer). The spin rate may correlate with the size of the second cell cluster that re-aggregates. The spin rate correlates with the size of the second cell cluster being similar to that of endogenous pancreatic islets. It can be controlled to allow for this. In some cases, the spin velocity is controlled so that the size of the second cell cluster can be approximately 75 μm to approximately 250 μm. The spin velocity of a dynamic cell culture vessel (e.g., a spinner flask) can be approximately 20 revolutions per minute (rpm) to approximately 100 rpm, for example, approximately 30 rpm to approximately 90 rpm, approximately 40 rpm to approximately 60 rpm, or approximately 45 rpm to approximately 50 rpm. In some cases, the spin velocity can be approximately 50 rpm.

[0238]

[0297] The Stage 6 cells provided herein may or may not be subjected to the separation and reaggregation processes as described herein. In some cases, cell clusters containing insulin-positive endocrine cells may be reaggregated. Reaggregation of cell clusters can enrich the insulin-positive endocrine cells. In some cases, the insulin-positive endocrine cells in the cell clusters may further mature into pancreatic β-cells. For example, after reaggregation, the second cell cluster may exhibit in vitro GSIS, similar to native pancreatic islets. For example, after reaggregation, the second cell cluster may contain non-native pancreatic β-cells exhibiting in vitro GSIS. In some embodiments, the reaggregation process may be carried out in accordance with the disclosure of PCT application PCT / US2018 / 043179, which is incorporated herein in whole by reference.

[0239]

[0298] Stage 6 cells obtained according to the methods provided herein may have a high recovery rate after cryopreservation and re-aggregation procedures. In some cases, Stage 6 cells obtained in differentiation processes involving treatment with BMP signaling pathway inhibitors (e.g., DMH-1 or LDN) and growth factors from the TGF-β superfamily (e.g., activin A) in Stage 3, and treatment with epigenetic modification compounds (e.g., histone methyltransferase inhibitors, e.g., EZH2 inhibitors, e.g., DZNep) in Stage 5 may have a higher recovery rate after cryopreservation following Stage 5 compared to the corresponding cell population without such treatment. In some cases, Stage 6 cells obtained through differentiation processes involving treatment with BMP signaling pathway inhibitors (e.g., DMH-1 or LDN) and growth factors from the TGF-β superfamily (e.g., activin A) in Stage 3, and treatment with epigenetic modification compounds (e.g., histone methyltransferase inhibitors, e.g., EZH2 inhibitors, e.g., DZNep) in Stage 5, may have a higher recovery rate after cryopreservation following Stage 5 compared to the corresponding cell population without treatment with BMP signaling pathway inhibitors (e.g., DMH-1 or LDN) and growth factors from the TGF-β superfamily (e.g., activin A) in Stage 3. In some cases, stage 6 cells obtained through differentiation processes involving treatment with BMP signaling pathway inhibitors (e.g., DMH-1 or LDN) and growth factors from the TGF-β superfamily (e.g., activin A) in stage 3, and treatment with epigenetic modification compounds (e.g., histone methyltransferase inhibitors, e.g., EZH2 inhibitors, e.g., DZNep) in stage 5, may have a recovery rate of at least approximately 35%, 37.5%, 40%, 42.5%, 45%, 47.5%, 48%, 49%, or 50% after cryopreservation following stage 5. Recovery rate can be calculated as the percentage of cells that survive and form reaggregated cell clusters after cryopreservation, thawing, retrieval, and reaggregation procedures, compared to cells before cryopreservation.

[0240]

[0299] In some embodiments, this disclosure relates to the cryopreservation of non-native pancreatic β-cells or their precursors obtained using methods provided herein. In some embodiments, a cell population including non-native pancreatic β-cells can be preserved by cryopreservation. For example, a cell population including non-native β-cells, e.g., stage 6 cells, may in some cases be isolated into a cell suspension, e.g., a single-cell suspension, and the cell suspension can be cryopreserved, e.g., frozen in a cryopreservation solution. Cell isolation can be performed using any of the techniques given herein. Alternatively, this can be done, for example, by enzymatic treatment. Cells can be frozen at temperatures up to -20°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -100°C, -110°C, -120°C, -130°C, -140°C, -150°C, -160°C, -170°C, -180°C, -190°C, or up to -200°C. In some cases, cells are frozen at approximately -80°C. In some cases, cells are frozen at approximately -195°C. Any cooling method can be used to achieve the low temperatures required for cryopreservation, including but not limited to electric freezers, solid carbon dioxide, and liquid nitrogen. In some cases, any cryopreservation solution available to those skilled in the art, including both custom-made and commercially available solutions, can be used to incubate cells for low-temperature storage. For example, a solution containing a cryoprotective substance may be used. The cryoprotective substance may be an agent configured to protect cells from freezing damage. For example, the cryoprotective substance may be a substance that can lower the glass transition temperature of the cryopreservation solution. Exemplary cryoprotective substances that can be used include DMSO (dimethyl sulfoxide), glycols (e.g., ethylene glycol, propylene glycol, and glycerol), dextran (e.g., dextran-40), and trehalose. Additional agents may be added to the cryopreservation solution for other purposes. In some cases, commercially available cryopreservation solutions, such as FrostaLife®, pZerve®, Prime-XV®, Gibco Synth-a-Freeze Cryopreservation Medium, STEM-CELLBANKER®, CryoStor® Freezing Media, HypoThermosol® FRS Preservation Media, and CryoDefend® Stem Cells Media, can be used in the methods provided herein.

[0241]

[0300] During the differentiation process, cells can be subjected to irradiation treatments provided herein. In some cases, a cell population or cluster in Stage 6, for example, a cell population or cluster having cells that differentiate from insulin-positive endocrine cells to pancreatic β-cells, is irradiated for a certain period of time. In some cases, a Stage 6 cell population after re-aggregation following retrieval from cryopreservation is irradiated for a certain period of time. In some cases, cryopreserved cells (e.g., cells cryopreserved at the end of Stage 5) are irradiated for a specific period of time before thawing and retrieval for the next differentiation process.

[0242]

[0301] V. Differentiation factor

[0302] Aspects of this disclosure relate to contacting progenitor cells (e.g., stem cells, e.g., iPS cells, endoderm cells, gastrulatinous cells, Pdx1-positive pancreatic progenitor cells, NKX6.1-positive pancreatic progenitor cells, insulin-positive endocrine cells) with β-cell differentiation factors to induce the maturation of insulin-positive endocrine cells or the differentiation of other progenitor cells into SC-β cells (e.g., mature pancreatic β-cells). In some embodiments, the differentiation factors can induce the differentiation of pluripotent cells (e.g., iPSCs or hESCs) into endoderm cells, for example, according to the method described herein. In some embodiments, the differentiation factors can induce the differentiation of endoderm cells into gastrulatinous cells, for example, according to the method described herein. In some embodiments, the differentiation factors can induce the differentiation of gastrulatinous cells into Pdx1-positive pancreatic progenitor cells, for example, according to the method described herein. In some embodiments, the differentiation factors can induce the differentiation of Pdx1-positive pancreatic progenitor cells into NKX6-1-positive pancreatic progenitor cells, for example, according to the method described herein. In some embodiments, the differentiation factor can induce the differentiation of NKX6-1-positive pancreatic progenitor cells into insulin-positive endocrine cells, for example, according to the method described herein. In some embodiments, the differentiation factor can induce the maturation of insulin-positive endocrine cells into SC-β cells, for example, according to the method described herein.

[0243]

[0303] At least one differentiation factor described herein can be used alone or in combination with other differentiation agents to produce SC-β cells according to the methods disclosed herein. In some embodiments, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten differentiation factors described herein are used in a method for producing SC-β cells.

[0244]

