Generation of human pluripotent stem cell derived functional beta cells showing glucose-dependent mitochondrial respiration and two-phase insulin secretion response

Abstract

To provide a method for producing in vitro functional beta-cells (functional pancreatic beta-cells) and populations of cells resulting from the differentiation of pluripotent stem cells.SOLUTION: A method of differentiating pancreatic endoderm cells into functional beta cells expressing PDX1, NKX6.1, MAFA, UCN3, and SLC2A1, comprises culturing immature pancreatic beta cells in a medium supplemented with (a) an effective amount of T3 and (b) an effective amount of 3-deazaneplanocin A (DEZA), thereby producing functional beta cells expressing insulin.SELECTED DRAWING: None

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application is a direct sequel to U.S. Provisional Patent Application No. 62 / 352,968 (filed June 21, 2016). Priority is claimed to the same which is incorporated by reference in its entirety.

[0002] FIELD OF THE INVENTION The present invention provides a method for producing functional pancreatic beta cells and populations resulting from the differentiation of pluripotent stem cells in vitro. In particular, the present invention relates to a method for producing mitochondrial respiration / activity response and It relates to a beta cell or population of beta cells that exhibits a phasic insulin secretory response. [Background technology]

[0003] With advances in cell replacement therapy for type 1 diabetes mellitus and a shortage of transplantable islets of Langerhans, Attention has focused on developing sources of insulin-producing cells, or beta (β) cells, suitable for engraftment. One approach is to generate cells from pluripotent stem cells, such as embryonic stem cells or induced pluripotent stem cells. Some produce functional beta cells.

[0004] During vertebrate embryonic development, pluripotent cells differentiate into three types in a process known as gastrulation. They give rise to a group of cells composed of different germ layers (ectoderm, mesoderm, and endoderm). For example, the thyroid Tissues such as the thymus, pancreas, intestine, and liver develop from the endoderm through intermediate stages.

[0005] D'Amour et al. reported that human embryonic stem cell-derived activin-1 (HES-1) cells were cultured in the presence of high concentrations of activin and low serum. described the production of an enriched medium for definitive endoderm from the mouse (Nature Biotechnology 2005, 23:1534-1541; U.S. Patent No. 7,704,738). Transplantation of these cells under the kidney capsule resulted in the development of more mature cells with characteristics of endodermal tissue. In vivo differentiation of human embryonic stem cells into blastocysts occurred (U.S. Patent No. 7,704,738). The endodermal cells were further differentiated into PDX1-positive cells after the addition of FGF-10 and retinoic acid. (U.S. Patent Application Publication No. 2005 / 0266554) Subsequent transplantation of these pancreatic progenitor cells in the fat pad resulted in the development of 3-4 months of maturation. The formation of functional pancreatic endocrine cells occurred (see U.S. Patent Nos. 7,534,608 and 7,999). No. 3,920).

[0006] Small molecule inhibitors have been used for the induction of pancreatic endocrine progenitor cells. Small molecule inhibitors of β receptors and BMP receptors (Development 2011, 138 :861~871;Diabetes 2011,60:239~247) is a pancreatic endocrine In addition, small molecule activators have also been used to enhance the number of cells in the definitive endoderm. It has been used to generate pancreatic endoderm cells (Curr.Opin.Cell Biol., 2009, 21:727~732). Nature Chem.Biol .,2009,5:258~265).

[0007] In general, the differentiation of progenitor cells into functional beta cells occurs through various stages. Advance and improve protocols for generating pancreatic cells from progenitor cells such as human pluripotent stem cells Despite these advances in research, there are still many challenges in differentiating progenitor cells. Each step in the process presents unique challenges. There remains a need to develop further differentiation protocols, especially for the purpose of generating functional beta cells. In particular, the rapid and regulated glucose-stimulated β-cell response observed in functional beta cells Inhibition of glucose-responsive insulin-producing cells to enable insulin secretion ("GSIS"). It would be desirable to provide in vitro production of insulin, particularly phase 1 and phase 2 insulin. In vitro generation of functional beta cells that exhibit increased mitochondrial respiration / activity after secretion It is desirable to provide a method for

[0008] GSIS is a glucose transporter (solute carrier family 2, member 1; SLC2A1; or commonly referred to as glucose transporter 1; GLUT1 in human beta cells), and Glucose is converted into pyruvate through a process called glycolysis. The uptake of pyruvate into the mitochondria is initiated by the uptake of pyruvate into the beta cells. uptake, TCA (tricarboxylic acid), and the subsequent electron transport chain ("ETC," herein The activation of the mitochondrial cycle (called "mitochondrial activity" or "mitochondrial respiration") Its metabolism through insulin is tightly coupled to insulin exocytosis, resulting in rapid and Ensures the correct amount of insulin is released.

[0009] Functional beta cells within the pancreatic islets respond to rapid increases in glucose concentration in two consecutive phases. It has been shown that insulin secretion occurs in the periphery of the rectum (Henquin et al., Diabe (2009) 52(5):739-751). The amplitude and duration of both phases During this time, intracellular Ca 2+ It is regulated by signals or the dynamics of additional secreted binding factors. phase(1 st ) insulin secretion occurs through the binding of small, readily releasable insulin granules. The second phase (2 nd ) insulin secretion involves mobilizing granules from the granule storage pool and releasing The docking / priming of these cells for maturation is crucial for the maturation of beta cells. Biphasic GSIS, a key marker, is not detected until the postnatal phase of human development, suggesting immature basal GSIS. contrast to the monophasic GSIS seen in T cells (Otonkoski et al. Diabetes (1988) 37:286-291).

[0010] In type 2 diabetes, the first phase of GSIS is absent and the second phase of GSIS is also reduced. Type 1 diabetes, in which the number of tumor cells is significantly reduced due to autoimmune attack, is characterized by a robust biphasic GSI. It has been reported that S is absent (Krogvold et al. Diabetes (2 015)64:2506~2512). Summary of the Invention [Means for solving the problem]

[0011] As embodied and fully described, the present invention provides functional cells resulting from the differentiation of pluripotent stem cells. Methods for producing beta cells (functional pancreatic beta cells) and populations of cells in vitro - Patent Application 20070122997 In particular, the present invention provides methods for controlling mitochondrial respiration / activity response and biphasic insulin secretion. Responsive functional pancreatic beta cells (insulin-producing cells) or a population of functional beta cells Regarding the generation of

[0012] One aspect of the present invention is a method for differentiating pluripotent stem cells into functional beta cells. Certain embodiments of the present invention comprise producing pancreatic endoderm cells by expressing PDX1, NKX6.1, MAFA, UCN3, and and SLC2A1-expressing functional beta cells. In this method, the pancreatic endocrine cells are selected from the group consisting of UNC0638, UNC0642, UCN0646 , TC-E5003, A366, PF03814735, ZM447439, SB747 651A, PFI1, LY303511, MS436, AZT, DEZA, Pyroxamide , CI9994, or MC1568. This involves culturing in a medium.

[0013] An embodiment of the present invention is directed to a single hormone derived from in vitro differentiation of pancreatic endocrine cells. Functional pancreatic beta cells expressing insulin, PDX1, NKX6.1, and MAFA In some embodiments, the population of functional beta cells is an in vitro population of cells. In some embodiments, MAFA expression is not observed. In an embodiment, the in vitro expression of functional beta cells is increased compared to mature beta cells. The populations of pancreatic endocrine cells were identified as UNC0638, UNC0642, UCN0646, and TC- E5003, A366, PF03814735, ZM447439, SB747651A , PFI1, LY303511, MS436, AZT, DEZA, Pyroxamide, CI9 994, or MC1568. This is obtained by:

[0014] A further embodiment of the present invention relates to a method for producing pluripotent stem cells by expressing PDX1, NKX6.1, MAFA, U This is a method for differentiating CN3 and SLC2A-expressing functional beta cells. The method includes the steps of differentiating pluripotent stem cells into pancreatic endocrine cells, and differentiating the pancreatic endocrine cells with PDX1. Functional beta cells expressing NKX6.1, MAFA, UCN3, and SLC2A1 In an embodiment, MAFA expression is indicative of differentiation into immature beta cells. In an embodiment, the method comprises inducing pancreatic endocrine cells to express UNC0638, UNC0642, UCN0646, TC-E5003, A366, PF03814735 , ZM447439, SB747651A, PFI1, LY303511, MS436, One or two of AZT, DEZA, pyroxamide, CI9994, or MC1568 The method includes culturing the cells in a culture medium supplemented with one or more small molecules.

[0015] In each of the above-described embodiments, the culture medium contains ZM447439, heparin, N - supplemented with acetylcysteine and one or more of formulation I. In some embodiments, the culture medium contains one or more of T3, T4, and their analogs. In the above embodiment, the culture medium is further supplemented with an ALK5 inhibitor. In some embodiments, the culture medium lacks an ALK5 inhibitor. In some embodiments, the culture medium is supplemented with AZT or DEZA. In this embodiment, the culture medium is supplemented with both AZT and DEZA.

[0016] In the above-described embodiment of the invention, the medium is further supplemented with a gamma secretase inhibitor. In some of the above embodiments, the culture medium is supplemented with T3. In some embodiments, T3 is in the range of 1 nM to 1 μM. T3 ranges from 1 nM to 100 nM.

[0017] In a further embodiment of the invention described above, functional beta cells are of the following group: cells expressing markers characteristic of definitive endoderm; cells expressing markers characteristic of early intestinal cells; cells expressing markers characteristic of foregut endoderm cells; cells expressing markers characteristic of pancreatic endoderm cells cells expressing specific markers; pancreatic endocrine progenitor cells; and immature beta cells (pancreatic endocrine cells) One or more stages of cells selected from cells expressing markers characteristic of It is obtained by chemical differentiation.

[0018] In each of the above-described embodiments, the method includes culturing cells at an air-liquid interface. In some embodiments described above, the method comprises culturing cells in suspension, e.g. It involves culturing cell clusters in suspension.

[0019] In the above embodiment, differentiated functional beta cells have a higher In an embodiment, differentiated functional beta cells express MAFA at levels similar to those of immature beta cells. In some embodiments, the IL-16 receptor agonist expresses UCN3 at a higher level than beta cells. The expression of MAFA and UCN3 is increased compared to that in immature beta cells.

[0020] In the above embodiment of the invention, the population of functional beta cells is a glucose-stimulated insulin-dependent In the above-described embodiment, the cells exhibit insulin secretion and glucose-dependent mitochondrial respiration. In this study, glucose-stimulated insulin secretion and glucose-dependent mitochondrial respiration were observed in the human In the above-described embodiment of the invention, functional beta cells are expressed in a manner similar to that of human pancreatic islet cells. Cells secrete insulin in multiple phases.

[0021] In the above-described embodiment, glucose stimulation of glucose-dependent mitochondrial respiration The subsequent maximum oxygen consumption rate response ranges from approximately 20% to approximately 80% above the basal oxygen consumption rate. In an embodiment, the oxygen consumption rate response occurs at least 15 minutes after glucose stimulation. Ta.

[0022] In the above embodiment, glucose-stimulated insulin secretion is a measure of insulin secretion in response to glucose stimulation. In embodiments, the first phase of biphasic insulin secretion is Phase 1 is an increase of at least 4 to at least 8 times the basal secretion, and is the first phase of biphasic insulin secretion. The two phases are increased by at least two to at least four times the basal secretion. Sucrine secretion occurs at least 5 to at least 10 minutes after glucose stimulation.

[0023] In some embodiments, the method of differentiating pluripotent stem cells comprises differentiating the pluripotent stem cells from an embryo. Differentiation into cells expressing markers characteristic of definitive endoderm ("stage 1 cells") and stage 1 cells were classified as cells expressing markers characteristic of early intestinal cells ("stage 2"). and differentiation of stage 2 cells into foregut endoderm cells ("stage 2 cells"). and differentiating the stage 3 cells into cells expressing the Differentiation into cells expressing markers characteristic of germ layer cells ("stage 4 cells") and stage 4 cells were classified as pancreatic endoderm / endocrine progenitor cells (pancreatic endoderm cells and pancreatic endocrine progenitor cells). Differentiation into cells expressing one or both characteristic markers ("Stage 5"). and differentiates stage 5 cells into immature beta cells (pancreatic endocrine cells, "stage 6"). and differentiation of stage 6 cells into functional beta cells (pancreatic endocrine cells, "stage 7"). and

[0024] In an embodiment, the differentiation method comprises supplementing pluripotent stem cells with an MCX compound and GDF-8. By culturing in a medium containing the above-mentioned compound, pluripotent stem cells can be differentiated into stage 1 cells. Includes:

[0025] In an embodiment, the method comprises culturing Stage 1 cells supplemented with FGF7 and ascorbic acid. By culturing in a medium containing Includes:

[0026] In an embodiment, the method comprises inducing Stage 2 cells with FGF7, retinoic acid, SANT- 1. Culturing in a medium supplemented with PKC activators, BMP inhibitors, and ascorbic acid The method includes differentiating stage 2 cells into stage 3 cells by:

[0027] In an embodiment, the method comprises inducing stage 3 cells with FGF7, retinoic acid, SANT- 1. Culturing in a medium supplemented with PKC activators, BMP inhibitors, and ascorbic acid The method includes differentiating stage 3 cells into stage 4 cells by:

[0028] In an embodiment, the method comprises inducing stage 4 cells with SANT-1, PKC activators, BMPs, Stage 4 cells were cultured in medium supplemented with the inhibitor, ascorbic acid. In an embodiment, the medium contains T3, T4, or analogs thereof. Therefore, the medium is further supplemented with an ALK5 inhibitor.

[0029] In an embodiment, the method comprises treating stage 5 cells with a BMP inhibitor, ascorbic acid, T3 , T4, or one or more of their analogs. Thus, the present invention includes culturing stage 5 cells into pancreatic endocrine cells. The base is supplemented with T3. In some embodiments, T3 is in the range of 1 nM to 1 μM. In certain embodiments, T3 is in the range of 1 nM to 100 nM. In some embodiments, the medium is further supplemented with an ALK5 inhibitor. In some embodiments, the medium is not supplemented with an ALK5 inhibitor. In certain embodiments, the medium is further supplemented with a secretase inhibitor. 447439, heparin, N-acetylcysteine, and one or more of Preparation I In an embodiment, the medium is supplemented with AZT or DEZA. In some embodiments, the culture medium is supplemented with both AZT and DEZA. In some embodiments, the method comprises culturing cells at an air-liquid interface. In this method, the cells in suspension, for example, cell clusters in suspension, are cultured. Includes:

[0030] In each of the embodiments of the differentiation methods described above, pancreatic endoderm cells (“Stage 4 cells”) In some embodiments, the pancreatic endoderm cells may be transformed into pancreatic endocrine progenitor cells. Methods for differentiation into cells include culturing cryopreserved cells.

[0031] In each of the above-described embodiments, the pluripotent stem cells are human pluripotent stem cells, such as human embryonic stem cells. In one embodiment, the pluripotent stem cells are human H1 or H9 cells. It's okay to have it.

[0032] Another embodiment of the present invention relates to a method for producing pluripotent stem cells by expressing PDX1, NKX6.1, MAFA, UC The method is to differentiate into functional beta cells that express N3 and SLC2A, and Pluripotent stem cells were cultured in a medium supplemented with activin A and WNT3A. The resulting cells are then differentiated into cells that express markers characteristic of definitive endoderm. and (b) differentiation of cells expressing markers characteristic of definitive endoderm into immature beta cells. (c)UNC0638, UNC0642, UCN0646, TC-E50 03, A366, PF03814735, ZM447439, SB747651A, PF I1, LY303511, MS436, AZT, DEZA, pyroxamide, CI9994 Immature beta cells were cultured in a medium supplemented with one or more of MC1568 or MC1568. By culturing immature beta cells, PDX1, NKX6.1, MAFA, and UC and differentiating the cells into functional beta cells that express N3 and SLC2A1. The pluripotent stem cells used in the method may be human pluripotent stem cells, such as human embryonic stem cells. In one embodiment, the pluripotent stem cells are human CyT49 cells.

[0033] These functional beta cells contain glucose-dependent mitochondria similar to human pancreatic islet cells. Functional beta cells may also have the ability to stimulate adiposity or glucose-stimulated insulin secretion. Insulin may be secreted in multiple phases.

[0034] In certain embodiments of the method, the culture medium lacks an ALK5 inhibitor. In embodiments, the culture medium contains heparin, N-acetylcysteine, formulations I and T In other embodiments, the compound is further supplemented with one or more of T3, T4, or an analog thereof. In an alternative embodiment, the culture medium is supplemented with T3. 9. Heparin, N-acetylcysteine, and one of T3, T4, or their analogs The medium is supplemented with two or more of the following and does not contain an ALK5 inhibitor. DEZA may be supplemented.

[0035] The method can be carried out at an air-liquid interface, in suspended clusters, in roller bottles, or in microcarriers. In one embodiment, the method may include culturing immature beta cells on a substrate. In another embodiment, the method comprises culturing immature beta cells in a roller bottle. involves culturing immature beta cells in roller bottles on microcarriers.

[0036] In the present method, differentiating pluripotent stem cells into immature beta cells comprises (a) differentiation of pluripotent stem cells into immature beta cells; By culturing stem cells in a medium supplemented with activin A and WNT3A, pluripotency was enhanced. The development of sexual stem cells into cells expressing markers characteristic of definitive endoderm ("stage 1 cells"). (b) stage 1 cells express markers characteristic of early intestinal cells. (c) differentiation of the stage 2 cells into cells that are expressed in the foregut ("stage 2 cells"); Differentiation into cells expressing markers characteristic of germ layer cells ("stage 3 cells") and (d) stage 3 cells, which express markers characteristic of pancreatic endoderm cells ("stage 3 cells"). (e) differentiating stage 4 cells into pancreatic endocrine progenitor cells (f) differentiation into cells expressing characteristic markers (“stage 5 cells”); and differentiating stage 5 cells into immature beta cells. In an embodiment, steps e. and f. are carried out at the air-liquid interface, in the suspended clusters, in the roller balls. In another embodiment, steps e. and f. and f. comprise culturing the cells in roller bottles. Figures e and f show immature beta cells cultured in roller bottles on microcarriers. This includes:

[0037] In certain embodiments, the method comprises treating Stage 1 cells with FGF7 and ascorbic acid. Stage 1 cells differentiate into stage 2 cells by culturing them in acid-supplemented medium. In another embodiment, the method also includes inducing Stage 2 cells with FGF7, Cultured in medium supplemented with thiamin, SANT-1, PKC activators, and ascorbic acid. This also includes differentiating stage 2 cells into stage 3 cells by culturing them. In certain embodiments, the medium lacks a BMP inhibitor. The method involves treating stage 3 cells with FGF7, retinoic acid, SANT-1, PKC activators, and ATP. Stage 3 cells were cultured in medium supplemented with scorbic acid to differentiate into stage 4 cells. Again, in certain embodiments, the medium comprises BM P inhibitors are lacking.

[0038] In another embodiment, the method further comprises treating stage 4 cells with SANT-1, a PKC activator, a BMP Stage 4 cells were cultured in medium supplemented with α- and α-ascorbic acid. The differentiation of cells into stage 5 cells is carried out in a medium containing T3, T4, or their analogs. may be further supplemented with one or more of the above.

[0039] In an alternative embodiment, the method further comprises treating the stage 5 cells with a BMP inhibitor, ascorbic acid. The cells are cultured in a medium supplemented with one or more of the following: T3, T4, or their analogs. The process involves culturing stage 5 cells into immature beta cells by culturing the cells in the medium. The method may be further supplemented with an ALK5 inhibitor or a gamma secretase inhibitor. Mature beta cells lacked ALK5 inhibitors, ZM447439, AZT, and N-acetylcysteine. Tein, DEZA, Formulation I, and one or more of T3, T4, or their analogs Immature beta cells were cultured in a medium supplemented with PDX1, NKX6, and .1, MAFA, UCN3, and SLC2A-expressing functional beta cells This may include:

[0040] In each of the embodiments of the differentiation methods described above, the culture is a suspension culture or a cell aggregate suspension. In certain embodiments, the culture is performed in a roller bottle. The method may be carried out on microcarriers or on microcarriers in roller bottles. [Brief explanation of the drawings]

[0041] Figures 1A-1H show small molecules that upregulate MAFA or UCN3 expression at stage 7. The results of all screenings are shown. [Figure 1A]Stage 7 expression of MAFA was observed in S6D7, i.e., untreated cultures, or in cultures treated with UNC0638 (selective G9a and GLP histone lysine methyltransferase inhibitor), UNC0646 (potent and selective G9a / GLP inhibitor), UNC0642 (potent and selective G9a and GLP histone lysine methyltransferase inhibitor), TC-E5003 (selective PRMT1 arginine methyltransferase inhibitor), A366 (potent and selective G9a / GLP histone lysine methyltransferase inhibitor), PF03814735 (selective PRMT1 arginine methyltransferase inhibitor), and PF03814735 (selective PRMT1 arginine methyltransferase inhibitor). We demonstrate that DMSO-treated cultures were significantly upregulated at stage 7 after treatment with DMSO-treated α-amyloid β ... [Figure 1B]Stage 7 expression of MAFA was observed in S6D7, i.e., untreated cultures, or in cultures treated with UNC0638 (selective G9a and GLP histone lysine methyltransferase inhibitor), UNC0646 (potent and selective G9a / GLP inhibitor), UNC0642 (potent and selective G9a and GLP histone lysine methyltransferase inhibitor), TC-E5003 (selective PRMT1 arginine methyltransferase inhibitor), A366 (potent and selective G9a / GLP histone lysine methyltransferase inhibitor), PF03814735 (selective PRMT1 arginine methyltransferase inhibitor), and PF03814735 (selective PRMT1 arginine methyltransferase inhibitor). We demonstrate that DMSO-treated cultures were significantly upregulated at stage 7 after treatment with DMSO-treated α-amyloid β ... [Figure 1C]Stage 7 expression of MAFA was observed in S6D7, i.e., untreated cultures, or in cultures treated with UNC0638 (selective G9a and GLP histone lysine methyltransferase inhibitor), UNC0646 (potent and selective G9a / GLP inhibitor), UNC0642 (potent and selective G9a and GLP histone lysine methyltransferase inhibitor), TC-E5003 (selective PRMT1 arginine methyltransferase inhibitor), A366 (potent and selective G9a / GLP histone lysine methyltransferase inhibitor), PF03814735 (selective PRMT1 arginine methyltransferase inhibitor), and PF03814735 (selective PRMT1 arginine methyltransferase inhibitor). We demonstrate that DMSO-treated cultures were significantly upregulated at stage 7 after treatment with DMSO-treated α-amyloid β ... [Figure 1D]Stage 7 expression of MAFA was observed in S6D7, i.e., untreated cultures, or in cultures treated with UNC0638 (selective G9a and GLP histone lysine methyltransferase inhibitor), UNC0646 (potent and selective G9a / GLP inhibitor), UNC0642 (potent and selective G9a and GLP histone lysine methyltransferase inhibitor), TC-E5003 (selective PRMT1 arginine methyltransferase inhibitor), A366 (potent and selective G9a / GLP histone lysine methyltransferase inhibitor), PF03814735 (selective PRMT1 arginine methyltransferase inhibitor), and PF03814735 (selective PRMT1 arginine methyltransferase inhibitor). We demonstrate that DMSO-treated cultures were significantly upregulated at stage 7 after treatment with DMSO-treated α-amyloid β ... [Figure 1E] We demonstrate that stage 7 expression of UCN3 was increased in S6D7, i.e., untreated cultures, or after the addition of 5-azacytidine ("AZT") (a DNA methyltransferase inhibitor), pyroxamide (a histone deacetylase inhibitor), and CI994 (a histone deacetylase inhibitor), compared to DMSO-treated cultures at stage 7. All conditions are S7D7, ALI cluster. [Figure 1F] We demonstrate that stage 7 expression of UCN3 was increased in S6D7, i.e., untreated cultures, or after the addition of 5-azacytidine ("AZT") (a DNA methyltransferase inhibitor), pyroxamide (a histone deacetylase inhibitor), and CI994 (a histone deacetylase inhibitor), compared to DMSO-treated cultures at stage 7. All conditions are S7D7, ALI cluster. [Figure 1G]We demonstrate that stage 7 expression of UCN3 was increased in S6D7, i.e., untreated cultures, or after the addition of 5-azacytidine ("AZT") (a DNA methyltransferase inhibitor), pyroxamide (a histone deacetylase inhibitor), and CI994 (a histone deacetylase inhibitor), compared to DMSO-treated cultures at stage 7. All conditions are S7D7, ALI cluster. [Figure 1H] We demonstrate that stage 7 expression of UCN3 was increased in S6D7, i.e., untreated cultures, or after the addition of 5-azacytidine ("AZT") (a DNA methyltransferase inhibitor), pyroxamide (a histone deacetylase inhibitor), and CI994 (a histone deacetylase inhibitor), compared to DMSO-treated cultures at stage 7. All conditions are S7D7, ALI cluster. [Figure 2A] Figures 2A and 2B demonstrate the robustness of the small molecules selected as upregulators of either MAFA or UCN3, as their effects were maintained across different stage 7 conditioning protocols. 3-Deazaneplanocin A ("DEZA") was found to be an effective upregulator of MAFA, but not UCN3 (Figure 2A). AZT was confirmed as an upregulator of UCN3, but not MAFA, during stage 7 (Figure 2B). All conditions were in the S7D7, ALI cluster. [Figure 2B] Figures 2A and 2B demonstrate the robustness of the small molecules selected as upregulators of either MAFA or UCN3, as their effects were maintained across different stage 7 conditioning protocols. 3-Deazaneplanocin A ("DEZA") was found to be an effective upregulator of MAFA, but not UCN3 (Figure 2A). AZT was confirmed as an upregulator of UCN3, but not MAFA, during stage 7 (Figure 2B). All conditions were in the S7D7, ALI cluster. [Figure 3A]This figure demonstrates that gene expression of a set of maturation markers in ALI cell clusters increased to levels observed in human islet cells after 7 days of modified stage 7 conditioning. The modifications used during stage 7 differentiation included: (i) removal of ALK5 inhibitor II; (ii) reduction of T3 concentration; (iii) addition of DEZA; (iv) addition of AZT; (v) addition of ZM; (vi) reduction of glucose concentration to 5.56 mM; and (vii) addition of a defined cocktail of vitamins, non-essential amino acids, lipids, sodium pyruvate, and trace elements (Formulation I - Table XII). Figure 3A shows that INHBB was enriched above expression seen in human islet cells in the ALK5 inhibitor II condition, and removal of the ALK5 inhibitor in the presence or absence of AZT / DEZA reduced INHBB to levels seen in human islet cells. All conditions were S7D7, ALI cluster. [Figure 3B] Figure 3B demonstrates that gene expression of a set of maturation markers in ALI cell clusters increased to levels observed in human islet cells after 7 days of modified stage 7 conditioning. The modifications used during stage 7 differentiation included: (i) removal of ALK5 inhibitor II; (ii) reduction in T3 concentration; (iii) addition of DEZA; (iv) addition of AZT; (v) addition of ZM; (vi) reduction in glucose concentration to 5.56 mM; and (vii) addition of a defined cocktail of vitamins, non-essential amino acids, lipids, sodium pyruvate, and trace elements (Formulation I - Table XII). Figure 3B shows that INHA expression increased in ALI clusters to levels observed in human islet cells upon removal of ALK5 inhibitor II. All conditions were S7D7, ALI clusters. [Figure 3C]We demonstrate that gene expression of a set of maturation markers in ALI cell clusters increased to levels observed in human islet cells after 7 days of modified stage 7 conditioning. The modifications used during stage 7 differentiation included: (i) removal of ALK5 inhibitor II; (ii) reduction of T3 concentration; (iii) addition of DEZA; (iv) addition of AZT; (v) addition of ZM; (vi) reduction of glucose concentration to 5.56 mM; and (vii) addition of a defined cocktail of vitamins, non-essential amino acids, lipids, sodium pyruvate, and trace elements (Formulation I - Table XII). MAFA and SLC2A1 expression, shown in Figures 3C and 3D, respectively, was reduced by removal of ALK5 inhibitor II, while the addition of AZT / DEZA rescued MAFA and SLC2A1 expression to human islet cell levels. All conditions were S7D7, ALI clusters. [Figure 3D] We demonstrate that gene expression of a set of maturation markers in ALI cell clusters increased to levels observed in human islet cells after 7 days of modified stage 7 conditioning. The modifications used during stage 7 differentiation included: (i) removal of ALK5 inhibitor II; (ii) reduction of T3 concentration; (iii) addition of DEZA; (iv) addition of AZT; (v) addition of ZM; (vi) reduction of glucose concentration to 5.56 mM; and (vii) addition of a defined cocktail of vitamins, non-essential amino acids, lipids, sodium pyruvate, and trace elements (Formulation I - Table XII). MAFA and SLC2A1 expression, shown in Figures 3C and 3D, respectively, was reduced by removal of ALK5 inhibitor II, while the addition of AZT / DEZA rescued MAFA and SLC2A1 expression to human islet cell levels. All conditions were S7D7, ALI clusters. [Figure 3E]We demonstrate that gene expression of a set of maturation markers in ALI cell clusters increased to levels observed in human islet cells after 7 days of modified stage 7 conditioning. The modifications used during stage 7 differentiation included: (i) removal of ALK5 inhibitor II; (ii) reduction in T3 concentration; (iii) addition of DEZA; (iv) addition of AZT; (v) addition of ZM; (vi) reduction in glucose concentration to 5.56 mM; and (vii) addition of a defined cocktail of vitamins, non-essential amino acids, lipids, sodium pyruvate, and trace elements (Formulation I - Table XII). UCN3 expression increased to or above the levels of human islet cells with the addition of AZT / DEZA and removal of ALK5 inhibitor II in ALI clusters. All conditions were S7D7, ALI clusters. [Figure 3F] We demonstrate that gene expression of a set of maturation markers in ALI cell clusters increased to levels observed in human islet cells after 7 days of modified stage 7 conditioning. The modifications used during stage 7 differentiation included: (i) removal of ALK5 inhibitor II; (ii) reduction of T3 concentration; (iii) addition of DEZA; (iv) addition of AZT; (v) addition of ZM; (vi) reduction of glucose concentration to 5.56 mM; and (vii) addition of a defined cocktail of vitamins, non-essential amino acids, lipids, sodium pyruvate, and trace elements (Formulation I - Table XII). G6PC2 (glucose-6-phosphatase catalytic subunit 2) expression increased to or above the levels of human islet cells with the addition of AZT / DEZA and removal of ALK5 inhibitor II in ALI clusters. All conditions were S7D7, ALI clusters. [Figure 3G]We demonstrate that gene expression of a set of maturation markers in ALI cell clusters increased to levels observed in human islet cells after 7 days of modified stage 7 conditioning. The modifications used during stage 7 differentiation included: (i) removal of ALK5 inhibitor II; (ii) reduction of T3 concentration; (iii) addition of DEZA; (iv) addition of AZT; (v) addition of ZM; (vi) reduction of glucose concentration to 5.56 mM; and (vii) addition of a defined cocktail of vitamins, non-essential amino acids, lipids, sodium pyruvate, and trace elements (Formulation I - Table XII). PDK1 (pyruvate dehydrogenase kinase 1) expression increased to or above the levels of human islet cells with the addition of AZT / DEZA and removal of ALK5 inhibitor II in ALI clusters. All conditions were S7D7, ALI clusters. [Figure 3H] We demonstrate that gene expression of a set of maturation markers in ALI cell clusters increased to levels observed in human islet cells after 7 days of modified stage 7 conditioning. The modifications used during stage 7 differentiation included: (i) removal of ALK5 inhibitor II; (ii) reduction of T3 concentration; (iii) addition of DEZA; (iv) addition of AZT; (v) addition of ZM; (vi) reduction of glucose concentration to 5.56 mM; and (vii) addition of a defined cocktail of vitamins, non-essential amino acids, lipids, sodium pyruvate, and trace elements (Formulation I - Table XII). INS (insulin) expression was gradually increased in a stepwise manner, first by removal of ALK5 inhibitor II, and then by addition of AZT / DEZA. All conditions were for the S7D7 ALI cluster. [Figure 3I]We demonstrate that gene expression of a set of maturation markers in ALI cell clusters increased to levels observed in human islet cells after 7 days of modified stage 7 conditioning. The modifications used during stage 7 differentiation included: (i) removal of ALK5 inhibitor II; (ii) reduction of T3 concentration; (iii) addition of DEZA; (iv) addition of AZT; (v) addition of ZM; (vi) reduction of glucose concentration to 5.56 mM; and (vii) addition of a defined cocktail of vitamins, non-essential amino acids, lipids, sodium pyruvate, and trace elements (Formulation I - Table XII). Expression of GJD2 (gap junction protein, delta 2; also CX36 / connexin 36) was gradually and stepwise increased, first by removal of ALK5 inhibitor II and then by addition of AZT / DEZA. All conditions were for the S7D7 ALI cluster. [Figure 3J] We demonstrate that gene expression of a set of maturation markers in ALI cell clusters increased to levels observed in human islet cells after 7 days of modified stage 7 conditioning. The modifications used during stage 7 differentiation included: (i) removal of ALK5 inhibitor II; (ii) reduction of T3 concentration; (iii) addition of DEZA; (iv) addition of AZT; (v) addition of ZM; (vi) reduction of glucose concentration to 5.56 mM; and (vii) addition of a defined cocktail of vitamins, non-essential amino acids, lipids, sodium pyruvate, and trace elements (Formulation I - Table XII). Expression of SIX2 (SIX homeobox 2) was gradually increased in a stepwise manner, first by removal of ALK5 inhibitor II, and then by addition of AZT / DEZA. All conditions were for the S7D7 ALI cluster. [Figure 3K]We demonstrate that gene expression of a set of maturation markers in ALI cell clusters increased to levels observed in human islet cells after 7 days of modified stage 7 conditioning. The modifications used during stage 7 differentiation included: (i) removal of ALK5 inhibitor II; (ii) reduction of T3 concentration; (iii) addition of DEZA; (iv) addition of AZT; (v) addition of ZM; (vi) reduction of glucose concentration to 5.56 mM; and (vii) addition of a defined cocktail of vitamins, non-essential amino acids, lipids, sodium pyruvate, and trace elements (Formulation I - Table XII). PDX1 expression was gradually and stepwise increased, first by removal of ALK5 inhibitor II, and then by addition of AZT / DEZA. All conditions were for the S7D7 ALI cluster. [Figure 3L] We demonstrate that gene expression of a set of maturation markers in ALI cell clusters increased to levels observed in human islet cells after 7 days of modified stage 7 conditioning. The modifications used during stage 7 differentiation included: (i) removal of ALK5 inhibitor II; (ii) reduction in T3 concentration; (iii) addition of DEZA; (iv) addition of AZT; (v) addition of ZM; (vi) reduction in glucose concentration to 5.56 mM; and (vii) addition of a defined cocktail of vitamins, non-essential amino acids, lipids, sodium pyruvate, and trace elements (Formulation I - Table XII). While NKX6.1 expression did not change significantly across conditions, its expression in ALI clusters consistently met or exceeded levels observed in human islet cells. All conditions were S7D7, ALI clusters. [Figure 3M]We demonstrate that gene expression of a set of maturation markers in ALI cell clusters increased to levels observed in human islet cells after 7 days of modified stage 7 conditioning. The modifications used during stage 7 differentiation included: (i) removal of ALK5 inhibitor II; (ii) reduction in T3 concentration; (iii) addition of DEZA; (iv) addition of AZT; (v) addition of ZM; (vi) reduction in glucose concentration to 5.56 mM; and (vii) addition of a defined cocktail of vitamins, non-essential amino acids, lipids, sodium pyruvate, and trace elements (Formulation I - Table XII). While expression of GLP1R (glucagon-like peptide 1 receptor) did not change significantly across conditions, its expression in ALI clusters consistently met or exceeded levels observed in human islet cells. All conditions were S7D7, ALI clusters. [Figure 4A] From a protein abundance perspective, we demonstrate the generation of C-peptide cells in ALI cell clusters co-expressing maturation markers: PDX1 (Figure 4A), NKX6.1 (Figure 4B), MAFA (Figure 4C), SLC2A1 (Figure 4D), and UCN3 (Figure 4E) in S7D7. Representative human islet cell staining is shown in the left column. The no ALK5, low T3, ZM, H, NAC, AZT / DEZA, FI, and BLAR001 condition is shown in the middle column. The no ALK5, low T3, ZM, H, NAC, AZT / DEZA, FI, and BLAR004 condition is shown in the right column. All conditions are S7D7, ALI clusters. [Figure 4B] From a protein abundance perspective, we demonstrate the generation of C-peptide cells in ALI cell clusters co-expressing maturation markers: PDX1 (Figure 4A), NKX6.1 (Figure 4B), MAFA (Figure 4C), SLC2A1 (Figure 4D), and UCN3 (Figure 4E) in S7D7. Representative human islet cell staining is shown in the left column. The no ALK5, low T3, ZM, H, NAC, AZT / DEZA, FI, and BLAR001 condition is shown in the middle column. The no ALK5, low T3, ZM, H, NAC, AZT / DEZA, FI, and BLAR004 condition is shown in the right column. All conditions are S7D7, ALI clusters. [Figure 4C]From a protein abundance perspective, we demonstrate the generation of C-peptide cells in ALI cell clusters co-expressing maturation markers: PDX1 (Figure 4A), NKX6.1 (Figure 4B), MAFA (Figure 4C), SLC2A1 (Figure 4D), and UCN3 (Figure 4E) in S7D7. Representative human islet cell staining is shown in the left column. The no ALK5, low T3, ZM, H, NAC, AZT / DEZA, FI, and BLAR001 condition is shown in the middle column. The no ALK5, low T3, ZM, H, NAC, AZT / DEZA, FI, and BLAR004 condition is shown in the right column. All conditions are S7D7, ALI clusters. [Figure 4D] From a protein abundance perspective, we demonstrate the generation of C-peptide cells in ALI cell clusters co-expressing maturation markers: PDX1 (Figure 4A), NKX6.1 (Figure 4B), MAFA (Figure 4C), SLC2A1 (Figure 4D), and UCN3 (Figure 4E) in S7D7. Representative human islet cell staining is shown in the left column. The no ALK5, low T3, ZM, H, NAC, AZT / DEZA, FI, and BLAR001 condition is shown in the middle column. The no ALK5, low T3, ZM, H, NAC, AZT / DEZA, FI, and BLAR004 condition is shown in the right column. All conditions are S7D7, ALI clusters. [Figure 4E] From a protein abundance perspective, we demonstrate the generation of C-peptide cells in ALI cell clusters co-expressing maturation markers: PDX1 (Figure 4A), NKX6.1 (Figure 4B), MAFA (Figure 4C), SLC2A1 (Figure 4D), and UCN3 (Figure 4E) in S7D7. Representative human islet cell staining is shown in the left column. The no ALK5, low T3, ZM, H, NAC, AZT / DEZA, FI, and BLAR001 condition is shown in the middle column. The no ALK5, low T3, ZM, H, NAC, AZT / DEZA, FI, and BLAR004 condition is shown in the right column. All conditions are S7D7, ALI clusters. [Figure 5A]We demonstrate the generation of C-peptide cells in S7D7 cells that exhibit glucose-dependent mitochondrial respiration kinetics similar to human islets at ALI, specifically through stage 7-specific "no ALK5, low T3, ZM, H, NAC, AZT, DEZA, FI, BLAR001" conditioning. Figure 5A shows that human islets rapidly responded to high D-glucose and maintained a high OCR over time (131.5% ± 11.32; 72 min post-injection), as demonstrated by an OCR exceeding baseline by 123.3% ± 12.92 at 15 min post-injection ("ip"). Conversely, S6D7 ALI clusters enriched for immature C-peptide-positive cells lacked a rapid OCR response to high D-glucose (104.4% ± 3.37; 15 min post-injection) and exhibited a relatively weak OCR response over time (113.3% ± 4.51; 72 min post-injection). [Figure 5B] We demonstrate the generation of C-peptide cells in S7D7 cells that exhibit glucose-dependent mitochondrial respiration kinetics similar to those of human islets in ALI by conditioning with a specific "no ALK5, low T3, ZM, H, NAC, AZT, DEZA, FI, BLAR001" strategy specifically for stage 7. The S7D7 ALI cluster conditions exhibited glucose-dependent mitochondrial kinetics indistinguishable from immature S6D7 ALI clusters: ALK5, T3, ZM, H, NAC, FI, BLAR001 (100.3% ± 4.04 at 15 min post-injection; 107.8% ± 6.51 at 72 min post-injection). [Figure 5C] We demonstrate the generation of C-peptide cells in S7D7 cells with glucose-dependent mitochondrial respiration kinetics similar to those of human islets in ALI by conditioning with the stage 7-specific "no ALK5, low T3, ZM, H, NAC, AZT, DEZA, FI, BLAR001" strategy. The S7D7 ALI cluster conditions exhibited glucose-dependent mitochondrial kinetics indistinguishable from immature S6D7 ALI clusters: "no ALK5, low T3, ZM, H, NAC, FI, BLAR001" (96.9% ± 3.06 at 15 min post-injection; 109.0% ± 4.58 at 72 min post-injection). [Figure 5D]We demonstrate the generation of C-peptide cells in S7D7 cells that exhibit glucose-dependent mitochondrial respiratory kinetics similar to that of human islets in ALI, specifically through the stage 7-specific "no ALK5, low T3, ZM, H, NAC, AZT, DEZA, FI, BLAR001" conditioning. Figure 5D demonstrates that among the S7D7 ALI clusters, only the "no ALK5, low T3, ZM, H, NAC, AZT / DEZA, FI, BLAR001" condition exhibited glucose-dependent mitochondrial respiratory kinetics similar to that of human islets (112.4% ± 3.25 at 15 min postinjection; 129.5% ± 3.78 at 72 min postinjection). [Figure 5E] We demonstrate the generation of C-peptide cells in S7D7 cells with glucose-dependent mitochondrial respiration kinetics similar to those of human islets in ALI by conditioning with the stage 7-specific "no ALK5, low T3, ZM, H, NAC, AZT, DEZA, FI, BLAR001" strategy. The S7D7 ALI cluster conditions exhibited glucose-dependent mitochondrial kinetics indistinguishable from immature S6D7 ALI clusters: "no ALK5, low T3, ZM, H, NAC, FI, BLAR004" (103.4% ± 4.76 at 15 min postinjection; 113.6% ± 6.72 at 72 min postinjection). [Figure 5F] We demonstrate that the stage 7-specific "no ALK5, low T3, ZM, H, NAC, AZT, DEZA, FI, BLAR001" conditioning generates C-peptide cells in S7D7 cells that exhibit glucose-dependent mitochondrial respiration kinetics similar to those of human islets in ALI. The S7D7 ALI cluster conditions exhibited glucose-dependent mitochondrial kinetics indistinguishable from immature S6D7 ALI clusters: "no ALK5, low T3, ZM, H, NAC, AZT / DEZA, FI, BLAR004" (102.1% ± 4.04 at 15 min postinjection; 112.7% ± 3.38 at 72 min postinjection). [Figure 6A]Figure 6A demonstrates Aggrewell™ cell clusters differentiated into immature pancreatic beta cells in suspension culture. Figure 6A depicts the procedure through which (i) fresh S4D3 monolayers or S4D3 cryopreserved cells are assembled into (ii) cell clusters by the Aggrewell™ method. [Figure 6B] Figure 6B demonstrates Aggrewell™ cell clusters differentiated into immature pancreatic beta cells in suspension culture. High protein abundance of the pancreatic endoderm TFs PDX1 (top row, left) and NKX6.1 (top row, center) was detected in S4D5 Aggrewell™ clusters, while low protein abundance of the endocrine TF NEUROD1 (top row, right), the alternative non-pancreatic endoderm lineage-allocating TFs SOX2 (bottom row, center), and CDX2 (bottom row, right) was detected. [Figure 6C] Figure 6C demonstrates Aggrewell™ cell clusters differentiated into immature pancreatic beta cells in suspension culture. FACS analysis shows that 99.3±0.1% of cells were PDX1+ (left), 84.4±0.1% were NKX6.1+ (center), while 2.2±0.4% were NKX6.1+ NEUROD1+ (right), or 1.35±0.55% were NKX6.1+ CHGA+ (center) (S4DS Aggrewell™ clusters). [Figure 6D] Figure 6D demonstrates Aggrewell™ cell clusters differentiated into immature pancreatic beta cells in suspension culture. Compared to S4D3 monolayers, Figure 6D shows that robust pancreatic endoderm characteristics were maintained in S4D4 Aggrewell™ clusters derived from S4D3 cryopreserved cells, as high gene expression of PDX1 (top row, left), NKX6.1 (top row, right), and low expression of NEUROD1 (bottom row, left) and CHGA (bottom row, right) was maintained. [Figure 6E]Demonstrating Aggrewell™ cell clusters differentiated into immature pancreatic beta cells in suspension culture. Figure 6E depicts the procedure through which (i) fresh S4D3 monolayers or S4D3 cryopreserved cells are (ii) assembled into cell clusters by the Aggrewell™ method, (iii) differentiated in suspension culture by S6D6, and (iv) differentiated into immature beta cells. [Figure 6F] Figure 6 demonstrates Aggrewell™ cell clusters differentiated into immature pancreatic beta cells in suspension culture. FACS analysis of S6D6 Aggrewell™ clusters reveals a robust immature beta cell protein profile: 78.5 ± 1.87% of cells were NKX6.1+ CHGA+ (left, Figure 6F), 73.6 ± 4.34% were NKX6.1+ NEUROD1+ (right, Figure 6F), and 38.4 ± 5.96% were NKX6.1+ insulin+ (left, Figure 6G). The majority of the insulin-positive population (50.2 ± 6.92% of total cells were insulin+) was NKX6.1+ (left, Figure 6G), whereas the glucagon-positive population (7.43 ± 1.49% insulin+ glucagon+; right, Figure 6G) was not. [Figure 6G] Figure 6 demonstrates Aggrewell™ cell clusters differentiated into immature pancreatic beta cells in suspension culture. FACS analysis of S6D6 Aggrewell™ clusters reveals a robust immature beta cell protein profile: 78.5 ± 1.87% of cells were NKX6.1+ CHGA+ (left, Figure 6F), 73.6 ± 4.34% were NKX6.1+ NEUROD1+ (right, Figure 6F), and 38.4 ± 5.96% were NKX6.1+ insulin+ (left, Figure 6G). The majority of the insulin-positive population (50.2 ± 6.92% of total cells were insulin+) was NKX6.1+ (left, Figure 6G), whereas the glucagon-positive population (7.43 ± 1.49% insulin+ glucagon+; right, Figure 6G) was not. [Figure 6H]Figure 6H demonstrates Aggrewell™ cell clusters differentiated into immature pancreatic beta cells in suspension culture, where at S6D6 the majority of C-peptide positive cells in the Aggrewell™ cluster were both PDX1+ (left) and NKX6.1+ (center). [Figure 6I] Figure 6I demonstrates Aggrewell™ cell clusters differentiated into immature pancreatic beta cells in suspension culture. The protein presence of the maturation gatekeeper TF MAFA (right) was already readily detected at S6D6 and was expressed at higher levels than in previous ALI clusters at S6D6-S6D7 (Nature Biotechnology, 2014(32)11, 1121-1133) (see also Figure 3C for ALI S7D7 comparison). [Figure 6J] Demonstration of Aggrewell™ cell clusters differentiated into immature pancreatic beta cells in suspension culture. Insulin expression was higher than in previous ALI clusters S6D6-S6D7. [Figure 6K] Demonstration of Aggrewell™ cell clusters differentiated into immature pancreatic beta cells in suspension culture. PDX1 expression was higher than in previous ALI clusters S6D6-S6D7. [Figure 6L] Demonstration of Aggrewell™ cell clusters differentiated into immature pancreatic beta cells in suspension culture. NKX6.1 expression was higher than in previous ALI clusters S6D6-S6D7. [Figure 7A]