[0304] Transforming Growth Factor-β (TGF-β) Superfamily

[0305] Aspects of this disclosure relate to the use of growth factors from the transforming growth factor-β (TGF-β) superfamily as differentiation factors. The "TGF-β superfamily" means proteins possessing the structural and functional characteristics of members of the known TGFβ family. Examples of TGFβ family proteins include the TGFβ series of proteins, inhibin (including inhibin A and inhibin B), activin (including activin A, activin B, and activin AB), MIS (Müllerian duct inhibitor), BMP (bone morphogenetic protein), dpp (decapentaplesic), Vg-1, and MNSF (monoclonal nonspecific repressor). The activity of proteins in this family may be based on specific binding to particular receptors in various cell types. Members of this family may share regions of sequence identity that correlate with their function, particularly at the C-terminus. The TGFβ family can include more than 100 distinct proteins, all of which share at least one region of amino acid sequence identity. Members of the family that can be used in the methods disclosed herein include, but are not limited to, the following proteins, which are GenBank accessions: P07995, P18331, P08476, Q04998, P03970, P43032, P55102, P27092, P42917, P09529, P27093, P04088, Q04999, P17491, P55104, Q9WUK5, P55103, O88959, O08717, P58166, O61643, P35621, P09534, P48970, Q9NR23, P25703, P30884, P12643, P49001, P21274, O46564, O19006, P22004, P20722, Q04 906, Q07104, P30886, P18075, P23359, P22003, P34821, P49003, Q90751, P21275, Q06826, P30885, P34820, Q29607, P12644, Q90752, O46576, P27539, P48969, Q26 974, P07713, P91706, P91699, P27091, O42222, Q24735, P20863, O18828, P55106,Q9PTQ2、O14793、O08689、O42221、O18830、O18831、O18836、O35312、O42220、P43026、P43027、P43029、O95390、Q9R229、O93449、Q9Z1W4、Q9BDW8、P43028、Q7Z4P5、P50414、P17246、P54831、P04202、P01137、P09533、P18341、O19011、Q9Z1Y6、P07200、Q9Z217、O95393、P55105、P30371、Q9MZE2、Q07258、Q96S42、P97737、AAA97415.1、NP-776788.1、NP-058824.1、EAL24001.1、1 S4Y、NP-001009856.1、NP-1-032406.1、NP-999193.1、XP-519063.1、AAG17260.1、CAA40806.1、NP-1-001009458.1、AAQ55808.1、AAK40341.1、AAP33019.1、AAK21265.1、AAC59738.1、CAI46003.1、B40905、AAQ55811.1、AAK40342.1、XP-540364.1、P55102、AAQ55810.1、NP-990727.1、CAA51163.1、AAD50448.1、JC4862、PN0504、BAB17600.1、AAH56742.1、BAB17596.1、CAG06183.1、CAG05339.1、BAB17601.1、 、CAB43091.1、A36192、AAA49162.1、AAT42200.1、NP-789822.1、AAA59451.1、AAA59169.1、XP-541000.1、NP-990537.1、NP-1-002184.1、AAC14187.1、AAP83319.1、AAA59170.1、BAB16973.1、AAM66766.1、WFPGBB、1201278C、AAH30029.1、CAA49326.1、XP-344131.1、AA-148845.1、XP-1-148966.3、148235、B41398、AAH77857.1、AAB26863.1、1706327A、BAA83804.1、NP-571143.1、CAG00858.1、BAB17599.1、BAB17602.1、AAB61468.1、PN0505、PN0506、CAB43092.1、BAB17598.1、BAA22570.1、BAB16972.1、BAC81672.1、BAA12694.1、BAA08494.1、B36192、C36192、BAB16971.1、NP-034695.1、AAA49160.1、CAA62347.1、AAA49161.1、AAD30132.1、CAA58290.1、NP-005529.1、XP-522443.1、AAM27448.1、XP-538247.1、AAD30133. I、AAC36741.1、AAH10404.1、NP-032408.1、AAN03682.1、XP-509161.1、AAC32311.1、NP-651942.2、AAL51005.1、AAC39083.1、AAH85547.1、NP-571023.1、CAF94113.1、EAL29247.1、AAW30007.1、AAH90232.1、A29619、NP-001007905.1、AAH73508.1、AADO2201.1、NP-999793.1、NP-990542.1、AAF19841.1、AAC97488.1、AAC60038.1、NP 989197.1、NP-571434.1、EAL41229.1、AAT07302.1、CAI19472.1、NP-031582.1、AAA40548.1、XP-535880.1、NP-1-037239.1、AAT72007.1、XP-418956.1、CAA41634.1、BAC30864.1、CAA38850.1、CAB81657.2、CAA45018.1、CAA45019.1、BAC28247.1、NP-031581.1、NP-990479.1、NP-999820.1、AAB27335.1、S45355、CAB82007.1、XP-534351.1、NP-058874.1、NP-031579.1、1REW、AAB96785.1、AAB46367.1、CAA05033.1、BAA89012.1、IES7、AAP20870.1、BAC24087.1、AAG09784.1、BAC06352.1、AAQ89234.1、AAM27000.1、AAH30959.1、CAGO1491.1、NP-571435.1、1REU、AAC60286.1、BAA24406.1、A36193、AAH55959.1、AAH54647.1、AAH90689.1、CAG09422.1、BAD16743.1、NP-032134.1、XP-532179.1、AAB24876.1、AAH57702.1、AAA82616.1、CAA40222.1、CAB90273.2、XP-342592.1、XP-534896.1、XP-534462.1、1LXI、XP-417496.1、AAF34179.1、AAL73188.1、CAF96266.1、AAB34226.1、AAB33846.1、AAT12415.1、AA033819.1、AAT72008.1、AAD38402.1、BAB68396.1、CAA45021.1、AAB27337.1、AAP69917.1、AATI2416.1、NP-571396.1、CAA53513.1、AA033820.1、AAA48568.1、BAC02605.1、BAC02604.1、BAC02603.1、BAC02602.1、BAC02601.1、BAC02599.1、BAC02598.1、BAC02597.1、BAC02595.1、BAC02593.1、BAC02592.1、BAC02590.1、AAD28039.1、AAP74560.1、AAB94786.1、NP-001483.2、XP-528195.1、NP-571417.1、NP-001001557. I、AAH43222.1、AAM33143.1、CAG10381.1、B. AA31132.1、EAL39680.1、EAA12482.2、P34820、AAP88972.1、AAP74559.1、CAI16418.1、AAD30538.1、XP-345502.1、NP-1-038554.1、CAG04089.1、CAD60936.2、NP-031584.1、B55452、AAC60285.1、BAA06410.1、AAH52846.1、NP-031580.1、NP-1-036959.1、CAA45836.1、CAA45020.1、Q29607、AAB27336.1、XP-547817.1、AAT12414.1、AAM54049.1、AAH78901.1、AA025745.1、NP-570912.1、XP-392194.1、AAD20829.1、AAC97113.1、AAC61694.1、AAH60340.1、AAR97906.1、BAA32227.1、BAB68395.1、BAC02895.1、AAWS 1451.1、AAF82188.1、XP-544189.1、NP-990568.1、BAC80211.1、AAW82620.1、AAF99597.1、NP-571062.1、CAC44179.1、AAB97467.1、AAT99303.1、AAD28038.1、AAH52168.1、NP-001004122.1、CAA72733.1、NP-032133.2、XP-394252.1、XP-224733.2、JH0801、AAP97721.1、NP-989669.1、S43296、P43029、A55452、AAH32495.1、XP-542974.1、NP-032135.1、AAK30842.1、AAK27794.1、BAC30847.1、EAA12064.2、AAP97720.1、XP-525704.1、AAT07301.1、BAD07014.1、CAF94356.1、AAR27581.1、AAG13400.1、AAC60127.1、CAF92055.1、XP-540103.1、AA020895.1、CAF97447.1、AAS01764.1、BAD08319.1、CAA10268.1、NP-998140.1、AAR03824.1、AAS48405.1、AAS48403.1、AAK53545.1、AAK84666.1、XP-395420.1、AAK56941.1、AAC47555.1、AAR88255.1、EAL33036.1、AAW47740.1、AAW29442.1、NP-722813.1、AARO8901.1、AAO 15420.2、CAC59700.1、AAL26886.1、AAK71708.1、AAK71707.1、CAC51427.2、AAK67984.1、AAK67983.1、AAK28706.1、P07713、P91706、P91699、CAG02450.1、AAC47552.1、NP-005802.1、XP-343149.1、AW34055.1、XP-538221.1、AAR27580.1、XP-125935.3、AAF21633.1、AAF21630.1、AAD05267.1、Q9Z1 W4、NP-1-031585.2、NP-571094.1、CAD43439.1、CAF99217.1、CAB63584.1、NP-722840.1、CAE46407.1、XP-1-417667.1、BAC53989.1、BAB19659.1、AAM46922.1、AAA81169. .1、AAK28707.1、AAL05943.1、AAB17573.1、CAH25443.1、CAG10269.1、BAD16731.1、EAA00276.2、AAT07320.1、AAT07300.1、AAN15037.1、CAH25442.1、AAK08152.2、2009388A、AAR12161.1、CAGO1961.1、CAB63656.1、CAD67714.1、CAF94162.1、NP-477340.1、EAL24792.1、NP-1-001009428.1、AAB86686.1、AAT40572.1、AAT40571.1、AAT40569.1、NP-033886.1、AAB49985.1、AAG39266.1、Q26974、AAC77461.1、AAC47262.1、BAC05509.1、NP-055297.1、XP-546146.1、XP-525772.1、NP-060525.2、AAH33585.1、AAH69080.1、CAG12751.1、AAH74757.2、NP-034964.1、NP-038639.1、042221、 AAF02773.1, NP-062024.1, AAR18244.1, AAR14343.1, XP-228285.2, AAT40573.1, AAT94456.1, AAL35278.1, AAL35277.1, AAL17640.1, AAC08035.1, AAB86692.1 CAB40844.1、BAC38637.1、BAB16046.1、AAN63522.1、NP-571041.1、AAB04986.2、 AAC26791.1、AAB95254.1、BAA11835.1、AAR18246.1、XP-538528.1、BAA31853.1、 AAK18000.1、XP-1-420540.1、AAL35276.1、AAQ98602.1、CAE71944.1、AAW50585. 1、AAV63982.1、AAW29941.1、AAN87890.1、AAT40568.1、CAD57730.1、AAB81508.1 , AAS00534.1, AAC59736.1, BAB79498.1, AAA97392.1, AAP85526.1, NP-999600.2, NP-878293.1, BAC82629.1, CAC60268.1, CAG04919.1, AAN10123.1, CAA07707.1, AAK20912.1, AAR88254.1, CAC34629.1, AAL35275.1, AAD46997. NP-990153 .1、AAF78069.1、AAK49790.1、NP-919367.2、NP-001192.1、XP-544948.1、AAQ18013.1、AAV38739.1、NP-851298.1、CAA676 85.1、AAT67171.1、AAT37502.1、AAD27804.1、AAN76665.1、BAC11909.1、XP-1-421648.1、CAB63704.1、NP-037306.1、A557 06.Identified by 1, B36193, CAA33024.1, NP-1-001009400.1, AAP36538.1, XP-512687.1, XP-510080.1, AAH05513.1, 1KTZ, AAH14690.1, and AAA31526.1.

[0245]

[0306] The growth factors from the TGF-β superfamily in the methods and compositions provided herein may be naturally occurring or recombinant. In some embodiments, the growth factors from the TGF-β superfamily include activin A. The term “activin A” may include fragments and derivatives of activin A. An exemplary sequence of activin A is disclosed in SEQ ID NO: 1 in U.S. Patent Application Publication No. 2009 / 0155218 ('218). Other non-limiting examples of activin A are provided in SEQ ID NOs: 2-16 in '218, and non-limiting examples of nucleic acids encoding activin A are provided in SEQ ID NOs: 33-34 in '218. In some embodiments, the growth factor from the TGF-β superfamily may include a polypeptide having an amino acid sequence identical to SEQ ID NO: 1 of Publication 218 by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, or a higher percentage.

[0246]

[0307] In some embodiments, the growth factors from the TGF-β superfamily include growth differentiation factor 8 (GDF8). The term "GDF8" may include fragments and derivatives of GDF8. The sequence of the GDF8 polypeptide is available to those skilled in the art. In some embodiments, the growth factors from the TGF-β superfamily include polypeptides having an amino acid sequence that is identical to the human GDF8 polypeptide sequence (GenBank contract EAX10880) by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, or higher.

[0247]

[0308] In some embodiments, the growth factors from the TGF-β superfamily include growth factors closely related to GDF8, such as growth differentiation factor 11 (GDF11). In some embodiments, the growth factors from the TGF-β superfamily include polypeptides having an amino acid sequence that is identical to the human GDF11 polypeptide sequence (GenBank contract AAF21630) by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, or higher.

[0248]

[0309] In some embodiments, growth factors from the TGF-β superfamily can be replaced with agents that mimic at least one growth factor from the TGF-β superfamily. Exemplary agents that mimic at least one growth factor from the TGF-β superfamily include, but are not limited to, IDE1 and IDE2.

[0249]

[0310] Bone morphogenetic protein (BMP) signaling pathway inhibitors

[0311] Aspects of this disclosure relate to the use of BMP signaling pathway inhibitors as β-cell differentiation factors. The BMP signaling family is a diverse subset of the TGF-β superfamily (Sebald et al. Biol. Chem. 385: pp. 697-710, 2004). More than 20 known BMP ligands are recognized by three distinct type II (BMPRII, ActRIIa, and ActRIIb) and at least three type I (ALK2, ALK3, and ALK6) receptors. Dimeric ligands promote the assembly of receptor heteromers, causing phosphorylation of type I receptor serine / threonine kinases to constitutively active type II receptor serine / threonine kinases. Activated type I receptors phosphorylate BMP-responsive (BR-)SMAD effectors (SMAD1, 5, and 8) to facilitate nuclear translocation in complex with SMAD4, a co-SMAD that also promotes TGF signaling. Furthermore, BMP signaling can activate intracellular effectors such as MAPK p38 in a SMAD-independent manner (Nohe et al., Cell Signal 16: pp. 291-299, 2004). Soluble BMP antagonists, such as noggin, chordin, gremlin, and follistatin, restrict BMP signaling by ligand sequestration.

[0250]

[0312] In some embodiments, the BMP signaling pathway inhibitors in the methods and compositions provided herein include DMH-1, or its derivatives, analogues, or variants. In some embodiments, the BMP signaling pathway inhibitors in the methods and compositions provided herein include the following compounds or derivatives, analogues, or variants of the following compounds:

[0251] [ka]

[0252]

[0313] In some embodiments, the BMP signaling pathway inhibitors in the methods and compositions provided herein include LDN193189 (also known as LDN193189, 1062368-24-4, LDN-193189, DM 3189, DM-3189, IUPAC name: 4-[6-(4-piperazine-1-ylphenyl)pyrazolo[1,5-a]pyrimidine-3-yl]quinolone). In some embodiments, the BMP signaling pathway inhibitors in the methods and compositions provided herein include the following compounds or derivatives, analogues, or variants of the following compounds:

[0253] [ka]

[0254]

[0314] In some cases, DMH-1 may be more selective than LDN193189. In some embodiments of this disclosure, DMH-1 may be particularly useful for the methods provided herein. In some embodiments, the methods and compositions provided herein exclude the use of LDN193189. In some embodiments, the methods and compositions provided herein exclude the use of LDN193189, or its derivatives, analogs, or variants, for producing Pdx1-positive pancreatic progenitor cells from gastrulatic cells. In some embodiments, the methods and compositions provided herein relate to the use of DMH-1, or its derivatives, analogs, or variants, for producing Pdx1-positive pancreatic progenitor cells from gastrulatic cells.

[0255]

[0315] In some embodiments, B in the methods and compositions provided herein MP signaling pathway inhibitors include analogs or derivatives of LDN193189, such as salts, hydrates, solvents, esters, or prodrugs of LDN193189. In some embodiments, the derivatives of LDN193189 (e.g., salts) include LDN193189 hydrochloride.

[0256]

[0316] In some embodiments, the BMP signaling pathway inhibitors in the methods and compositions provided herein include compounds of formula I from U.S. Patent Application Publication No. 2011 / 0053930.

[0257]

[0317] TGF-β signaling pathway inhibitors

[0318] Aspects of this disclosure relate to the use of TGF-β signaling pathway inhibitors as β-cell differentiation factors.

[0258]

[0319] In some embodiments, the TGF-β signaling pathway includes TGF-β receptor type I kinase (TGF-β RI) signaling. In some embodiments, the TGF-β signaling pathway inhibitor includes ALK5 inhibitor II (CAS 446859-33-2, also known as RepSox, an ATP competitive inhibitor of TGF-β RI kinase, IUPAC name: 2-[5-(6-methylpyridine-2-yl)-1H-pyrazole-4-yl]-1,5-naphthiridine). In some embodiments, the TGF-β signaling pathway inhibitor is an analog or derivative of ALK5 inhibitor II.

[0259]

[0320] In some embodiments, an analog or derivative of ALK5 inhibitor II (also referred to as "ALK5i") is a compound of formula I described in U.S. Patent Application Publication No. 2012 / 0021519, which is incorporated entirely herein by reference.

[0260]

[0321] In some embodiments, the TGF-β signaling pathway inhibitor in the methods and compositions provided herein is a TGF-β receptor inhibitor described in U.S. Patent Application Publication 2010 / 0267731. In some embodiments, the TGF-β signaling pathway inhibitor in the methods and compositions provided herein includes ALK5 inhibitors described in U.S. Patent Application Publications 2009 / 0186076 and 2007 / 0142376. In some embodiments, the TGF-β signaling pathway inhibitor in the methods and compositions provided herein is A 83-01. In some embodiments, the TGF-β signaling pathway inhibitor in the methods and compositions provided herein is not A 83-01. In some embodiments, the compositions and methods described herein exclude A 83-01. In some embodiments, the TGF-β signaling pathway inhibitor in the methods and compositions provided herein is SB 431542. In some embodiments, the TGF-β signaling pathway inhibitor is SB Not 431542. In some embodiments, the compositions and methods described herein exclude SB 431542. In some embodiments, the TGF-β signaling pathway inhibitor in the methods and compositions provided herein is D 4476. In some embodiments, the TGF-β signaling pathway inhibitor is not D 4476. In some embodiments, the compositions and methods described herein exclude D 4476. In some embodiments, the TGF-β signaling pathway inhibitor in the methods and compositions provided herein is GW 788388. In some embodiments, the TGF-β signaling pathway inhibitor is not GW 788388. In some embodiments, the compositions and methods described herein exclude GW 788388. In some embodiments, the TGF-β signaling pathway inhibitor in the methods and compositions provided herein is LY 364947. In some embodiments, the TGF-β signaling pathway inhibitor is not LY 364947. In some embodiments, the compositions and methods described herein exclude LY 364947. In some embodiments, provided herein The TGF-β signaling pathway inhibitor in the methods and compositions provided herein is LY 580276. In some embodiments, the TGF-β signaling pathway inhibitor is not LY 580276. In some embodiments, the compositions and methods described herein exclude LY 580276. In some embodiments, the TGF-β signaling pathway inhibitor in the methods and compositions provided herein is SB 525334. In some embodiments, the TGF-β signaling pathway inhibitor is not SB 525334. In some embodiments, the compositions and methods described herein exclude SB 525334. In some embodiments, the TGF-β signaling pathway inhibitor in the methods and compositions provided herein is SB 505124. In some embodiments, the TGF-β signaling pathway inhibitor is not SB 505124. In some embodiments, the compositions and methods described herein exclude SB 505124. In some embodiments, the TGF-β signaling pathway inhibitor in the methods and compositions provided herein is SD 208. In some embodiments, the TGF-β signaling pathway inhibitor is not SD 208. In some embodiments, the compositions and methods described herein exclude SD 208. In some embodiments, the TGF-β signaling pathway inhibitor in the methods and compositions provided herein is GW 6604. In some embodiments, the TGF-β signaling pathway inhibitor is not GW 6604. In some embodiments, the compositions and methods described herein exclude GW 6604. In some embodiments, the TGF-β signaling pathway inhibitor in the methods and compositions provided herein is GW 788388. In some embodiments, the TGF-β signaling pathway inhibitor in the methods and compositions provided herein is GW Not 788388. In some embodiments, the compositions and methods described herein exclude GW 788388.

[0261]

[0322] From the above collection of compounds, the following can be obtained from various sources: Sigma, PO Box 14508, St. Louis, Mo. LY-364947, SB-525334, SD-208, and SB-505124 available from 63178-9916; 616452 and 616453 available from Calbiochem (EMD Chemicals, Inc.), 480 S. Democrat Road, Gibbstown, NJ, 08027; GW788388 and GW6604 available from GlaxoSmithKline, 980 Great West Road, Brentford, Middlesex, TW8 9GS, United Kingdom; Lilly Research, Available from Indianapolis, Ind. 46285 (LY580276) and Biogen Idec, PO Box 14627, 5000 Davis Drive, Research Triangle Park, NC, 27709-4627 (SM16).

[0262]

[0323] WNT signaling pathway

[0324] Aspects of this disclosure relate to the use of WNT signaling pathway activators as β-cell differentiation factors.