[0049] Figure 7A illustrates the generation of S7D7 Aggrewell™ clusters by suspension culture to mature pancreatic beta cells. Figure 7A depicts the steps through which (i) new S4D3 monolayers or S4D3 cryopreserved cells assemble into Aggrewell™ clusters, (ii & iii) conditioning in suspension culture through stages 5, 6, and 7, and (iv) generating S7D7 Aggrewell™ clusters. [Figure 7B]We demonstrate the generation of S7D7 Aggrewell™ clusters from suspension culture to mature pancreatic beta cells. S7D7 Aggrewell™ clusters cultured during stage 7 with BLAR001 (no ALK5, low T3, ZM, H, NAC, DEZA, AZT, FI) maintained baseline beta cell protein profiles (Figures 7B-7C). 89% of cells were NKX6.1+ CHGA+ (left, Figure 7B), and 77.7% were NKX6.1+ NEUROD1+ (right, Figure 7B). [Figure 7C] We demonstrate the generation of S7D7 Aggrewell™ clusters by suspension culture into mature pancreatic beta cells. S7D7 Aggrewell™ clusters cultured with BLAR001 (no ALK5, low T3, ZM, H, NAC, DEZA, AZT, FI) during stage 7 maintained baseline beta cell protein profiles (Figures 7B-7C). 40.8% of cells were NKX6.1+ insulin+ (left, Figure 7C). The majority of the insulin-positive population (46.4% of all cells were insulin+) was NKX6.1+ (left, Figure 7C), and none were glucagon-positive (3.9% insulin+ glucagon+; (right, Figure 7C). [Figure 7D] We demonstrate the generation of S7D7 Aggrewell™ clusters from suspension culture to mature pancreatic beta cells. Gene expression of maturation markers MAFA (FIG. 7D), UCN3 (FIG. 7E), SLC2A1 (FIG. 7F), G6PC2 (FIG. 7G), insulin (FIG. 7H), and NKX6.1 (FIG. 7I) was at or above human islet levels in S7D7 Aggrewell™ clusters conditioned with stage 7 "no ALK5, low T3, ZM, H, NAC, DEZA, AZT, FI, BLAR001, or BLAR004." The expression levels of maturation markers such as MAFA (Figure 3C), G2PC2 (Figure 3F), insulin (Figure 3H), and NKX6.1 (Figure 3L) in Aggrewell™ ("No ALK5, Low T3, ZM, H, NAC, DEZA, AZT, FI, BLAR001 or BLAR004") S7D7 were much higher than those in the ALI cluster. [Figure 7E] We demonstrate the generation of S7D7 Aggrewell™ clusters from suspension culture to mature pancreatic beta cells. Gene expression of maturation markers MAFA (FIG. 7D), UCN3 (FIG. 7E), SLC2A1 (FIG. 7F), G6PC2 (FIG. 7G), insulin (FIG. 7H), and NKX6.1 (FIG. 7I) was at or above human islet levels in S7D7 Aggrewell™ clusters conditioned with stage 7 "no ALK5, low T3, ZM, H, NAC, DEZA, AZT, FI, BLAR001, or BLAR004." The expression levels of maturation markers such as MAFA (Figure 3C), G2PC2 (Figure 3F), insulin (Figure 3H), and NKX6.1 (Figure 3L) in Aggrewell™ ("No ALK5, Low T3, ZM, H, NAC, DEZA, AZT, FI, BLAR001 or BLAR004") S7D7 were much higher than those in the ALI cluster. [Figure 7F] We demonstrate the generation of S7D7 Aggrewell™ clusters from suspension culture to mature pancreatic beta cells. Gene expression of maturation markers MAFA (FIG. 7D), UCN3 (FIG. 7E), SLC2A1 (FIG. 7F), G6PC2 (FIG. 7G), insulin (FIG. 7H), and NKX6.1 (FIG. 7I) was at or above human islet levels in S7D7 Aggrewell™ clusters conditioned with stage 7 "no ALK5, low T3, ZM, H, NAC, DEZA, AZT, FI, BLAR001, or BLAR004." The expression levels of maturation markers such as MAFA (Figure 3C), G2PC2 (Figure 3F), insulin (Figure 3H), and NKX6.1 (Figure 3L) in Aggrewell™ ("No ALK5, Low T3, ZM, H, NAC, DEZA, AZT, FI, BLAR001 or BLAR004") S7D7 were much higher than those in the ALI cluster. [Figure 7G]We demonstrate the generation of S7D7 Aggrewell™ clusters from suspension culture to mature pancreatic beta cells. Gene expression of maturation markers MAFA (FIG. 7D), UCN3 (FIG. 7E), SLC2A1 (FIG. 7F), G6PC2 (FIG. 7G), insulin (FIG. 7H), and NKX6.1 (FIG. 7I) was at or above human islet levels in S7D7 Aggrewell™ clusters conditioned with stage 7 "no ALK5, low T3, ZM, H, NAC, DEZA, AZT, FI, BLAR001, or BLAR004." The expression levels of maturation markers such as MAFA (Figure 3C), G2PC2 (Figure 3F), insulin (Figure 3H), and NKX6.1 (Figure 3L) in Aggrewell™ ("No ALK5, Low T3, ZM, H, NAC, DEZA, AZT, FI, BLAR001 or BLAR004") S7D7 were much higher than those in the ALI cluster. [Figure 7H] We demonstrate the generation of S7D7 Aggrewell™ clusters from suspension culture to mature pancreatic beta cells. Gene expression of maturation markers MAFA (FIG. 7D), UCN3 (FIG. 7E), SLC2A1 (FIG. 7F), G6PC2 (FIG. 7G), insulin (FIG. 7H), and NKX6.1 (FIG. 7I) was at or above human islet levels in S7D7 Aggrewell™ clusters conditioned with stage 7 "no ALK5, low T3, ZM, H, NAC, DEZA, AZT, FI, BLAR001, or BLAR004." The expression levels of maturation markers such as MAFA (Figure 3C), G2PC2 (Figure 3F), insulin (Figure 3H), and NKX6.1 (Figure 3L) in Aggrewell™ ("No ALK5, Low T3, ZM, H, NAC, DEZA, AZT, FI, BLAR001 or BLAR004") S7D7 were much higher than those in the ALI cluster. [Figure 7I]We demonstrate the generation of S7D7 Aggrewell™ clusters from suspension culture to mature pancreatic beta cells. Gene expression of maturation markers MAFA (FIG. 7D), UCN3 (FIG. 7E), SLC2A1 (FIG. 7F), G6PC2 (FIG. 7G), insulin (FIG. 7H), and NKX6.1 (FIG. 7I) was at or above human islet levels in S7D7 Aggrewell™ clusters conditioned with stage 7 "no ALK5, low T3, ZM, H, NAC, DEZA, AZT, FI, BLAR001, or BLAR004." The expression levels of maturation markers such as MAFA (Figure 3C), G2PC2 (Figure 3F), insulin (Figure 3H), and NKX6.1 (Figure 3L) in Aggrewell™ ("No ALK5, Low T3, ZM, H, NAC, DEZA, AZT, FI, BLAR001 or BLAR004") S7D7 were much higher than those in the ALI cluster. [Figure 7J] Figures 7J-7M demonstrate the generation of S7D7 Aggrewell™ clusters in suspension culture into mature pancreatic beta cells. Addition of DEZA and AZT to the "BLAR004 with no ALK5, low T3, ZM, H, NAC, FI" stage 7 conditioning regimen generated significant numbers of non-glucagon (Figure 7J; bottom right) C-peptide cells (co-expressed in S7D7 in terms of protein abundance), PDX1 (Figure 7J; bottom left), NKX6.1 (Figure 7J; bottom center), MAFA (Figure 7K; bottom left), UCN3 (Figure 7K; bottom center), and SLC2A1 (Figure 7K; bottom right). [Figure 7K] Figures 7J-7M demonstrate the generation of S7D7 Aggrewell™ clusters in suspension culture into mature pancreatic beta cells. Addition of DEZA and AZT to the "BLAR004 with no ALK5, low T3, ZM, H, NAC, FI" stage 7 conditioning regimen generated significant numbers of non-glucagon (Figure 7J; bottom right) C-peptide cells (co-expressed in S7D7 in terms of protein abundance), PDX1 (Figure 7J; bottom left), NKX6.1 (Figure 7J; bottom center), MAFA (Figure 7K; bottom left), UCN3 (Figure 7K; bottom center), and SLC2A1 (Figure 7K; bottom right). [Figure 7L] Figures 7J-7M demonstrate the generation of S7D7 Aggrewell™ clusters in suspension culture into mature pancreatic beta cells. Addition of DEZA and AZT to the "BLAR004 with no ALK5, low T3, ZM, H, NAC, FI" stage 7 conditioning regimen generated significant numbers of non-glucagon (Figure 7J; bottom right) C-peptide cells (co-expressed in S7D7 in terms of protein abundance), PDX1 (Figure 7J; bottom left), NKX6.1 (Figure 7J; bottom center), MAFA (Figure 7K; bottom left), UCN3 (Figure 7K; bottom center), and SLC2A1 (Figure 7K; bottom right). Figures 7L-7M demonstrate the generation of C-peptide cells by the "BLAR004 with no ALK5, low T3, ZM, H, NAC, FI" specific stage 7 conditioning, which are co-expressed by S7D7 maturation markers; PDX1 (Figure 7L; bottom row, bottom), NKX6.1 (Figure 7L; bottom row, center), MAFA (Figure 7M; bottom row, bottom), and SLC2A1 (Figure 7M; bottom row, right), but not by glucagon (Figure 7L; bottom row, right) or UCN3 (Figure 7M; bottom row, center), in terms of protein abundance. [Figure 7M]Figures 7J-7M demonstrate the generation of S7D7 Aggrewell™ clusters in suspension culture into mature pancreatic beta cells. Addition of DEZA and AZT to the "BLAR004 with no ALK5, low T3, ZM, H, NAC, FI" stage 7 conditioning regimen generated significant numbers of non-glucagon (Figure 7J; bottom right) C-peptide cells (co-expressed in S7D7 in terms of protein abundance), PDX1 (Figure 7J; bottom left), NKX6.1 (Figure 7J; bottom center), MAFA (Figure 7K; bottom left), UCN3 (Figure 7K; bottom center), and SLC2A1 (Figure 7K; bottom right). Figures 7L-7M demonstrate the generation of C-peptide cells by the "BLAR004 with no ALK5, low T3, ZM, H, NAC, FI" specific stage 7 conditioning, which are co-expressed by S7D7 maturation markers; PDX1 (Figure 7L; bottom row, bottom), NKX6.1 (Figure 7L; bottom row, center), MAFA (Figure 7M; bottom row, bottom), and SLC2A1 (Figure 7M; bottom row, right), but not by glucagon (Figure 7L; bottom row, right) or UCN3 (Figure 7M; bottom row, center), in terms of protein abundance. [Figure 8A]We demonstrate the generation of pluripotent stem cell-derived mature pancreatic beta cells in suspension culture with glucose-dependent mitochondrial respiration and GSIS kinetics similar to those of human islet cells. Figures 8A-8B both show that human islet cells responded rapidly to high D-glucose and maintained a high OCR (131.5% ± 11.32; 72 min post-injection) over time, as demonstrated by an OCR exceeding baseline by 123.3% ± 12.92 at 15 min post-injection. S6D7 ALI clusters enriched for immature C-peptide-positive cells lacked the rapid OCR response to high D-glucose (104.4% ± 3.37; 15 min post-injection) and exhibited a relatively weak OCR response over time (113.3% ± 4.51; 72 min post-injection). Glucose-dependent mitochondrial respiration dynamics similar to those of human islets were observed in the S7D13 "No ALK5, Low T3, ZM, H, NAC, FI, BLAR004" condition in the context of Aggrewell™ clusters in suspension (110.7% ± 2.46 at 15 min post-injection; 125.9% ± 2.27 at 72 min post-injection) (Figure 8A). Aggrewell™ clusters in suspension "No ALK5, Low T3, ZM, H, NAC, AZT, DEZA, FI, BLAR004" consumed oxygen above human islet cell levels in response to high glucose stimulation (162.0% ± 11.51 at 15 min post-injection; 177.1% ± 0.99 at 72 min post-injection) (Figure 8B). Figure 8C shows that mature beta cells within human islet cells exhibited the ability to undergo multiple rounds of rapid biphasic insulin secretion in response to glucose stimulation. Human islet cells demonstrated the ability to undergo multiple rounds of "on-off" insulin secretion switching. All conditions tested exhibited a strong insulin secretory response to KCl (Figures 8C-8I). The addition of exendin-4 did not increase the magnitude of the GSIS response in the human islets shown, but was included for comparison to the ALI or Aggrewell™ GSIS profiles (Figure 8D).S7D21-ALI clusters conditioned during stage 7 with "no ALK5, low T3, ZM, H, NAC, FI" in BLAR001 (Fig. 8E) or BLAR004 (Fig. 8F) showed a first biphasic GSIS response (approximately 4-10-fold the first phase of the first GSIS response), but not a second biphasic GSIS response, and a relatively slow biphasic GSIS response. The ALI clusters were unable to block the second phase of insulin secretion upon reperfusion with 3 mM D-glucose after stimulation. S7D14 Aggrewell™ clusters conditioned during stage 7 with "no ALK5, low T3, ZM, H, NAC, FI, BLAR004" exhibited a strong initial biphasic GSIS (the first phase of the initial GSIS response was approximately 5-fold), followed by the ability to completely block GSIS, and a weaker second monophasic response (Figure 8G). By adding DEZA and AZT to the "no ALK5, low T3, ZM, H, NAC, FI, BLAR004" conditioning, S7D14 Aggrewell™ clusters exhibited multiple rounds of biphasic GSIS (the first phase of the initial GSIS response was approximately 5-7-fold), similar to human islets, and the ability to completely block GSIS during high glucose pulses (Figures 8H-8I). [Figure 8B]We demonstrate the generation of pluripotent stem cell-derived mature pancreatic beta cells in suspension culture with glucose-dependent mitochondrial respiration and GSIS kinetics similar to those of human islet cells. Figures 8A-8B both show that human islet cells responded rapidly to high D-glucose and maintained a high OCR (131.5% ± 11.32; 72 min post-injection) over time, as demonstrated by an OCR exceeding baseline by 123.3% ± 12.92 at 15 min post-injection. S6D7 ALI clusters enriched for immature C-peptide-positive cells lacked the rapid OCR response to high D-glucose (104.4% ± 3.37; 15 min post-injection) and exhibited a relatively weak OCR response over time (113.3% ± 4.51; 72 min post-injection). Glucose-dependent mitochondrial respiration dynamics similar to those of human islets were observed in the S7D13 "No ALK5, Low T3, ZM, H, NAC, FI, BLAR004" condition in the context of Aggrewell™ clusters in suspension (110.7% ± 2.46 at 15 min post-injection; 125.9% ± 2.27 at 72 min post-injection) (Figure 8A). Aggrewell™ clusters in suspension "No ALK5, Low T3, ZM, H, NAC, AZT, DEZA, FI, BLAR004" consumed oxygen above human islet cell levels in response to high glucose stimulation (162.0% ± 11.51 at 15 min post-injection; 177.1% ± 0.99 at 72 min post-injection) (Figure 8B). Figure 8C shows that mature beta cells within human islet cells exhibited the ability to undergo multiple rounds of rapid biphasic insulin secretion in response to glucose stimulation. Human islet cells demonstrated the ability to undergo multiple rounds of "on-off" insulin secretion switching. All conditions tested exhibited a strong insulin secretory response to KCl (Figures 8C-8I). The addition of exendin-4 did not increase the magnitude of the GSIS response in the human islets shown, but was included for comparison to the ALI or Aggrewell™ GSIS profiles (Figure 8D).S7D21-ALI clusters conditioned during stage 7 with "no ALK5, low T3, ZM, H, NAC, FI" in BLAR001 (Fig. 8E) or BLAR004 (Fig. 8F) showed a first biphasic GSIS response (approximately 4-10-fold the first phase of the first GSIS response), but not a second biphasic GSIS response, and a relatively slow biphasic GSIS response. The ALI clusters were unable to block the second phase of insulin secretion upon reperfusion with 3 mM D-glucose after stimulation. S7D14 Aggrewell™ clusters conditioned during stage 7 with "no ALK5, low T3, ZM, H, NAC, FI, BLAR004" exhibited a strong initial biphasic GSIS (the first phase of the initial GSIS response was approximately 5-fold), followed by the ability to completely block GSIS, and a weaker second monophasic response (Figure 8G). By adding DEZA and AZT to the "no ALK5, low T3, ZM, H, NAC, FI, BLAR004" conditioning, S7D14 Aggrewell™ clusters exhibited multiple rounds of biphasic GSIS (the first phase of the initial GSIS response was approximately 5-7-fold), similar to human islets, and the ability to completely block GSIS during high glucose pulses (Figures 8H-8I). [Figure 8C]We demonstrate the generation of pluripotent stem cell-derived mature pancreatic beta cells in suspension culture with glucose-dependent mitochondrial respiration and GSIS kinetics similar to those of human islet cells. Figures 8A-8B both show that human islet cells responded rapidly to high D-glucose and maintained a high OCR (131.5% ± 11.32; 72 min post-injection) over time, as demonstrated by an OCR exceeding baseline by 123.3% ± 12.92 at 15 min post-injection. S6D7 ALI clusters enriched for immature C-peptide-positive cells lacked the rapid OCR response to high D-glucose (104.4% ± 3.37; 15 min post-injection) and exhibited a relatively weak OCR response over time (113.3% ± 4.51; 72 min post-injection). Glucose-dependent mitochondrial respiration dynamics similar to those of human islets were observed in the S7D13 "No ALK5, Low T3, ZM, H, NAC, FI, BLAR004" condition in the context of Aggrewell™ clusters in suspension (110.7% ± 2.46 at 15 min post-injection; 125.9% ± 2.27 at 72 min post-injection) (Figure 8A). Aggrewell™ clusters in suspension "No ALK5, Low T3, ZM, H, NAC, AZT, DEZA, FI, BLAR004" consumed oxygen above human islet cell levels in response to high glucose stimulation (162.0% ± 11.51 at 15 min post-injection; 177.1% ± 0.99 at 72 min post-injection) (Figure 8B). Figure 8C shows that mature beta cells within human islet cells exhibited the ability to undergo multiple rounds of rapid biphasic insulin secretion in response to glucose stimulation. Human islet cells demonstrated the ability to undergo multiple rounds of "on-off" insulin secretion switching. All conditions tested exhibited a strong insulin secretory response to KCl (Figures 8C-8I). The addition of exendin-4 did not increase the magnitude of the GSIS response in the human islets shown, but was included for comparison to the ALI or Aggrewell™ GSIS profiles (Figure 8D).S7D21-ALI clusters conditioned during stage 7 with "no ALK5, low T3, ZM, H, NAC, FI" in BLAR001 (Fig. 8E) or BLAR004 (Fig. 8F) showed a first biphasic GSIS response (approximately 4-10-fold the first phase of the first GSIS response), but not a second biphasic GSIS response, and a relatively slow biphasic GSIS response. The ALI clusters were unable to block the second phase of insulin secretion upon reperfusion with 3 mM D-glucose after stimulation. S7D14 Aggrewell™ clusters conditioned during stage 7 with "no ALK5, low T3, ZM, H, NAC, FI, BLAR004" exhibited a strong initial biphasic GSIS (the first phase of the initial GSIS response was approximately 5-fold), followed by the ability to completely block GSIS, and a weaker second monophasic response (Figure 8G). By adding DEZA and AZT to the "no ALK5, low T3, ZM, H, NAC, FI, BLAR004" conditioning, S7D14 Aggrewell™ clusters exhibited multiple rounds of biphasic GSIS (the first phase of the initial GSIS response was approximately 5-7-fold), similar to human islets, and the ability to completely block GSIS during high glucose pulses (Figures 8H-8I). [Figure 8D]We demonstrate the generation of pluripotent stem cell-derived mature pancreatic beta cells in suspension culture with glucose-dependent mitochondrial respiration and GSIS kinetics similar to those of human islet cells. Figures 8A-8B both show that human islet cells responded rapidly to high D-glucose and maintained a high OCR (131.5% ± 11.32; 72 min post-injection) over time, as demonstrated by an OCR exceeding baseline by 123.3% ± 12.92 at 15 min post-injection. S6D7 ALI clusters enriched for immature C-peptide-positive cells lacked the rapid OCR response to high D-glucose (104.4% ± 3.37; 15 min post-injection) and exhibited a relatively weak OCR response over time (113.3% ± 4.51; 72 min post-injection). Glucose-dependent mitochondrial respiration dynamics similar to those of human islets were observed in the S7D13 "No ALK5, Low T3, ZM, H, NAC, FI, BLAR004" condition in the context of Aggrewell™ clusters in suspension (110.7% ± 2.46 at 15 min post-injection; 125.9% ± 2.27 at 72 min post-injection) (Figure 8A). Aggrewell™ clusters in suspension "No ALK5, Low T3, ZM, H, NAC, AZT, DEZA, FI, BLAR004" consumed oxygen above human islet cell levels in response to high glucose stimulation (162.0% ± 11.51 at 15 min post-injection; 177.1% ± 0.99 at 72 min post-injection) (Figure 8B). Figure 8C shows that mature beta cells within human islet cells exhibited the ability to undergo multiple rounds of rapid biphasic insulin secretion in response to glucose stimulation. Human islet cells demonstrated the ability to undergo multiple rounds of "on-off" insulin secretion switching. All conditions tested exhibited a strong insulin secretory response to KCl (Figures 8C-8I). The addition of exendin-4 did not increase the magnitude of the GSIS response in the human islets shown, but was included for comparison to the ALI or Aggrewell™ GSIS profiles (Figure 8D).S7D21-ALI clusters conditioned during stage 7 with "no ALK5, low T3, ZM, H, NAC, FI" in BLAR001 (Fig. 8E) or BLAR004 (Fig. 8F) showed a first biphasic GSIS response (approximately 4-10-fold the first phase of the first GSIS response), but not a second biphasic GSIS response, and a relatively slow biphasic GSIS response. The ALI clusters were unable to block the second phase of insulin secretion upon reperfusion with 3 mM D-glucose after stimulation. S7D14 Aggrewell™ clusters conditioned during stage 7 with "no ALK5, low T3, ZM, H, NAC, FI, BLAR004" exhibited a strong initial biphasic GSIS (the first phase of the initial GSIS response was approximately 5-fold), followed by the ability to completely block GSIS, and a weaker second monophasic response (Figure 8G). By adding DEZA and AZT to the "no ALK5, low T3, ZM, H, NAC, FI, BLAR004" conditioning, S7D14 Aggrewell™ clusters exhibited multiple rounds of biphasic GSIS (the first phase of the initial GSIS response was approximately 5-7-fold), similar to human islets, and the ability to completely block GSIS during high glucose pulses (Figures 8H-8I). [Figure 8E]We demonstrate the generation of pluripotent stem cell-derived mature pancreatic beta cells in suspension culture with glucose-dependent mitochondrial respiration and GSIS kinetics similar to those of human islet cells. Figures 8A-8B both show that human islet cells responded rapidly to high D-glucose and maintained a high OCR (131.5% ± 11.32; 72 min post-injection) over time, as demonstrated by an OCR exceeding baseline by 123.3% ± 12.92 at 15 min post-injection. S6D7 ALI clusters enriched for immature C-peptide-positive cells lacked the rapid OCR response to high D-glucose (104.4% ± 3.37; 15 min post-injection) and exhibited a relatively weak OCR response over time (113.3% ± 4.51; 72 min post-injection). Glucose-dependent mitochondrial respiration dynamics similar to those of human islets were observed in the S7D13 "No ALK5, Low T3, ZM, H, NAC, FI, BLAR004" condition in the context of Aggrewell™ clusters in suspension (110.7% ± 2.46 at 15 min post-injection; 125.9% ± 2.27 at 72 min post-injection) (Figure 8A). Aggrewell™ clusters in suspension "No ALK5, Low T3, ZM, H, NAC, AZT, DEZA, FI, BLAR004" consumed oxygen above human islet cell levels in response to high glucose stimulation (162.0% ± 11.51 at 15 min post-injection; 177.1% ± 0.99 at 72 min post-injection) (Figure 8B). Figure 8C shows that mature beta cells within human islet cells exhibited the ability to undergo multiple rounds of rapid biphasic insulin secretion in response to glucose stimulation. Human islet cells demonstrated the ability to undergo multiple rounds of "on-off" insulin secretion switching. All conditions tested exhibited a strong insulin secretory response to KCl (Figures 8C-8I). The addition of exendin-4 did not increase the magnitude of the GSIS response in the human islets shown, but was included for comparison to the ALI or Aggrewell™ GSIS profiles (Figure 8D).S7D21-ALI clusters conditioned during stage 7 with "no ALK5, low T3, ZM, H, NAC, FI" in BLAR001 (Fig. 8E) or BLAR004 (Fig. 8F) showed a first biphasic GSIS response (approximately 4-10-fold the first phase of the first GSIS response), but not a second biphasic GSIS response, and a relatively slow biphasic GSIS response. The ALI clusters were unable to block the second phase of insulin secretion upon reperfusion with 3 mM D-glucose after stimulation. S7D14 Aggrewell™ clusters conditioned during stage 7 with "no ALK5, low T3, ZM, H, NAC, FI, BLAR004" exhibited a strong initial biphasic GSIS (the first phase of the initial GSIS response was approximately 5-fold), followed by the ability to completely block GSIS, and a weaker second monophasic response (Figure 8G). By adding DEZA and AZT to the "no ALK5, low T3, ZM, H, NAC, FI, BLAR004" conditioning, S7D14 Aggrewell™ clusters exhibited multiple rounds of biphasic GSIS (the first phase of the initial GSIS response was approximately 5-7-fold), similar to human islets, and the ability to completely block GSIS during high glucose pulses (Figures 8H-8I). [Figure 8F]We demonstrate the generation of pluripotent stem cell-derived mature pancreatic beta cells in suspension culture with glucose-dependent mitochondrial respiration and GSIS kinetics similar to those of human islet cells. Figures 8A-8B both show that human islet cells responded rapidly to high D-glucose and maintained a high OCR (131.5% ± 11.32; 72 min post-injection) over time, as demonstrated by an OCR exceeding baseline by 123.3% ± 12.92 at 15 min post-injection. S6D7 ALI clusters enriched for immature C-peptide-positive cells lacked the rapid OCR response to high D-glucose (104.4% ± 3.37; 15 min post-injection) and exhibited a relatively weak OCR response over time (113.3% ± 4.51; 72 min post-injection). Glucose-dependent mitochondrial respiration dynamics similar to those of human islets were observed in the S7D13 "No ALK5, Low T3, ZM, H, NAC, FI, BLAR004" condition in the context of Aggrewell™ clusters in suspension (110.7% ± 2.46 at 15 min post-injection; 125.9% ± 2.27 at 72 min post-injection) (Figure 8A). Aggrewell™ clusters in suspension "No ALK5, Low T3, ZM, H, NAC, AZT, DEZA, FI, BLAR004" consumed oxygen above human islet cell levels in response to high glucose stimulation (162.0% ± 11.51 at 15 min post-injection; 177.1% ± 0.99 at 72 min post-injection) (Figure 8B). Figure 8C shows that mature beta cells within human islet cells exhibited the ability to undergo multiple rounds of rapid biphasic insulin secretion in response to glucose stimulation. Human islet cells demonstrated the ability to undergo multiple rounds of "on-off" insulin secretion switching. All conditions tested exhibited a strong insulin secretory response to KCl (Figures 8C-8I). The addition of exendin-4 did not increase the magnitude of the GSIS response in the human islets shown, but was included for comparison to the ALI or Aggrewell™ GSIS profiles (Figure 8D).S7D21-ALI clusters conditioned during stage 7 with "no ALK5, low T3, ZM, H, NAC, FI" in BLAR001 (Fig. 8E) or BLAR004 (Fig. 8F) showed a first biphasic GSIS response (approximately 4-10-fold the first phase of the first GSIS response), but not a second biphasic GSIS response, and a relatively slow biphasic GSIS response. The ALI clusters were unable to block the second phase of insulin secretion upon reperfusion with 3 mM D-glucose after stimulation. S7D14 Aggrewell™ clusters conditioned during stage 7 with "no ALK5, low T3, ZM, H, NAC, FI, BLAR004" exhibited a strong initial biphasic GSIS (the first phase of the initial GSIS response was approximately 5-fold), followed by the ability to completely block GSIS, and a weaker second monophasic response (Figure 8G). By adding DEZA and AZT to the "no ALK5, low T3, ZM, H, NAC, FI, BLAR004" conditioning, S7D14 Aggrewell™ clusters exhibited multiple rounds of biphasic GSIS (the first phase of the initial GSIS response was approximately 5-7-fold), similar to human islets, and the ability to completely block GSIS during high glucose pulses (Figures 8H-8I). [Figure 8G]We demonstrate the generation of pluripotent stem cell-derived mature pancreatic beta cells in suspension culture with glucose-dependent mitochondrial respiration and GSIS kinetics similar to those of human islet cells. Figures 8A-8B both show that human islet cells responded rapidly to high D-glucose and maintained a high OCR (131.5% ± 11.32; 72 min post-injection) over time, as demonstrated by an OCR exceeding baseline by 123.3% ± 12.92 at 15 min post-injection. S6D7 ALI clusters enriched for immature C-peptide-positive cells lacked the rapid OCR response to high D-glucose (104.4% ± 3.37; 15 min post-injection) and exhibited a relatively weak OCR response over time (113.3% ± 4.51; 72 min post-injection). Glucose-dependent mitochondrial respiration dynamics similar to those of human islets were observed in the S7D13 "No ALK5, Low T3, ZM, H, NAC, FI, BLAR004" condition in the context of Aggrewell™ clusters in suspension (110.7% ± 2.46 at 15 min post-injection; 125.9% ± 2.27 at 72 min post-injection) (Figure 8A). Aggrewell™ clusters in suspension "No ALK5, Low T3, ZM, H, NAC, AZT, DEZA, FI, BLAR004" consumed oxygen above human islet cell levels in response to high glucose stimulation (162.0% ± 11.51 at 15 min post-injection; 177.1% ± 0.99 at 72 min post-injection) (Figure 8B). Figure 8C shows that mature beta cells within human islet cells exhibited the ability to undergo multiple rounds of rapid biphasic insulin secretion in response to glucose stimulation. Human islet cells demonstrated the ability to undergo multiple rounds of "on-off" insulin secretion switching. All conditions tested exhibited a strong insulin secretory response to KCl (Figures 8C-8I). The addition of exendin-4 did not increase the magnitude of the GSIS response in the human islets shown, but was included for comparison to the ALI or Aggrewell™ GSIS profiles (Figure 8D).S7D21-ALI clusters conditioned during stage 7 with "no ALK5, low T3, ZM, H, NAC, FI" in BLAR001 (Fig. 8E) or BLAR004 (Fig. 8F) showed a first biphasic GSIS response (approximately 4-10-fold the first phase of the first GSIS response), but not a second biphasic GSIS response, and a relatively slow biphasic GSIS response. The ALI clusters were unable to block the second phase of insulin secretion upon reperfusion with 3 mM D-glucose after stimulation. S7D14 Aggrewell™ clusters conditioned during stage 7 with "no ALK5, low T3, ZM, H, NAC, FI, BLAR004" exhibited a strong initial biphasic GSIS (the first phase of the initial GSIS response was approximately 5-fold), followed by the ability to completely block GSIS, and a weaker second monophasic response (Figure 8G). By adding DEZA and AZT to the "no ALK5, low T3, ZM, H, NAC, FI, BLAR004" conditioning, S7D14 Aggrewell™ clusters exhibited multiple rounds of biphasic GSIS (the first phase of the initial GSIS response was approximately 5-7-fold), similar to human islets, and the ability to completely block GSIS during high glucose pulses (Figures 8H-8I). [Figure 8H]We demonstrate the generation of pluripotent stem cell-derived mature pancreatic beta cells in suspension culture with glucose-dependent mitochondrial respiration and GSIS kinetics similar to those of human islet cells. Figures 8A-8B both show that human islet cells responded rapidly to high D-glucose and maintained a high OCR (131.5% ± 11.32; 72 min post-injection) over time, as demonstrated by an OCR exceeding baseline by 123.3% ± 12.92 at 15 min post-injection. S6D7 ALI clusters enriched for immature C-peptide-positive cells lacked the rapid OCR response to high D-glucose (104.4% ± 3.37; 15 min post-injection) and exhibited a relatively weak OCR response over time (113.3% ± 4.51; 72 min post-injection). Glucose-dependent mitochondrial respiration dynamics similar to those of human islets were observed in the S7D13 "No ALK5, Low T3, ZM, H, NAC, FI, BLAR004" condition in the context of Aggrewell™ clusters in suspension (110.7% ± 2.46 at 15 min post-injection; 125.9% ± 2.27 at 72 min post-injection) (Figure 8A). Aggrewell™ clusters in suspension "No ALK5, Low T3, ZM, H, NAC, AZT, DEZA, FI, BLAR004" consumed oxygen above human islet cell levels in response to high glucose stimulation (162.0% ± 11.51 at 15 min post-injection; 177.1% ± 0.99 at 72 min post-injection) (Figure 8B). Figure 8C shows that mature beta cells within human islet cells exhibited the ability to undergo multiple rounds of rapid biphasic insulin secretion in response to glucose stimulation. Human islet cells demonstrated the ability to undergo multiple rounds of "on-off" insulin secretion switching. All conditions tested exhibited a strong insulin secretory response to KCl (Figures 8C-8I). The addition of exendin-4 did not increase the magnitude of the GSIS response in the human islets shown, but was included for comparison to the ALI or Aggrewell™ GSIS profiles (Figure 8D).S7D21-ALI clusters conditioned during stage 7 with "no ALK5, low T3, ZM, H, NAC, FI" in BLAR001 (Fig. 8E) or BLAR004 (Fig. 8F) showed a first biphasic GSIS response (approximately 4-10-fold the first phase of the first GSIS response), but not a second biphasic GSIS response, and a relatively slow biphasic GSIS response. The ALI clusters were unable to block the second phase of insulin secretion upon reperfusion with 3 mM D-glucose after stimulation. S7D14 Aggrewell™ clusters conditioned during stage 7 with "no ALK5, low T3, ZM, H, NAC, FI, BLAR004" exhibited a strong initial biphasic GSIS (the first phase of the initial GSIS response was approximately 5-fold), followed by the ability to completely block GSIS, and a weaker second monophasic response (Figure 8G). By adding DEZA and AZT to the "no ALK5, low T3, ZM, H, NAC, FI, BLAR004" conditioning, S7D14 Aggrewell™ clusters exhibited multiple rounds of biphasic GSIS (the first phase of the initial GSIS response was approximately 5-7-fold), similar to human islets, and the ability to completely block GSIS during high glucose pulses (Figures 8H-8I). [Figure 8I]We demonstrate the generation of pluripotent stem cell-derived mature pancreatic beta cells in suspension culture with glucose-dependent mitochondrial respiration and GSIS kinetics similar to those of human islet cells. Figures 8A-8B both show that human islet cells responded rapidly to high D-glucose and maintained a high OCR (131.5% ± 11.32; 72 min post-injection) over time, as demonstrated by an OCR exceeding baseline by 123.3% ± 12.92 at 15 min post-injection. S6D7 ALI clusters enriched for immature C-peptide-positive cells lacked the rapid OCR response to high D-glucose (104.4% ± 3.37; 15 min post-injection) and exhibited a relatively weak OCR response over time (113.3% ± 4.51; 72 min post-injection). Glucose-dependent mitochondrial respiration dynamics similar to those of human islets were observed in the S7D13 "No ALK5, Low T3, ZM, H, NAC, FI, BLAR004" condition in the context of Aggrewell™ clusters in suspension (110.7% ± 2.46 at 15 min post-injection; 125.9% ± 2.27 at 72 min post-injection) (Figure 8A). Aggrewell™ clusters in suspension "No ALK5, Low T3, ZM, H, NAC, AZT, DEZA, FI, BLAR004" consumed oxygen above human islet cell levels in response to high glucose stimulation (162.0% ± 11.51 at 15 min post-injection; 177.1% ± 0.99 at 72 min post-injection) (Figure 8B). Figure 8C shows that mature beta cells within human islet cells exhibited the ability to undergo multiple rounds of rapid biphasic insulin secretion in response to glucose stimulation. Human islet cells demonstrated the ability to undergo multiple rounds of "on-off" insulin secretion switching. All conditions tested exhibited a strong insulin secretory response to KCl (Figures 8C-8I). The addition of exendin-4 did not increase the magnitude of the GSIS response in the human islets shown, but was included for comparison to the ALI or Aggrewell™ GSIS profiles (Figure 8D).S7D21-ALI clusters conditioned during stage 7 with "no ALK5, low T3, ZM, H, NAC, FI" in BLAR001 (Fig. 8E) or BLAR004 (Fig. 8F) showed a first biphasic GSIS response (approximately 4-10-fold the first phase of the first GSIS response), but not a second biphasic GSIS response, and a relatively slow biphasic GSIS response. The ALI clusters were unable to block the second phase of insulin secretion upon reperfusion with 3 mM D-glucose after stimulation. S7D14 Aggrewell™ clusters conditioned during stage 7 with "no ALK5, low T3, ZM, H, NAC, FI, BLAR004" exhibited a strong initial biphasic GSIS (the first phase of the initial GSIS response was approximately 5-fold), followed by the ability to completely block GSIS, and a weaker second monophasic response (Figure 8G). By adding DEZA and AZT to the "no ALK5, low T3, ZM, H, NAC, FI, BLAR004" conditioning, S7D14 Aggrewell™ clusters exhibited multiple rounds of biphasic GSIS (the first phase of the initial GSIS response was approximately 5-7-fold), similar to human islets, and the ability to completely block GSIS during high glucose pulses (Figures 8H-8I). [Figure 9A]