[0263]

[0325] In some embodiments, the WNT signaling pathway activator in the methods and compositions provided herein comprises CHIR99021. In some embodiments, the WNT signaling pathway activator in the methods and compositions provided herein comprises derivatives of CHIR99021, for example, salts of CHIR99021, for example, trihydrochloride and hydrochloride salts of CHIR99021. In some embodiments, the WNT signaling pathway activator in the methods and compositions provided herein comprises Wnt3a recombinant protein. In some embodiments, the WNT signaling pathway activator in the methods and compositions provided herein comprises glycogen synthase quina This includes GSK3 inhibitors. Exemplary GSK3 inhibitors include, but are not limited to, 3F8, A 1070722, AR-A 014418, BIO, BIO-acetoxime, FRATide, 10Z-himenialdisine, indirubin-3'oxime, Kaempaulon, L803, L803-mts, lithium carbonate, NSC 693868, SB 216763, SB 415286, TC-G 24, TCS 2002, TCS 21311, TWS 119, and any analogs or derivatives thereof. In certain embodiments, the methods, compositions, and kits disclosed herein exclude WNT signaling pathway activators.

[0264]

[0326] Fibroblast Growth Factor (FGF) Family

[0327] A part of this disclosure relates to the use of growth factors from the FGF family as β-cell differentiation factors.

[0265]

[0328] In some embodiments, the growth factors from the FGF family in the methods and compositions provided herein include keratinocyte growth factor (KGF). KGF polypeptide sequences are available to those skilled in the art. In some embodiments, the growth factors from the FGF family include polypeptides having amino acid sequences that are identical to the human KGF polypeptide sequence (GenBank contract AAB21431) by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, or higher.

[0266]

[0329] In some embodiments, the growth factors from the FGF family in the methods and compositions provided herein include FGF2. The polypeptide sequence of FGF2 is available to those skilled in the art. In some embodiments, the growth factors from the FGF family include polypeptides having an amino acid sequence that is identical to the human FGF2 polypeptide sequence (GenBank contract NP_001997) by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, or higher.

[0267]

[0330] In some embodiments, the growth factors from the FGF family in the methods and compositions provided herein include FGF8B. The polypeptide sequence of FGF8B is available to those skilled in the art. In some embodiments, the growth factors from the FGF family include polypeptides having an amino acid sequence that is identical to the human FGF8B polypeptide sequence (GenBank contract AAB40954) by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, or higher.

[0268]

[0331] In some embodiments, the growth factors from the FGF family in the methods and compositions provided herein include FGF10. The polypeptide sequence of FGF10 is available to those skilled in the art. In some embodiments, the growth factors from the FGF family include polypeptides having an amino acid sequence that is identical to the human FGF10 polypeptide sequence (GenBank contract CAG46489) by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, or higher.

[0269]

[0332] In some embodiments, the methods and compositions provided herein include at least one growth factor from the FGF family, which comprises FGF21. Lipeptide sequences are available to those skilled in the art. In some embodiments, growth factors from the FGF family include polypeptides having amino acid sequences identical to the human FGF21 polypeptide sequence (GenBank contract AAQ89444.1) by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, or higher.

[0270]

[0333] Sonic Hedgehog (SHH) Signaling Pathway

[0334] Aspects of this disclosure relate to the use of SHH signaling pathway inhibitors as β-cell differentiation factors.

[0271]

[0335] In some embodiments, the SHH signaling pathway inhibitor in the methods and compositions provided herein includes Sant1. In some embodiments, the SHH signaling pathway inhibitor in the methods and compositions provided herein includes SANT2. In some embodiments, the SHH signaling pathway inhibitor in the methods and compositions provided herein includes SANT3. In some embodiments, the SHH signaling pathway inhibitor in the methods and compositions provided herein includes SANT4. In some embodiments, the SHH signaling pathway inhibitor includes Cur61414. In some embodiments, the SHH signaling pathway inhibitor in the methods and compositions provided herein includes forskolin. In some embodiments, the SHH signaling pathway inhibitor in the methods and compositions provided herein includes tomatidine. In some embodiments, the SHH signaling pathway inhibitor in the methods and compositions provided herein includes AY9944. In some embodiments, the SHH signaling pathway inhibitor in the methods and compositions provided herein includes tripanol. In some embodiments, the SHH signaling pathway inhibitor in the methods and compositions provided herein includes compound A or compound B (disclosed in U.S. Patent Application Publication 2004 / 0060568). In some embodiments, the SHH signaling pathway inhibitors in the methods and compositions provided herein include steroidal alkaloids (e.g., cyclopamine or its derivatives) that antagonistize hedgehog signaling as disclosed in U.S. Patent Application Publication 2006 / 0276391. In certain embodiments, the methods, compositions, and kits disclosed herein exclude SHH signaling pathway inhibitors.

[0272]

[0336] Rho kinase (ROCK) signaling pathway

[0337] Aspects of this disclosure relate to the use of ROCK signaling pathway inhibitors (ROCK inhibitors) as β-cell differentiation factors.

[0273]

[0338] In some embodiments, the ROCK inhibitor in the methods and compositions provided herein comprises Y-27632 or thiazovibin. In some embodiments, the ROCK inhibitor in the methods and compositions provided herein comprises thiazovibin. In some embodiments, the ROCK inhibitor in the methods and compositions provided herein comprises Y-27632. In some cases, the ROCK inhibitor in the methods and compositions provided herein comprises the following compounds or derivatives thereof:

[0274] [ka]

[0275]

[0339] In some cases, the ROCK inhibitors in the methods and compositions provided herein include the following compounds or derivatives thereof:

[0276] [ka]

[0277]

[0340] Non-limiting examples of ROCK inhibitors that can be used in the methods and compositions provided herein include thiazovibin, Y-27632, and fasudil / H Examples include A1077, H-1152, ripasudil, Y39983, Wf-536, SLx-2119, azabenzimidazole-aminoflazan, DE-104, olefins, isoquinolines, indazoles, and pyridine alkene derivatives, ROKα inhibitors, XD-4000, HMN-1152, 4-(1-aminoalkyl)-N-(4-pyridyl)cyclohexane-carboxamide, Rhostatin, BA-210, BA-207, BA-215, BA-285, BA-1037, Ki-23095, VAS-012, and quinazoline.

[0278]

[0341] Retinoic acid signaling pathway

[0342] Aspects of this disclosure relate to the use of retinoic acid signaling modulators as β-cell differentiation factors.

[0279]

[0343] In some embodiments, the retinoic acid signaling modulator in the methods and compositions provided herein comprises a retinoic acid signaling activator. In some embodiments, the RA signaling pathway activator in the methods and compositions provided herein comprises retinoic acid. In some embodiments, the RA signaling pathway activator in the methods and compositions provided herein comprises a retinoic acid receptor agonist. Exemplary retinoic acid receptor agonists in the methods and compositions provided herein include, but are not limited to, CD 1530, AM 580, TTNPB, CD 437, Ch 55, BMS 961, AC 261066, AC 55649, AM 80, BMS 753, tazarotene, adapalene, and CD 2314 is one example.

[0280]

[0344] In some embodiments, the methods and compositions provided herein are used Modulators of tinoic acid signaling include inhibitors of retinoic acid signaling. In some embodiments, the retinoic acid signaling pathway inhibitor includes DEAB (IUPAC name: 2-[2-(diethylamino)ethoxy]3-propa-2-enylbenzaldehyde). In some embodiments, the retinoic acid signaling pathway inhibitor includes analogs or derivatives of DEAB.

[0281]

[0345] In some embodiments, the retinoic acid signaling pathway inhibitors in the methods and compositions provided herein include retinoic acid receptor antagonists. In some embodiments, the retinoic acid receptor antagonists in the methods and compositions provided herein include (E)-4-[2-(5,6-dihydro-5,5-dimethyl-8-phenyl-2-naphthalenyl)ethenyl]benzoic acid, (E)-4-[[(5,6-dihydro-5,5-dimethyl-8-phenylethynyl)-2-naphthalenyl]ethenyl]benzoic acid, (E)-4-[2-[5,6-dihydro-5,5-dimethyl-8-(2-naphthalenyl)-2-naphthalenyl]ethenyl]benzoic acid, and (E)-4-[2-[5,6-dihydro-5,5-dimethyl-8-(4-methoxyphenyl)-2-naphthalenyl]ethenyl]benzoic acid. In some embodiments, the retinoic acid receptor antagonist includes BMS 195614 (CAS No. 253310-42-8), ER 50891 (CAS No. 187400-85-7), BMS 493 (CAS No. 170355-78-9), CD 2665 (CAS No. 170355-78-9), LE 135 (CAS No. 155877-83-1), BMS 453 (CAS No. 166977-43-1), or MM 11253 (CAS No. 345952-44-5).

[0282]

[0346] In certain embodiments, the methods, compositions, and kits disclosed herein exclude retinoic acid signaling modulators. In certain embodiments, the methods, compositions, and kits disclosed herein exclude retinoic acid signaling pathway activators. In certain embodiments, the methods, compositions, and kits disclosed herein exclude retinoic acid signaling pathway inhibitors.

[0283]

[0347] Protein kinase C

[0348] Aspects of this disclosure relate to the use of protein kinase C activators as β-cell differentiation factors. Protein kinase C is one of the largest families of protein kinase enzymes and consists of various isoforms. Conventional isoforms include α, βI, βII, and γ; novel isoforms include δ, ε, η, and Θ; and atypical isoforms include ξ and ι / λ. PKC enzymes are primarily cytosolic but translocate to the membrane upon activation. In the cytoplasm, PKC is phosphorylated or autophosphorylated by other kinases. To be activated, some PKC isoforms (e.g., PKC-ε) require molecules to bind to a diacylglycerol ("DAG") or phosphatidylserine ("PS") binding site. Others can be activated without requiring any secondary binding messengers. Examples of PKC activators that bind to the DAG site include, but are not limited to, bryostatin, picologue, phorbol ester, apriciatoxin, and gunidimacrine. Examples of PKC activators that bind to the PS site include, but are not limited to, polyunsaturated fatty acids and their derivatives. Any protein kinase C activator capable of inducing the differentiation of at least one insulin-producing endocrine cell or its precursor into SC-β cells, either alone or in combination with one or more other β-cell differentiation factors, is intended to be used in the methods, compositions, and kits described herein.