[0039] Figure 9A demonstrates the generation of pancreatic endoderm from CyT49 hESCs in multiple suspension cultures. Figure 9A depicts a stepwise differentiation protocol for generating pancreatic endoderm from CyT49 hESCs. Key transcription factors ("TFs") are indicated for each stage. [Figure 9B] We demonstrate the generation of pancreatic endoderm from CyT49 hESCs in multiple suspension cultures. Figure 9B shows the number of S4D3 cells generated relative to hESC input in millions of cells per mL. [Figure 9C]We demonstrate the generation of pancreatic endoderm from CyT49 hESCs in multiple suspension cultures. Figure 9C shows the S4D3 yield for 0.1 PBS Mini and 0.5 PBS Mini. For example, the 0.5 PBS suspension culture format generated 4.08 ± 0.854 (4.08 million ± 854,000) cells per hESC, significantly more than PEC-01 d12 (2.23 ± 0.090). Figure 9C demonstrates a nearly six-fold increase (million cells) in total S4D3 cell yield when scaling up the medium volume from 100 mL (0.1 PBS) to 500 mL (0.5 PBS). [Figure 9D] We demonstrate the generation of pancreatic endoderm from CyT49 hESCs in multiple suspension cultures. Figures 9D and 9E depict phase-contrast images and diameters (in micrometers) of S4D3 aggregates from 2-liter roller bottles (Figure 9D, right), 0.1 PBS (Figure 9E, center), and 0.5 PBS (Figure 9E, right) suspension formats. PEC-01 d12 (Figure 9D, left) and PEC-01 d15 (Figure 9E, left) produced by 2-liter roller bottles are shown as prior art examples. S4D3 aggregates produced by 0.1 PBS and 0.5 PBS suspension cultures are uniformly smaller than those produced by roller bottles. [Figure 9E] We demonstrate the generation of pancreatic endoderm from CyT49 hESCs in multiple suspension cultures. Figures 9D and 9E depict phase-contrast images and diameters (in micrometers) of S4D3 aggregates from 2-liter roller bottles (Figure 9D, right), 0.1 PBS (Figure 9E, center), and 0.5 PBS (Figure 9E, right) suspension formats. PEC-01 d12 (Figure 9D, left) and PEC-01 d15 (Figure 9E, left) produced by 2-liter roller bottles are shown as prior art examples. S4D3 aggregates produced by 0.1 PBS and 0.5 PBS suspension cultures are uniformly smaller than those produced by roller bottles. [Figure 9F]We demonstrate the generation of pancreatic endoderm from CyT49 hESCs in multiple suspension cultures. Figures 9F-9L show gene expression for S4D3 aggregates. Gene expression of NKX6.1 (Figure 9F), PTF1A (Figure 9G), PDX1 (Figure 9H), SOX2 (Figure 9I), CDX2 (Figure 9J), NEUROD1 (Figure 9K), and CHGA (Figure 9L) for CyT49 hESCs is shown for S4D3 aggregates generated in 2-liter roller bottles, 0.1 PBS, and 0.5 PBS, as well as for PEC-01 d15 generated by 2-liter roller bottles. Figures 9F-9L demonstrate robust induction of the pancreatic endoderm gene program while restricting expression of alternative endoderm lineages and early pancreatic endocrine differentiation compared to PEC-01 d15 (prior art). [Figure 9G] We demonstrate the generation of pancreatic endoderm from CyT49 hESCs in multiple suspension cultures. Figures 9F-9L show gene expression for S4D3 aggregates. Gene expression of NKX6.1 (Figure 9F), PTF1A (Figure 9G), PDX1 (Figure 9H), SOX2 (Figure 9I), CDX2 (Figure 9J), NEUROD1 (Figure 9K), and CHGA (Figure 9L) for CyT49 hESCs is shown for S4D3 aggregates generated in 2-liter roller bottles, 0.1 PBS, and 0.5 PBS, as well as for PEC-01 d15 generated by 2-liter roller bottles. Figures 9F-9L demonstrate robust induction of the pancreatic endoderm gene program while restricting expression of alternative endoderm lineages and early pancreatic endocrine differentiation compared to PEC-01 d15 (prior art). [Figure 9H]We demonstrate the generation of pancreatic endoderm from CyT49 hESCs in multiple suspension cultures. Figures 9F-9L show gene expression for S4D3 aggregates. Gene expression of NKX6.1 (Figure 9F), PTF1A (Figure 9G), PDX1 (Figure 9H), SOX2 (Figure 9I), CDX2 (Figure 9J), NEUROD1 (Figure 9K), and CHGA (Figure 9L) for CyT49 hESCs is shown for S4D3 aggregates generated in 2-liter roller bottles, 0.1 PBS, and 0.5 PBS, as well as for PEC-01 d15 generated by 2-liter roller bottles. Figures 9F-9L demonstrate robust induction of the pancreatic endoderm gene program while restricting expression of alternative endoderm lineages and early pancreatic endocrine differentiation compared to PEC-01 d15 (prior art). [Figure 9I] We demonstrate the generation of pancreatic endoderm from CyT49 hESCs in multiple suspension cultures. Figures 9F-9L show gene expression for S4D3 aggregates. Gene expression of NKX6.1 (Figure 9F), PTF1A (Figure 9G), PDX1 (Figure 9H), SOX2 (Figure 9I), CDX2 (Figure 9J), NEUROD1 (Figure 9K), and CHGA (Figure 9L) for CyT49 hESCs is shown for S4D3 aggregates generated in 2-liter roller bottles, 0.1 PBS, and 0.5 PBS, as well as for PEC-01 d15 generated by 2-liter roller bottles. Figures 9F-9L demonstrate robust induction of the pancreatic endoderm gene program while restricting expression of alternative endoderm lineages and early pancreatic endocrine differentiation compared to PEC-01 d15 (prior art). [Figure 9J]We demonstrate the generation of pancreatic endoderm from CyT49 hESCs in multiple suspension cultures. Figures 9F-9L show gene expression for S4D3 aggregates. Gene expression of NKX6.1 (Figure 9F), PTF1A (Figure 9G), PDX1 (Figure 9H), SOX2 (Figure 9I), CDX2 (Figure 9J), NEUROD1 (Figure 9K), and CHGA (Figure 9L) for CyT49 hESCs is shown for S4D3 aggregates generated in 2-liter roller bottles, 0.1 PBS, and 0.5 PBS, as well as for PEC-01 d15 generated by 2-liter roller bottles. Figures 9F-9L demonstrate robust induction of the pancreatic endoderm gene program while restricting expression of alternative endoderm lineages and early pancreatic endocrine differentiation compared to PEC-01 d15 (prior art). [Figure 9K] We demonstrate the generation of pancreatic endoderm from CyT49 hESCs in multiple suspension cultures. Figures 9F-9L show gene expression for S4D3 aggregates. Gene expression of NKX6.1 (Figure 9F), PTF1A (Figure 9G), PDX1 (Figure 9H), SOX2 (Figure 9I), CDX2 (Figure 9J), NEUROD1 (Figure 9K), and CHGA (Figure 9L) for CyT49 hESCs is shown for S4D3 aggregates generated in 2-liter roller bottles, 0.1 PBS, and 0.5 PBS, as well as for PEC-01 d15 generated by 2-liter roller bottles. Figures 9F-9L demonstrate robust induction of the pancreatic endoderm gene program while restricting expression of alternative endoderm lineages and early pancreatic endocrine differentiation compared to PEC-01 d15 (prior art). [Figure 9L]We demonstrate the generation of pancreatic endoderm from CyT49 hESCs in multiple suspension cultures. Figures 9F-9L show gene expression for S4D3 aggregates. Gene expression of NKX6.1 (Figure 9F), PTF1A (Figure 9G), PDX1 (Figure 9H), SOX2 (Figure 9I), CDX2 (Figure 9J), NEUROD1 (Figure 9K), and CHGA (Figure 9L) for CyT49 hESCs is shown for S4D3 aggregates generated in 2-liter roller bottles, 0.1 PBS, and 0.5 PBS, as well as for PEC-01 d15 generated by 2-liter roller bottles. Figures 9F-9L demonstrate robust induction of the pancreatic endoderm gene program while restricting expression of alternative endoderm lineages and early pancreatic endocrine differentiation compared to PEC-01 d15 (prior art). [Figure 9M] We demonstrate the generation of pancreatic endoderm from CyT49 hESCs in multiple suspension cultures. Figures 9N–9P show that, compared with PEC-01 d12 aggregates (Figure 9M) and PEC-01 d15 aggregates (Figure 9Q) generated by the new protocol in 2 L roller bottles (Figure 9N), 0.1 PBS (Figure 9O), and 0.5 PBS (Figure 9P), pancreatic endoderm markers (e.g., PDX1, NKX6.1) showed strong protein colocalization, whereas protein markers of alternative endodermal lineages (e.g., SOX2, CDX2) and markers for endocrine differentiation (e.g., CHGA) did not. [Figure 9N]We demonstrate the generation of pancreatic endoderm from CyT49 hESCs in multiple suspension cultures. Figures 9N–9P show that, compared with PEC-01 d12 aggregates (Figure 9M) and PEC-01 d15 aggregates (Figure 9Q) generated by the new protocol in 2 L roller bottles (Figure 9N), 0.1 PBS (Figure 9O), and 0.5 PBS (Figure 9P), pancreatic endoderm markers (e.g., PDX1, NKX6.1) showed strong protein colocalization, whereas protein markers of alternative endodermal lineages (e.g., SOX2, CDX2) and markers for endocrine differentiation (e.g., CHGA) did not. [Figure 9O] We demonstrate the generation of pancreatic endoderm from CyT49 hESCs in multiple suspension cultures. Figures 9N–9P show that, compared with PEC-01 d12 aggregates (Figure 9M) and PEC-01 d15 aggregates (Figure 9Q) generated by the new protocol in 2 L roller bottles (Figure 9N), 0.1 PBS (Figure 9O), and 0.5 PBS (Figure 9P), pancreatic endoderm markers (e.g., PDX1, NKX6.1) showed strong protein colocalization, whereas protein markers of alternative endodermal lineages (e.g., SOX2, CDX2) and markers for endocrine differentiation (e.g., CHGA) did not. [Figure 9P] We demonstrate the generation of pancreatic endoderm from CyT49 hESCs in multiple suspension cultures. Figures 9N–9P show that, compared with PEC-01 d12 aggregates (Figure 9M) and PEC-01 d15 aggregates (Figure 9Q) generated by the new protocol in 2 L roller bottles (Figure 9N), 0.1 PBS (Figure 9O), and 0.5 PBS (Figure 9P), pancreatic endoderm markers (e.g., PDX1, NKX6.1) showed strong protein colocalization, whereas protein markers of alternative endodermal lineages (e.g., SOX2, CDX2) and markers for endocrine differentiation (e.g., CHGA) did not. [Figure 9Q]We demonstrate the generation of pancreatic endoderm from CyT49 hESCs in multiple suspension cultures. Figures 9N–9P show that, compared with PEC-01 d12 aggregates (Figure 9M) and PEC-01 d15 aggregates (Figure 9Q) generated by the new protocol in 2 L roller bottles (Figure 9N), 0.1 PBS (Figure 9O), and 0.5 PBS (Figure 9P), pancreatic endoderm markers (e.g., PDX1, NKX6.1) showed strong protein colocalization, whereas protein markers of alternative endodermal lineages (e.g., SOX2, CDX2) and markers for endocrine differentiation (e.g., CHGA) did not. [Figure 10A]