[0284]

[0349] In some embodiments, the PKC activator in the methods and compositions provided herein includes PdbU. In some embodiments, the method provided herein includes PdbU. The PKC activator in the law and composition includes TPB. In some embodiments, the PKC activators in the methods and compositions provided herein include, as described in WIPO Publication WO / 2013 / 071282, cyclopropanated polyunsaturated fatty acids, cyclopropanated monounsaturated fatty acids, cyclopropanated polyunsaturated fatty alcohols, cyclopropanated monounsaturated fatty alcohols, cyclopropanated polyunsaturated fatty acid esters, cyclopropanated monounsaturated fatty acid esters, cyclopropanated polyunsaturated fatty acid sulfates, cyclopropanated monounsaturated fatty acid sulfates, cyclopropanated polyunsaturated fatty acid phosphates, cyclopropanated monounsaturated fatty acid phosphates, macrocyclic lactones, DAG derivatives, isoprenoids, octyrindractam V, gunidimacrine, iriparidal, ingenol, naphthalene sulfonamide, diacylglycerol kinase inhibitors, fibroblast growth factor 18 (FGF-18), insulin growth factor, hormones, and growth factor activators. In some embodiments, bryostatin includes bryostatin-1, bryostatin-2, bryostatin-3, bryostatin-4, bryostatin-5, bryostatin-6, bryostatin-7, bryostatin-8, bryostatin-9, bryostatin-10, bryostatin-11, bryostatin-12, bryostatin-13, bryostatin-14, bryostatin-15, bryostatin-16, bryostatin-17, or bryostatin-18. In certain embodiments, the methods, compositions, and kits disclosed herein exclude protein kinase C activators.

[0285]

[0350] γ-secretase inhibitors

[0351] Aspects of this disclosure relate to the use of γ-secretase inhibitors as β-cell differentiation factors.

[0286]

[0352] In some embodiments, the γ-secretase inhibitor in the methods and compositions provided herein includes XXI. In some embodiments, the γ-secretase inhibitor in the methods and compositions provided herein includes DAPT. Additional exemplary γ-secretase inhibitors in the methods and compositions provided herein include, but are not limited to, those described in U.S. Patents 7,049,296, 8,481,499, 8,501,813, and WIPO Publication WO / 2013 / 052700. In certain embodiments, the methods, compositions, and kits disclosed herein exclude the γ-secretase inhibitor.

[0287]

[0353] Thyroid hormone signaling pathway activators

[0354] Aspects of this disclosure relate to the use of thyroid hormone signaling pathway activators as β-cell differentiation factors.

[0288]

[0355] In some embodiments, the thyroid hormone signaling pathway activator in the methods and compositions provided herein comprises triiodothyronine (T3). In some embodiments, the thyroid hormone signaling pathway activator in the methods and compositions provided herein comprises GC-1. In some embodiments, the thyroid hormone signaling pathway activator in the methods and compositions provided herein comprises T3 or analogs or derivatives of GC-1. Exemplary analogs of T3 in the methods and compositions provided herein include, but are not limited to, selective and non-selective thyromimetics, TRβ selective agonists GC-1, GC-24, 4-hydroxy-PCB 106, MB07811, MB07344, 3,5-diiodotyropropionic acid (DITPA); selective TR-β agonist GC-1; 3-iodothyronamine (T(1)AM) and 3,3',5-triiodothyroacetate (Triac) (biologically active metabolite of the hormone thyroxine (T(4)); KB-2115 and KB-141; thyronamine; SKF L-94901; DIBIT; 3'-AC -T2; tetraiodothyroacetate (Tetrac) and triiodothyroacetate (Triac) (by oxidative deamination and decarboxylation of thyroxine [T4] and triiodothyronine [T3] alanine chains), 3,3',5'-triiodothyronine (rT3) (by deiodination of T4 and T3), 3,3'-diiodothyronine (3,3'-T2) and 3,5-diiodothyronine (T2) (by deiodination of T4, T3, and rT3), and Examples of structural analogues of TH include 3-iodothyronamine (T1AM) and thyronamine (T0AM) (by T4 and T3 deiodination and amino acid decarboxylation), as well as 3,5,3'-triiodotyropropionic acid (Triprop), 3,5-dibulmo-3-pyridazinone-1-thyronine (L-940901), N-[3,5-dimethyl-4-(4'-hydroxy-3'-isopropylphenoxy)-phenyl]oxamic acid (CGS 23425), 3,5-dimethyl-4-[(4'-hydroxy-3'-isopropylbenzyl)-phenoxy]acetic acid (GC-1), 3,5-dichloro-4-[(4-hydroxy-3-isopropylphenoxy)phenyl]acetic acid (KB-141), and 3,5-diiodotyropropionic acid (DITPA).

[0289]

[0356] In some embodiments, the thyroid hormone signaling pathway activators in the methods and compositions provided herein include T3 prodrugs or prohormones, such as T4 thyroid hormones (e.g., thyroxine or L-3,5,3',5'-tetraiodothyronine).

[0290]

[0357] In some embodiments, the thyroid hormone signaling pathway activator in the methods and compositions provided herein is the iodothyronine composition described in U.S. Patent No. 7,163,918.

[0291]

[0358] Epidermal growth factor (EGF) family

[0359] Aspects of this disclosure relate to the use of growth factors from the EGF family as β-cell differentiation factors.

[0292]

[0360] In some embodiments, at least one growth factor from the EGF family in the methods and compositions provided herein comprises beta-cellulin. In some embodiments, at least one growth factor from the EGF family in the methods and compositions provided herein comprises EGF. Epidermal growth factor (EGF) is a 53-amino acid cytokine cleaved by proteolysis from a large membrane-bound protein precursor. In some embodiments, the growth factor from the EGF family in the methods and compositions provided herein comprises a variant EGF polypeptide, for example, an isolated epidermal growth factor polypeptide having...

Claims

1. A method comprising the step of contacting a population of pancreatic progenitor cells or their precursors with an epigenetic modification compound, wherein the contact step results in a population of endocrine cells in which the proportion of chromogranin A-positive (CHGA+) cells is increased, or the proportion of C-peptide-positive and NKX6.1-positive (C-PEP+,NKX6.1+) cells is increased, compared to a corresponding population of endocrine cells that have not been contacted with the epigenetic modification compound.

2. A method comprising the step of contacting a population of pancreatic progenitor cells or their precursors with an epigenetic modification compound, wherein the contact step results in a population of endocrine cells in which the proportion of cells expressing VMAT or Cdx2 is reduced compared to a corresponding population of endocrine cells that have not been contacted with the epigenetic modification compound.

3. The method according to claim 1 or 2, wherein the epigenetic modification compound comprises one or more of the following: a DNA methylation inhibitor, a histone acetyltransferase inhibitor, a histone deacetylase inhibitor, a histone methyltransferase inhibitor, or a bromodomain inhibitor.

4. The method according to claim 3, wherein the epigenetic modification compound comprises a histone methyltransferase inhibitor.

5. The method according to claim 4, wherein the histone methyltransferase inhibitor is an EZH2 inhibitor.

6. The method according to claim 4 or 5, wherein the histone methyltransferase inhibitor is selected from the group consisting of DZNep, GSK126, and EPZ6438.

7. The method according to claim 6, wherein the histone methyltransferase inhibitor is DZNep.

8. The method according to claim 7, wherein the concentration of DZNep brought into contact with the population of pancreatic progenitor cells or their precursors is about 0.05 μM to about 50 μM, about 0.1 μM to about 10 μM, about 0.5 μM to about 5 μM, about 0.75 μM to about 2.5 μM, or about 1 μM to about 2 μM.

9. The method according to claim 8, wherein the concentration of DZNep is at least about 0.5 μM.

10. The method according to claim 8, wherein the concentration of DZNep is approximately 1 μM.

11. The method according to any one of claims 1 to 10, wherein the epigenetic modification compound comprises a histone deacetylase (HDAC) inhibitor.

12. The method according to claim 11, wherein the HDAC inhibitor is a class I HDAC inhibitor, a class II HDAC inhibitor, or a combination thereof.

13. The method according to claim 12, wherein the HDAC inhibitor is selected from the group consisting of KD5170, MC1568, and TMP195.

14. The method according to claim 13, wherein the HDAC inhibitor is KD5170.

15. The epigenetic modified compound includes an HDAC inhibitor and an EZH2 inhibitor. The method described in item 1 or 2.

16. The method according to claim 1 or 2, wherein the epigenetic modification compound comprises DZNep and KD5170.

17. The method according to any one of claims 1 to 16, which is carried out in vitro.

18. The method according to any one of claims 1 to 17, further comprising the step of contacting the population of pancreatic progenitor cells or their precursors with a drug selected from the group consisting of (i) an SHH pathway inhibitor, (ii) a retinoic acid (RA) signaling pathway activator, (iii) a γ-secretase inhibitor, (iv) a growth factor from the epidermal growth factor (EGF) family, (v) a bone morphogenetic protein (BMP) signaling pathway inhibitor, (vi) a TGF-β signaling pathway inhibitor, (vii) a thyroid hormone signaling pathway activator, (viiii) a protein kinase inhibitor, and (ix) a ROCK inhibitor.

19. (A) The SHH pathway inhibitor includes SANT1, (B) The RA signaling pathway activator contains retinoic acid, (C) The γ-secretase inhibitor comprises XXI, (D) The growth factor from the EGF family includes beta-cell phosphate, (E) The BMP signaling pathway inhibitor includes LDN, (F) The TGF-β signaling pathway inhibitor comprises Alk5i II, (G) The thyroid hormone signaling pathway activator comprises GC-1, (H) The protein kinase inhibitor contains staurosporine, or (I) The ROCK inhibitor contains thiazobinin, The method according to claim 18.

20. The method according to claim 18 or 19, comprising the step of contacting the pancreatic progenitor cells or a population of their precursors with a drug selected from the group consisting of beta-cellulin, thiazobinin, retinoic acid, SANT1, XXI, Alk5i II, GC-1, LDN, and staurosporine.