[0033] Figure 10 demonstrates the generation of insulin-producing cells from CyT49 hESCs in multiple types of suspension culture. Figure 10A depicts the disclosed stepwise differentiation protocol for generating non-functional insulin-producing cells from CyT49 hESCs. Starting with the input of S4D3 CyT49 hESC-derived cells to stage 5 conditioning, key transcription factors ("TFs") are shown for each stage. [Figure 10B] We demonstrate the generation of insulin-producing cells from CyT49 hESCs in multiple suspension cultures. Figure 10B shows the number of S6D7 cells generated per hESC input in millions of cells per mL. For example, the 0.5 PBS suspension culture format using the new protocol generated 2.4 ± 0.169 S6D7 cells per hESC cell. [Figure 10C] Figure 10 demonstrates the generation of insulin-producing cells from CyT49 hESCs in multiple suspension cultures. Figure 10C shows S6D7 yields from 0.1 PBS and 0.5 PBS cultures. Figure 10C demonstrates a 7.5-fold increase (million cells) in total S6D7 cell yield when the medium volume was scaled up from 100 mL (0.1 PBS) to 500 mL (0.5 PBS). [Figure 10D]We demonstrate the generation of insulin-producing cells from CyT49 hESCs in multiple suspension cultures. Figure 10D depicts phase-contrast images of S5D3 aggregates from 2 liter roller bottles (Figure 10D, left), 0.1 PBS (Figure 10D, center), and 0.5 PBS (Figure 10D, right) suspension formats. [Figure 10E] We demonstrate the generation of insulin-producing cells from CyT49 hESCs in multiple suspension cultures. Figure 10E depicts phase-contrast images of S6D7 aggregates from 2 liter roller bottles (Figure 10E, left), 0.1 PBS (Figure 10E, center), and 0.5 PBS (Figure 10E, right) suspension formats, with the aggregate diameter indicated below each photograph. [Figure 10F] We demonstrate the generation of insulin-producing cells from CyT49 hESCs in multiple suspension cultures. Figure 10F depicts phase-contrast images of S7D13 aggregates from 2-liter roller bottle (Figure 10F, left) and 0.1 PBS (Figure 10F, right) suspension cultures. Suspension cultures in 0.1 PBS and 0.5 PBS maintained a tight aggregate structure through stage 7. Conversely, stage 6-7 aggregates were loose and formed sheets of cells in 2-liter roller bottle suspension cultures. [Figure 10G] We demonstrate the generation of insulin-producing cells from CyT49 hESCs in multiple suspension cultures. Figures 10G-10N show gene expression at S4D3, S5D3, S6D7, and S7D7. Figures 10G-10N demonstrate that all suspension culture methods using the new protocol induced robust pancreatic monohormone insulin-producing cell gene signatures. The 2-liter roller bottle, 0.1 PBS, and 0.5 PBS suspension methods induced relatively equal expression of PDX1 (Figure 10G), NKX6.1 (Figure 10H), CHGA (Figure 10I), NEUROD1 (Figure 10J), NGN3 (Figure 10K), insulin (Figure 10L), MAFA (Figure 10M), and glucagon (Figure 10N). [Figure 10H]We demonstrate the generation of insulin-producing cells from CyT49 hESCs in multiple suspension cultures. Figures 10G-10N show gene expression at S4D3, S5D3, S6D7, and S7D7. Figures 10G-10N demonstrate that all suspension culture methods using the new protocol induced robust pancreatic monohormone insulin-producing cell gene signatures. The 2-liter roller bottle, 0.1 PBS, and 0.5 PBS suspension methods induced relatively equal expression of PDX1 (Figure 10G), NKX6.1 (Figure 10H), CHGA (Figure 10I), NEUROD1 (Figure 10J), NGN3 (Figure 10K), insulin (Figure 10L), MAFA (Figure 10M), and glucagon (Figure 10N). [Figure 10I] We demonstrate the generation of insulin-producing cells from CyT49 hESCs in multiple suspension cultures. Figures 10G-10N show gene expression at S4D3, S5D3, S6D7, and S7D7. Figures 10G-10N demonstrate that all suspension culture methods using the new protocol induced robust pancreatic monohormone insulin-producing cell gene signatures. The 2-liter roller bottle, 0.1 PBS, and 0.5 PBS suspension methods induced relatively equal expression of PDX1 (Figure 10G), NKX6.1 (Figure 10H), CHGA (Figure 10I), NEUROD1 (Figure 10J), NGN3 (Figure 10K), insulin (Figure 10L), MAFA (Figure 10M), and glucagon (Figure 10N). [Figure 10J] We demonstrate the generation of insulin-producing cells from CyT49 hESCs in multiple suspension cultures. Figures 10G-10N show gene expression at S4D3, S5D3, S6D7, and S7D7. Figures 10G-10N demonstrate that all suspension culture methods using the new protocol induced robust pancreatic monohormone insulin-producing cell gene signatures. The 2-liter roller bottle, 0.1 PBS, and 0.5 PBS suspension methods induced relatively equal expression of PDX1 (Figure 10G), NKX6.1 (Figure 10H), CHGA (Figure 10I), NEUROD1 (Figure 10J), NGN3 (Figure 10K), insulin (Figure 10L), MAFA (Figure 10M), and glucagon (Figure 10N). [Figure 10K]We demonstrate the generation of insulin-producing cells from CyT49 hESCs in multiple suspension cultures. Figures 10G-10N show gene expression at S4D3, S5D3, S6D7, and S7D7. Figures 10G-10N demonstrate that all suspension culture methods using the new protocol induced robust pancreatic monohormone insulin-producing cell gene signatures. The 2-liter roller bottle, 0.1 PBS, and 0.5 PBS suspension methods induced relatively equal expression of PDX1 (Figure 10G), NKX6.1 (Figure 10H), CHGA (Figure 10I), NEUROD1 (Figure 10J), NGN3 (Figure 10K), insulin (Figure 10L), MAFA (Figure 10M), and glucagon (Figure 10N). [Figure 10L] We demonstrate the generation of insulin-producing cells from CyT49 hESCs in multiple suspension cultures. Figures 10G-10N show gene expression at S4D3, S5D3, S6D7, and S7D7. Figures 10G-10N demonstrate that all suspension culture methods using the new protocol induced robust pancreatic monohormone insulin-producing cell gene signatures. The 2-liter roller bottle, 0.1 PBS, and 0.5 PBS suspension methods induced relatively equal expression of PDX1 (Figure 10G), NKX6.1 (Figure 10H), CHGA (Figure 10I), NEUROD1 (Figure 10J), NGN3 (Figure 10K), insulin (Figure 10L), MAFA (Figure 10M), and glucagon (Figure 10N). [Figure 10M] We demonstrate the generation of insulin-producing cells from CyT49 hESCs in multiple suspension cultures. Figures 10G-10N show gene expression at S4D3, S5D3, S6D7, and S7D7. Figures 10G-10N demonstrate that all suspension culture methods using the new protocol induced robust pancreatic monohormone insulin-producing cell gene signatures. The 2-liter roller bottle, 0.1 PBS, and 0.5 PBS suspension methods induced relatively equal expression of PDX1 (Figure 10G), NKX6.1 (Figure 10H), CHGA (Figure 10I), NEUROD1 (Figure 10J), NGN3 (Figure 10K), insulin (Figure 10L), MAFA (Figure 10M), and glucagon (Figure 10N). [Figure 10N]We demonstrate the generation of insulin-producing cells from CyT49 hESCs in multiple suspension cultures. Figures 10G-10N show gene expression at S4D3, S5D3, S6D7, and S7D7. Figures 10G-10N demonstrate that all suspension culture methods using the new protocol induced robust pancreatic monohormone insulin-producing cell gene signatures. The 2-liter roller bottle, 0.1 PBS, and 0.5 PBS suspension methods induced relatively equal expression of PDX1 (Figure 10G), NKX6.1 (Figure 10H), CHGA (Figure 10I), NEUROD1 (Figure 10J), NGN3 (Figure 10K), insulin (Figure 10L), MAFA (Figure 10M), and glucagon (Figure 10N). [Figure 10O] We demonstrate the generation of insulin-producing cells from CyT49 hESCs in multiple suspension cultures. Figures 10O-10T show the gradual formation of protein colocalization of insulin-producing cell markers, including CHGA, NKX6.1, and insulin, but not glucagon, for S5D3 in 0.5 PBS (Figures 10O-10P), S6D7 in 2 L roller bottles (Figure 10Q), S6D7 in 0.1 PBS (Figure 10R), S7D13 in 2 L roller bottles (Figure 10S), and S7D14 in 0.1 PBS (Figure 10T). [Figure 10P] We demonstrate the generation of insulin-producing cells from CyT49 hESCs in multiple suspension cultures. Figures 10O-10T show the gradual formation of protein colocalization of insulin-producing cell markers, including CHGA, NKX6.1, and insulin, but not glucagon, for S5D3 in 0.5 PBS (Figures 10O-10P), S6D7 in 2 L roller bottles (Figure 10Q), S6D7 in 0.1 PBS (Figure 10R), S7D13 in 2 L roller bottles (Figure 10S), and S7D14 in 0.1 PBS (Figure 10T). [Figure 10Q]We demonstrate the generation of insulin-producing cells from CyT49 hESCs in multiple suspension cultures. Figures 10O-10T show the gradual formation of protein colocalization of insulin-producing cell markers, including CHGA, NKX6.1, and insulin, but not glucagon, for S5D3 in 0.5 PBS (Figures 10O-10P), S6D7 in 2 L roller bottles (Figure 10Q), S6D7 in 0.1 PBS (Figure 10R), S7D13 in 2 L roller bottles (Figure 10S), and S7D14 in 0.1 PBS (Figure 10T). [Figure 10R] We demonstrate the generation of insulin-producing cells from CyT49 hESCs in multiple suspension cultures. Figures 10O-10T show the gradual formation of protein colocalization of insulin-producing cell markers, including CHGA, NKX6.1, and insulin, but not glucagon, for S5D3 in 0.5 PBS (Figures 10O-10P), S6D7 in 2 L roller bottles (Figure 10Q), S6D7 in 0.1 PBS (Figure 10R), S7D13 in 2 L roller bottles (Figure 10S), and S7D14 in 0.1 PBS (Figure 10T). [Figure 10S] We demonstrate the generation of insulin-producing cells from CyT49 hESCs in multiple suspension cultures. Figures 10O-10T show the gradual formation of protein colocalization of insulin-producing cell markers, including CHGA, NKX6.1, and insulin, but not glucagon, for S5D3 in 0.5 PBS (Figures 10O-10P), S6D7 in 2 L roller bottles (Figure 10Q), S6D7 in 0.1 PBS (Figure 10R), S7D13 in 2 L roller bottles (Figure 10S), and S7D14 in 0.1 PBS (Figure 10T). [Figure 10T]We demonstrate the generation of insulin-producing cells from CyT49 hESCs in multiple suspension cultures. Figures 10O-10T show the gradual formation of protein colocalization of insulin-producing cell markers, including CHGA, NKX6.1, and insulin, but not glucagon, for S5D3 in 0.5 PBS (Figures 10O-10P), S6D7 in 2 L roller bottles (Figure 10Q), S6D7 in 0.1 PBS (Figure 10R), S7D13 in 2 L roller bottles (Figure 10S), and S7D14 in 0.1 PBS (Figure 10T). [Figure 10U] We demonstrate the generation of insulin-producing cells from CyT49 hESCs in multiple suspension cultures. Figure 10U shows the prevalence of NKX6.1 cells co-expressing CHGA, insulin, and glucagon, as well as the prevalence of cells co-expressing insulin and Glucagon, for PEC-01-derived S6D7 cells. Figure 10U shows the high prevalence of polyhormone insulin-producing cells and, consequently, the low number of monohormone insulin-positive cells for PEC-01-derived S6D7 aggregates in 2-liter roller bottles. [Figure 10V] Figure 10 demonstrates the generation of insulin-producing cells from CyT49 hESCs in multiple suspension cultures. Figure 10V shows the colocalization of glucagon and NKX6.1 for PEC-01-derived S6D7, 0.1PBS S7D14, and roller bottle (RB) S7D13. Figure 10V demonstrates that all insulin + glucagon + cells are negative for the presence of NKX6.1 in all conditions tested (PEC-01-derived S6D7, 0.1PBS S7D14 using the new protocol, and RB S7D13). [Figure 10W]We demonstrate the generation of insulin-producing cells from CyT49 hESCs in multiple suspension cultures. Figures 10W-10AB show protein colocalization of insulin-producing cell markers, such as synaptophysin (a pan-endocrine marker), NKX6.1, insulin, and MAFA, at S6D7 in 0.1L PBS (Figures 10W-10X), S7D16 in 0.1L PBS (Figures 10Y-10Z), and S7D23 in 2-liter roller bottles (Figures 10AA-10AB). By stage 7, robust protein colocalization between synaptophysin, NKX6.1, insulin, and MAFA was observed, indicating nonfunctional monohormonal insulin-producing cells. [Figure 10X] We demonstrate the generation of insulin-producing cells from CyT49 hESCs in multiple suspension cultures. Figures 10W-10AB show protein colocalization of insulin-producing cell markers, such as synaptophysin (a pan-endocrine marker), NKX6.1, insulin, and MAFA, at S6D7 in 0.1L PBS (Figures 10W-10X), S7D16 in 0.1L PBS (Figures 10Y-10Z), and S7D23 in 2-liter roller bottles (Figures 10AA-10AB). By stage 7, robust protein colocalization between synaptophysin, NKX6.1, insulin, and MAFA was observed, indicating nonfunctional monohormonal insulin-producing cells. [Figure 10Y] We demonstrate the generation of insulin-producing cells from CyT49 hESCs in multiple suspension cultures. Figures 10W-10AB show protein colocalization of insulin-producing cell markers, such as synaptophysin (a pan-endocrine marker), NKX6.1, insulin, and MAFA, at S6D7 in 0.1L PBS (Figures 10W-10X), S7D16 in 0.1L PBS (Figures 10Y-10Z), and S7D23 in 2-liter roller bottles (Figures 10AA-10AB). By stage 7, robust protein colocalization between synaptophysin, NKX6.1, insulin, and MAFA was observed, indicating nonfunctional monohormonal insulin-producing cells. [Figure 10Z]We demonstrate the generation of insulin-producing cells from CyT49 hESCs in multiple suspension cultures. Figures 10W-10AB show protein colocalization of insulin-producing cell markers, such as synaptophysin (a pan-endocrine marker), NKX6.1, insulin, and MAFA, at S6D7 in 0.1L PBS (Figures 10W-10X), S7D16 in 0.1L PBS (Figures 10Y-10Z), and S7D23 in 2-liter roller bottles (Figures 10AA-10AB). By stage 7, robust protein colocalization between synaptophysin, NKX6.1, insulin, and MAFA was observed, indicating nonfunctional monohormonal insulin-producing cells. [Figure 10AA] We demonstrate the generation of insulin-producing cells from CyT49 hESCs in multiple suspension cultures. Figures 10W-10AB show protein colocalization of insulin-producing cell markers, such as synaptophysin (a pan-endocrine marker), NKX6.1, insulin, and MAFA, at S6D7 in 0.1L PBS (Figures 10W-10X), S7D16 in 0.1L PBS (Figures 10Y-10Z), and S7D23 in 2-liter roller bottles (Figures 10AA-10AB). By stage 7, robust protein colocalization between synaptophysin, NKX6.1, insulin, and MAFA was observed, indicating nonfunctional monohormonal insulin-producing cells. [Figure 10AB] We demonstrate the generation of insulin-producing cells from CyT49 hESCs in multiple suspension cultures. Figures 10W-10AB show protein colocalization of insulin-producing cell markers, such as synaptophysin (a pan-endocrine marker), NKX6.1, insulin, and MAFA, at S6D7 in 0.1L PBS (Figures 10W-10X), S7D16 in 0.1L PBS (Figures 10Y-10Z), and S7D23 in 2-liter roller bottles (Figures 10AA-10AB). By stage 7, robust protein colocalization between synaptophysin, NKX6.1, insulin, and MAFA was observed, indicating nonfunctional monohormonal insulin-producing cells. DETAILED DESCRIPTION OF THE INVENTION

[0042] The following detailed description of the present invention will be better understood when read in conjunction with the accompanying drawings. The drawings are provided to illustrate certain embodiments of the invention. However, the invention is not limited to the precise arrangements, embodiments, and instrumentalities shown. Therefore, and without limitation, the Detailed Description is intended to provide a specific understanding of the invention. The present invention is divided into subsections that describe or illustrate features, embodiments, or uses of the present invention.

[0043] A.Definition Stem cells are undifferentiated cells defined by both the ability to self-renew and the ability to differentiate at the single cell level. Stem cells include self-renewing progenitor cells, non-renewing progenitor cells, and terminally differentiated cells. Stem cells can also give rise to multiple germ layers (endoderm, mesoderm, and ectoderm). They are characterized by their ability to differentiate in vitro from stem cells (e.g., stem cells) into functional cells of various cell lineages. The blastocysts also give rise to tissues of multiple germ layers after implantation, and after injection into blastocysts, virtually all (if not all) stem cells contribute to most tissues (if not all) by virtue of their developmental potential. Pluripotent stem cells are capable of giving rise to all embryonic cell types.

[0044] Differentiation is unspecialized ("uncommitted") ") or relatively unspecialized cells are transformed into specialized cells, e.g., nerve cells or muscles. The process by which a cell acquires specific characteristics. Differentiated cells are more specific within a cell lineage. These are cells that take on a specialized ("committed") position. The term "committed" means that the cells continue to differentiate into a particular cell type or subset of cell types under normal circumstances, and under normal circumstances differentiate into different cell types or relatively undifferentiated cell types. A cell that has progressed in the differentiation pathway to a point where it cannot revert to differentiation. "Dimerization" refers to the return of cells to a state where they are relatively unspecialized (or uncommitted) within a cell lineage. As used herein, the lineage of a cell refers to the hereditary nature of that cell, i.e. , from which cell it comes, and what kind of cells it can give rise to. The lineage of a cell defines its position within a genetic scheme of development and differentiation. Lineage-specific markers are those that are specifically associated with the phenotype of cells of a lineage of interest. It refers to a characteristic of cells that can be used to assess the differentiation of uncommitted cells into a lineage of interest. It can be used.

[0045] As used herein, a "marker" refers to a nucleic acid that is differentially expressed in a cell of interest. or polypeptide molecules. In this regard, differential expression refers to the differentiation of undifferentiated or differentiated cells. Positive markers represent the level of proliferation compared to cells at the other stage, while negative markers represent the level of proliferation. For a marker, this means a reduced level. is sufficiently high or low in the target cells compared to other cells, Identifying the cells of interest from other cells using any of a variety of methods known in the art and it is possible to distinguish between them.

[0046] As used herein, "cell density" and "seeding density" are used interchangeably herein. used to calculate the number of cells seeded per unit area of a solid or semi-solid planar or curved substrate. Point.

[0047] As used herein, a "suspension culture" refers to a cell that is suspended in a medium rather than attached to a surface. Refers to a culture of suspended cells, single cells, or clusters.

[0048] As used herein, a cell is considered to be a cell of interest when specific markers are sufficiently detected within the cell. Similarly, cells are "positive for" a specific marker, "positive" or "+". When the marker is not sufficiently detected in the cells, the cells are classified as "negative for" the specific marker; " or "-". In particular, by fluorescence activated cell sorting cytometry ("FACS") The FACS threshold for positivity is usually below about 1%, while the FACS threshold for positivity is usually above about 2%. Positive results by enzyme chain reaction cytometry ("PCR") usually occur at approximately 30 cycles (Ct s) or less, but negative PCR results usually exceed about 31 cycles.

[0049] Replicating Differentiation of Pluripotent Stem Cells into Functional Pancreatic Endocrine Cells in Static In Vitro Cell Culture In attempts to do so, the differentiation process can be seen to proceed through a number of successive stages. In particular, the differentiation process is generally viewed as proceeding through multiple stages. In this stepwise differentiation, "Stage 1" refers to the first step in the differentiation process, the differentiation of pluripotent stem cells. Differentiation of cells into cells expressing markers characteristic of definitive endoderm ("stage 1 cells"). "Stage 2" refers to the second step, where cells express markers characteristic of definitive endoderm cells. Differentiation of cells into cells expressing markers characteristic of early intestinal cells ("stage 2 cells"). "Stage 3" refers to the third step, in which markers characteristic of early intestinal cells are expressed. cells into cells expressing markers characteristic of foregut endoderm cells ("stage 3 cells"). "Stage 4" refers to the fourth step, the expression of markers characteristic of foregut endoderm cells. cells that express markers characteristic of pancreatic endoderm cells ("stage 4 cells"). "Stage 5" refers to the fifth step, the expression of markers characteristic of pancreatic endoderm cells. markers characteristic of one or both of pancreatic endoderm cells and pancreatic endocrine precursor cells, -expressing cells (collectively "stage 5 cells" or alternatively "pancreatic endoderm / endocrine precursors" Stage 6 refers to the sixth step, differentiation into pancreatic endocrine progenitor cells. Cells expressing markers characteristic of immature beta cells ("Stage 6 cells"). In and for this purpose, a seventh step, "Stage 7," is used to differentiate immature beta cells. These cells express markers characteristic of pancreatic endocrine cells (beta cells), which are functional beta cells and stem cells. Cells expressing markers characteristic of cells with a more mature phenotype compared to Ge 6 cells This refers to the differentiation of “functional beta cells with a more mature phenotype” or “stage 7 The cells were single hormone insulin+, MAFA+, and IFN-γ-glucan-1 (IFN-γ) cells compared to stage 6 cells. NKX6.1+, UCN3+, SLC2A1+, and PDX1+ as well as more immature Pancreatic endocrine cells express MAFA at higher levels than mature pancreatic endocrine cells, especially immature beta cells. It means cells.

[0050] It is important to note that not all cells in a particular population progress through these stages at the same rate. In conclusion, in in vitro cell culture, especially at the late differentiation stage, aggregation They may be less advanced in the differentiation pathway than the majority of cells present in the population, or may be more advanced than the majority. For example, in the culture of stage 5 cells, During cultivation, it is not uncommon to observe the appearance of markers characteristic of pancreatic endocrine cells. For purposes of this discussion, characteristics of the various cell types associated with the above-identified stages are described herein. do.

[0051] As used herein, "definitive endoderm" refers to the layer of the embryo that arises from the epiblast during gastrulation and is responsible for the development of the gastrointestinal tract. Definitive endoderm cells are cells that possess the characteristics of cells that form the ducts and their derivatives. The following markers were identified: FOXA2 (hepatocyte nuclear factor 3-β (“HNF3-β”)) also known as GATA4, SOX17, CXCR4, and Brachy ury), Cerberus, OTX2, Goosecoid, C-Kit, CD99, and MIXL 1. Markers characteristic of definitive endoderm cells include CXCR 4, FOXA2, and SOX17. Thus, definitive endoderm cells express CXCR4, In addition, cells can be characterized by the expression of FOXA2 and SOX17. Depending on the length of time allowed to remain at 1, an increase in HNF4α can be observed.

[0052] "Primitive intestinal cells," as used herein, are cells derived from the definitive endoderm and are used in the development of the lung, liver, pancreas, and other organs. These cells are capable of giving rise to all endodermal organs, such as the peritoneum, the stomach, and the intestine. Characterized by substantially increased expression of HNF4α relative to that expressed by definitive endoderm cells For example, a 10- to 40-fold increase in HNF4α mRNA expression can be observed in stage 1. 2.

[0053] "Foregut endoderm cells," as used herein, include cells from the esophagus, lung, stomach, liver, pancreas, bladder, Foregut endoderm cells are cells that give rise to the peritoneum and part of the duodenum. at least one of PDX1, FOXA2, CDX2, SOX2, and HNF4α Foregut endoderm cells are characterized by increased expression of PDX1 compared to intestinal cells. For example, in stage 3 cultures, more than 50 percent of the cells typically undergo PD. Expresses X1.

[0054] "Pancreatic endoderm cells," as used herein, are defined as cells expressing the following markers: PDX1 , NKX6.1, HNF1β, PTF1α, HNF6, HNF4α, SOX9, NGN3 , gastrin, HB9, or PROX1. Pancreatic endoderm cells can be characterized by a substantial lack of expression of CDX2 or SOX2.

[0055] "Pancreatic endocrine precursor cells," as used herein, are cells that can become pancreatic hormone-expressing cells. Pancreatic endocrine progenitor cells are characterized by the following markers: NGN3 , NKX2.2, NeuroD1, ISL1, PAX4, PAX6, or ARX Pancreatic endocrine progenitor cells express at least one of NKX2.2 and NeuroD1. It can be characterized by the present.

[0056] "Pancreatic endocrine cells," as used herein, are cells that produce the following hormones: insulin; at least one of glucagon, somatostatin, ghrelin, and pancreatic polypeptide In addition to these hormones, pancreatic endocrine cells are characterized by Typical markers include NeuroD1, ISL1, PDX1, NKX6.1, and PAX4. , ARX, NKX2.2, HB9, and PAX6.

[0057] "Beta cells" ("β cells") have the ability to express insulin, but not glucagon. , pancreatic endocrine cells that lack the ability to express somatostatin, ghrelin, and pancreatic polypeptide Pancreatic endocrine cells expressing markers characteristic of β cells are responsible for insulin and the following: transcription factors, namely, PDX1, NKX2.2, NKX6.1, NeuroD1, IS L1, HNF3β, HB9, MAFA, and PAX6. can be assigned.

[0058] "Functional beta cells" are those that are capable of rapid, regulated glucose-stimulated insulin secretion ("GS"). Specifically, phase 1 after increased mitochondrial respiration / activity and a second phase of insulin secretion ("biphasic GSIS"), In detail, functional beta cells are pancreatic endocrine cells that exhibit biphasic GSIS. The properties of (i) coupling of mitochondrial respiration / activity with insulin secretion (ii) respond to increased demand (defined herein as high glucose concentrations) (iii) a rapid insulin secretory response, and (iv) a rapid cessation of insulin secretion after demand subsides. (iv) the ability to switch insulin secretion "on and off" multiple times. (v) the ability to secrete the correct amount of insulin as determined by demand; and (vi) Multiple insulin secretagogues (e.g., exendin-4, or amino acid L-glucan) The ability to respond to L-glutamine and L-arginine. Targeted beta cells express insulin and the following transcription factors: PDX1, NKX2.2, NKX6.1, NeuroD1, ISL1, HNF3β, HB9, PAX6, MAFA, Characterized by expression of at least one of SLC2A1, UCN3, and GLP1R That's fine.

[0059] Immature beta cells have glucose-dependent mitochondrial respiration / activity and biphasic G These are pancreatic endocrine cells that do not show SIS. Immature β cells express markers characteristic of β cells. The tumor cells express insulin and the following transcription factors: PDX1, NKX2.2, and NKX 6.1, NeuroD1, ISL1, HNF3β, HB9, MAFA, and PAX6 It can be characterized by at least one of these.

[0060] "Air-liquid interface" or "ALI" as used herein refers to an open culture vessel or culture The term "air-liquid interface" refers to the air-liquid interface that exists in a culture vessel that is partially filled with liquid. Although referred to above as "air," the present invention is directed to the mixture of gases and compounds found in the ambient environment. The present invention is not limited to the specific contemplated, e.g., enriched in a particular component or Gases with a composition different from the surrounding environment, including mixtures in which certain components have been depleted or eliminated Includes mixtures.

[0061] As used herein, "d1", "1d", and "day 1", "d2", "2d", and "2 These number combinations are used interchangeably to represent the stepwise differentiation process of the present application. These refer to specific days of culture at different stages during the protocol.

[0062] Phase 1 (1 st) insulin secretion occurs when glucose levels increase rapidly. , representing rapid exocytosis of a small pool of readily releasable insulin granules.

[0063] The second phase (2 nd ) Insulin secretion in the nucleus involves mobilizing granules from the storage pool of granules and releasing them. It refers to binding / priming these.

[0064] OCR is defined as the rate of oxygen consumption, specifically via the electron transport chain ("ETC"). It is also an indicator of mitochondrial respiration and a direct measure of mitochondrial activity.

[0065] An "effective amount" or "therapeutic amount" or its equivalent is an amount that induces some degree of hESC differentiation or are partially differentiated hESCs (e.g., those that have been subjected to one or more previous stages of differentiation) In additional examples, the compound may be present in the culture medium of hESCs or during any proliferation stage of hESCs. In some embodiments, a compound, drug, small molecule, or growth factor may be added to C. Using factors, definitive endoderm cells, foregut cells, pancreatic foregut cells, and pancreatic hormone-secreting cells In certain instances, hepatocytes are produced, which are capable of expressing any differentiated cell type. exposed to compounds, drugs, small molecules, or growth factors prior to or during the first stage of differentiation. In other instances, hepatocytes may be differentiated into intermediate cell types, such as definitive endoderm. They may be differentiated first and then exposed to a compound, drug, small molecule, or growth factor.

[0066] The "compounds," "small molecule compounds," or their equivalents of the present invention are those disclosed herein. "Compounds" refers to compounds encompassed by the general formulas (e.g., Table XI) and whose structures are disclosed herein. The compounds of the present invention include any specific compound or analog thereof in the formulation. They may be identified by either chemical structure or chemical name. If the chemical structure and chemical name do not match, In this case, the chemical structure determines the identity of the compound. and may contain chiral centers or double bonds, resulting in stereoisomers, e.g., double bond isomers ( That is, they may exist as geometric isomers, enantiomers, or diastereomers. Thus, the chemical structures depicted herein represent all possible mirror images of the exemplified compounds. It includes isomers and stereoisomers, including stereoisomerically pure forms (e.g., geometrically enantiomerically pure, enantiomerically pure, or diastereomerically pure), and Enantiomeric and stereoisomeric mixtures are included. Enantiomeric and stereoisomeric mixtures are known in the art. their constituent enantiomers or stereoisomers using separation techniques or chiral synthesis techniques well known to those skilled in the art. The compounds of the present invention can also be decomposed into their constituent isomers, where one or more atoms are This includes isotopically labeled compounds having atomic masses different from those found in nature. Examples of isotopes that may be incorporated into compounds of the invention include 2H, 3H, 13C, 14C, 1 Examples include 5N, 18O, 17O, 31P, 32P, 35S, 18F, and 36Cl. Furthermore, it is to be understood that the partial structures of the compounds of the present invention are not limited to these. When used in conjunction with a substituted or unsubstituted aryl group, the parentheses indicate the point of attachment of the substructure to the rest of the molecule.

[0067] B. Characterization of Pluripotent Stem Cells The pluripotent stem cells are cultured using one or two of the designated TRA-1-60 and TRA-1-81 antibodies. They may express one or more of the following genes (Thomson et al., 1998, Science 282 :1145-1147). In vitro differentiation of pluripotent stem cells was performed using TRA-1-60 and T This results in the loss of RA-1-81 expression. Undifferentiated pluripotent stem cells are typically expressed as 4 After fixation with 100% paraformaldehyde, the cells were cultured in a 5% CO2-free medium according to the manufacturer's instructions (Vector Laboratories, es, Inc., Burlingame, California) Alkaline phosphatase sold under the trademark VECTOR® Red is Alkaline phosphatase, which can be detected by generating it with a phosphatase substrate kit Undifferentiated pluripotent stem cells also typically have reverse transcription polymerase chain reaction (RTP) activity. OCT4 and TERT are also expressed, as detected by RT-PCR.

[0068] Another desirable phenotype of the expanded pluripotent stem cells is the differentiation of three embryonic germ layers: endoderm, mesoderm, and ectoderm. The pluripotency of stem cells is their ability to differentiate into all cell types of the lamina. Teratomas formed in SCID mice were treated with 4% paraformaldehyde. The cells were then fixed in a PBS solution and examined histologically to determine the origin of these three germ layer-derived cell types. Alternatively, pluripotency can be confirmed by the formation of embryoid bodies, which can then be divided into three germ layers. This can be determined by assessing for the presence of markers associated with

[0069] The expanded pluripotent stem cell lines were karyotyped using standard G-banding techniques and then Cells can be compared with established karyotypes of the corresponding primate species. Cells with a "normal karyotype" It is desirable to obtain a "normal karyotype" when the cell is euploid and the human chromosomes are It means that everything is there and there are no noticeable changes.

[0070] C. Sources of Pluripotent Stem Cells Any pluripotent stem cells can be used in the methods of the present invention. Exemplary types include any time during pregnancy (usually, but not necessarily, around 10th pregnancy) Includes pre-embryonic tissue (e.g., blastocysts), embryonic tissue, or fetal tissue collected before 12 weeks of age and established lines of pluripotent cells. Non-limiting examples include human embryonic stem cells ("hESCs"). ) or an established line of human embryonic germ cells, human embryonic stem cell line H1 (NIH code: WA01 ), H7 (NIH code: WA07), H9 (NIH code: WA09) (WiCell Research Institute, Madison, WI, USA), and SA 002(Cellartis AB Corporation,Goteburg,Sw eden), etc.

[0071] Cells taken from a pluripotent stem cell population already cultured in the absence of feeder cells are also suitable. Many pluripotency-inducing genes, including OCT4, NANOG, SOX2, KLF4, and ZFP42, are involved in the regulation of pluripotency. Using forced expression of transcription factors related to pluripotent stem cells (ITCs), we have generated induced pluripotent cells (ITCs) derived from adult somatic cells. PS) or reprogrammed pluripotent cells (Annu Rev Genomics H um Genet 2011,12:165~185, IPS,Cell,126(4) :663-676) can also be used. Human embryonic stem cells can also be prepared as described by Thomson et al. (US Pat. No. 6,429,199). No. 5,843,780, Science, 1998, 282: 1145~1147; Curr Top Dev Biol 1998,38:133~165;Proc N atl Acad Sci USA 1995, 92:7844~7848). B.G. Mutant human embryonic stem cells, such as 01v (BresaGen, Athens, Georgia). Cell lines, or Takahashi et al., Cell 131:1-12 (2007 In certain embodiments, cells derived from human somatic cells, such as those disclosed in In some embodiments, pluripotent stem cells suitable for use in the present invention are those described by Li et al. (Cell Stem Cell Cell 4:16~19,2009), Maherali et al. ell 1:55~70, 2007), Stadtfeld et al. ell 2:230~240), Nakagawa et al. (Nature Biotechn ol 26:101~106,2008), Takahashi et al. (Cell 131: 861-872, 2007), and U.S. Patent Application Publication No. 2011 / 0104805. In certain embodiments, the use of the present invention Pluripotent stem cells suitable for are considered to be "naive" and can be cultured using the methods described below, i.e. , Gafni et al. (Nature, 504:282, 2013), and Ware et al. (PNA S, 111: 4484-4489, 2014). All of the literature, patents, and patent applications relate, inter alia, to the isolation, culture, expansion, and propagation of pluripotent cells. and differentiation, which are incorporated herein by reference in their entireties.

[0072] Other sources of pluripotent stem cells include induced pluripotent stem cells (IPS, Cell, 1 26(4):663-676). Still other sources of suitable cells include human umbilical cord tissue-derived cells. Examples of such cells include human amniotic fluid-derived cells, human placenta-derived cells, and human parthenogenetic organisms. In this embodiment, umbilical cord tissue-derived cells are cultured by the method described in U.S. Pat. No. 7,510,873. In another embodiment, placental tissue-derived cells may be obtained by the method described in U.S. Patent Application Publication No. 2003 / 0129994. In another embodiment, the amniotic fluid-derived The cells may be obtained using the methods described in U.S. Patent Application Publication No. 2007 / 0122903. The disclosure of each of these patent applications is pertinent as it relates to the isolation and characterization of cells. , which is incorporated herein in its entirety. In certain embodiments, the pluripotent stem cells are May be of non-viviparous origin.

[0073] D. Proliferation and Culture of Pluripotent Stem Cells Many different known methods for growing and culturing pluripotent stem cells can be used in the claimed invention. For example, pluripotent stem cells may be plated onto a suitable culture substrate. In one embodiment, a suitable culture substrate is one derived from a basement membrane or one that accepts adhesion molecules. Suitable extracellular matrix components include those that can form part of a ligand-binding protein. The culture substrate is manufactured under the trademark MATRIGEL(TM) (Corning Incorporate d, Corning, New York) MATRIGEL™ is a treatment for Engelbreth-Holm Swarm tumors. It is a soluble cell-derived preparation that gels at room temperature to form a reconstituted basement membrane.

[0074] Other extracellular matrix components and component mixtures known in the art are alternatives. Depending on the cell type being grown, this may be laminin, fibronectin, protease inhibitors, or other suitable substrates. Proteoglycan, entactin, heparin sulfate, and the like, alone or It may be included in various combinations.

[0075] Pluripotent stem cells, in their preferred distribution, promote cell survival, proliferation, and maintenance of desired characteristics All of these characteristics allow careful consideration of seeding distribution. These procedures will benefit from careful consideration and can be readily determined by one skilled in the art. A suitable culture medium may be prepared using the following ingredients: Registered trademark GIBCO by Grand Island, New York Dulbecco's modified egg, sold under the product name (catalog number 11965-092) DMEM (“DMEM”), Life Technologies Corporatio Sold by GIBCO under the trademark GIBCO® (Cat. No. 10829-018) Knockout Dulbecco's Modified Eagle Medium ("KO DMEM"), Ham F1 2 / 50% DMEM Basal Medium, trademark GIB by Life Technologies CO (registered trademark) (catalog number 25030-081) M L-Glutamine, trademarked by GIBCO Life Technologies A non-essential amino acid solution sold under the trademark (catalog number 11140-050) , β-mercaptoethanol, Sigma-Aldrich Company, LLC Saint Louis, MO (Catalog No. M7522), Life Technology Trademarked by GIBCO® (Cat. No. 13256-029) It is made from human recombinant basic fibroblast growth factor ("bFGF"), which is sold under the Large-scale expansion and controlled differentiation of human embryonic stem cells in suspension bioreactors This can also be achieved using

[0076] E. Differentiation of Pluripotent Stem Cells As pluripotent cells differentiate towards functional β cells, they pass through various stages. These differentiate into various types, each of which can be characterized by the presence or absence of specific markers. Differentiation of cells into stages involves the presence or absence of certain factors added to the culture medium. This differentiation is achieved by specific culture conditions. Generally, this differentiation is achieved by the differentiation of pluripotent stem cells into definitive embryos. These cells then give rise to the early intestinal tract. These cells can further differentiate into blastocysts, which can then in turn differentiate into foregut endoderm cells. , which can be differentiated into pancreatic endoderm cells, and then into pancreatic endocrine progenitor cells or pancreatic endoderm / pancreatic endocrine progenitor cells. These cells can be further differentiated into pancreatic hormone-producing or secretory cells. This application is directed to an air-liquid exchange membrane that is preferably present in a culture vessel partially filled with medium. By culturing cells at the body interface or in suspension, it is possible to achieve a specific stage, particularly one of stages 5-7. By culturing cells at the air-liquid interface or in suspension at two or more It provides stepwise differentiation of stem cells toward pancreatic endocrine cells.

[0077] the thyroid hormones triiodothyronine ("T3") and thyroxine ("T4"), and One or more of these analogs may be used alone or in further combination with an ALK-5 inhibitor. In combination, at one or more of differentiation stages 1 to 7, preferably at stage 5 Alternatively, ALK-5 inhibitors can be used in cells cultured at each of the following stages of differentiation: In one or more stages, but preferably in each of stages 5 to 7, more preferably Preferably, it can be used alone at each of stages 5 and 6. More preferably, it is used as a thyroid hormone. One or more of the ALK5 inhibitors or their analogs may be used in combination with one or more of the In the above differentiation stages, preferably at each of stages 5 to 7, more preferably at stage Suitable thyroid hormone analogs include, without limitation, GC-1 (Sobertirome)(R&D Systems, Inc.Minneapolis) s, Minnesota), 3,5-diiodothyropropionic acid ("DIP TA”), J.Steroid Biochem.Mol.Biol.,2008,11 1:262-267 and Proc.Natl.Acad.Sci.US 2003,10 KB-141, Proc. Natl. Ac. MB discussed in ad.Sci.US 2007,104:15490~15495 07344, J. Lipid Res., May 2009, 50:938 and Endo T0681 discussed in cr.Pract.2012,18(6):954~964 (the disclosures of which are incorporated herein by reference in their entireties). Useful ALK5 inhibitors include ALK5 inhibitor II, which is also a preferred ALK5 inhibitor. (Enzo Life Sciences, Inc., Farmingdale, New York), ALK5i (Axxora, Inc., San Diego, California) ornia), SD208 (R&D Systems), TGF-β inhibitor SB4315 42(Xcess Biosciences, Inc., San Diego, Cali fornia), ITD-1 (Xcess Biosciences), LY21097 61 (Xcess Biosciences), A83-01 (Xcess Biosc iences), LY2157299 (Xcess Biosciences), TGF -β receptor inhibitor V (EMD Millipore Chemical, Gibstow n, New Jersey), TGF-β receptor inhibitor I (EMD Millipore ), TGF-β receptor inhibitor IV (EMD Millipore), TGF-β receptor inhibitor Inhibitor VII (EMD Millipore), TGF-β receptor inhibitor VIII (EMD Millipore), TGF-β receptor inhibitor II (EMD Millipore) , TGF-β receptor inhibitor VI (EMD Millipore), and TGF-β receptor Inhibitor VI (EMD Millipore).

[0078] In additional preferred embodiments of the invention, these methods include administering to a subject a composition comprising vitamin E, acetylcysteine, tein, vitamin C, antioxidant supplement (catalog no. A1345, Sigma-Aldrich) Company, LLC Saint Louis, Missouri, Group Antioxidants such as thiocyanate, superoxide dismutase, catalase, and the like and a combination thereof. Treating cells at stage 7, but preferably treating cells during stage 8 In an even more preferred embodiment, when performing stage 6, gamma secretion is performed. A gamma secretase inhibitor, gamma secretase inhibitor XX (EMD Millipo re), gamma secretase inhibitor XXI (EMD Millipore), gammasecretase Lipase inhibitor XVI (EMD Millipore), N-[(3,5-difluoromethyl)- [phenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl DAPT ester ("DAPT") (Cat. No. 2634, Tocris Biosciences e, Bristol, United Kingdom), and similar, and A useful amount of a gamma secretase inhibitor can be about 50 nM The amount of antioxidant can be about 0 to 5000 nM, preferably about 50 nM to 500 nM. 0.1 to 100 μM, alternatively about 0.1 to 20 μM, and preferably about 1 to 10 μM. Alternatively, useful amounts of antioxidants may range from about 100 nM to 5 mM, about 1000 nM to 2 mM, or about 1000 nM to 2 mM. It may be mM, and preferably about 0.1 to 1 mM.

[0079] In embodiments of the present invention, certain small molecules are useful in inhibiting one or more stages of differentiation. The medium is preferably used in one or both of stages 6 and 7. Small molecules inhibit Aurora kinase, p90 ribosomal S6 kinase, or methyltransferase The enzyme is capable of inhibiting the DOT1L receptor, and is preferably used in cultured cells. It is used in conjunction with antioxidants to reduce oxidative stress in cells. Aurora kinase inhibitor II and RSK inhibitor II. Aurora kinase inhibitor II is (4 -(4'-benzamidoanilino)-6,7-dimethoxyquinazoline RSK inhibitor II is a cell-permeable compound. Fluoro-4-hydroxy-anilino)-8-isopentyl-5,7-dimethyl-7H- It is a racemic mixture of pteridin-6-ones. Examples of the DOT1L protein include ZM447439 and PF03814735. Methyltransferase receptor inhibitors, particularly EPZ-5676, are also of interest. 5676 inhibits S-adenosylmethionine in the protein methyltransferase DOT1L. This compound has been shown to inhibit cell proliferation. DOT1L (disruptor of telomere silencing-1-like) ncing 1-like) methyltransferase inhibitor. -6-amine, 9-[5-deoxy-5-[[cis-3-[2-[6-(1,1-dimethyl ethylethyl)-1H-benzimidazol-2-yl]ethyl]cyclobutyl](1-methylethyl)-1H-benzimidazol-2-yl]ethyl]cyclobutyl] (2R,3R,4S,5R) methylethylamino]-β-D-ribofuranosyl]-, and (2R,3R,4S,5R) -2-(6-aminopurin-9-yl)-5-[[[3-[2-(6-tert-butyl -1H-benzimidazol-2-yl)ethyl]cyclobutyl]-propan-2-yl Aminomethyloxolane-3,4-diol is known. Further targeted inhibition agents include DNA methyltransferase inhibitors, such as 5-azacytidine ("AZT"); Histone deacetylase inhibitors such as pyroxamide and CI994. Additional small molecules include UNC0638, UNC0646, UNC0642, and A366 (G 9a and GLP histone lysine methyltransferase inhibitor), TC-E5003 ( PRMT1 arginine methyltransferase inhibitor), SB747651A dihydro chloride (MSK1 inhibitor, also inhibits other AGC kinases), PFI1 (BET blocker) romodomain inhibitor), LY303511 (BRD2, BRD3, and BRD4 inhibitor) , MS436 (BRD4 bromodomain inhibitor), and MC1568 (class II H These include 3-deazaneplanocin A ("DEZA"), which selectively inhibits DAC, and 3-deazaneplanocin A ("DEZA"). can be done.

[0080] In a preferred embodiment of the present invention, the small molecule is More preferably, the medium is used in stage 7 or more, more preferably in stage 8 medium. Select the amount that shows the best expression of maturation markers and which amount does not cause toxic effects. Typically, a useful amount is about 500 nM to 10 μM, alternatively The target concentration is about 500 nM to 5 μM, and preferably about 500 nM to 2 μM.

[0081] Pluripotent cells have a mature phenotype characteristic of pancreatic endocrine cells (functional beta cells) Differentiation into cells expressing markers The properties of pluripotent stem cells are well known to those skilled in the art, and further properties of pluripotent stem cells continue to be identified. Markers of pluripotent stem cells include, for example, the following: ABC G2, cripto, FOXD3, CONNEXIN43, CONNEXIN45, OC T4, SOX2, NANOG, hTERT, UTF1, ZFP42, SSEA-3, SS EA-4, TRA-1-60, and TRA-1-81. These may be detectable by RT-PCR.

[0082] Exemplary pluripotent stem cells include the human embryonic stem cell line H9 (NIH code: WA09), Human embryonic stem cell line H1 (NIH code: WA01), human embryonic stem cell line H7 (NIH code: WA07), and human embryonic stem cell line SA002. The following markers were identified: ABCG2, cripto, CD9, FOXD3, CO NNEXIN43, CONNEXIN45, OCT4, SOX2, NANOG, hTER T, UTF1, ZFP42, SSEA-3, SSEA-4, TRA-1-60, and TR Also suitable are cells that express at least one of A-1-81.

[0083] Cells expressing at least one marker characteristic of the definitive endoderm lineage are considered to be markers of the present invention. In one embodiment of the present invention, the present invention provides a method for the production of a medicament for the preparation of a medicament for the production ... In an alternative embodiment, the cells specific to the definitive endoderm lineage are primitive streak precursor cells. The cells expressing characteristic markers are mesendoderm cells. Cells expressing markers characteristic of the endodermal lineage are definitive endoderm cells.

[0084] Cells expressing at least one of these markers characteristic of the pancreatic endoderm lineage are also present. In one aspect of the present invention, a mammalian cell lineage characteristic of the pancreatic endoderm lineage is also suitable for use in the present invention. The cells expressing the markers are pancreatic endoderm cells, and the expression of PDX1 and NKX6.1 is The expression of DX2 and SOX2 is substantially higher. In certain embodiments, the expression of DX2 and SOX2 is As determined by the NIH / ... The expression of PDX1 and NKX6.1 is higher than that of CDX2. Or cells with at least two-fold higher expression of SOX2 are particularly useful.

[0085] Cells expressing at least one of these markers characteristic of the pancreatic endocrine lineage are also present. In one embodiment of the present invention, a method for producing a pancreatic endocrine system-specific Cells that express characteristic markers are pancreatic endocrine cells. insulin, glucagon, somatostatin, ghrelin, or pancreatic polypeptides The cells may be pancreatic hormone-expressing cells capable of expressing at least one of the following hormones: In a preferred embodiment, the pancreatic endocrine cells are insulin-producing beta cells.

[0086] In certain embodiments of the present invention, functional beta cells (pancreatic endocrine Starting with pluripotent stem cells to arrive at cells expressing markers characteristic of beta cells A protocol is used that includes the following:

[0087] [Table 1]

[0088] In certain embodiments, the present invention provides methods for the production of pluripotent stem cells (e.g., pre-stage 1 cells). ) into stage 7 cells, but the present invention also encompasses differentiation of cells at other stages. The present invention also encompasses differentiation of stage 4 cells towards stage 7. This process involves the differentiation of stage 7 cells into stage 8 cells. However, the processing and progression of cells through the differentiation process can be sequential or continuous. Differentiation of reproductive stem cells into stage 6 or stage 7 cells can be achieved in suspension culture. Cut.

[0089] Differentiation efficiency is determined by dividing the treated cell population into protein markers expressed by the differentiated cells of interest. The degree of cell proliferation may be determined by exposing the cells to an agent, such as an antibody, that specifically recognizes the cell. Methods for assessing the expression of protein and nucleic acid markers in isolated cells are well known in the art. These methods include RT-PCR, Northern blot, and in situ hybridization. tubing hybridization (see, e.g., Current Protocols in Microbiology). Olecular Biology(Ausubelet al., eds.2001 supplement), and immunoassays, e.g., immunoassays of material sections. Immunohistochemical analysis, Western blot, and analysis of markers accessible within intact cells In this case, flow cytometry analysis (FACS) (e.g., Harlow and Lan e,Using Antibodies:A Laboratory Manual,N ew york:Cold Spring Harbor Laboratory Pr. ess (1998)).

[0090] The differentiated cells may be further purified. For example, pluripotent stem cells may be purified by treating them with the methods of the present invention. The treated cell population is then purified using a protein marker that is characteristically expressed by the differentiated cells. Differentiated cells may be purified by exposing them to agents (e.g., antibodies) that specifically recognize the target protein. In certain embodiments, the differentiated cells are not purified.

[0091] Sufficient amounts of vitamins, minerals, salts, glucose, amino acids, and other nutrients required for cell differentiation Any suitable growth medium containing somatic proteins can be used for the various stages 1-7. Preferably, the following: Stage 1-MCDB-131 (Life Technology Available from Aologies Corporation, Grand Island, NY (available from Sigma-Aldrich) or RPMI (available from Sigma-Aldrich), Stage 2-MCDB -131 or Dulbecco's Modified Eagle's Medium F12 ("DMEM-F12"), stage 3 ~5-MCDB-131, BLAR (Table 1 and Table IV), or DMEM, and stage 6 and 7-BLAR or CMRL (Life Technologies) are used Preferably, the glucose concentration of the medium is maintained at about 10 mM for stages 1 to 4. or more preferably kept lower, and for stages 5-7, about 10 mM or lower. Preferably, for stage 7, the medium contains sufficient amounts of vitamins, Contains non-essential amino acids, lipids, sodium pyruvate, and trace elements, e.g., Formulation I. do.

[0092] Stage 1: Differentiation of pluripotent cells into cells expressing markers characteristic of definitive endoderm cells transformation Pluripotent stem cells can be obtained by methods known in the art or by the methods proposed in the present invention. These cells can be differentiated into cells that express markers characteristic of definitive endoderm cells. These methods are useful for differentiating cells into cells that express markers characteristic of the definitive endoderm lineage. The method reported to be effective is that of D'Amour et al., Nature Biotech chnology 23,1534~1541(2005), Shinozaki et al., Development 131, 1651~1662 (2004), McL ean et al., Stem Cells 25, 29~38 (2007), and D 'Amour et al.,Nature Biotechnology 24,13 92-1401 (2006). Additional suitable differentiation methods are disclosed in U.S. Pat. Publication No. 2007 / 0254359, U.S. Patent Application Publication No. 2009 / 0170198, U.S. Patent Application Publication No. 2011 / 0091971, U.S. Patent Application Publication No. 2010 / 001 5711, U.S. Patent Application Publication No. 2012 / 0190111, U.S. Patent Application Publication No. 20 No. 12 / 0190112 and U.S. Patent Application Publication No. 2012 / 0196365. These disclosures are not intended to be limiting because they relate to the differentiation of pluripotent stem cells into definitive endoderm cells. and are hereby incorporated by reference in their entireties.

[0093] In one embodiment, the pluripotent cells are grown in a suitable growth medium, preferably MCDB-131 or The medium is preferably RPMI-treated. The medium preferably contains a growth differentiation factor, such as growth differentiation factor 8 (" GDF8") and glycogen synthase kinase-3β ("GSK3β") inhibitors, See, e.g., U.S. Patent Application Publication No. 2010 / 0015711, the entire contents of which are incorporated herein by reference. The embryos are supplemented with a cyclic aniline-pyridine triazine compound disclosed in Induce differentiation into cells expressing markers characteristic of definitive endoderm cells. Preferred: GSK3 β-blockers include 14-prop-2-en-1-yl-3,5,7,14,17,23,27 -Heptaazatetracyclo[19.3.1.1~2,6~.1~8,12~]heptacosa -1(25),2(27),3,5,8(26),9,11,21,23-Nonaene-1 6-one ("MCX compound"). The treatment involves treating pluripotent stem cells with a concentration of about 50 ng / mL to about 150 ng / mL, alternatively about 75 ng / mL to about 125 ng / mL, preferably about 10 This treatment may require contact with medium supplemented with 0 ng / mL of GDF8. Alternatively, the cells are treated with about 0.1 to about 5 μM, alternatively about 0.5 to about 2.5 μM, preferably about 1 It may also be necessary to contact the pluripotent cells with 1 μM of MCX compound. The pluripotent cells are preferably cultured for approximately 2-5 days. Preferably, the cells are cultured for about 2 to 3 days, and the cells express markers characteristic of definitive endoderm cells. The differentiation of the cells into cells that are capable of expressing the pluripotent stem cells may be promoted.

[0094] In a preferred embodiment, cells are cultured in the presence of GDF8 and MCX compounds for 1 day. and then cultured for 1 day in the presence of GDF8 and lower concentrations of MCX compounds. The cells were cultured for one day in the presence of DF8 but in the absence of MCX compounds. The cells were cultured for 1 day in the presence of MCX compound at about 0.8 and about 1 μM, followed by culture in the presence of GDF8 and about 0.1 μM MCX compounds were cultured for 1 day, followed by incubation in the presence of GDF8 and the non-MCX compounds. Alternatively, the cells are cultured for 1 day in the presence of GDF8 and about 1 μM of the MCX compound. The cells were cultured for 1 day in the presence of GDF8 and approximately 0.1 μM of the MCX compound. It may be cultivated.

[0095] Alternatively, pluripotent stem cells are cultured in medium containing activin A in the absence of serum. The cells can then be cultured with activin A and serum, as described by D'Amour et al. al.,Nature Biotechnology 23,1534~1541(20 05) cells were cultured with different concentrations of activin A and serum. In another alternative, pluripotent stem cells are cultured in a medium containing activin A in the absence of serum. The cells were cultured and then analyzed by D'Amour et al., Nature Biotechnology Cells were cultured with serum and activin A as disclosed in Technology, 2005. By culturing the cells in this manner, they differentiate into cells that express markers characteristic of definitive endoderm cells. The pluripotent stem cells may also be cultured in a medium containing activin A and a WNT ligand, without serum. Pluripotent stem cells were cultured in the absence of WNT ligands and then removed, and D'Amour et al. t al.,Nature Biotechnology 24,1392~1401( As disclosed in

[2006] , cells were cultured with activin A in combination with serum. They may also be differentiated into cells expressing markers characteristic of the definitive endoderm lineage.

[0096] In one embodiment of the present invention, pluripotent stem cells are treated with activin A and WNT3A. The treatment results in the formation of cells that express markers characteristic of definitive endoderm cells. The reproductive stem cells are treated with a serotonin-containing solution at a concentration of about 50 ng / mL to about 150 ng / mL, alternatively about 75 ng / mL to about 1 Alternatively, the antibody may be contacted with approximately 25 ng / mL of activin A, or alternatively, approximately 100 ng / mL of activin A. This treatment may also involve precipitating the cells at a concentration of about 10 ng / mL to about 50 ng / mL, alternatively about 1 Contact with 5 ng / mL to about 30 ng / mL, alternatively about 20 ng / mL, of WNT3A. The pluripotent cells may need to be cultured for about 3 days to reach definitive endoderm cells. In one embodiment, the cells are cultured in the presence of activin A and WNT3A for 1 day, followed by The remainder are cultured in the presence of activin A (but not WNT3A).

[0097] To detect the formation of cells expressing markers characteristic of definitive endoderm cells, specific promoters were used. Cells may be tested for the presence of markers before and after the protocol is performed. Therefore, differentiation of pluripotent cells is not possible if the cells are definitive embryos. They can be detected when they begin to express characteristic markers in the leaves.

[0098] Stage 2: Cells expressing markers characteristic of definitive endoderm cells are differentiated into early intestinal cells. Differentiation into cells expressing characteristic markers Cells expressing markers characteristic of definitive endoderm cells were cultured in MCDB-131 or DMEM. -Further differentiation into cells expressing markers characteristic of intestinal cells in a growth medium such as F12 In one embodiment, the formation of cells expressing markers characteristic of intestinal cells can be achieved by: Cells expressing markers characteristic of definitive endoderm cells were cultured using fibroblast growth factor (FGF) ), preferably by culturing the cells in a medium containing FGF7 or FGF10, to differentiate the cells. For example, the cell culture may be cultured at a concentration of about 10 ng / mL to about 75 ng / mL, alternatively about 25 ng / mL to about 75 ng / mL, or alternatively about 30 ng / mL to about 60 ng / mL mL, alternatively about 50 ng / mL of fibroblast growth factor, preferably FGF7 or FGF 10, more preferably FGF7, and most preferably about 25 ng / mL FGF7. The cells can be cultured under these conditions for about 2 to 3 days, preferably about 2 days.

[0099] In another embodiment, the formation of cells expressing markers characteristic of intestinal cells is achieved in vivo. Cells expressing markers characteristic of germ layer lineages can be cultured using fibroblast growth factors, preferably FGF This involves culturing the cells using FGF7 or FGF10, and ascorbic acid (vitamin C). The nutrient medium may contain from about 0.1 mM to about 0.5 mM ascorbic acid, alternatively from about 0.2 mM to about 0 The cells may contain about 0.4 mM ascorbic acid, alternatively about 0.25 mM ascorbic acid. The culture may also contain from about 10 ng / mL to about 35 ng / mL, alternatively from about 15 ng / mL to about 3 0 ng / mL, alternatively about 25 ng / mL of fibroblast growth factor, preferably FGF7 or The medium may also contain FGF10, and more preferably FGF7. For example, the cell culture may contain about 0.25 1 mM ascorbic acid and about 25 ng / mL FGF7. , stage 1 cells are treated with FGF7 and ascorbic acid for 2 days.

[0100] Stage 3: Cells expressing markers characteristic of early intestinal cells are differentiated into foregut endoderm cells. Differentiation into cells expressing characteristic markers The early intestinal cells arising from stage 2 were cultured in MCDB-131, DMEM, or custom media such as BLAR (Table I). can further differentiate into stage 3 cells, i.e., cells that express markers characteristic of foregut endoderm The medium may contain (i) a fibroblast growth factor, preferably FGF7 or FGF10, and more preferably (ii) retinoic acid ("RA"), (iii) 1-piperazine amine N-[(3,5-dimethyl-1-phenyl-1H-pyrazol-4-yl)methylene ]-4-(phenylmethyl)- or ((E)-4-benzyl-N-((3,5-dimethyl (1-phenyl-1H-pyrazol-4-yl), ethylene-piperazin-1-amine ), sonic hedgehog ("SHH") signaling pathway antagonists (e.g., Smoothened antagonist ("SANT-1"), 2-methoxyethyl 1,4,5,6,7,8-hexahydro-4-(3-hydroxyphenyl)-7-(2- methoxyphenyl)-2-methyl-5-oxo-3-quinolinecarboxylate, HPI-1, and preferably SANT-1; (iv) protein kinase C ("PKC" ) activator, e.g., ((2S,5S)-(E,E)-8-(5-(4-(trifluoromethyl) (Tetyl)phenyl)-2,4-pentadienylamino)benzolactam ("TPB") ), phorbol-12,13-dibutyrate ("PDBu"), phorbol-12-milli phthalocyanine-13-acetate ("PMA") or indolactam V ("ILV") and preferably preferably TPB, (v) bone morphogenetic protein (“BMP”) inhibitors, such as LDN-1 93189, Noggin, or Chordin, and preferably LDN-193189, and vi) Ascorbic acid may be supplemented. Alternatively, Smoothened ("SMO") receptor inhibitors may be used. Toxicants (e.g., MRT10 (N[[[3-benzoylamino)phenyl]amino]thioxanthate) [2,3,4,5-trimethoxybenzamide] or cyclopamine may also be used. For example, the cell culture may contain about 100 nM to about 500 nM, alternatively about 100 nM. The cells may contain about 400 nM to about 400 nM, alternatively about 200 nM, of a PKC activator. growth factors, small molecule agonists, and antagonists for about 2 to 4 days, preferably The can be cultured for about 2 to 3 days, more preferably for about 2 days.

[0101] Alternatively, stage 3 cells may be treated with SMO receptor inhibitors, SANT-1, retinoic acid, and Stage 2 cells were obtained by culturing these cells in culture medium supplemented with ATP and Noggin. The cells may be cultured for about 2 to 4 days, preferably about 2 days.

[0102] In one embodiment, the medium contains from about 10 ng / mL to about 35 ng / mL, alternatively about 15 ng / mL to about 30 ng / mL, alternatively about 25 ng / mL fibroblast growth factor, preferred or FGF7 or FGF10, more preferably FGF7, about 0.1 mM to about 0.5 mM ascorbic acid, alternatively about 0.2 mM to about 0.4 mM, alternatively about 0.25 mM Corbic acid, about 0.1 μM to about 0.4 μM SANT-1, about 100 to about 300 nM TPB, and about 50 nM to about 200 nM and about 100 nM of LDN-193189 In another embodiment, the medium is supplemented with about 25 ng / mL FGF-7, about 1 μM retinoic acid, about 0.25 μM SANT-1, about 200 nM TPB, about 100 nM of LDN-193189, and about 0.25 mM ascorbic acid.

[0103] In one embodiment, the medium contains about 0.1 μM to about 0.3 μM SANT-1, about 0.5 μM μM to about 3 μM retinoic acid, and about 75 ng / mL to about 125 ng / mL noggin. will be replenished.

[0104] Stage 4: Cells expressing markers characteristic of foregut endoderm cells are differentiated into cells specific to pancreatic endoderm cells. Differentiation into cells expressing characteristic markers. In one embodiment, the method of the present invention involves growing stage 3 cells in any suitable growth medium. Preferably, custom media such as MCDB-131, DMEM, or BLAR (Table I) The induction of stage 4 cells involves treatment with a differentiation medium containing: , namely, (a) TGF-β receptor inhibitor V, TGF-β receptor inhibitor I, TGF-β Receptor inhibitor IV, TGF-β receptor inhibitor VII, TGF-β receptor inhibitor VIII, TGF-β receptor inhibitor II, TGF-β receptor inhibitor VI, TGF-β receptor inhibitor I II, TGF-β inhibitors SB431542, SD-208, ITD-1, and LY21097 61, A83-01, LY2157299, consisting of ALK5i and ALK5 inhibitor II (b) an ALK5 inhibitor selected from the group consisting of T3, T4, an analog of T3, an analog of T4, and (c) a thyroid hormone selected from the group consisting of SANT-1 or H an SHH signaling pathway antagonist selected from IP-1, (d) LDN-193; 189, noggin, or chordin, (e) TPB, P (f) a PKC activator selected from PBu, PMA, and ILV; (f) FGF-7 or FG F-10, (g) retinoic acid, and (h) ascorbic acid. The compound may be supplemented with one or more of the following: MCDB131 or a preferred Preferably, the growth medium, such as BLAR, contains an SHH signaling pathway antagonist (SANT -1 or HPI-1, etc.), BMP inhibitors (LDN-193189, Noggin, or Chordin, etc.), ascorbic acid, and PKC activators (TPB, PDBu, PMA) , or ILVs, etc.) to provide a useful differentiation medium.

[0105] Stage 3 cells are cultured in such a medium for about 2 to 4 days, preferably for about 2 to 3 days, more preferably for about 1 to 2 days. Preferably, culturing for about 3 days usually allows stage 3 cells to differentiate into stage 4 cells. In another embodiment, the medium contains an SMO inhibitor and an SHH signaling inhibitor. In a preferred embodiment, stage 3 cells are , approximately 0.25μM SANT-1, approximately 100nM RA, approximately 2ng / mL FGF7, approximately 100 nM LDN-193189, about 0.25 mM ascorbic acid, and about 200 nM The cells can be treated with medium supplemented with 100 mg of TPB for 3 days.

[0106] At stage 4, cells may grow for the entire stage or for approximately 2-3 days after plating. Specifically, the present invention provides a method for culturing cells that are multipotent at an air-liquid interface. An in vitro cell culture for differentiating cells derived from sexual stem cells is provided, which comprises: (a) a culture vessel; and (b) a volume within the vessel sufficient to fill only a portion of the vessel's volume. (c) air in the container filling a portion of the container adjacent to the medium; and (d) the medium. (e) a porous substrate located at the interface between the soil and air, and (f) a medium that is only partially on the surface of the cells. and cells derived from pluripotent stem cells arranged on the surface of the substrate so as to contact the Alternatively, stage 4 can be performed entirely in plate cultures.

[0107] Alternatively, at stage 4, after about 2-3 days of plating, the cells form cell clusters. Specifically, the present invention relates to a method for producing a cell suspension prepared as an aggregated cell such as a cell cluster. The present invention provides an in vitro cell culture for differentiating selected cells, which comprises (a) aggregation or differentiation of cells. (b) a culture vessel that promotes the formation of cell clusters; and (c) a volume of growth medium (culture medium) within the vessel. (c) Cells are induced to aggregate and form clusters in a container. and cells derived from the cultured pluripotent stem cells. In an embodiment, the container comprises a well or Plates with microwells, such as Aggrewell™ plates (microwells) Crowell plate, STEMCELL Technologies Inc., Van Specifically, the cell clusters are, for example, Agg Use rewell™ plates to form cell aggregates of uniform size and shape In an embodiment, the plate is prepared in a microwell for carrying out the measurement. To prepare aggregated cells or cell clusters, approximately 1000 μg of cellulose per well or microwell is used. 500 to 2000 cells are seeded. Specifically, about 5 cells per well or microwell. 0 to about 3000 cells are seeded, preferably about 50 to about 2000 cells, and more preferably about 50 to about 1000 cells. cells, about 50 to about 900 cells, about 50 to about 800 cells, about 100 to about 800 cells, about 25 More preferably, 0 to about 800 cells, or about 500 to about 800 cells, are seeded in the well. Alternatively, about 700 to about 800 cells are seeded per microwell.

[0108] In a further embodiment, the cell clusters formed by the method of aggregating cells They may also be cultured in suspension (suspension culture), which means that aggregated cells or cell classes Removing the target from the well or microwell and removing the aggregated cells / cell clusters In an embodiment, the aggregated cells / cell clusters are seeded in a suspension culture vessel. - is approximately 0.75 x 10 6cells / mL ~ approx. 2.0×10 6 cells / mL, preferably about 1x 10 6 cells / mL ~ approx. 2.0×10 6 cells / mL, more preferably about 1.5 x 10 6 cell / mL ~ approx. 2.0×10 6 The cells are seeded in suspension culture at a cell density of 1000 cells / mL. Any known suspension culture system may be useful for suspending clusters of aggregated cells. Suspension culture systems involve the growth of aggregated cells or cell clusters in a spinner flask or spinner holder. This may involve seeding in a flask, such as a Yell flask.

[0109] In a further embodiment, the cells at the completion of stage 4 (after 2-3 days of culture) are Rh o-related kinase ("ROCK") inhibitors, e.g., Y27632 ((1R,4r)-4- ((R)-1-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxy amide), GSK269962 (N-[3-[[2-(4-amino-1,2,5-oxa Diazol-3-yl)-1-ethyl-1H-imidazo[4,5-c]pyridin-6-yl -4-[2-(4-morpholinyl)ethoxy]benzamide), H1152((S)-(+)-2-Methyl-1-[(4-methyl-5-isoquinolinyl) sulfonyl]homopiperazine, 2HCl), and SR3677 (N-[2-[2-(dimethylamino)methyl] [1H-pyrazol-4-yl]phenyl-2,3-dihydroxy dro-1,4-benzodioxin-2-carboxamide dihydrochloride) In certain embodiments, about 1 to 20 μM, about 1 to 15 μM, or about 1 to 10 μM , or about 10 μM of a ROCK inhibitor may be used.

[0110] In certain embodiments of the invention herein, late stage 4 cells, e.g., adherent Only cells grown in plate culture for 1-2 days or 1-3 days were required for completion of stage 4. The cells may then be cultured at the air-liquid interface, or aggregated cells such as cell clusters. In one embodiment of the invention herein, the cells may be cultured in a culture medium containing a ROCK inhibitor. Only the late stage 4 cells are cultured at the air-liquid interface. ~Approx. 0.75×10 5 Cells / microliter are seeded and cultured at the air-liquid interface Alternatively, about 2-6 x 10 6 Cells are seeded and cultured at the air-liquid interface. Morphologically, late stage 4 cells treated with ROCK inhibitors were cultured to aggregate cells. In an embodiment, the cells are allowed to grow in a cell-free zone to form aggregated cells, such as cell clusters. The cells are seeded in a container that promotes aggregation or cell clusters (three-dimensional aggregates of cells). In some embodiments, the container is a plate having wells or microwells. Specifically, cell clusters can be grown on, for example, Aggrewell™ plates (ST EMCELL Technologies Inc., Vancouver, Canada a) is used to form cell aggregates of uniform size and shape. In an embodiment, the cells are prepared in a plate having a cell cluster. To prepare the cells, approximately 50-3,000 cells are seeded per well or microwell. Specifically, about 50 to about 3,000 cells are seeded per well or microwell. Preferably, the number of cells is about 50 to about 2000, about 50 to 1000, about 50 to 900, or about 5 0 to 800 cells, 100 to 800 cells, 250 to 800 cells, or about 500 to about 800 cells More preferably, about 700 to 800 cells are seeded per well or microwell. In another embodiment, the aggregated cells or cell clusters are further seeded in suspension. In certain embodiments, the adherent cells in the plate are cultured at an air-liquid interface. Before culturing in T cells or culturing to aggregate cells, such as to form cell clusters, Tissues such as rypLE™, Accutase™, or Dispase™ The cells may be treated with a cell detachment solution, such as a solution containing proteinolytic and collagenolytic enzymes. .

[0111] In an alternative embodiment, the stage 4 cells are treated with an ALK5 inhibitor, noggin, and PKC activity. Treatment of stage 3 cells with differentiation medium containing growth medium supplemented with factors such as TPB In certain embodiments, the medium may be obtained from stage 3 cells by 0.1 μM ALK5 inhibitor, about 100 ng / mL Noggin, and about 500 nM TPB The cell culture may be in a monolayer format. Treatment continues for a total of about 3 days. In certain embodiments, cells are treated for two days and then protein-treated on the final day. The cells are treated with proteolytic enzymes, collagenolytic enzymes, or both to produce a single-cell suspension. The resulting cells can be seeded at the air-liquid interface or aggregated cells. Alternatively, cells can be seeded to form cell clusters. Single cells can aggregate to form clusters of approximately 100 microns. After the cells were clustered into submicrometer diameter cells, ALK5 inhibitors and LD In one particular embodiment, the cells are cultured in the presence of about 100 micrograms of N-193189. Cell clusters with diameters of less than one meter were observed at approximately 200 nM ALK5 inhibitor and approximately The cells may be cultured in medium supplemented with 100 nM LDN-193189. In this study, culturing stage 4 cells at the air-liquid interface or in suspension was shown to be effective in detecting endocrine-related myeloma. Along with the markers, pancreatic endoderm markers can be significantly enhanced.

[0112] Stage 5: Cells expressing markers characteristic of pancreatic endoderm cells become pancreatic endocrine progenitor cells. Differentiation into cells expressing characteristic markers. In one embodiment, the method of the present invention involves growing stage 4 cells in any suitable growth medium. Preferably, custom media such as MCDB-131, DMEM, or BLAR (Table I) The differentiation medium contains: , namely, (a) TGF-β receptor inhibitor V, TGF-β receptor inhibitor I, TGF-β Receptor inhibitor IV, TGF-β receptor inhibitor VII, TGF-β receptor inhibitor VIII, TGF-β receptor inhibitor II, TGF-β receptor inhibitor VI, TGF-β receptor inhibitor I II, TGF-β inhibitors SB431542, SD-208, ITD-1, and LY21097 61, A83-01, LY2157299, ALK5i, and ALK5 inhibitor II (b) an ALK5 inhibitor selected from the group consisting of T3, T4, an analog of T3, and an analog of T4; and mixtures thereof; (c) SANT-1 or an SHH signaling pathway antagonist selected from HIP-1; (d) LDN-19; 3189, noggin, or chordin; (e) a BMP receptor inhibitor selected from retinoids; (f) ascorbic acid, (g) heparin, and (h) zinc sulfate. One or more supplements may be added, and the cells are incubated at the air-liquid interface for about 2-4 days, preferably about The cells are cultured for 3 days to differentiate into stage 5 cells. In addition, SMO inhibitors (such as MRT10 or cyclopamine) and FGF-7 or FGF- F-10 or both of the fibroblast growth factors are supplemented. The treatment of the cells is carried out for about 2 to 4 days, preferably about 3 days, to differentiate the cells into stage 5 cells. To make.

[0113] In a preferred embodiment, stage 4 cells are treated with about 0.1 μM to about 0.4 μM SAN. T-1 and preferably about 0.25 μM SANT-1, about 50 nM RA, about 0.1 mM to about 0.5 mM ascorbic acid, alternatively about 0.2 mM to about 0.4 mM and preferably about 0.25 mM ascorbic acid, about 50 nM to about 200 nM and preferably about 100 nM LDN-193189, approximately 1 μM T3, and approximately 10,000 nM ALK5 inhibitor, More preferably, by treating the cells with medium supplemented with ALK5 inhibitor II, In yet another embodiment, the cells are also differentiated into 5-cells, which are optionally preferred. or about 1 to 15 μM, alternatively about 1 to 10 μM, alternatively about 5 to 10 μM, preferably about 10 μM zinc sulfate (ZnSO4), and about 1 to 100 μg / mL, preferably about 10 μ Treatment of stage 4 cells is preferably performed for approximately 2-4 days. This takes place over a period of approximately 3 days and allows the cells to differentiate into stage 5 cells.