21. The method according to any one of claims 18 to 20, wherein the contact step is for at least three days.

22. The method according to claim 21, wherein the contact step comprises contacting a population of pancreatic progenitor cells or their precursors with the epigenetic modification compound for a period of more than three days, and removing the SHH pathway inhibitor, the RA signaling pathway activator, or the growth factor from the EGF family after the contact step with the population of pancreatic progenitor cells or their precursors during the first three days of the period.

23. The method according to any one of claims 18 to 22, wherein the contact step is for at least 5 days.

24. The method according to any one of claims 18 to 23, wherein the contact step is approximately 7 days.

25. The method according to any one of claims 1 to 24, wherein at least one cell in the population of pancreatic progenitor cells expresses at least one of PDX1 and NKX6-1.

26. Of the aforementioned population of pancreatic progenitor cells, at least one cell is both PDX1 and NKX6-1. The method according to any one of claims 1 to 25, which expresses the method.

27. The method according to any one of claims 1 to 26, wherein at least one cell in the population of endocrine cells expresses CHGA.

28. The method according to any one of claims 1 to 27, wherein at least one cell in the population of endocrine cells expresses C-peptide and NKX6.

1.

29. The method according to any one of claims 1 to 28, wherein the population of endocrine cells comprises, as measured by flow cytometry, a proportion of CHGA+ cells that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 180%, 200%, 220%, 250%, 280%, 300%, 320%, 350%, 380%, 400%, 420%, 450%, 480%, or 500% higher than the corresponding population of endocrine cells that have not come into contact with the epigenetic modification compound.

30. The method according to any one of claims 1 to 29, wherein the population of endocrine cells comprises, as measured by flow cytometry, a proportion of C-PEP+, NKX6.1+ cells that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 180%, 200%, 220%, 250%, 280%, 300%, 320%, 350%, 380%, 400%, 420%, 450%, 480%, or 500% higher than the corresponding population of endocrine cells that have not come into contact with the epigenetic modification compound.

31. The method according to any one of claims 1 to 30, wherein the population of endocrine cells comprises cells expressing VMAT or Cdx2 at a rate at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 180%, 200%, 220%, 250%, 280%, 300%, 320%, 350%, 380%, or 400% lower than the corresponding population of endocrine cells not in contact with the at least one epigenetic modification compound, as measured by flow cytometry.

32. Cells produced by the method according to any one of claims 1 to 31.

33. A composition comprising a cell population, wherein the cell population is measured by flow cytometry, (a) cells expressing at least about 20% C-peptide and NKX6.1, (b) At least about 60% of cells express CHGA, (c) At most about 20% of cells express Cdx2, or (d) At most about 45% of cells express VMAT1 A composition containing the following:

34. A composition comprising a cell population that, as measured by flow cytometry, contains at least about 30% ISL1-positive, NKX6.1-positive cells and at most about 20% ISL1-negative, NKX6.1-negative cells.

35. The composition according to claim 34, wherein the cell population comprises at least about 35% ISL1-positive and NKX6.1-positive cells.

36. The composition according to claim 34, wherein the cell population comprises at least about 40% ISL1-positive and NKX6.1-positive cells.

37. The composition according to any one of claims 34 to 36, wherein the cell population comprises at most about 15% ISL1-negative and NKX6.1-negative cells.

38. Measured by flow cytometry, (a) cells expressing at least about 20% C-peptide and NKX6.1, (b) at least about 60% of cells expressing CHGA, and (c) At most about 20% of cells express Cdx2 A composition according to any one of claims 33 to 37, comprising:

39. The composition according to claim 38, comprising at most about 45% of cells expressing VMAT1, as measured by flow cytometry.

40. The composition according to any one of claims 33 to 39, further comprising an epigenetic modification compound.

41. The composition according to claim 40, wherein the epigenetic modification compound comprises one or more of the following: a DNA methylation inhibitor, a histone acetyltransferase inhibitor, a histone deacetylase inhibitor, a histone methyltransferase inhibitor, or a bromodomain inhibitor.

42. The composition according to claim 40, wherein the epigenetic modification compound comprises a histone methyltransferase inhibitor.

43. The composition according to claim 42, wherein the histone methyltransferase inhibitor is an EZH2 inhibitor.

44. The composition according to claim 42 or 43, wherein the histone methyltransferase inhibitor is selected from the group consisting of DZNep, GSK126, and EPZ6438.

45. The composition according to claim 44, wherein the histone methyltransferase inhibitor is DZNep.

46. The composition according to claim 45, wherein the concentration of DZNep brought into contact with the population of pancreatic progenitor cells or their precursors is about 0.05 μM to about 50 μM, about 0.1 μM to about 10 μM, about 0.5 μM to about 5 μM, about 0.75 μM to about 2.5 μM, or about 1 μM to about 2 μM.

47. The composition according to claim 46, wherein the concentration of DZNep is at least about 0.5 μM.

48. The composition according to claim 46, wherein the concentration of DZNep is about 1 μM.

49. The composition according to any one of claims 40 to 48, wherein the epigenetic modification compound comprises a histone deacetylase (HDAC) inhibitor.

50. The composition according to claim 49, wherein the HDAC inhibitor is a class I HDAC inhibitor, a class II HDAC inhibitor, or a combination thereof.

51. The composition according to claim 50, wherein the HDAC inhibitor is selected from the group consisting of KD5170, MC1568, and TMP195.

52. The composition according to claim 50, wherein the HDAC inhibitor is KD5170.

53. The composition according to claim 40, wherein the epigenetic modification compound comprises an HDAC inhibitor and an EZH2 inhibitor.

54. The composition according to claim 40, wherein the epigenetic modification compound comprises DZNep and KD5170.

55. The composition according to any one of claims 33 to 54, further comprising an agent selected from the group consisting of (i) an SHH pathway inhibitor, (ii) a retinoic acid (RA) signaling pathway activator, (iii) a γ-secretase inhibitor, (iv) a growth factor from the epidermal growth factor (EGF) family, (v) a bone morphogenetic protein (BMP) signaling pathway inhibitor, (vi) a TGF-β signaling pathway inhibitor, (vii) a thyroid hormone signaling pathway activator, (viiii) a protein kinase inhibitor, and (ix) a ROCK inhibitor.

56. (A) The SHH pathway inhibitor includes SANT1, (B) The RA signaling pathway activator contains retinoic acid, (C) The γ-secretase inhibitor comprises XXI, (D) The growth factor from the EGF family includes beta-cell phosphate, (E) The BMP signaling pathway inhibitor includes LDN, (F) Whether the TGF-β signaling pathway inhibitor contains Alk5i II (G) The thyroid hormone signaling pathway activator comprises GC-1, (H) The protein kinase inhibitor contains staurosporine, or (I) The ROCK inhibitor contains thiazobinin, The composition according to claim 55.

57. The composition according to claim 55 or 56, comprising a drug selected from the group consisting of beta-cellulin, thiazobinin, retinoic acid, SANT1, XXI, Alk5i II, GC-1, LDN, and staurosporine.

58. A composition comprising pancreatic progenitor cells and at least one of a histone deacetylase (HDAC) inhibitor or a histone methyltransferase inhibitor.

59. The composition according to claim 58, further comprising endocrine cells.

60. The composition according to claim 58 or 59, wherein the HDAC inhibitor is a class I HDAC inhibitor, a class II HDAC inhibitor, or a combination thereof.

61. The composition according to claim 60, wherein the HDAC inhibitor is selected from the group consisting of KD5170, MC1568, and TMP195.

62. The composition according to claim 61, wherein the HDAC inhibitor is KD5170.

63. The composition according to claim 62, wherein the concentration of KD5170 in the composition is about 0.05 μM to about 50 μM, about 0.1 μM to about 10 μM, about 0.5 μM to about 5 μM, about 0.75 μM to about 2.5 μM, or about 1 μM to about 2 μM.

64. The composition according to claim 63, wherein the concentration of KD5170 is at least 0.5 μM.

65. The composition according to claim 64, wherein the concentration of KD5170 is approximately 1 μM.

66. The composition according to any one of claims 58 to 65, wherein the histone methyltransferase inhibitor is an EZH2 inhibitor.

67. The composition according to claim 66, wherein the histone methyltransferase inhibitor is selected from the group consisting of DZNep, GSK126, and EPZ6438.

68. The composition according to claim 67, wherein the histone methyltransferase inhibitor is DZNep.

69. The composition according to claim 68, wherein the concentration of DZNep in the composition is about 0.05 μM to about 50 μM, about 0.1 μM to about 10 μM, about 0.5 μM to about 5 μM, about 0.75 μM to about 2.5 μM, or about 1 μM to about 2 μM.

70. The composition according to claim 69, wherein the concentration of DZNep is at least about 0.5 μM.

71. The composition according to claim 69, wherein the concentration of DZNep is about 1 μM.

72. The composition according to claim 58, wherein the HDAC inhibitor is KD5170 and the histone methyltransferase inhibitor is DZNep.

73. The composition according to any one of claims 58 to 72, which is an in vitro composition.

74. The composition according to any one of claims 58 to 73, further comprising an agent selected from the group consisting of (i) an SHH pathway inhibitor, (ii) a retinoic acid (RA) signaling pathway activator, (iii) a γ-secretase inhibitor, (iv) a growth factor from the epidermal growth factor (EGF) family, (v) a bone morphogenetic protein (BMP) signaling pathway inhibitor, (vi) a TGF-β signaling pathway inhibitor, (vii) a thyroid hormone signaling pathway activator, (viiii) a protein kinase inhibitor, and (ix) a ROCK inhibitor.

75. (A) The SHH pathway inhibitor contains SANT1, (B) The RA signaling pathway activator contains retinoic acid, (C) The γ-secretase inhibitor contains XXI, (D) The growth factor from the EGF family contains beta-cellulin, (E) The BMP signaling pathway inhibitor includes LDN, (F) The TGF-β signaling pathway inhibitor contains Alk5i II, (G) The thyroid hormone signaling pathway activator contains GC-1, (H) The protein kinase inhibitor contains staurosporine, or (I) The ROCK inhibitor contains thiazobinin, The composition according to claim 74.