[0114] In yet another embodiment, the method of the present invention further comprises treating stage 4 cells with heparin, SMO inhibitors, or the like. inhibitors, or SHH signaling pathway antagonists, RA, BMP receptor inhibitors, and A The cells were treated with medium supplemented with LK5 inhibitors and cultured at the air-liquid interface for approximately 3 days. and differentiating the cells into stage 5 cells to obtain stage 5 cells. In an alternative embodiment, the medium comprises RA, a BMP receptor inhibitor, and an ALK5 inhibitor. In addition to the agent, both an SMO inhibitor and an SHH signaling pathway antagonist may be supplemented. Thus, in one embodiment, stage 4 cells are cultured in a medium containing heparin, ZnSO, SM O inhibitor or SHH signaling pathway antagonist, RA, LDN-193189, and Stage 4 cells were treated with medium supplemented with ALK5 inhibitor II and ALK5 inhibitor II. In an alternative embodiment, the medium contains an SMO inhibitor and an SHH inhibitor. In one embodiment, both a stage 4 signaling pathway antagonist and a stage 5 signaling pathway antagonist may be substituted. The cells were incubated with approximately 10 μg / mL heparin, approximately 0.25 μM SANT-1, and approximately 50 nM R A, about 50 nM LDN-193189, about 10 nM T3, and about 1000 nM AL Treatment of cells with medium supplemented with K5 inhibitor led to differentiation into stage 5 cells. Suitable ALK5 inhibitors include SD-208, ALK5 inhibitor II, and TGF-β receptor. TGF-β receptor inhibitor V, TGF-β receptor inhibitor I, TGF-β receptor inhibitor IV, TGF-β receptor TGF-β receptor inhibitor VII, TGF-β receptor inhibitor VIII, TGF-β receptor inhibitor II, T GF-β receptor inhibitor VI, TGF-β receptor inhibitor III, and their combinations Treatment of stage 4 cells is preferably carried out for about 2 to 4 days. This process takes approximately three days and allows the cells to differentiate into stage 5 cells.

[0115] In a preferred embodiment, the ALK5 inhibitor is ALK5 inhibitor II. In a preferred embodiment, about 10,000 nM of ALK5 inhibitor II is used. In a preferred embodiment, the stage 4 cells are treated with about 10 μg / mL heparin, about 0.25 μg / mL heparin, μM SANT-1, about 50 nM RA, about 100 nM LDN-193189, and about Treatment was carried out in a medium supplemented with 10,000 nM (10 mM) of ALK5 inhibitor II. In another alternative embodiment, the method of the present invention comprises treating stage 4 cells with an SMO inhibitor or Treatment with medium supplemented with HH signaling pathway antagonist, RA, and ALK5 inhibitor and allowing the cells to stand, preferably at the air-liquid interface or in suspension, for about 2-4 days. and culturing the cells for about 3 days to differentiate the cells into stage 5 cells. In this study, the medium was supplemented with both an SMO inhibitor and an SHH signaling pathway antagonist. In one embodiment, stage 4 cells may be supplemented with about 0.25 μM SANT-1, About 50 nM RA, about 50 nM LDN-193189, about 1 μM T3, and about 100 Stage 5 cells by treating the cells with medium supplemented with 0 nM ALK5 inhibitor It is differentiated into.

[0116] The amount of cells seeded for culturing at the air-liquid interface can vary. For example, For culturing at the air-liquid interface, approximately 0.5–6 × 10 5 Single cell suspension containing cells / µL Droplets of the suspension can be seeded onto a porous substrate (e.g., a filter). The suspension can be approximately 2 x 10 5 cells / μL ~ approx. 6×10 5 cells / μL, approximately 4 x 10 5 cells / μL ~ approx. 6×10 5 cell / μL, approximately 5×10 5 cells / μL ~ approx. 6×10 5 cells / μL, approximately 5 x 10 5 cells / μL ~ approx. 6×10 5 cells / μL, approximately 2 × 10 5 cells / μL~approx. 5×10 5 cells / μL, approximately 2 × 10 5 cells / μL ~ approx. 4×10 5 cells / µL, or approximately 3 x 10 5 cells / μL, and air - can be seeded onto a porous substrate, such as a filter, that sits at the liquid interface. At about 0.5 × 10 5 cells / μL ~ approx. 0.75×10 5 cells / μL, approximately 0.6×1 0 5 cells / μL ~ approx. 0.75×10 5 cells / µL, or approximately 0.5 x 10 5 cells / μL~approx. 0.6×10 5 Droplets of single-cell suspension containing cells / µL are to be cultured at the ALI It is seeded onto a porous support.

[0117] For suspension culture, cell clusters are generated in plates with microwells. This is then inoculated into a suspension culture. Aggregation is induced using a target plate, where aggregated cells or cell clusters are prepared. To prepare the cells, approximately 50 to 3,000 cells are seeded per well or microwell. Typically, about 50 to about 3,000 cells are seeded per well or microwell, preferably. Approximately 50 to 2000 cells, approximately 50 to 1000 cells, approximately 50 to 900 cells, approximately 50 to about 800 cells, about 100 to about 800 cells, about 250 to about 800 cells, or about 500 to about 8 More preferably, about 700 to about 1000 cells are seeded per well or microwell. 800 cells are seeded. The aggregated cells / cell clusters are approximately 0.75 x 10 6 cell / mL~approx. 2.0×10 6 cells / mL, preferably about 1 x 10 6 cells / mL ~ approx. 2.0×1 0 6 cells / mL, approximately 1.25×10 6 cells / mL ~ approx. 2.0×10 6 Cells / mL and More preferably about 1.5 x 10 6 cells / mL ~ approx. 2.0×10 6 Suspension at a cell density of 100 cells / mL It is inoculated into suspension culture.

[0118] In another embodiment, the method of the present invention involves treating stage 4 cells with a BMP receptor inhibitor (e.g., For example, LDN-193189, Noggin, or Chordin) and ALK5 inhibitors are supplemented. The treatment involves treating the stage 4 cells with a medium containing the above-mentioned marker for approximately one day to differentiate the stage 4 cells into stage 5 cells. For example, the medium may contain about 100 nM LDN-193189 and about 100 nM ALK5. The inhibitor and about 1 μM T3 may be supplemented. The cells may be in adherent plate culture or In certain embodiments, the cells may be in the form of clusters. Before culturing or culturing to form aggregated cells or cell clusters, The cells may be treated with a cell detachment solution, such as a solution containing protein- and collagen-degrading enzymes. do.

[0119] In accordance with the foregoing method, the present invention provides a method for isolating cells expressing markers characteristic of pancreatic endoderm from pancreatic endoderm. Differentiation into cells expressing markers characteristic of pancreatic endoderm / pancreatic endocrine precursor cells The present invention further provides a cell culture for culturing cells, the cell culture comprising: (a) a culture vessel; and (b) a vessel containing the cells. (c) a volume of growth medium sufficient to fill only a portion of the volume of a container adjacent to the medium; (d) the porous substrate located at the interface between the medium and the air; and (e) placed on the surface of the substrate so that the medium contacts only a portion of the surface of the cells. and cells expressing markers characteristic of pancreatic endoderm cells induced from the pluripotent stem cells. nothing.

[0120] Alternatively, in accordance with the foregoing method, the present invention provides cells expressing markers characteristic of pancreatic endoderm. These cells express markers characteristic of pancreatic endocrine precursor cells (pancreatic endoderm / pancreatic endocrine precursor cells). Further provided is a cell culture for differentiation into cells, which may include (a) cell aggregation or cell clusters. (b) a culture vessel that promotes star formation; and (b) a volume of growth medium (culture medium) within the vessel. (c) Pluripotent cells placed in a container so that they can be induced to aggregate and form clusters. (d) cells derived from sexual stem cells and the resulting cell clones placed in suspension (suspension culture). In an embodiment, the container for cell aggregation comprises a well or a micro-organism. Plates with wells, such as Aggrewell™ plates (microwell plates) Le Plate, STEMCELL Technologies Inc., Vancouver In an embodiment, the suspension culture system is a suspension culture of aggregated cells or cells. The clusters are placed in flasks, such as spinner flasks or spinner wheel flasks. The method may include inoculating the cells into a suspension culture vessel.

[0121] Stage 6: Immature beta cells expressing markers characteristic of pancreatic endocrine progenitor cells Differentiation into cells expressing markers characteristic of blastocysts. In one embodiment, the method of the present invention comprises growing stage 5 cells in any suitable growth medium, preferably Preferably, a custom medium such as MCDB-131 or CMRL, more preferably BLAR ( The method involves obtaining stage 6 cells by treating them with differentiation medium, which may be (Table I). The medium contains the following: (a) TGF-β receptor inhibitor V, TGF-β receptor inhibitor I, TGF-β receptor inhibitor Agent IV, TGF-β receptor inhibitor VII, TGF-β receptor inhibitor VIII, TGF-β Receptor inhibitor II, TGF-β receptor inhibitor VI, TGF-β receptor inhibitor III, TG F-β inhibitors SB431542, SD-208, ITD-1, LY2109761, A8 3-01, LY2157299, ALK5i, and ALK5 inhibitor II. (b) an ALK5 inhibitor selected from T3, T4, their analogs, and mixtures thereof; (c) a thyroid hormone selected from the group consisting of preferably LDN-193189, Noggin (d) a BMP receptor inhibitor selected from the group consisting of α- and β-gamma-secretase inhibitors; XX, gamma secretase inhibitor XXI, gamma secretase inhibitor XVI, or DAP gamma secretase inhibitors such as T, (e) ascorbic acid, (f) heparin, and ( g) zinc sulfate. The cells are preferably air- The cells are cultured in a liquid interface or suspension culture for about 2 to 4 days, preferably about 3 days, to reach stage 5. The cells can be differentiated into stage 6 cells. Optionally, the medium contains an SHH signaling pathway. tract antagonists, smoothened receptor inhibitors, fibroblast growth factors, and retinoic acid One or more of the above may be further supplemented.

[0122] In a preferred embodiment, stage 5 cells are treated with about 50 nM RA, about 0.25 mM A Corbic acid, approximately 100 nM LDN-193189, approximately 10,000 nM ALK5 inhibition agent and preferably ALK5 inhibitor II, 1 μM T3, about 100 nM gamma secretion inhibitor The cells were differentiated into stage 6 cells by treatment with medium supplemented with enzyme inhibitors for approximately 7 days. Alternatively, stage 5 cells may be cultured in a medium containing about 0.25 μM SANT-1, about 50 nM RA, , about 0.25 mM ascorbic acid, about 1000 nM ALK5 inhibitor, and 1 μM T3 The cells can be differentiated into stage 6 cells by treatment with a medium supplemented with PEG for about 3 days. The cells can be cultured in such medium for an additional 2 days or more, if necessary.

[0123] Alternatively, stage 5 cells may be expressed in the presence of heparin, SMO inhibitors, or SHH signaling pathway inhibitors. antagonists, BMP inhibitors, T3, T4, their analogs and mixtures thereof, and Treat with a medium supplemented with an ALK5 inhibitor, preferably at the air-liquid interface or in suspension; Stage 6 cells are cultured for approximately 1-7 days, alternatively for approximately 6 days, or alternatively for approximately 7 days. In an alternative embodiment, the medium contains an SMO inhibitor and an SHH signaling inhibitor. For example, cells may be supplemented with approximately 10 μg / mL of heparin or a combination of heparin and heparin. Valine, approximately 0.25 μM SANT-1, approximately 100 nM LDN-193189, approximately 10 00 nM T3, and about 500 to about 10,000 nM, alternatively about 500 nM, alternatively in medium supplemented with about 1000 mM, and alternatively about 10,000 nM, of an ALK5 inhibitor Suitable ALK5 inhibitors include SD-208, ALK5 inhibitor II, TGF-β receptor inhibitor V, TGF-β receptor inhibitor I, TGF-β receptor inhibitor IV, TGF-β receptor inhibitor VII, TGF-β receptor inhibitor VIII, TGF-β receptor inhibitor TGF-β receptor inhibitor II, TGF-β receptor inhibitor VI, TGF-β receptor inhibitor III, and their Combinations include, but are not limited to:

[0124] In a preferred embodiment, the ALK5 inhibitor is ALK5 inhibitor II. In a preferred embodiment, about 10,000 nM (10 mM) of ALK5 inhibitor II is used. Thus, in one embodiment, stage 5 cells are treated with heparin, an SMO inhibitor or SHH signaling pathway antagonists, BMP inhibitors, T3, T4, their analogs, and and their mixtures, as well as medium supplemented with ALK5 inhibitors, and can be differentiated into stage 6 cells by culturing in suspension, preferably for about 7 days. In an alternative embodiment, the medium contains an SMO inhibitor and an SHH signaling pathway antagonist. In certain embodiments, cells can be replenished at either the air-liquid interface or Before culturing in suspension, the cells were treated with a solution containing proteolytic enzymes and collagenolytic enzymes. The cells may be treated with a cell detachment solution such as

[0125] In another embodiment, the stage 5 cells are treated with heparin, an SMO inhibitor, or an SHH signaling inhibitor. The drug was supplemented with a leukemia pathway antagonist, a BMP inhibitor, T3, and an ALK5 inhibitor. Treat with medium and at the air-liquid interface for about 5 days to about 7 days, alternatively for about 5 days, alternatively for about 6 days Alternatively, they can be differentiated into stage 6 cells by culturing for about 7 days. In an embodiment, the medium contains about 10 μg / mL heparin, about 0.25 μM SANT- 1, about 100 nM LDN-193189, about 1000 nM T3, and about 10,000 In certain embodiments, the medium may be supplemented with 100 nM of ALK5 inhibitor II. Zinc (ZnSO4) may be further supplemented. For example, the medium may contain about 10 mM ZnSO4 In an alternative embodiment, the medium may be further supplemented with an SMO inhibitor and an SHH signaling inhibitor. Both the vasopressin and vasopressin pathway antagonists can be supplemented.

[0126] In a particularly preferred embodiment of the present invention, an Aurora kinase inhibitor, preferably an Aurora a RSK inhibitor, preferably RSK inhibitor II, and DOT1L one of the protein methyltransferase inhibitors, preferably EPZ-5676 Or two or more of them are added to the medium. The amounts added are Aurora kinase and RSK inhibitors. about 100-5000 nM, alternatively about 1000-5000 nM, alternatively about 2 000-5000 nM, alternatively about 3000-5000 nM, preferably about 1000-2 000 nM, and for DOT1L inhibitors, about 100 to 1000 nM, more preferably about 1 It can be from 1 μM to about 10 nM.

[0127] In accordance with the foregoing method, the present invention provides a method for producing a pancreatic endoderm / endocrine progenitor cell expressing markers characteristic of pancreatic endoderm / endocrine progenitor cells. The cells were then cultured to differentiate into cells expressing markers characteristic of immature beta cells. Further provided is a cell culture, comprising: (a) a culture vessel; (b) a cell culture vessel containing a cell culture medium; and (c) a cell culture vessel containing a cell culture medium, the cell culture medium comprising a cell culture medium, and a cell culture medium containing a cell culture medium. (c) a volume of growth medium sufficient to fill only a portion of the container adjacent to the medium; (d) the air in the container, (e) the porous substrate located at the interface between the medium and the air, and The cells are derived from pluripotent stem cells arranged on the surface of a substrate so that only a portion of the surface of the cells is in contact with the cells. These cells contain cells that express markers characteristic of the developing pancreatic endoderm / endocrine progenitor cells.

[0128] In accordance with the foregoing method, alternatively, the present invention provides a method for producing pancreatic endocrine precursor cells (or pancreatic endoderm / pre-endocrine cells). cells expressing markers characteristic of progenitor cells and those expressing markers characteristic of immature beta cells Further provided is a cell culture for differentiation into cells expressing the gene, which may be (a) a cell aggregation or (b) a culture vessel that promotes the formation of cell clusters; (c) a volume of growth medium (culture (c) cells are placed in a container so that they can be induced to aggregate and form clusters. (d) cells derived from pluripotent stem cells, and (e) cells generated and placed in suspension (suspension culture). In an embodiment, the container for cell aggregation is a well or a microwell. Plates with multiple wells, such as Aggrewell™ plates. In embodiments, suspension culture systems involve the growth of aggregated cells or cell clusters in a flask, e.g. , seeding in suspension culture vessels such as spinner flasks or spinner wheel flasks may include:

[0129] In one embodiment, stage 5 cells cultured according to embodiments of the invention are used, In other embodiments, the cells are differentiated into stage 6 cells, while in other embodiments, the cells are differentiated into stage 6 and stage Stage 5 cells cultured according to other protocols to obtain stage 7 cells were used in this method. It may be possible.

[0130] In an embodiment, the method of the present invention provides for the formation of stage 6 cells that are single hormone positive. Thus, in one embodiment, the method of the present invention involves the use of NKX6.1, insulin The resulting stage 6 cells co-express chromogranin, chromogranin, and PDX1. In one embodiment, the method of the present invention comprises culturing stage 6 cells that co-express NKX6.1 and insulin. In certain embodiments of the present invention, the method includes steps 4 to 6 or later. At stages 4 to 6, or at stages 5 and 6, the custom medium BLAR (see Table I) was used. The medium can be changed daily, or alternatively every other day.

[0131] In another embodiment, the present invention provides a stem cell that co-expresses NKX6.1 and chromogranin. In a method for forming stage 6 cells, stage 4, preferably late stage 4 cells are cultured in air. In yet another embodiment, the cells are cultured at a liquid interface or in suspension to stage 6 cells. In this regard, the present invention provides a method for culturing stage 4, preferably late stage 4, cells at an air-liquid interface or NKX6.1 stage 6 cells were generated by culturing in suspension to stage 6 cells. The present invention relates to a method for generating single hormone-expressing insulin-positive cells.

[0132] Stage 7: Biphasic GSIS and differentiation of cells expressing markers characteristic of immature beta cells and cells expressing markers characteristic of functional beta cells capable of mitochondrial respiratory response. Differentiation into. In one embodiment, the method of the present invention comprises growing stage 6 cells in any suitable growth medium, preferably Preferably, MCDB-131 or CMRL, or more preferably, BLAR001 (Table 1 ) or a custom medium such as BLAR004 (Table IV) This includes treatment with for 7 days.

[0133] The medium was the following: 2.7g / 1000mL sodium bicarbonate, 1:2 Supplemented with 0.00 dilution of ITS-X; 1x GlutaMAX™; 2% FAF -BSA; 10 μg / mL heparin ("H"); 10 nM T3 ("low T3"); 1 m 0.5 μM N-acetylcysteine ("NAC"); 0.5 μM ZM447439 ("ZM" and the following ingredients comprising Formulation I ("FI") (Table XII): 1:200 Dilute RPMI vitamin supplement 1:200. Dilute MEM non-essential amino acid supplement 1:2 1:000 dilution of chemically defined lipid concentrate; 1:200 dilution of sodium pyruvate; 1:200 Trace Elements A (Corning, Cat. No. 25-021) at 0 dilution; 1:2000 dilution One or more of the following trace elements: B (Corning, Cat. No. 25-022) Additional compounds that may be added to obtain stage 7 include approximately 10 nM T 3 (“low T3”); 5 μM 5-azacytidine (“AZT”) (Sigma Aldri ch, Catalog No. A2385); or about 1 μM 3-deazaneplanocin A ("DEZ A) (Biovision, Inc., Cat. No. 2060). In an embodiment for obtaining the 7 cells, the medium does not contain an ALK5 inhibitor.

[0134] In one embodiment, stage 6 cells are treated with about 10 nM T3, about 0.5 μM Aurora one or more of kinase inhibitors II and about 1 mM N-acetylcysteine Alternatively, stage 7 cells can be differentiated by treatment with medium supplemented with Stage 6 cells are treated with heparin, T3, T4, their analogs or mixtures, antioxidants and Aurora kinase inhibitors, or their mixtures, and treated with medium supplemented with air- Liquid interface or suspension culture for about 7 to 21 days, alternatively about 7 to 10 days, preferably about 7 days By culturing, they can be differentiated into stage 7 cells.

[0135] In accordance with the foregoing method, the present invention provides a method for producing cells that express markers characteristic of pancreatic endocrine precursor cells. , and culture the cells for differentiation into cells expressing markers characteristic of functional beta cells. Further provided is a method for producing a culture vessel, the method comprising: (a) a culture vessel; and (b) a method for producing a culture vessel within the vessel, the method comprising: (c) a container that fills a portion of the container adjacent to the medium; (d) the porous substrate located at the interface between the medium and the air; and (e) the medium that is a pluripotent stem cell-derived pancreatic cell arranged on the surface of a substrate so as to contact only a portion of the surface of the and cells expressing markers characteristic of endodermal / endocrine precursor cells.

[0136] In one embodiment, stage 6 cells cultured according to embodiments of the invention are used, In other embodiments, to obtain stage 7 cells, Alternatively, stage 6 cells cultured according to other protocols may be used in this method. In an embodiment, the methods of the present invention result in the formation of stage 7 cells that are single hormone positive. Thus, in one embodiment, the method of the present invention involves the detection of NKX6.1, chromogranin, Stage 7 cells co-expressing IFN-γ, PDX1, UCN3, SLC2A1, and MAFA were also identified. In another embodiment, the method of the present invention provides a method for the production of NKX6.1, PDX1, insulin. , resulting in stage 7 cells that co-express UCN3, SLC2A1, and MAFA. In another embodiment, each of the cells comprises at least about 10% of the cell population, alternatively less than about 10% of the cell population. at least about 20%, alternatively at least about 30%, alternatively at least about 40%, alternatively at least at least about 50%, alternatively at least about 60%, alternatively at least about 70%, alternatively at least about 80%, or alternatively at least about 90%, of the A population of cells expressing KX6.1, UCN3, SLC2A1, and MAFA results. Glass.

[0137] In some embodiments, at least 10% of the cells in the resulting cell population are express insulin, PDX1, NKX6.1, UCN3, SLC2A1, and MAFA In other embodiments, at least 20% of the cells in the population express insulin, PDX1, N In another embodiment, the cells express KX6.1, UCN3, SLC2A1, and MAFA. , at least 30% of the cells in the population express insulin, PDX1, NKX6.1, UCN3, In yet another embodiment, a small proportion of the cells in the population express SLC2A1, and MAFA. At least 40% of the genes involved in insulin, PDX1, NKX6.1, UCN3, SLC2A1, and In yet another embodiment, at least 50% of the cells in the population express IMAFA. express insulin, PDX1, NKX6.1, UCN3, SLC2A1, and MAFA In yet other embodiments, at least 60%, 70%, 80%, or 90% of the cells of the population 0% for insulin, PDX1, NKX6.1, UCN3, SLC2A1, and MAFA In an alternative embodiment, the cells of the population express at least 91, 92, 93, 94, 95, 96, 97, 98, or 99% of patients were insulin, PDX1, NKX6.1, or UCN. 3, expressing SLC2A1 and MAFA.

[0138] In certain preferred embodiments of the present invention, the method comprises administering to a subject in stages 4-7 or later stages. Use custom medium BLAR (Table I) at stages 4 to 7, or at stages 5, 6, and 7. The medium may be changed preferably every day, or alternatively every other day.

[0139] In another embodiment, the present invention relates to a method for producing a medicament for the treatment of NKX6.1, PDX1, MAFA, UCN3, S The present invention relates to a method for generating stage 7 cells that co-express LC2A1 and chromogranin. The method comprises culturing stage 4, preferably late stage 4, cells at the air-liquid interface or in suspension. In yet another embodiment, the present invention provides a method for culturing a cell line comprising culturing the cell line to stage 7 cells. Stage 4, preferably late stage 4 cells, stage 7 cells at the air-liquid interface or in suspension By culturing the cells in the presence of NKX6.1, PDX1, UCN3, SLC2A1, and M AFA stage 7 cells expressing single hormone insulin positive cells (functional beta cells) The present invention relates to a method for forming

[0140] The present invention provides a method for detecting vacancies at all stages in the pathway from pluripotent cells to pancreatic endocrine cells. Although the invention contemplates culturing at the air-liquid interface, the invention also contemplates culturing in plate or submerged culture. Stage 1 to stage 4 cells, as well as culturing cells at the air-liquid interface or in suspension culture In another embodiment, the present invention provides for the formation of stage 5, 6, and 7 cells by: Differentiation of pluripotent cells, including culturing stage 4, 5, and 6 cells at the air-liquid interface In certain embodiments, the cells cultured during stages 4 to 7 are The cells may be cultured at an air-liquid interface. In another embodiment, late stage 4 to stage Stage 6 cells, or only stage 5 and stage 6 cells, cultured at the air-liquid interface or in suspension In yet another alternative embodiment, stages 1 to 4 are performed by culturing cells in plate culture. Stages 5-7, or stages 6-7, or stage 7 only is carried out by culturing in suspension culture.

[0141] In addition, cultures during one or all of stages 5, 6, and 7 may be cultured at T3, T4, or These analogs and one or more of ALK5 inhibitors, or T3, T4, and and one or more of their analogs, or an ALK5 inhibitor. In a preferred embodiment, one or more of stages 5, 6, and 7, and and preferably all of the culturing is carried out in the presence of a T3 and ALK5 inhibitor, and more preferably Preferably, the treatment is carried out in the presence of T3 and ALK5 inhibitor II. Therefore, the culture during stage 7 is carried out in the presence of low concentrations of T3. Therefore, culture during stage 7 is performed without ALK5 inhibitors.

[0142] When cells are cultured at the air-liquid interface ("ALI"), they are cultured on a porous substrate. The cells can be placed in a container such that they are in contact with air on the top side and with cell culture medium on the bottom side. For example, a sufficient volume of medium may be placed in a culture vessel containing a porous substrate (e.g., a filter insert). The medium may be added to the bottom of the substrate so that it contacts the bottom of the cells residing on the substrate. They do not encapsulate or immerse. A suitable porous substrate will not adversely affect cell growth and differentiation. An exemplary porous substrate is polyethylene terephthalate (PET). It is made of a polymer such as PET, polyester, or polycarbonate. The porous substrate may be coated or uncoated. In another embodiment of the present invention, the coating may be MATRIGEL™. In the method, the porous substrate is a porous film that can be coated with MATRIGEL™. In another embodiment of the present invention, the porous substrate is an uncoated filter insert. The porosity of the matrix maintains cell viability and promotes cell differentiation. should be sufficient.

[0143] Culturing cells at the air-liquid interface involves placing the cells on a porous substrate, such as a porous filter insert. In certain embodiments, the substrate pore size is about 0.3 to about 3 mm. Seeding can be done as single cells from monolayer cultures or in the micrometer range. The release of cells from monolayer cultures as clusters into suspension followed by single cell suspension or can be achieved by dispensing suspended cell cultures onto porous substrates at the ALI. Cells are porosigenic from suspensions with approximately 1,000 cells / µL to approximately 100,000 cells / µL. The cells may be seeded onto a substrate containing individual cells or aggregates or clusters of cells. In certain embodiments, the cells may be seeded as droplets of a cell suspension having The method disclosed in the patent application 2014 / 0186305 is used to The present disclosure relates to the culture and differentiation of pluripotent stem cells at an air-liquid interface. and is incorporated herein by reference.

[0144] The medium can be changed or refreshed every other day or preferably every day. The cells grown on top are generally not single cells, but rather they are in the form of a sheet. Cells cultured at the ALI are either in a cluster or aggregated cell clusters. They may experience a higher oxygen tension compared to submerged cells.

[0145] In certain embodiments, the methods of the present invention involve growing cells as clusters in suspension culture. This may be achieved by culturing and differentiating pluripotent stem cells. Exemplary suitable methods for this are described in U.S. Patent Application Publication Nos. 2014 / 0242693 and 2014 / 0242694. These disclosures use suspended clusters. The present invention is incorporated herein with respect to the culture and differentiation of pluripotent stem cells. The present invention provides an in vitro cell culture in which cells are induced to aggregate and form clusters. Aggregation or cell clusters of cells with growth medium (culture medium) to form stars and seeding cells into culture vessels that promote the formation of aggregates such as cell clusters. In an embodiment, the method further comprises differentiating the cells prepared as the aggregated cells. Useful vessels for inducing the A are plates with wells or microwells, e.g. The resulting aggregated cells or cell clusters are The stars are further cultured in suspension (suspension culture), which results in aggregated cells / cell clusters. The cells were removed from the microwells and seeded into suspension culture vessels, thereby forming aggregates. The method involves differentiating the cells in suspension.

[0146] Embodiments of the present invention are directed to the late stages 4-7, preferably at the air-liquid interface or in suspension. Stages 5 to 7 encompass the formation of cells. Cells are formed by differentiating pluripotent stem cells. or by further differentiating stage 3, 4, 5, or 6 cells. Stage 4 cells can be cultured entirely at the air-liquid interface, or the cells can be cultured at stage 4 During the early part of the incubation period (approximately 1-2 days), the cells can be cultured in submerged plates and then transferred to stage 4. The later part of the experiment (around day 2-3) was cultured at the air-liquid interface or in suspension. Preferably, stage 4 is not performed in ALI or suspension, but in submerged culture. This is carried out.

[0147] In one embodiment, the present invention provides a method for producing a cell that expresses markers characteristic of functional beta cells. The present invention provides a method for producing a pluripotent stem cell from a pluripotent stem cell, the method comprising culturing the pluripotent stem cell. and differentiation of pluripotent stem cells into cells expressing markers characteristic of pancreatic endoderm. and express markers characteristic of pancreatic endoderm when cultured at the air-liquid interface or in suspension. and differentiating the cells into cells expressing markers characteristic of pancreatic endocrine cells. The method includes administering to a subject a therapeutically effective amount of (i) T3, T4, or an analog thereof, and (ii) an ALK5 inhibitor. The method may include treatment with a medium supplemented with (i) or (ii), or with a medium supplemented with both (i) and (ii). Cells expressing markers characteristic of gut endoderm cells (stage 3 cells) were classified as (i) T3, T 4, or one or both of their analogs, (ii) an ALK5 inhibitor, or (i) and (ii) Intrapancreatic leukocytes were cultured in a medium supplemented with both leukocytes and plate cultures. This may include differentiating the cells into cells that express markers characteristic of the germ layer (stage 4 cells). The method also provides for the production of cells expressing markers characteristic of pancreatic endoderm cells (stage 4 cells). (i) T3, T4, or one or both of their analogs; (ii) ALK5 inhibition; Treatment with medium supplemented with the agent, or both (i) and (ii), and plating or empty culture Cultivation at the air-liquid interface or in suspension cultures results in characteristic growth of immature beta cells. The method may include differentiating the cells into cells expressing a specific marker (stage 6 cells). , stage 6 cells were treated with (i) one or both of T3, T4, or their analogs, ( ii) an ALK5 inhibitor, or both (i) and (ii) together with an Aurora kinase inhibitor agents, RSK inhibitors, and inhibitors of protein methyltransferase DOT1L and optionally an antioxidant, such as vitamin E or acetylcysteine. Treatment with medium supplemented with tein resulted in a more mature phenotype compared to stage 6. to cells expressing markers characteristic of functional beta cells (stage 7 cells) The preferred amount of acetylcysteine useful is about 0.1 to about 2 The preferred amount of vitamin E is about 0.1 to about 10 μM. In one embodiment, the method comprises treating stage 5 cells with (i) T3, T4, or an analog thereof. (ii) an ALK5 inhibitor, or both (i) and (ii); In addition, gamma secretase inhibitors, RSK inhibitors, and protein methyltransferase inhibitors Treatment with medium supplemented with one or more of the inhibitors of the enzyme DOT1L Thus, further comprising performing stage 6. In yet another embodiment, stage 6 comprises: Stage 5 cells were treated with (i) T3, T4, or one or both of their analogs; On the other hand, (ii) an ALK5 inhibitor, or both (i) and (ii) in combination with a gamma secretion inhibitor enzyme inhibitors, RSK inhibitors, and inhibition of protein methyltransferase DOT1L treatment with a medium supplemented with one or more of the agents, followed by (i) one or both of T3, T4, or analogs thereof, (ii) an ALK5 inhibitor or both (i) and (ii), in combination with an Aurora kinase inhibitor, an RSK inhibitor, a tyrosine kinase ... one or more of the following inhibitors of protein methyltransferase DOT1L, and and optionally supplemented with antioxidants, such as vitamin E or acetylcysteine. Stage 7 is performed by the processing.

[0148] One embodiment of the present invention is directed to the production of functional beta cells (i.e., cells expressing markers characteristic of mature beta cells). This method forms pancreatic endocrine cells (stage 7 cells) that are characteristic of pancreatic endoderm cells. Marker-expressing cells (stage 4 cells) are cultured at the air-liquid interface or in suspension. This involves differentiation into cells that express markers characteristic of stage 7 cells by Cells expressing markers characteristic of functional beta cells with a more mature phenotype are present in PD. X1, as well as the following transcription factors: NKX2.2, NKX6.1, and NeuroD1 , ISL1, HNF3β, MAFA, UCN3, SLC2A1, PAX4, HB9, and In one embodiment, the method of the present invention comprises expressing at least one of N Formation of stage 6 cells positive for KX6.1, PDX1, HB9, and MAFA Preferably, during at least stages 5-7, the method includes the steps of: or treatment with medium supplemented with an analog thereof, an ALK5 inhibitor, or both. Stage 6 cells are positive for NKX6.1, PDX1, HB9, and MAFA In other embodiments, the stage 6 or 7 cells are single hormone positive cells. For example, stage 6 and 7 cells express (a) NKX6.1 and chromogranin. (b) co-expression of NKX6.1 and insulin, or (c) NKX6 The cells may also co-express PDX1, MAFA, and the single hormone insulin. Stage 7 cells exhibit increased levels and proliferation within the cell population compared to stage 6 cells. At the increased cell numbers, they express the single hormones insulin and MAFA.