76. The composition according to claim 74 or 75, further comprising an agent selected from the group consisting of beta-cellulin, thiazobinin, retinoic acid, SANT1, XXI, Alk5i II, GC-1, LDN, and staurosporine.

77. A step of contacting a cell population containing pancreatic progenitor cells or their precursors with a histone methyltransferase inhibitor to produce a cell population containing endocrine cells, and The cell population including the aforementioned endocrine cells is matured and subjected to the glucose challenge in vi Step 1: Obtain at least one pancreatic β-cell that exhibits a glucose-stimulated insulin secretion response. A method that includes this.

78. The method according to claim 77, further comprising the step of contacting the cell population with a drug selected from the group consisting of (i) an SHH pathway inhibitor, (ii) a retinoic acid (RA) signaling pathway activator, (iii) a γ-secretase inhibitor, (iv) a growth factor from the epidermal growth factor (EGF) family, (v) a bone morphogenetic protein (BMP) signaling pathway inhibitor, (vi) a TGF-β signaling pathway inhibitor, (vii) a thyroid hormone signaling pathway activator, (viiii) a protein kinase inhibitor, and (ix) a ROCK inhibitor.

79. (A) The SHH pathway inhibitor includes SANT1, (B) The RA signaling pathway activator contains retinoic acid, (C) The γ-secretase inhibitor comprises XXI, (D) The growth factor from the EGF family includes beta-cell phosphate, (E) The BMP signaling pathway inhibitor includes LDN, (F) The TGF-β signaling pathway inhibitor comprises Alk5i II, (G) The thyroid hormone signaling pathway activator comprises GC-1, (H) The protein kinase inhibitor contains staurosporine, or (I) The ROCK inhibitor contains thiazobinin, The method according to claim 78.

80. The method according to claim 78 or 79, comprising the step of contacting the cell population with a drug selected from the group consisting of beta-cellulin, thiazobinin, retinoic acid, SANT1, XXI, Alk5i II, GC-1, LDN, and staurosporine.

81. The method according to any one of claims 77 to 80, further comprising the step of contacting the cell population with a histone deacetylase (HDAC) inhibitor.

82. The method according to claim 81, wherein the HDAC inhibitor is KD5170.

83. The method according to any one of claims 77 to 82, wherein the histone methyltransferase inhibitor is selected from the group consisting of DZNep, GSK126, and EPZ6438.

84. The method according to claim 83, wherein the histone methyltransferase inhibitor is DZNep.

85. The method according to any one of claims 77 to 84, wherein the step of contacting with the histone methyltransferase inhibitor results in a population comprising the endocrine cells, wherein the proportion of chromogranin A-positive (CHGA+) cells is increased, or the proportion of C-peptide-positive and NKX6.1-positive (C-PEP+,NKX6.1+) cells is increased, compared to a corresponding population of endocrine cells that have not been contacted with the histone methyltransferase inhibitor.

86. The method according to any one of claims 77 to 85, wherein the step of contacting with the histone methyltransferase inhibitor results in a population comprising the endocrine cells, in which the proportion of cells expressing VMAT or Cdx2 is reduced compared to a corresponding population of endocrine cells that have not been contacted with the histone methyltransferase inhibitor.

87. The method according to any one of claims 77 to 86, wherein at least one of the pancreatic progenitor cells or its precursors expresses both Pdx1 and NKX6.

1.

88. The method according to any one of claims 77 to 87, further comprising the step of differentiating a plurality of stem cells in vitro to obtain the cell population containing the pancreatic progenitor cells or their precursors.

89. Pancreatic β-cells produced by the method according to any one of claims 77 to 88.

90. (a) A step of contacting a population of Pdx1-negative NKX6.1-negative gastrullary cells with a bone morphogenetic protein (BMP) signaling pathway inhibitor and growth factors from the transformation growth factor β (TGF-β) superfamily to produce a cell population including Pdx1-positive, NKX6.1-positive pancreatic progenitor cells, and (b) The step of contacting the cell population, which includes the Pdx1-positive and NKX6.1-positive pancreatic progenitor cells, with an epigenetic modification compound to produce a cell population including endocrine cells. A method that includes this.

91. The method according to claim 90, wherein the BMP signaling pathway inhibitor comprises DMH-1, its derivatives, analogs, or variants.

92. The method according to claim 91, wherein the concentration of DMH-1 brought into contact with the population of Pdx1-negative, NKX6.1-negative gastrullous cells is approximately 0.01 μM to approximately 10 μM, approximately 0.05 μM to approximately 5 μM, approximately 0.1 μM to approximately 1 μM, or approximately 0.15 μM to approximately 0.5 μM.

93. The method according to claim 91, wherein the concentration of DMH-1 brought into contact with the population of Pdx1-negative, NKX6.1-negative gastrullous cells is approximately 0.25 μM.

94. The method according to any one of claims 91 to 93, wherein the growth factor from the TGF-β superfamily comprises activin A.

95. The method according to claim 94, wherein the concentration of activin A brought into contact with the population of Pdx1-negative, NKX6.1-negative gastrulline cells is approximately 0.5 ng / mL to approximately 200 ng / mL, approximately 1 ng / mL to approximately 100 ng / mL, approximately 2 ng / mL to approximately 50 ng / mL, or approximately 5 ng / mL to approximately 30 ng / mL.

96. The method according to claim 94, wherein the concentration of activin A brought into contact with the population of Pdx1-negative, NKX6.1-negative gastrullous cells is at least about 5 ng / mL or at least about 10 ng / mL.

97. The method according to claim 94, wherein the concentration of activin A that is brought into contact with the population of Pdx1-negative, NKX6.1-negative gastrullous cells is approximately 20 ng / mL.

98. The method according to any one of claims 90 to 97, wherein the step of contacting the population of Pdx1-negative, NKX6.1-negative gastrullous cells further comprises the step of contacting the population with a drug selected from the group consisting of growth factors from the FGF family, SHH pathway inhibitors, RA signaling pathway activators, protein kinase C activators, and ROCK inhibitors.

99. The epigenetic modification compound is a DNA methylation inhibitor, a histone acetyltransferase inhibitor, a histone deacetylase inhibitor, or a histone methyltransferase inhibitor. The method according to any one of claims 90 to 98, comprising a compound selected from the group consisting of -ase inhibitors and bromodomain inhibitors.

100. The method according to claim 99, wherein the epigenetic modification compound comprises a histone methyltransferase inhibitor.

101. The method according to claim 100, wherein the histone methyltransferase inhibitor is an EZH2 inhibitor.

102. The method according to claim 100 or 101, wherein the histone methyltransferase inhibitor is selected from the group consisting of DZNep, GSK126, and EPZ6438.

103. The method according to claim 102, wherein the histone methyltransferase inhibitor is DZNep.

104. The method according to claim 103, wherein the concentration of DZNep brought into contact with the population of pancreatic progenitor cells or their precursors is about 0.05 μM to about 50 μM, about 0.1 μM to about 10 μM, about 0.5 μM to about 5 μM, about 0.75 μM to about 2.5 μM, or about 1 μM to about 2 μM.

105. The method according to claim 104, wherein the concentration of DZNep is at least about 0.5 μM.

106. The method according to claim 104, wherein the concentration of DZNep is approximately 1 μM.

107. The method according to any one of claims 90 to 106, wherein the epigenetic modification compound comprises a histone deacetylase (HDAC) inhibitor.

108. The method according to claim 107, wherein the HDAC inhibitor is a class I HDAC inhibitor, a class II HDAC inhibitor, or a combination thereof.

109. The method according to claim 108, wherein the HDAC inhibitor is selected from the group consisting of KD5170, MC1568, and TMP195.

110. The method according to claim 109, wherein the HDAC inhibitor is KD5170.

111. The method according to any one of claims 90 to 110, which is carried out in vitro.

112. The method according to any one of claims 90 to 111, further comprising the step of contacting the population comprising Pdx1-positive, NKX6.1-positive pancreatic progenitor cells with a drug selected from the group consisting of (i) an SHH pathway inhibitor, (ii) a retinoic acid (RA) signaling pathway activator, (iii) a γ-secretase inhibitor, (iv) a growth factor from the epidermal growth factor (EGF) family, (v) a bone morphogenetic protein (BMP) signaling pathway inhibitor, (vi) a TGF-β signaling pathway inhibitor, (vii) a thyroid hormone signaling pathway activator, (viiii) a protein kinase inhibitor, and (ix) a ROCK inhibitor.

113. (A) The SHH pathway inhibitor includes SANT1, (B) The RA signaling pathway activator contains retinoic acid, (C) The γ-secretase inhibitor comprises XXI, (D) The growth factor from the EGF family includes beta-cell phosphate, (E) The BMP signaling pathway inhibitor includes LDN, (F) The TGF-β signaling pathway inhibitor contains Alk5i II, (G) The thyroid hormone signaling pathway activator comprises GC-1, (H) The protein kinase inhibitor contains staurosporine, or (I) The ROCK inhibitor contains thiazobinin, The method according to claim 112.

114. The method according to claim 112 or 113, comprising the step of contacting the population comprising the Pdx1-positive, NKX6.1-positive pancreatic progenitor cells with a drug selected from the group consisting of beta-cellulin, thiazobinin, retinoic acid, SANT1, XXI, Alk5i II, GC-1, LDN, and staurosporine.

115. The method according to any one of claims 112 to 114, wherein the contact step is for at least three days.

116. The method according to claim 115, wherein the contact step comprises contacting the population, including the Pdx1-positive, NKX6.1-positive pancreatic progenitor cells, with the epigenetic modification compound for a period of more than three days, and removing the SHH pathway inhibitor, the RA signaling pathway activator, or a growth factor from the EGF family after the contact step with the population of pancreatic progenitor cells or their precursors during the first three days of the period.