[0149] In another embodiment, the present invention provides a method for the production of, for example, a population of PDX1 and NKX6.1 co-expressing cells. The cells are preferably cultured and differentiated at the air-liquid interface or in suspension to produce single cells. Insulin-positive cells (e.g., cells co-expressing NKX6.1 and insulin, or NKX6 The present invention provides a method for enhancing the number of cells that co-express chromogranin. In this embodiment, the pancreatic endoderm cells cultured at the air-liquid interface or in suspension are , ALK5 inhibitor, BMP inhibitor, gamma secretase inhibitor, ephrin ligand, Ep hB inhibitors, PKC inhibitors, EGFr inhibitors, retinoic acid, vitamin C, T3 / T4, Glucose, cell cycle regulators, WNT regulators, SHH inhibitors, Aurora inhibitors, antioxidants a compound selected from a hydroxybenzoate, vitamin E, acetyl-cysteine, or a combination thereof; The cells are further differentiated into functional beta cells by treatment with

[0150] In a further embodiment, the present invention relates to a stepwise method for differentiating pluripotent cells, the method comprising: The method involves inoculating stage 4 to stage 6 cells with sufficient amounts of (i) T3, T4, and their analogs. (ii) an ALK5 inhibitor, or both (i) and (ii). Stage 6 cells were cultured in a medium containing an Aurora kinase inhibitor, an RSK inhibitor, and and one or more inhibitors of protein methyltransferase DOT1L; and further culturing in a medium optionally containing an antioxidant to produce insulin, PDX1, N Functional beta cells (developing) expressing KX6.1, UCN3, SLC2A1, and MAFA This involves generating a population of mature phenotype pancreatic endocrine cells and functional beta cells.

[0151] Stage 6 and 7 cells generated according to the methods described herein also produce pancreatic hormones. and in screening compounds for their effects on the secretion of endocrine markers. It is also suitable for use in, among other things, stage 4 bacteria grown at ALI or in suspension culture. ~Stage 7 cells can be tested in different culture formats, from 384 to 6-well formats. These patterns are responsible for the subsequent expression of pancreatic endoderm, pancreatic endocrine precursors, pancreatic endocrine, and pancreatic β-cell markers. Allows evaluation of various small molecules or biologics at various doses and time intervals for expression Such assessment can be performed by PCR for gene expression and FACS or immunoassay for protein expression. Secretion of factors by cells influenced by immunostaining or by the addition of small molecules / biological agents This can be achieved by measuring by ELISA.

[0152] F. Cells obtainable by the methods of the present invention The present invention relates to stage 7 cells, or stage 7 cells, obtainable by the method of the present invention. In certain embodiments, the cells or cell populations are purified after differentiation. In certain embodiments, these stage 7 cells are not transfected with a single hormone. Expresses surin and is positive for PDX1, NKX6.1, UCN3, SLC2A1, and MAFA In addition, these cells have higher MAFA expression levels than stage 6 cells. The cells express UCN3 at levels higher than stage 6 cells (immature beta cells). The resulting cell population is composed of MAFA-positive cells and cells expressing the single hormone insulin. and are both at a higher percentage than stage 6 cells. expression (preferably greater than about 30%), PDX1 expression (preferably greater than about 30%), UCN3 expression (preferably greater than about 10%), SLC2A1 expression (preferably greater than about 10%), and MAFA Functional beta cells (of a mature phenotype) characterized by expression (preferably greater than about 10%) of Insulin-positive cells or insulin-positive cells expressing markers characteristic of pancreatic endocrine cells A population of cells is also provided.

[0153] In an embodiment of the invention, the stage 7 cells or cell populations are insulin producing cells or A population of insulin-producing cells. Insulin-producing cells are myocytes similar to human pancreatic islet cells. Functionally mature cells exhibiting mitochondrial respiration / activity and GSIS response to glucose. In an embodiment of the present invention, functionally mature beta cells are obtained by culturing beta cells in suspension. Produced in culture.

[0154] In embodiments, functional beta cells are those capable of glucose-stimulated insulin secretion and glucose regulation. In an embodiment, the cells exhibit glucose-stimulated insulin-dependent mitochondrial respiration. Secretion and glucose-dependent mitochondrial respiration are similar to those of human pancreatic islet cells In an embodiment of the present invention, functional beta cells secrete insulin in multiple phases. do.

[0155] In an embodiment, glucose-dependent mitochondrial respiration is maximal after glucose stimulation. The oxygen consumption rate response is about 20% to about 80%, preferably about 20% to about 80%, of the basal oxygen consumption rate. About 70%, 20% to about 60%, more preferably about 20%, about 25%, about 30%, about 3 5%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 7 In an embodiment, the oxygen consumption rate response is in the range of about 5%, or about 80% or more. After the stimulation, the temperature is maintained for at least 10 to about 15 minutes, preferably about 10 minutes, about 11 minutes, about 12 minutes, or about In embodiments, the oxygen consumption is faster than the basal OCR. The speed is at least 60 minutes to at least 80 minutes, preferably at least 70 minutes to at least At least 80 minutes, more preferably at least 70, 71, 72, 73, 74, 75, 76, 77 , 78, 79, or 80 minutes.

[0156] In the above embodiment, glucose-stimulated insulin secretion is a measure of insulin secretion in response to glucose stimulation. In embodiments, the first phase of biphasic insulin secretion is The first phase is an increase of at least 4 to at least 8 times the basal secretion, preferably a decrease of the basal secretion. In an embodiment, the biphasic insulin is increased by at least 4-fold, 5-fold, 6-fold, 7-fold, or 8-fold. The second phase of insulin secretion is increased by at least two to at least four times the basal secretion, preferably by more than the basal secretion. In an embodiment, insulin secretion is increased by at least two, three, or four times. At least 5 to 10 minutes after glucose stimulation, preferably at least 10 minutes after glucose stimulation at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, or occurs in at least 10 minutes.

[0157] Tables A and B illustrate exemplary culture conditions suitable for use in embodiments of the methods of the present invention. When used in Tables A and B below, "MCX" refers to the MCX compound, and "AA" refers to the active ingredient. Bin, "ALK5inh." is an ALK5 inhibitor, "RA" is retinoic acid, "Vitamin C " is ascorbic acid, "inh." is an inhibitor, and "act." is an activator. In certain embodiments, one stage (e.g., stage 1, 2, 3, 4, 5, 6, or any one of 7) may be processed in another stage (e.g. For example, any one of stages 1, 2, 3, 4, 5, 6, or 7) In other embodiments, methods other than those in Table A may be used in combination with any one of the methods described above. Stage 4 cells obtained by the method described above were cultured under the culture conditions shown in Table B. In other embodiments, the methods in Tables A and B may be used to differentiate the cells into stage 5 to stage 7 cells. Stage 5 cells obtained by different methods were cultured using the culture conditions shown in Table B. In yet another embodiment, the cells of Tables A and B may be differentiated into stage 6 or stage 7 cells. Stage 6 cells obtained by a method different from that in Table B were cultured under the culture conditions shown in Table B. The cells may be differentiated into stage 7 cells using the conditions described above.

[0158] [Table 2]

[0159] [Table 3]

[0160] Publications cited throughout this specification are incorporated herein by reference in their entirety. The present invention is further illustrated by the following non-limiting examples. It is not more limited. [Example]

[0161] The sources of materials and compounds used in the following examples are identified in Table X.

[0162] Example 1 Screening and identification of small molecules that upregulate MAFA or UCN3 expression. The following examples illustrate the use of MAFA (v-maf avian myofascial fibrosarcoma oncogene homolog A) or Increased gene expression of mature beta cell markers, including UCN3 (urocortin 3) and identifying small molecules that can enhance the maturation status of pancreatic beta cells. Cells of the human embryonic stem cell line H1 ("H1-hESC") in EZ8 medium at passage 28 were cultured. Dulbecco's Modified Eagle's Medium Nutrient Mixture F-12 ("DMEM-F12"), 1:100 Diluted ("1x") GlutaMAX™, 0.25 mM ascorbic acid, 10 0 ng / mL fibroblast growth factor 2 ("FGF2"), 1 ng / mL transforming growth factor beta ("TGFβ"), insulin-transferrin-selenium-ene diluted 1:100 ethanolamine ("ITS-X"), 2% fatty acid-free bovine serum albumin ("FAF-B") SA"), and 20 ng / mL insulin-like growth factor-1 ("IGF-1"), in medium supplemented with 1 μM of the Rock inhibitor Y-27632 ("Y compound"). 0.094 x 10 cells onto a 1:30 dilution of MATRIGEL™ in a dish containing 6 cell / c m 2 Y compounds were added only during the first 24 hours after seeding. 48 hours after seeding, cultures were transferred to incomplete PBS (containing no magnesium or calcium). The cells were washed in phosphate-buffered saline (PBS) without HCl.

[0163] For Figures 1A-1H, the cultures were differentiated using the following protocol. During stages 1 to 4, cultures were maintained on planar adherent culture. a. Stage 1 (3 days): Cells were cultured in the following Stage 1 medium: 2.7g / 10 00 mL sodium bicarbonate (Sigma-Aldrich Co. LLC, St. Louis, MO) (Missouri, Cat. No. 5761) with 0.5% FAF-BSA GlutaMAX™ at a 1:100 dilution ("1x concentration"), D-GlutaMAX™ at a concentration of 10 mM 4.5 mM D-glucose for obtaining glucose, 100 ng / mL growth differentiation factor 8 ( "GDF8"), and 1.5 μM 14-prop-2-en-1-yl-3,5,7,1 4,17,23,27-heptaazatetracyclo[19.3.1.1~2,6~.1~8 ,12~]Heptacosa-1(25),2(27),3,5,8(26),9,11,21 MCDB-131 medium supplemented with 23-nonane-16-one ("MCX compound"). The medium was then cultured for 1 day in a 0.5% sodium bicarbonate solution containing 2.7 g / 1000 mL of sodium bicarbonate. FAF-BSA, 1x GlutaMAX™, 10mM D-glucose 4.5 mM D-glucose, 100 ng / mL GDF8, and 0.1 Cells were cultured for an additional day in MCDB-131 medium supplemented with μM MCX compound. Next, 0.5% FAF-BSA containing 2.7 g / 1000 mL sodium bicarbonate, 4. To obtain a 1x concentration of GlutaMAX™ and a 10 mM concentration of D-glucose. MCDB-13 supplemented with 5 mM D-glucose and 100 ng / mL GDF8 The cells were cultured in 1 for an additional day. b. Stage 2 (2 days): Contains 2.7 g / 1000 mL sodium bicarbonate and 0. 5% FAF-BSA, 1x GlutaMAX™, 10 mM D-glucose 4.5 mM D-glucose, 0.25 mM ascorbic acid, and 50 ng Cells were cultured in MCDB-131 medium supplemented with 1 / mL fibroblast growth factor 7 ("FGF7"). The cells were treated for 2 days. c. Stage 3 (2 days): Contains 3.6 g / 1000 mL sodium bicarbonate and 1: 200% dilution of ITS-X; 4.5 mM D-glucose to obtain a concentration of 10 mM Glucose; 1x GlutaMAX™; 2% FAF-BSA; 25 ng / mL FGF7, 0.25 μM SANT-1 (N-[(3,5-dimethyl-1-phenyl-1 H-pyrazol-4-yl)methylene]-4-(phenylmethyl)-1-piperazine amine 1 μM retinoic acid (“RA”); 0.25 mM ascorbic acid, 300 nM P KC activator ((2S,5S-(E,E)-8-(5-(4-trifluoromethyl)phenyl) (2,4-pentadienoylamino)benzolactam ("TPB"); and bone morphology Morphogenetic protein ("BMP") receptor inhibitor LDN-193189-HCl ("LDN- BLAR001 custom medium (see Table I) supplemented with HCl) for 2 days. The cells were treated for 2 days. The concentration of LDN-HCl used on day 1 of stage 3 was 100 nM and for stage 3 day 2 it was 10 nM. d. Stage 4 (3 days): 3.6 g / 1000 mL sodium bicarbonate containing 1: 200% dilution of ITS-X; 4.5 mM D-glucose to obtain a concentration of 10 mM Glucose; 1x GlutaMAX™; 2% FAF-BSA; 0.25 μL M SANT-1, 50nM RA; 2ng / mL FGF7, 50nM LDN-HC l; BLAR001 supplemented with 0.25 mM ascorbic acid; and 200 nM TPB At the end of stage 4 (3 days), the cells were cultured in a plate. The cells were seeded at the air-liquid interface ("ALI"). Specifically, the cells were treated with 10 μM Y- 27632 for 4 hours, rinsed with PBS, and then treated with 1x TrypLE™ Ex After treating with the enzyme for approximately 2 minutes, remove the enzyme and gently tap the flask to remove the cells. The resulting cell suspension was removed from the MATRIGEL™ surface. 5~1.0×10 6 At a density of cells (in 5 μL aliquots) of 0.4 μg on a 10 cm plate Either 0.25 mm or 3.0 mm porosity cell culture filter inserts 8.0 mL of medium was added to the bottom of each insert and the tip of the filter or No additional medium was added to the top or sides. The medium was added to the sustained stage of stages 5, 6, and 7. It was changed daily during that time. e. Stage 5 (3 days): 2.7 g / 1000 mL sodium bicarbonate, 1: 200 dilution of ITS-X; 14.5 to achieve a final concentration of 20 mM D-glucose 1 mM D-glucose; 1x GlutaMAX™; 2% FAF-BSA; 10 μL g / mL heparin ("H"); 10 μM ZnSO4; 0.25 μM SANT-1; 50nM RA; 100nM LDN-HCl; 3,3',5-Triiodo-L-threo 1 μM T3 in the form of its sodium salt; 10 μM 2-(3-(6-methylpyridinyl) (1H-pyrazol-2-yl)-1,5-naphthyridine (ALK5 inhibitor) Cells were cultured at the ALI for 3 days in BLAR001 medium supplemented with ALK5 (anti-inflammatory drug II or ALK5). Treatment was carried out for days. f. Stage 6 (7 days): 2.7 g / 1000 mL sodium bicarbonate containing 1: 200 dilution of ITS-X; 14.5 to achieve a final concentration of 20 mM D-glucose 1 mM D-glucose; 1x GlutaMAX™; 2% FAF-BSA; 10 μL 10 μM ZnSO4; 100 nM LDN-HCl; 1 μM T 3; 10 μM ALK5 inhibitor II; and 100 nM (S,S)-2-[2-(3,5- difluorophenyl)acetylamino]-N-(5-methyl-6-oxo-6,7-dihydroxy dro-5H-dibenzo[b,d]azepin-7-yl)propionamide ("gammasec Cells were treated with BLAR001 medium supplemented with ATPase inhibitor XX for 7 days at the ALI. I understood. g. Stage 7 (7 days): Contains 2.7 g / 1000 mL sodium bicarbonate, 1: 200% diluted ITS-X; 1x GlutaMAX™; 2% FAF-BSA; 10 μg / mL heparin ("H"); 10 μM ZnSO4; 1 μM T3; 10 μM A LK5 inhibitor II; BLA supplemented with 1 mM N-acetylcysteine ("NAC"); Cells were treated with ALI in R001 medium. Additionally, during stage 7 (S7), as shown in Figures 1A-1H, cells were treated with 20 mM D- Conditioned with glucose and BME vitamin supplement (1:100 dilution) and shown The animals were exposed to all 79 small molecules listed and described in Table XI. (ii) lomeguatrib; (iii) 5-azacytidine; (iv) mitoxantrone dihydrochloride; (v) EGCG; (vi) fisetin; (v ii) SGI 1027; (viii) temozolomide; (ix) L002; (x) C64 6; and (xi) SGC0946 induced the expression of MAFA or UCN3.

[0164] For Figures 2A-2B, cultures were differentiated using the following protocol. During stages 1 to 4 of the le, the cultures were maintained on planar adherent culture. a. Stage 1 (3 days): Cells were cultured in the following Stage 1 medium: 2.7g / 10 Contains 00 mL sodium bicarbonate, 0.5% FAF-BSA, 1:100 dilution (1 GlutaMAX™ (4x concentration) to obtain a 10 mM D-glucose concentration 0.5 mM D-, 100 ng / mL growth differentiation factor 8 ("GDF8"), and 1.5 μM 14-prop-2-en-1-yl-3,5,7,14,17,23,27-heptaaza Tetracyclo[19.3.1.1~2,6~.1~8,12~]heptacosa-1(25) ,2(27),3,5,8(26),9,11,21,23-nonane-16-one (" The cells were cultured for one day in MCDB-131 medium supplemented with 2.7 MCX compounds. g / 1000mL sodium bicarbonate, 0.5% FAF-BSA, 1x concentration of G lutaMAX™, 4.5 mM D-glucose to obtain a concentration of 10 mM Supplemented with glucose, 100 ng / mL GDF8, and 0.1 μM MCX compound The cells were cultured for another day in MCDB-131 medium. Contains sodium carbonate, 0.5% FAF-BSA, 1x GlutaMAX (commercially available) 4.5 mM D-glucose to obtain a concentration of 10 mM D-glucose, and 1 Cells were cultured for an additional day in MCDB-131 supplemented with 0.00 ng / mL GDF8. . b. Stage 2 (2 days): Contains 2.7 g / 1000 mL sodium bicarbonate and 0. 5% FAF-BSA, 1x GlutaMAX™, 10 mM D-glucose 4.5 mM D-glucose, 0.25 mM ascorbic acid, and 50 ng Cells were cultured in MCDB-131 medium supplemented with 1 / mL fibroblast growth factor 7 ("FGF7"). The cells were treated for 2 days. c. Stage 3 (2 days): Contains 3.6 g / 1000 mL sodium bicarbonate and 1: 200% dilution of ITS-X; 4.5 mM D-glucose to obtain a concentration of 10 mM Glucose; 1x GlutaMAX™; 1% FAF-BSA; 25 ng / mL FGF7, 0.25 μM SANT-1 (N-[(3,5-dimethyl-1-phenyl-1 H-pyrazol-4-yl)methylene]-4-(phenylmethyl)-1-piperazine amine 1 μM retinoic acid (“RA”); 0.25 mM ascorbic acid, 300 nM P KC activator ((2S,5S-(E,E)-8-(5-(4-trifluoromethyl)phenyl) (2,4-pentadienoylamino)benzolactam ("TPB"); and bone morphology Morphogenetic protein ("BMP") receptor inhibitor LDN-193189-HCl ("LDN- Cells were treated with BLAR001 custom medium supplemented with HCl for 2 days. The concentration of LDN-HCl used on day 1 of stage 3 was 100 nM. On the second day, the concentration was 50 nM. d. Stage 4 (3 days): 3.6 g / 1000 mL sodium bicarbonate containing 1: 200% dilution of ITS-X; 4.5 mM D-glucose to obtain a concentration of 10 mM Glucose; 1x GlutaMAX™; 1% FAF-BSA; 0.25 μL M SANT-1, 50nM RA; 2ng / mL FGF7, 70nM LDN-HC l; BLAR001 supplemented with 0.25 mM ascorbic acid; and 200 nM TPB At the end of stage 4 (3 days), the cells were cultured in a plate. The cells were seeded at the air-liquid interface ("ALI"). Specifically, the cells were treated with 10 μM Y2 7632 for 4 hours, rinsed with PBS, and then treated with 1x the enzyme TrypLE™ Exp. After approximately 2 minutes of treatment with the enzyme, the enzyme was removed and the cells were gently shaken off. The resulting cell suspension was removed from the MATRIGEL™ surface. ~1.0×10 6 Cells (in 5 μL aliquots) were plated at a density of 0.4 μL in a 10 cm plate. 1.0 micrometer porous cell culture filter insert or 3.0 micrometer porous cell culture filter The culture medium was placed on top of one of the culture filter inserts. 8.0 mL of medium was added to the bottom of each insert. No additional medium was added to the apex, top, or sides of the filter. They were changed daily for the duration of stages 5, 6, and 7. e. Stage 5 (3 days): 2.7 g / 1000 mL sodium bicarbonate, 1: 200 dilution of ITS-X; 14.5 to achieve a final concentration of 20 mM D-glucose 1 mM D-glucose; 1x GlutaMAX™; 2% FAF-BSA; 10 μL g / mL heparin ("H"); 10 μM ZnSO4; 0.25 μM SANT-1; 50nM RA; 100nM LDN-HCl; 3,3',5-Triiodo-L-threo 1 μM T3 in the form of its sodium salt; 10 μM 2-(3-(6-methylpyridinyl) (1H-pyrazol-2-yl)-1,5-naphthyridine (ALK5 inhibitor) Cells were cultured at the ALI for 3 days in BLAR001 medium supplemented with ALK5 (anti-inflammatory drug II or ALK5). Treatment was carried out for days. f. Stage 6 (7 days): 2.7 g / 1000 mL sodium bicarbonate containing 1: 200 dilution of ITS-X; 14.5 to achieve a final concentration of 20 mM D-glucose 1 mM D-glucose; 1x GlutaMAX™; 2% FAF-BSA; 10 μL g / mL heparin ("H"); 10 μM ZnSO4; 100 nM LDN-HCl; 1 μM T3; 10 μM ALK5 inhibitor II; and 100 nM (S,S)-2-[2- (3,5-difluorophenyl)acetylamino]-N-(5-methyl-6-oxo-6 ,7-dihydro-5H-dibenzo[b,d]azepin-7-yl)propionamide (" Cells were cultured at the ALI in BLAR001 medium supplemented with gamma secretase inhibitor XX. The treatment was continued for 7 days. g. Stage 7 (7 days): Contains 2.7 g / 1000 mL sodium bicarbonate, 1: 200% diluted ITS-X; 1x GlutaMAX™; 2% FAF-BSA; 10 μg / mL heparin ("H"); 10 nM T3 ("low T3"); 1 mM N-acetyl Cells were treated at ALI with BLAR001 medium supplemented with nasal acetylcysteine (NAC). did. Further, for Figures 2A and 2B, 0.5 μM ZM447439 (“ZM”); and Formulation I ("FI") (Table XII) comprises the following components: RP at a 1:200 dilution MI Vitamin Supplement; 1:200 dilution MEM Non-Essential Amino Acid Supplement; 1:2000 dilution of defined lipid concentrate; 1:200 dilution of sodium pyruvate; 1:2000 dilution of microcrystalline cellulose Trace element A; and trace element B at a 1:2000 dilution were added for 7 days. Additional components included 5 μM 5-azacytidine (“AZT”); or 1 μM or 1 0 μM 3-deazaneplanocin A (“DEZA”) was included.

[0165] The embodiments described herein are based on Applicant's proprietary BLAR medium, specifically BLAR0. BLAR01 or BLAR004 medium is used. BLAR medium was filed on December 31, 2012. PCT Application No. 61 / 747,662 filed December 18, 2013, both of which claim the benefit of PCT Application No. First described in U.S. Patent Application Serial No. 13 / 998,884 and U.S. Patent Application Serial No. 13 / 75939 and then again in Rezania et al. (2014) Nature Biot ech,32(11)1124~1134,Supplemental Table 3 (published online September 11, 2014), the references of which are incorporated herein in their entirety. BLAR001 and BLAR004 are not intended to be limiting unless otherwise specified. The concentrations or levels of the agents are different.

[0166] [Table 4]

[0167] Generally, embodiments herein involve the use of human polymorphonuclear cells, as described above in stages 1-7. In stage 7, various small molecules are added to the cells to investigate their effects. The small molecules and their targets were evaluated by quantitative real-time PCR. Table II lists the small molecules and their targets. These small molecules were evaluated and added at 2 μM each at stage 7. The chemical names and structures of the compounds obtained are shown in Table XI.

[0168] [Table 5-1]

[0169] [Table 5-2]

[0170] [Table 5-3]

[0171] Quantification and characterization of differentiated cells: for quantification of gene expression at different stages , human pancreatic islets, H, substantially as described in Rezania et al. (2014), supra. 1-hESCs, stage 6 day 7 (S6D7), and stage 7 day 7-stage 7 Cells on day 14 (S7D7 to S7D14) were collected as ALI clusters. Expression was analyzed using custom Taqman Arrays (Applied Biosystems) The sequence was evaluated in cells using a 5'-dimer of 1 ... Detection software (Applied Biosystems, Foster City, , California) and analyzed the data. The ΔΔCt method was used to determine the undifferentiated H1- Normalization was performed using GAPDH as a housekeeping gene against hESCs. The timer details are outlined in Table III.

[0172] [Table 6]

[0173] Figures 1A-1D show quantitative real-time P changes in MAFA expression after treatment with small molecules. Graph showing data from a CR analysis in which various small molecules interact with S6D7, or S6D7. Compared to untreated or DMSO-treated cultures at stage 7 (S7D7), M UNC0638 (Fig. 1A; selectively inhibited G9a and GLP histochemical expression of AFA). lysine methyltransferase inhibitor), UNC0646 (Figure 1A; potent and selective potent and selective G9a / GLP inhibitor), UNC0642 (Fig. 1A; P histone lysine methyltransferase inhibitor), TC-E5003 (Figure 1B; selection a potent PRMT1 arginine methyltransferase inhibitor), A366 (Fig. 1B; and selective G9a / GLP histone lysine methyltransferase inhibitor), PF0 3814735 (Figure 1B; Aurora kinase A and B inhibitor), ZM447439 (Figure 1 B; inhibits Aurora kinase B), SB747651A dihydrochloride (Figure 1B; potent a potent inhibitor of MSK1; also inhibits other AGC family kinases), PFI1 (Fig. 1B; BET bromod main inhibitor), LY303511 (Figure 1B; BRD2, BRD3, and BRD4 inhibitor ), MS436 (Figure 1B; a potent and selective BRD4 bromodomain inhibitor), and MC Addition of 1568 (Figure 1C; selectively inhibits HDAC class II (IIa)) increased the S6 D7, or stage 7 untreated or DMSO-treated cultures compared to M The expression of AFA was significantly upregulated. Figures 1E-1H show the U after treatment with small molecules. 5 is a graph depicting data from quantitative real-time PCR analysis of CN3 expression; -azacytidine ("AZT") (Figure 1E; a DNA methyltransferase inhibitor), roxithromycin (Figure 1G; histone deacetylase inhibitor), and CI994 (Figure 1G; histone deacetylase inhibitor). The addition of a phosphodiesterase inhibitor (PDI) resulted in a significant improvement in the survival of untreated S6D7 or stage 7 UCN3 expression was significantly upregulated compared with cultures treated with DMSO or DMSO.

[0174] Figures 2A and 2B show the small molecules selected as upregulators of either MAFA or UCN3. We demonstrate the robustness of the molecules, which indicates their effects across different stage 7 conditioning protocols. This is to ensure that it remains standing.

[0175] The culture conditions shown in Figures 2A and 2B were the following changes at stage 7 (S7): (i) removal of ALK5 inhibitor II (“ALK5”); (ii) reduction of T3 levels (“ (iii) the addition of ZM447439 (“ZM”); and (iv) vitamin , by the addition of a cocktail of trace elements, lipids, and amino acids (Formulations I-XII) Specifically, the basal conditions tested were: (1) ALK5 inhibitor II ("ALK5" ) None; (2) Low T3; (3) Low ZM; (4) Low H; (5) Low NAC; (6) FI; and AZT up-regulates UCN3 (Figure 2B) during stage 7. However, it was not identified as an upregulator of MAFA (Figure 2A). In addition to the small molecules studied, 3-deazaneplanocin A ("DEZA"; Figure 2A) was also shown to induce MAFA. It was found to be an effective upregulator of β-glucan in the presence of ZM447439. Neither was an effective upregulator of UCN3 (Fig. 2B) expression.

[0176] In summary, this example demonstrates the expression of maturation markers MAFA or UCN during stage 7. We demonstrate the identification of small molecules that upregulate the expression of 3.

[0177] Example 2 Glucose-rich cells with improved maturation marker expression and similar to human pancreatic islets at the air-liquid interface Generation of endocrine cells with ATP-dependent mitochondrial respiratory dynamics. The following examples illustrate the use of the following maturation markers: PDX1 (pancreatic and duodenal homeostasis factor); box 1); NKX6.1 (NK6 homeobox 1); MAFA; UCN3, SLC 2A1 (solute carrier family 2 member 1; also known as GLUT1 / glucose transporter 1) The cells co-expressed the mitochondrial respiration protein (mRNA) and exhibited glucose-dependent mitochondrial respiration similar to that observed in human islet cells. C-peptide (a single chain connecting the insulin A and B chains in the proinsulin molecule) Demonstrate the generation of human 31 amino acid polypeptide cells in EZ8 medium for 28 passages. Cells of the embryonic stem cell line H1 ("H1-hESC") were cultured in Dulbecco's modified Eagle's medium with nutrient mixture. DMEM-F12 ("DMEM-F12"), 1:100 dilution ("1x concentration") of GlutaM AX™, 0.25 mM ascorbic acid, 100 ng / mL fibroblast growth factor 2 ( "FGF2"), 1ng / mL transforming growth factor beta ("TGFβ"), 1:100 Diluted insulin-transferrin-selenium-ethanolamine ("ITS-X"); 2% fatty acid-free bovine serum albumin ("FAF-BSA"), and 20 ng / mL of insulin 10 μM of the Rock inhibitor Y-27632 of insulin-like growth factor-1 ("IGF-1"); The cells were coated with a 1:30 dilution of MATRIGEL™ in medium supplemented with α-Y compound ("Y compound"). 0.094 x 10 6 cells / cm 2 The cells were seeded as single cells at 100°C. After 48 hours, cultures were resuspended in incomplete PBS (phosphate-buffered saline without magnesium or calcium). The cells were washed in acetic acid-buffered saline.

[0178] For Figures 3A-3M, 4A-4E, and 5A-5F, culture using the following protocol: During stages 1–4 of the protocol, the cultures were maintained on planar adherent culture. . a. Stage 1 (3 days): Cells were cultured in the following Stage 1 medium: 2.7g / 10 Contains 00 mL sodium bicarbonate, 0.5% FAF-BSA, 1:100 dilution (1 GlutaMAX™ (4x concentration) to obtain a 10 mM D-glucose concentration 0.5 mM D-glucose, 100 ng / mL growth differentiation factor 8 ("GDF8"), and 1 0.5 μM 14-prop-2-en-1-yl-3,5,7,14,17,23,27- Heptaazatetracyclo[19.3.1.1~2,6~.1~8,12~]heptacosa- 1(25),2(27),3,5,8(26),9,11,21,23-nonane-16 The mixture was cultured for one day in MCDB-131 medium supplemented with α-methyl-1-one ("MCX compound"). , containing 2.7g / 1000mL sodium bicarbonate, 0.5% FAF-BSA, 1x GlutaMAX™ concentration, 4.5 ml to obtain a 10 mM concentration of D-glucose 100 ng / mL D-glucose, 100 ng / mL GDF8, and 0.1 μM MCX compound were supplemented. The cells were cultured for another day in MCDB-131 medium supplemented with 2.7g / 100ml of MCDB-131 medium. Contains 0 mL sodium bicarbonate, 0.5% FAF-BSA, 1x GlutaM AX™, 4.5 mM D-glucose to obtain a concentration of 10 mM D-glucose The cells were incubated for an additional day in MCDB-131 supplemented with 100 ng / mL GDF8. Cultivated. b. Stage 2 (2 days): Contains 2.7 g / 1000 mL sodium bicarbonate and 0. 5% FAF-BSA, 1x GlutaMAX™, 10 mM D-glucose 4.5 mM D-glucose, 0.25 mM ascorbic acid, and 50 ng Cells were cultured in MCDB-131 medium supplemented with 1 / mL fibroblast growth factor 7 ("FGF7"). The cells were treated for 2 days. c. Stage 3 (2 days): Contains 3.6 g / 1000 mL sodium bicarbonate and 1: 200% dilution of ITS-X; 4.5 mM D-glucose to obtain a concentration of 10 mM Glucose; 1x GlutaMAX™; 1% FAF-BSA; 25 ng / mL FGF7, 0.25 μM SANT-1 (N-[(3,5-dimethyl-1-phenyl-1 H-pyrazol-4-yl)methylene]-4-(phenylmethyl)-1-piperazine amine 1 μM retinoic acid (“RA”); 0.25 mM ascorbic acid, 300 nM P KC activator ((2S,5S-(E,E)-8-(5-(4-trifluoromethyl)phenyl) (2,4-pentadienoylamino)benzolactam ("TPB"); and bone morphology Morphogenetic protein ("BMP") receptor inhibitor LDN-193189-HCl ("LDN- Cells were treated with BLAR001 custom medium supplemented with HCl for 2 days. The concentration of LDN-HCl used on day 1 of stage 3 was 100 nM. On the second day, the concentration was 50 nM. d. Stage 4 (3 days) and ALI transition at S4D3: 3.6g / 1000mL bicarbonate Contains sodium chloride, 1:200 dilution of ITS-X; 10 mM D-glucose 4.5 mM D-glucose to obtain 1x GlutaMAX™; 1% FAF-BSA; 0.25μM SANT-1, 50nM RA; 2ng / mL FGF 7, 70 nM LDN-HCl; 0.25 mM ascorbic acid; and 200 nM TPB The cells were treated with BLAR001 medium supplemented with β-glucan for 3 days. At the end of stage 4 (3 days), At the end of the incubation period, the cells cultured in the dish were seeded onto the air-liquid interface ("ALI"). For I migration, cells were treated with 10 μM Y compound for 4 h, rinsed with PBS, and incubated at 1x concentration. After treating with TrypLE™ Express enzyme for about 2 minutes, the enzyme was removed. The cells were removed from the MATRIGEL™ surface by gently tapping the flask. The resulting cell suspension was cultured at 0.5-1.0 x 10 6 At a density of 1,000 cells (in a 5 μL aliquot), 0.4 micrometer porous cell culture filter inserts in 3.0 cm plates or 8. The cells were seeded onto either a 0 micrometer porous cell culture filter insert. 100 mL of medium was added to the bottom of each insert, and no more was added to the apex, top, or sides of the filter. No additional medium was added. The medium was changed daily for the duration of stages 5, 6, and 7. . e. Stage 5 (3 days): 2.7 g / 1000 mL sodium bicarbonate, 1: 200 dilution of ITS-X; 14.5 to achieve a final concentration of 20 mM D-glucose 1 mM D-glucose; 1x GlutaMAX™; 2% FAF-BSA; 10 μL g / mL heparin ("H"); 10 μM ZnSO4; 0.25 μM SANT-1; 50nM RA; 100nM LDN-HCl; 3,3',5-Triiodo-L-threo 1 μM T3 in the form of thiamin sodium salt (Sigma Aldrich, catalog no. No. T6397; 10 μM 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-2-yl)- (4-azol-4-yl)-1,5-naphthyridine ("ALK5 inhibitor II" or "ALK5" ) and cells were treated at ALI for 3 days. f. Stage 6 (7 days): 2.7 g / 1000 mL sodium bicarbonate containing 1: 200 dilution of ITS-X; 14.5 to achieve a final concentration of 20 mM D-glucose 1 mM D-glucose; 1x GlutaMAX™; 2% FAF-BSA; 10 μL g / mL heparin ("H"); 10 μM ZnSO4; 100 nM LDN-HCl; 1 μM T3; 10 μM ALK5 inhibitor II; and 100 nM (S,S)-2-[2- (3,5-difluorophenyl)acetylamino]-N-(5-methyl-6-oxo-6 ,7-dihydro-5H-dibenzo[b,d]azepin-7-yl)propionamide (" Cells were cultured at the ALI in BLAR001 medium supplemented with gamma secretase inhibitor XX. The treatment was continued for 7 days. g. Stage 7 (7 days): Cells at the ALI were cultured in two types of custom BLAR medium. The first medium was treated with AR001 and BLAR004. ITS-X containing sodium phosphate at a 1:200 dilution; 1x GlutaMAX™; 2% FAF-BSA; 10 μg / mL heparin ("H"); 10 nM T3 ("Low T" 3"); 1 mM N-acetylcysteine ("NAC"); 0.5 μM ZM44743 9 ("ZM"); and the following components comprising Formulation I ("FI"): 1:200 Dilute RPMI vitamin supplement 1:200. Dilute MEM non-essential amino acid supplement 1:2 1:000 dilution of chemically defined lipid concentrate; 1:200 dilution of sodium pyruvate; 1:200 BLAR001 supplemented with trace element A at 0 dilution; trace element B at 1:2000 dilution for 7 days The medium (a list of ingredients is outlined in Table I) contains additional ingredients added during stage 7. , either 1 μM T3 or 10 nM T3 (“low T3”); 10 μM ALK5 inhibition Agent II; 5 μM 5-azacytidine (“AZT”); or 1 μM or 10 μM 3- Deazaneplanocin A ("DEZA") was included. The second medium contained 2.7 g / 1000 mL sodium bicarbonate at a 1:200 dilution. ITS-X; 1x GlutaMAX™; 2% FAF-BSA; 10 μg / mL Heparin ("H"); 1 mM N-acetylcysteine ("NAC"); 0.5 μM Z M447439 ("ZM"); and the following ingredients comprising Formulation I ("FI"): RPMI vitamin supplement at a 1:200 dilution; MEM non-essential amino acid supplement at a 1:200 dilution Additives: 1:2000 dilution of chemically defined lipid concentrate; 1:200 dilution of sodium pyruvate 1:2000 dilution of trace element A; 1:2000 dilution of trace element B supplemented for 7 days LAR004 (a list of ingredients is outlined in Table IV) medium. Additional components included either 1 μM T3 or 10 nM T3 (“low T3”); M ALK5 inhibitor II; 5 μM 5-azacytidine (“AZT”); or 1 μM or less contained 10 μM 3-deazaneplanocin A (“DEZA”).