117. The method according to any one of claims 112 to 116, wherein the contact step is for at least 5 days.

118. The method according to any one of claims 112 to 116, wherein the contact step is approximately 7 days.

119. The method according to any one of claims 90 to 118, wherein the cell population including the Pdx1-positive, NKX6.1-positive pancreatic progenitor cells contains at least about 10% of cells expressing Cdx2, as measured by flow cytometry.

120. The method according to any one of claims 90 to 119, wherein the cell population comprising the Pdx1-positive, NKX6.1-positive pancreatic progenitor cells comprises a smaller proportion of cells expressing Cdx2 compared to the corresponding cell population that has not been in contact with the bone morphogenetic protein (BMP) signaling pathway inhibitor and the growth factors from the transformation growth factor β (TGF-β) superfamily.

121. The method according to any one of claims 90 to 120, wherein the cell population including the endocrine cells comprises, as measured by flow cytometry, at least about 40% of the cells expressing ISL1 and NKX6.

1.

122. The method according to any one of claims 90 to 121, wherein the cell population including the endocrine cells comprises a greater proportion of cells expressing ISL1 and NKX6.1 compared to the corresponding cell population that has not come into contact with the bone morphogenetic protein (BMP) signaling pathway inhibitor and the growth factors from the transformation growth factor β (TGF-β) superfamily.

123. The method according to any one of claims 90 to 122, wherein the cell population including the endocrine cells contains at least about 15% ISL1-negative and NKX6.1-negative cells as measured by flow cytometry.

124. The method according to any one of claims 90 to 122, wherein the cell population including the endocrine cells comprises a smaller proportion of ISL1-negative, NKX6.1-negative cells compared to the corresponding cell population that has not been in contact with the bone morphogenetic protein (BMP) signaling pathway inhibitor and the growth factors from the transformation growth factor β (TGF-β) superfamily.

125. The method according to any one of claims 90 to 124, further comprising the step of cryopreserving a cell population including the endocrine cells.

126. The cell population including the endocrine cells is a cell cluster, and the method is (a) the step of separating a plurality of cells from the cell cluster, and (b) A step of culturing the plurality of cells from step (a) in a re-aggregation medium to form a second cell cluster in at least a portion of the plurality of cells. The method according to any one of claims 90 to 125, further comprising:

127. The method according to claim 126, wherein the separation step does not include the step of subjecting the plurality of cells to flow cytometry.

128. The method according to claim 126 or 127, wherein the reaggregation medium does not contain serum.

129. The method according to any one of claims 126 to 128, wherein the reaggregation medium does not contain exogenous differentiation factors.

130. The endocrine cells were matured in vitro and subjected to a glucose challenge. The method according to any one of claims 90 to 129, further comprising the step of obtaining at least one pancreatic β-cell that exhibits a vitro-glucose-stimulated insulin secretion response.

131. The method according to claim 130, wherein the maturation step is carried out in a serum-free medium.

132. The method according to claim 130 or 131, wherein the maturation step is carried out in a xenofree medium.

133. The method according to any one of claims 130 to 132, wherein the maturation step is carried out in a culture medium that does not contain exogenous differentiation factors.

134. The method according to any one of claims 130 to 133, wherein the maturation step is carried out in the presence of human serum albumin (HSA).

135. The method according to claim 134, wherein the HSA is present in a concentration of about 0.1% to about 5% and about 0.5% to about 2%.

136. The method according to claim 134, wherein the HSA is present at a concentration of about 1%.

137. (a) A step of differentiating pluripotent stem cells from a population into endoderm cells by contacting them with growth factors from the TGF-β superfamily and WNT signaling pathway activators, (b) A step of differentiating at least a portion of the endoderm cells into gastrulline cells by contacting the endoderm cells with growth factors from the FGF family, (c) The gastrulatum cells are subjected to a ROCK inhibitor, a growth factor from the FGF family, and BMP sigma. (d) Differentiating at least a portion of the gastrulatum cells into Pdx1-positive pancreatic progenitor cells by contacting them with a nal signaling pathway inhibitor, a PKC activator, a retinoic acid signaling pathway activator, an SHH pathway inhibitor, and growth factors from the TGF-β superfamily; (d) Differentiating at least a portion of the Pdx1-positive pancreatic progenitor cells into Pdx1-positive, NKX6.1-positive pancreatic progenitor cells by contacting the Pdx1-positive pancreatic progenitor cells with a ROCK inhibitor, growth factors from the TGFβ superfamily, growth factors from the FGF family, an RA signaling pathway activator, and an SHH pathway inhibitor; and (e) Differentiating at least a portion of the Pdx1-positive, NKX6.1-positive pancreatic progenitor cells into a cell population containing at least one NKX6.1+ and C-peptide+ cell by contacting them with a TGF-β signaling pathway inhibitor, a growth factor from the EGF family, an RA signaling pathway activator, an SHH pathway inhibitor, a TH signaling pathway activator, a γ-secretase inhibitor, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and an epigenetic modification compound. A method that includes this.

138. A cell population comprising endocrine cells produced by any one of claims 90 to 129 or the method described in claim 137.

139. A cell population comprising SC-β cells produced by the method according to any one of claims 130 to 136.

140. A method comprising the step of exposing an in vitro cell population, including endocrine cells, to irradiation at a dose of approximately 100 rad to approximately 100,000 rad for a time of approximately 1 minute to approximately 60 minutes.

141. A method for reducing cell proliferation, comprising the step of exposing a cell population including stem cells, endoderm cells of an embryo, gastrula cells, pancreatic progenitor cells, or endocrine cells to irradiation, wherein the irradiation results in a cell population with reduced proliferative capacity compared to the corresponding cell population that has not been irradiated.

142. The method according to claim 140 or 141, wherein the cell population is exposed to irradiation of approximately 100 rad to approximately 50,000 rad, approximately 100 rad to approximately 25,000 rad, approximately 100 rad to approximately 10,000 rad, approximately 250 rad to approximately 25,000 rad, approximately 500 rad to approximately 25,000 rad, approximately 1,000 rad to approximately 25,000 rad, approximately 2,500 rad to approximately 25,000 rad, approximately 5,000 rad to approximately 25,000 rad, or approximately 10,000 rad to approximately 15,000 rad.

143. The method according to claim 140 or 141, wherein the cell population is exposed to irradiation of about 10,000 rads.

144. The method according to any one of claims 140 to 143, wherein the cell population is exposed to irradiation for approximately 1 to 55 minutes, approximately 1 to 50 minutes, approximately 1 to 45 minutes, approximately 1 to 40 minutes, approximately 1 to 35 minutes, approximately 1 to 30 minutes, approximately 1 to 25 minutes, approximately 1 to 20 minutes, approximately 1 to 10 minutes, approximately 1 to 5 minutes, approximately 10 to 55 minutes, approximately 15 to 55 minutes, approximately 20 to 55 minutes, approximately 25 to 55 minutes, approximately 30 to 55 minutes, approximately 20 to 40 minutes, or approximately 25 to 35 minutes.

145. The method according to any one of claims 140 to 143, wherein the cell population is exposed to irradiation for about 30 minutes.

146. The method according to any one of claims 140 to 145, wherein the irradiation includes ionization irradiation. 。

147. The method according to any one of claims 140 to 146, wherein the ionization irradiation includes gamma rays, X-rays, ultraviolet radiation, alpha rays, beta rays, or neutron rays.

148. The method according to any one of claims 140 to 147, wherein the irradiation results in a cell population with reduced proliferation compared to the corresponding cell cluster that has not been subjected to the irradiation.

149. The method according to any one of claims 140 to 148, wherein the cell population includes a second cell cluster having a diameter of approximately 50 μm to approximately 500 μm, approximately 50 μm to approximately 300 μm, approximately 50 μm to approximately 200 μm, approximately 50 μm to approximately 150 μm, approximately 600 μm to approximately 150 μm, approximately 700 μm to approximately 150 μm, approximately 80 μm to approximately 150 μm, or approximately 60 μm to approximately 100 μm.

150. (a) the step of separating multiple cells from a first cell cluster, and (b) A step of culturing the plurality of cells from step (a) in a reaggregation medium to cause the second cell cluster to form in at least a portion of the plurality of cells. The method according to claim 149, further comprising:

151. The method according to claim 150, wherein the separation step does not include the step of subjecting the plurality of cells to flow cytometry.

152. The method according to claim 150 or 151, wherein the first cell cluster is obtained by separating a third cell cluster and culturing the cells separated from the third cell cluster to form the first cell cluster.

153. The method according to claim 149, wherein the cell cluster is cryopreserved before the irradiation, and the method further comprises the step of thawing the cryopreserved cell cluster before the irradiation.

154. The method according to claim 149, wherein the cell cluster is cryopreserved while being subjected to the irradiation.

155. The method according to claim 154, further comprising the step of thawing the cryopreserved cell cluster after irradiation and differentiating at least a portion of the endocrine cells.

156. The method according to any one of claims 140 to 155, further comprising the step of obtaining the cell population including the endocrine cells by differentiating pancreatic progenitor cells or their precursors in vitro.

157. The method according to any one of claims 140 to 156, further comprising the step of differentiating stem cells in vitro to produce the cell population including the endocrine cells.

158. The method according to any one of claims 140 to 157, further comprising the step of maturing at least a portion of the endocrine cells into pancreatic β-cells in vitro, thereby producing a cell population.

159. The method according to claim 158, further comprising the step of implanting the pancreatic β-cells into a subject that requires them.

160. The method according to claim 159, wherein the implanted pancreatic β-cells are configured to control blood glucose levels in the subject for at least about 50, 60, 70, 80, 90 days, or longer.

161. A cell population comprising endocrine cells produced by the method according to any one of claims 140 to 157.

162. A cell population comprising pancreatic β-cells produced by the method according to any one of claims 158 to 160.

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