[0179] [Table 7]

[0180] Characterization and quantification of differentiated cells: for quantification of gene expression at different stages , Rezania et al.,Nature Biotechnology,201 4;32(11):1121-1133, human pancreatic islet cells, H1- hESCs, stage 6 day 7 (S6D7), and stage 7 day 7 to stage 7 day 1 Cells on day 4 (S7D7–S7D14) were collected as ALI clusters. The results were analyzed using custom Taqman Arrays (Applied Biosystems, F The sequence was evaluated in cells using a ELISA kit (Oster City, California). Software for (Applied Biosystems, Foster City, CA) Data were analyzed using the ΔΔCt method to determine the undifferentiated H1-hE Normalization was performed using GAPDH as a housekeeping gene against SC. The details of this are outlined in Table V.

[0181] [Table 8]

[0182] To quantify protein colocalization at various stages, human pancreatic islets, S7D7 cells, were used. The cells were collected as ALI cell clusters and analyzed by immunofluorescence ("IF"). hESC-derived cells were cultured essentially as described in Rezania et al. (2014), supra. The frozen sections were prepared and stained as described above and with the antibodies listed in Table VI herein. To achieve this, the cells were rinsed with PBS and then fixed overnight in 4% PFA at 4°C. The FA was removed, the cells were rinsed twice with PBS, and then incubated overnight in a 30% sucrose solution at 4°C. These samples were cryopreserved in OCT solution and 5 μm sections were cut using Su Perfrost Plus Slides (VWR International, LLC) Radnor, PA, Catalog No. 48311-703).

[0183] For IF staining, primary antibodies were added at appropriate dilutions overnight at 4°C, while secondary antibodies were added at room temperature for 3 h. After 0 min, the sections were rinsed with PBS and then filled with Vectastain mounting reagent ( Vector Laboratories Inc., Burlingame, Cali fornia, Cat. No. H-1200) was added. A Nikon Ti fluorescence microscope ( (Nikon Instruments, Inc., Melville, NY) The pieces were visualized.

[0184] [Table 9]

[0185] For quantification of glucose-dependent mitochondrial activity at various stages of maturation Human pancreatic islets, S6D7 ALI clusters, and S7D7 ALI clusters were collected and Their oxygen consumption rates ("OCR") were measured using XF before and after injection of 20 mM D-glucose. e 24 Extracellular Flux Analyzer(Seahorse Bioscience, Cat. No. 102238-100). Stars were removed from their stage 7 conditioning and placed in a 37°C non-CO2 environment and baseline The cells were incubated for 2 hours in a medium designed to achieve OCR. The incubation medium consisted of XF base medium containing 1 mM D-glucose, 1 mM L -glutamine, and 1 mM sodium pyruvate. ,The ALI cluster is loaded onto a Seahorse machine, where the following measurements, i.e. (i) baseline OCR (before D-glucose injection) and (ii) D-glucose injection. Post-injection incubation was performed five times (72 min post-injection incubation time). All OCR measurements were , the DNA of individual samples of ALI or Aggrewell clusters in the spinner DNA was normalized to A content. DNA content was determined by NanoDr op 8000 UV-Vis Spectrophotometer(Thermo Scientific, catalogue number ND8000).

[0186] Figures 3A-3M show gene expression of a set of maturation markers in ALI cell clusters at 7 days. demonstrated that after stage 7 conditioning, the levels of ATP increased to levels observed in human islets. Maturation markers are the positive stimulators of rapid glucose-stimulated insulin secretion (GSIS). The following list is defined herein as genes required for ALI stimulation: S7D7 was observed to improve gene expression of maturation markers in stage 7 differentiation process. The changes in the protocol are detailed below: (i) removal of ALK5 inhibitor II; (ii) T3 concentration. (iii) Addition of DEZA; (iv) Addition of AZT; (v) Addition of ZM; (vi (vii) a reduction in glucose concentration to 5.56 mM; and (vii) vitamins, non-essential amino acids, Defined Cocktail of Lipids, Sodium Pyruvate, and Trace Elements (Formulation I-"FI") Similar findings and results were observed with BLAR001-based conditioning during stage 7 and BLAR The gene expression profile of mature genes for both 004 and 005 lineages was observed. Figure 3A showed that INHBB was observed in human pancreatic islet cells under conditions using ALK5 inhibitor II. The findings indicate that ALK5 inhibition in S7 cells was enriched over expression in the control group. Inhibition of TGFB1 (transforming growth factor beta 1) by agonist II inhibits the GSIS process. These findings suggest that TGF-beta signaling induces a negative TGF-beta signaling profile that is detrimental to the immune system. Removal of the ALK5 inhibitor in the presence or absence of T / DEZA inhibited INHBB in human islets. cells, resulting in a negative effect on GSIS during stage 7. It was observed that the effects of TGF-beta signaling were eliminated. In addition, Figure 3B shows that I NHA expression was upregulated in ALI clusters by removal of ALK5 inhibitor II in human islets. It is shown that an increase up to 1000 bp was observed.

[0187] The MAFA and SLC2A1 expression levels shown in Figures 3C and 3D, respectively, were significantly increased by ALK5 inhibitor I. It was observed that the removal of I reduced M AFA and SLC2A1 expression was rescued to human islet levels. 6PC2 (glucose-6-phosphatase catalytic subunit 2; Figure 3F), and PDK 1 (pyruvate dehydrogenase kinase 1; Figure 3G) expression was associated with the ALI cluster class The addition of AZT / DEZA and removal of ALK5 inhibitor II in the culture medium increased the expression of human islet cells. The levels of insulin increased to levels exceeding those of human pancreatic islet cells. ), GJD2 (gap junction protein, delta 2; together with CX36 / connexin 36; 3I), SIX2 (SIX homeobox 2; Fig. 3J), and PDX1 (Fig. 3K). Currently, the treatment is gradually improving, first by removing the ALK5 inhibitor II and then by adding AZT / DEZA. A stepwise increase in NKX6.1 (Fig. 3L) and GLP1R (glucan) was observed. Although no changes in expression of the gonadotropin-like peptide 1 receptor (GLP-1 receptor; Figure 3M) were observed across conditions, , their expression in ALI clusters is consistently at levels observed in human pancreatic islets. was at or above that level.

[0188] 4A-4E show the following maturation markers in terms of protein abundance: S7D7 In the present study, PDX1 (Figure 4A), NKX6.1 (Figure 4B), MAFA (Figure 4C), and SLC2 C-pe in ALI cell clusters co-expressing A1 (Fig. 4D) and UCN3 (Fig. 4E). For each of Figures 4A-4E, IF staining was performed in a single channel. As shown, the C-peptide is shown in the top row and the target mature protein is shown in the bottom row. Human islet staining in the left row, no ALK5, low T3, ZM, H, NAC, AZT / DEZA ,FI,BLAR001 conditioning in the middle row,no ALK5,low T3,ZM,H,NAC , AZT / DEZA, FI, BLAR004 conditions are shown in the right row. Even though most C-peptide-positive cells were PDX1-positive, as seen in human islets (Figure 4A). The C-peptide-positive cells were simultaneously positive for NKX6.1 and NKX6.1 (Fig. 4B). The rest of the data was analyzed using MAFA( 4C), SLC2A1 (Fig. 4D), and UCN3 (Fig. 4E). However, the expression of MAFA, SLC2A1, and UCN3 was significantly increased in the presence of C-peptide. was not restricted to time-positive cells.

[0189] Figures 5A-5E show the specific "ALK5 absent, low T3, ZM, H, N" tumors specific to stage 7. AC, AZT, DEZA, FI, BLAR001" conditioning by the air-liquid interface (" exhibit glucose-dependent mitochondrial respiratory kinetics similar to that of human islets in ALI (also known as AL1). Figure 1 shows the production of C-peptide in S7D7 cells, as expressed by the oxidative consumption rate ("OCR"). Mitochondrial respiration or activity measured at baseline and after 20 mM D-glucose injection Five OCR measurements over a 72-minute period were performed after injection of 20 mM D-glucose. The measurements were normalized by the DNA content of each individual sample. Figure 5A shows the OCR at 1 post-injection ("ip") and plotted as a percentage of the OCR over the 1 ip. demonstrated by a 123.3% ± 12.92% increase over baseline at 5 minutes ("minutes"). As shown, human islets (black circle line) responded rapidly to high D-glucose, resulting in a high OC over time. R (131.5% ± 11.32; 72 min after injection) was maintained. - S6D7 ALI cluster enriched for peptide-positive cells (grey triangular line) lacked a rapid OCR response to high D-glucose (104.4% ± 3.37; note 15 min after injection), exhibiting a relatively weak OCR response over time (113.3% ± 4.51; Figure 5D shows that ALK was significantly increased in the S7D7 ALI cluster. 5 None, low T3, ZM, H, NAC, AZT / DEZA, FI, BLAR001 conditions (gray Only the line with colored squares) exhibited glucose-dependent mitochondrial respiration kinetics similar to that of human islets ( 112.4% ± 3.25 min after injection; 129.5% ± 3.78 min after injection) The following S7D7 ALI cluster conditions (black squares) were observed: The square lines) show glucose-dependent phenotypes indistinguishable from immature S6D7 ALI clusters. Mitochondrial dynamics were observed: ALK5, T3, ZM, H, NAC, FI , BLAR001 (100.3% ± 4.04-15 minutes after injection; 107.8% ± 6.51- 72 min after injection) (Figure 5B); no ALK5, low T3, ZM, H, NAC, FI, BLAR 001 (96.9% ± 3.06 - 15 minutes after injection; 109.0% ± 4.58 - 72 minutes after injection ) (Figure 5C); no ALK5, low T3, ZM, H, NAC, FI, BLAR004 (10 3.4% ± 4.76 - 15 min after injection; 113.6% ± 6.72 - 72 min after injection) (Figure 5E ); and no ALK5, low T3, ZM, H, NAC, AZT / DEZA, FI, BLAR 004 (102.1%±4.04-15 minutes after injection; 112.7%±3.38-72 after injection minutes) (Figure 5F).

[0190] In summary, this example demonstrates the effectiveness of incorporating improvements in Stage 7 conditioning in ALI. We demonstrate that the maturation status of hESC-derived mature beta cells is enhanced by the addition of This example demonstrates that the β-cells co-express multiple beta cell maturation markers and C-peptide A cells exhibit beta cell-specific functions similar to those of human pancreatic islets, such as respiration. Shows generation in LI.

[0191] Example 3 Robust pancreatic endoderm cells and embryos in a cell cluster format suitable for scalable suspension culture Mature beta cell generation The following examples illustrate the use of Aggrewell™ cell classes suitable for scalable suspension culture. Demonstrate the generation of either robust pancreatic endoderm cells or immature beta cells in a pancreatic blastoma model The suspension culture used in this example was Aggrewel grown in spinner flasks. Aggrewell™ cluster ("Aggrewell™ cluster in spinner") Cells of the human embryonic stem cell line H1 ("H1-hESC") in EZ8 medium at passage 28 1:1 Dulbecco's Modified Eagle's Medium Nutrient Mixture F-12 ("DMEM-F12") 00 dilution ("1x concentration") of GlutaMAX™, 0.25 mM ascorbic acid, 100ng / mL fibroblast growth factor 2 ("FGF2"), 1ng / mL transforming growth factor transforming growth factor beta ("TGFβ"), insulin-transferrin-selenium at a 1:100 dilution -ethanolamine ("ITS-X"), 2% fatty acid-free bovine serum albumin ("FAF") -BSA"), and 20 ng / mL insulin-like growth factor-1 ("IGF-1"); 1:30 in medium supplemented with 10 μM Rock inhibitor Y-27632 (“Y compound”) Dilute 0.094 x 10 onto a MATRIGEL™ coated dish. 6 cell / cm 2 Y compounds were added only during the first 24 hours after seeding. 48 hours after seeding, the cultures were transferred to incomplete PBS (containing no magnesium or calcium). The mice were washed in phosphate-buffered saline (denoted as "- / -" meaning no phosphate buffered saline).

[0192] For Figures 6A-6L, cultures were differentiated using the following protocol. S6D6 ALI cell clusters shown in Figure 1 were cultured at day 3 of stage 4 in Aggrewe. II™ cell clusters were grown on fresh S4D3 monolayers or cryopreserved S4D3 cells. (described in more detail below) for an additional 48 hours (Day 5 of Stage 4, The cells were cultured as described above, except that they were prepared by culturing the cells in a 500-well plate (i.e., S4D5). The medium was changed daily throughout the differentiation protocol. a. Stage 1 (3 days): Cells were cultured in the following Stage 1 medium: 2.7g / 10 Contains 00 mL sodium bicarbonate, 0.5% FAF-BSA, 1:100 dilution (1 GlutaMAX™ (4x concentration) to obtain a 10 mM D-glucose concentration 0.5 mM D-glucose, 100 ng / mL growth differentiation factor 8 ("GDF8"), and 1 0.5 μM 14-prop-2-en-1-yl-3,5,7,14,17,23,27- Heptaazatetracyclo[19.3.1.1~2,6~.1~8,12~]heptacosa- 1(25),2(27),3,5,8(26),9,11,21,23-nonane-16 MCDB-131 medium supplemented with -one ("MCX compound") (GSK-3β inhibitor) The medium was then cultured for 1 day in a 0.5% sodium bicarbonate solution containing 2.7 g / 1000 mL of sodium bicarbonate. FAF-BSA, 1x GlutaMAX™, 10mM D-glucose 4.5 mM D-glucose, 100 ng / mL GDF8, and 0.1 Cells were cultured for an additional day in MCDB-131 medium supplemented with μM MCX compound. Next, 0.5% FAF-BSA containing 2.7 g / 1000 mL sodium bicarbonate, 4. To obtain a 1x concentration of GlutaMAX™ and a 10 mM concentration of D-glucose. MCDB-13 supplemented with 5 mM D-glucose and 100 ng / mL GDF8 The cells were cultured in 1 for an additional day. b. Stage 2 (2 days): Contains 2.7 g / 1000 mL sodium bicarbonate and 0. 5% FAF-BSA, 1x GlutaMAX™, 10 mM D-glucose 4.5 mM D-glucose, 0.25 mM ascorbic acid, and 50 ng Cells were cultured in MCDB-131 medium supplemented with 1 / mL fibroblast growth factor 7 ("FGF7"). The cells were treated for 2 days. c. Stage 3 (2 days): Contains 3.6 g / 1000 mL sodium bicarbonate and 1: 200% dilution of ITS-X; 4.5 mM D-glucose to obtain a concentration of 10 mM Glucose; 1x GlutaMAX™; 1% FAF-BSA; 25 ng / mL FGF7, 0.25 μM SANT-1 (N-[(3,5-dimethyl-1-phenyl-1 H-pyrazol-4-yl)methylene]-4-(phenylmethyl)-1-piperazine amine 1 μM retinoic acid (“RA”) (Sigma Aldrich, Cat. No. R 2625); 0.25 mM ascorbic acid, 300 nM PKC activator (2S,5S- (E,E)-8-(5-(4-trifluoromethyl)phenyl-2,4-pentadieno (amino)benzolactam ("TPB"); and bone morphogenetic protein ("BMP") BLA supplemented with the receptor inhibitor LDN-193189-HCl (“LDN-HCl”) Cells were treated with R001 custom medium (see Table I) for 2 days. The concentration of LDN-HCl used on day 1 of stage 3 was 100 nM, and on day 2 of stage 3 For the eye it was 10 nM. d. Stage 4 (3 days): 3.6 g / 1000 mL sodium bicarbonate containing 1: 200% dilution of ITS-X; 4.5 mM D-glucose to obtain a concentration of 10 mM Glucose; 1x GlutaMAX™; 1% FAF-BSA; 0.25 μL M SANT-1, 50nM RA; 2ng / mL FGF7, 70nM LDN-HC l; BLAR001 supplemented with 0.25 mM ascorbic acid; and 200 nM TPB The cells were treated with the medium for 3 days. e. Cryopreservation of stage 4 day 3 monolayer: Cryopreserved monolayer from S4D3 monolayer A cell bank was established by the following procedure. Briefly, S4D3 monolayers were grown at 10 μM The cells were treated with Y compound for 4 hours. Then, the cells were treated as a single cell suspension with TrypLE™ E After release by the express enzyme, the "release" medium (4 kU / mL DNase I and 10 μM Y compound) The enzyme was neutralized by PBS. Single cells were pelleted and resuspended in cold "release" medium. Counting was performed using a ocounter® NC-100. 0% KSR;15% BLAR001;5% HEPES(1M concentration);20% DM SO) was added in a 1:1 ratio to the single cell suspension in cold "release" medium. 5.0 x 10 in 5 mL of 1:1 "cryopreservation / release" medium 6 Cells frozen and stored in one 5mL tube The vial(s) were placed in a CRF (Controlled Rate Freezer). Freezer) (Planar PLC, Cat. No. Kryo 360) and Cells were frozen at 100°C using the following freezing profile and stored long-term in liquid nitrogen: CR The freezing procedure is shown in Table VII. f. Thawing of cryopreserved S4D3 monolayer cells: 5.0 x 10 6 S4D3 monolayer thin films A frozen 5 mL vial containing cells was thawed in a 37°C water bath for 2 minutes. The cells were collected in the following medium (detailed below in section "g") and stored in an Aggrewell™ 40 Cells were added at a density of approximately 787 cells per Aggrewell™ well of the 0EX plate. Ta. g. Stage 4 (2 days) for Aggrewell™ Cluster Migration: Ag For grownell™ cluster generation, stage 4 day 3 monolayer cells or Thawed stage 4 day 3 cryopreserved cells were treated with Y compound for 4 hours and then rinsed with PBS. After treating with Accutase cell detachment solution for 3 minutes, the enzyme was removed and the flask was gently The cells were removed from the MATRIGEL™ surface by tapping. The resulting cell suspension was plated onto an Aggrewell™ 400EX plate. ll™ at a density of approximately 787 cells per well and gently mix the plate at 100 x g. Aggrewell™ clusters were generated by settling the cells in a 100% aqueous solution. The amount of cells can range from 50 to 3,000 cells. Transfer the layered cells to Aggrewell™ clusters and use them for 48 hours of aggregation followed by culture. The culture medium ("post-S4D3 medium") was as follows: cells were cultured at 3.6 g / 1000 mL sodium bicarbonate, 1:200 dilution of ITS-X; 10 mM D-glucose 4.5 mM D-glucose to obtain a course; 1x GlutaMAX™ ;1% FAF-BSA;0.25μM SANT-1, 50nM RA;2ng / mL FGF7, 70 nM LDN-HCl; 0.25 mM ascorbic acid; and 200 nM The cells were treated with BLAR001 medium supplemented with TPB for 2 days. 2 μg / mL human recombinant laminin was added to the medium only during the first 24 hours. The Aggrewell™ cluster, herein referred to as S4D5, was then The solution was removed from the wells of the ewell™ plate and transferred to a PBS0.1 MAG spinner. ("Aggrewell clusters in suspension"). h. Stage 5 (3 days): S4D5 Aggrewell™ clusters were Cells were harvested from the grownell™ 400EX plates and cultured in Stage 5 medium for 3 days. Cell density: 1.5-2 million cells / mL, rotation speed: 27 rpm (revolutions per minute), PBS0 The mixture was transferred to a 1.1 MAG spinner containing 2.7 g / 1000 mL sodium bicarbonate and 1: 200 dilution of ITS-X; 14.5 to achieve a final concentration of 20 mM D-glucose 1 mM D-glucose; 1x GlutaMAX™; 2% FAF-BSA; 10 μL g / mL heparin ("H"); 10 μM ZnSO4; 0.25 μM SANT-1; 50nM RA; 100nM LDN-HCl; 3,3',5-Triiodo-L-threo 1 μM T3 in the form of thiamin sodium salt; and 10 μM 2-(3-(6-methylpiperidin) Lysin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine ("ALK Cells were treated with BLAR001 medium supplemented with ALK5 inhibitor II or ALK5 inhibitor II. 4 kU / mL DNase I and 5 μM Y compound were supplemented only on day 1 of stage 5. Ta. i. Stage 6 (6-8 days): Contains 2.7g / 1000mL sodium bicarbonate and 1:200 dilution of ITS-X to achieve a final concentration of 20 mM D-glucose 14.5 mM D-glucose; 1x GlutaMAX™; 2% FAF-BSA 10 μg / mL heparin ("H"); 10 μM ZnSO4; 100 nM LDN- HCl; 1 μM T3; 10 μM ALK5 inhibitor II), and 100 nM (S,S )-2-[2-(3,5-difluorophenyl)acetylamino]-N-(5-methyl- 6-oxo-6,7-dihydro-5H-benzo[b,d]azepin-7-yl)propionate in BLAR001 medium supplemented with acetamide ("gamma secretase inhibitor "XX"). Aggrewell™ clusters were treated with

[0193] [Table 10]

[0194] Quantification and characterization of differentiated cells: quantification of protein colocalization at different stages For S4D5 Aggrewell™ cluster and S6D6 Aggre Well™ clusters were collected and analyzed by immunofluorescence ("IF"). The characterization procedures and reagents used were as shown in Table VI of Example 2.

[0195] For quantification of gene expression at various stages, Aggrew at day 5 of stage 4 ell™ Cluster and Aggrewell™ Class on Day 6 of Stage 6 The target was collected and published in Nature Biotechnology, (32) 11, 1121. Reverse transcriptase quantitative polymerase chain reaction ("qRT-PCR") was performed as described in 1133. The characterization procedures and reagents used are shown in Table V of Example 2. It was.

[0196] For quantification of colocalization of proteins present, Aggrewel at day 5 of stage 4 l™ Cluster and Stage 6 D6 Aggrewell™ Cluster The cells were collected and analyzed by fluorescence activated flow cytometry ("FACS"). Staining is Nature Biotechnology, 2014(32)11,1121 1133 and using the antibodies listed in Table VII. The cells were incubated in TrypLE™ Express for 5-10 minutes at 37°C. After incubation and release into a single cell suspension, the cells were buffered with PBS containing 0.2% BSA. The cells were washed twice with buffer. Intracellular antibody staining was performed using LIVE / DEAD purple fluorescent reactive dye at 4°C. Fixation of the cells was achieved by applying PBS for 30 minutes followed by one wash with cold PBS. 0 μL of Cytofix / Cytoperm buffer followed by Perm / Wash buffer. The cells were then washed twice in buffer. The cells were then incubated with the appropriate antibody for 30 min at 4°C (unbound antibody) or After incubation for 1 hour (bound antibody), the cells were washed twice, and then the acquired events were analyzed. At least 30,000 cells were collected using BD FACS Diva software. Analysis was performed using a CS Canto II. During FACS analysis, non-viable cells were excluded and gated. was determined using an isotype antibody ("IgG").

[0197] [Table 11]

[0198] Figure 6A shows the results of (i) growing fresh S4D3 monolayers or S4D3 cryopreserved cells; (ii) by the Aggrewell™ method; (iii) by assembling into cell clusters; Figure 6B depicts the procedure for assembling an S4D5 Aggrewell™ cluster. Among them, the pancreatic endoderm TFs PDX1 (upper left) and NKX6.1 (upper center) showed high transcriptional activity. Protein presence was detected, along with the endocrine TF NEUROD1 (top row, right), and alternative non-pancreatic endoderm. Proteins low in the lineage-allocated TFs SOX2 (bottom, center) and CDX2 (bottom, right) FACS analysis (Figure 6C) showed that the presence of 99.3 ± 0.1% of the cells % PDX1 + (Left), and 84.4±0.1% was NKX6.1 + (Center) , 2.2±0.4% were NKX6.1 + NEUROD1 + (right), or 1.35±0.5 5% are NKX6.1 + CHGA + (Center). Figure 6D shows Compared with the 3 monolayer, the PDX1 (top row, left) and NKX6.1 (top row, right) genes were significantly higher. Gene expression and low expression of NEUROD1 (lower left) and CHGA (lower right) were maintained. The robust pancreatic endoderm characteristics were maintained in S4D4 Agg cells derived from S4D3 cryopreserved cells. Overall, the S4D5 Ag was maintained in the rewell™ cluster. Grewell™ clusters are described in Nature Biotechnology, 2 Compared to the stage 4 cells reported in 014(32)11,1121~1133 These results suggest that the cells exhibited a greater degree of non-endocrine pancreatic endoderm characteristics than did the cells of the Aggrewell Cluster™ is a scalable platform for the development of suspension culture-based differentiation towards beta cells. It is important because it provides a starting point.

[0199] Figure 6E shows that (i) fresh S4D3 monolayers or S4D3 cryopreserved cells (ii) assembly into cell clusters by the Aggrewell™ method; ii) S6D6 in suspension culture, and (iv) differentiation into immature beta cells. FACS analysis of S6D6 Aggrewell™ clusters revealed that Mature beta cell protein profile showed that 78.5±1.87% of cells were NKX6.1 + CHGA + (left, Figure 6F), and 73.6 ± 4.34% were NKX6.1 + NEU ROD1 + (Right, Figure 6F), 38.4 ± 5.96% were NKX6.1 + insulin + (left, The insulin-positive population (50.2 ± 6.92% of the total cells) was insulin-positive (Figure 6G). Phosphorus + The majority of the NKX6.1 + (left, Fig. 6G) and glucagon-positive (7 0.43±1.49% insulin + Glucagon + (right, Figure 6G) was not. 6H was identified by IF in S6D6 as a C-peptide in the Aggrewell™ cluster. The majority of PDX1-positive cells + (Left) and NKX6.1 + (Center) and Surprisingly, the maturation gatekeeper, TF MAFA (right), is present. was already easily detected in S6D6, and the ALI clusters so far in S6D6-S6D7 It was expressed at a higher level (Nature Biotechnology, 2014( 32)11,1121-1133) (Figure 6I; also Figure 3C for ALI S7D7 comparison) Similarly, insulin (Figure 6J), PDX1 (Figure 6K), and NKX6. The expression of 1 (Fig. 6L) was higher at S6D6–S6D7 than in previous ALI clusters. These results demonstrate that the S4D5 Aggrewell™ cluster is an immature base pair. These cells can be cultured in suspension culture towards a beta cell state and then transformed into functional mature beta cells. It has been proven to provide a scalable and ready starting point for culture-based differentiation. do.

[0200] In summary, this example demonstrates that stage 4 cells do not maintain their pancreatic endoderm phenotype. We demonstrate that these cells can be aggregated to form cell clusters. Stage 4 cell clusters are grown in expandable suspension culture to generate robust immature beta cell populations. They can further differentiate towards the present form (stage 6).

[0201] Example 4 Enhanced beta cell maturation marker expression and protein presence similar to mature human islets 1. Generation of Suspension Cultures of Endocrine Cells with The following examples show the maturation markers PDX1, NKX6.1, MAFA, UCN3, Co-expression of SLC2A1 and C-peptide protein by suspension culture of cells The H1-hESC cell line was cultured in EZ8 medium at passage 28. DMEM-F12 supplemented with 10 μM Y compound, diluted 1:100 ("1x concentration") GlutaMAX™, 0.25 mM ascorbic acid, 100 ng / mL FGF 2, 1 ng / mL TGFβ, 1:100 dilution of ITS-X, 2% FAF-BSA, and and 20 ng / mL IGF-1 in MATRIGEL™ medium at a 1:30 dilution On the coated dish, 0.094 x 10 6 cells / cm 2 were seeded as single cells at Y compounds were added only during the first 24 hours after seeding. 48 hours after seeding, the cultures were Washed in PBS(- / -).

[0202] For Figures 7A-7M, cultures were differentiated using the following protocol. During stages 1–4, cultures were maintained on planar adherent cultures. S4D3 monolayers or cryopreserved cultures were used. Aggrewell™ cell clusters were prepared starting from cultured S4D3 cells. The cells were cultured for an additional 48 hours (stage 4, day 5, or S4D5). During stages 5, 6, and 7, Aggrewell ( (trademark) clusters, except that stage 6 occurs over 7 days compared to 6 days , and cultivated in suspension culture as described in Example 3. a. Stage 1 (3 days): Cells were cultured in the following Stage 1 medium: 2.7g / 10 Contains 00 mL sodium bicarbonate, 0.5% FAF-BSA, 1:100 dilution (1 GlutaMAX™ (4x concentration) to obtain a 10 mM D-glucose concentration 0.5 mM D-glucose, 100 ng / mL GDF8, and 1.5 μM MCX compound The cells were cultured for one day in MCDB-131 medium supplemented with 2.7g / 1000mL of PEG-400. Contains sodium bicarbonate, 0.5% FAF-BSA, 1x GlutaMAX ( trademark), 4.5 mM D-glucose to obtain a concentration of 10 mM D-glucose, 10 MCDB- supplemented with 0 ng / mL GDF8 and 0.1 μM GSK-3β inhibitor The cells were cultured for another day in 131 medium. Then, 2.7 g / 1000 mL sodium bicarbonate was added. 0.5% FAF-BSA, 1x GlutaMAX™, 4.5 mM D-glucose to obtain 10 mM D-glucose, and 100 ng / Cells were cultured for an additional day in MCDB-131 supplemented with mL GDF8. b. Stage 2 (2 days): Contains 2.7 g / 1000 mL sodium bicarbonate and 0. 5% FAF-BSA, 1x GlutaMAX™, 10 mM D-glucose 4.5 mM D-glucose, 0.25 mM ascorbic acid, and 50 ng Cells were treated with MCDB-131 medium supplemented with 1 / mL FGF7 for 2 days. c. Stage 3 (2 days): Contains 3.6 g / 1000 mL sodium bicarbonate and 1: 200% dilution of ITS-X; 4.5 mM D-glucose to obtain a concentration of 10 mM Glucose; 1x GlutaMAX™; 1% FAF-BSA; 25 ng / mL FGF7, 0.25 μM SANT-1, 1 μM RA; 0.25 mM ascorbic acid, BLAR001 custom supplemented with 300 nM TPB and LDN-HCl for 2 days The cells were treated with 100% LDN-HCl for 2 days. The concentration of LDN-HCl used on day 1 of stage 3 was 100 nM and for day 2 of stage 3 it was 10 nM. d. Stage 4 (3 days): 3.6 g / 1000 mL sodium bicarbonate containing 1: 200% dilution of ITS-X; 4.5 mM D-glucose to obtain a concentration of 10 mM Glucose; 1x GlutaMAX™; 1% FAF-BSA; 0.25 μL M SANT-1, 50nM RA; 2ng / mL FGF7, 70nM LDN-HC l; BLAR001 supplemented with 0.25 mM ascorbic acid; and 200 nM TPB The cells were treated with the medium for 3 days. Cryopreservation of stage 4 day 3 monolayers: Cryopreserved cells from S4D3 monolayers The bank was established according to the procedures outlined in Example 3 and Table VII. e. Thawing of cryopreserved S4D3 monolayer cells: 5.0 x 10 6 S4D3 monolayer thin films Frozen 5 mL vials containing vesicles were thawed as described in Example 3. f. Stage 4 (2 days) for Aggrewell™ Cluster Migration: Ag Greweell™ clusters were cultured at stage 4, day 3, as described in Example 3. were generated from monolayer cells or thawed cryopreserved stage 4 day 3 cells. g. Stage 5 (3 days): S4D5 Aggrewell™ cluster Cells were harvested from the grownell™ 400EX plates and grown in Stage 5 medium for 3 days. The cells were cultured in PBS 0.5% CO₂ at a cell density of 1.5–2 million cells / mL and at a rotation speed of 27 rpm (revolutions per minute). Transferred to a 1MAG spinner. Containing 2.7g / 1000mL sodium bicarbonate, 1:2 00 dilution of ITS-X; 14.5mM to achieve a final concentration of 20mM D-glucose M D-glucose; 1x GlutaMAX™; 2% FAF-BSA; 10 μg / mL heparin ("H"); 10 μM ZnSO4; 0.25 μM SANT-1; 5 0 nM RA; 100 nM LDN-HCl; 1 μM T3; and 10 μM ALK5 inhibitor Cells were treated with BLAR001 medium supplemented with cytotoxic agent II. 4 kU / mL DNas eI and 5 μM Y compound were supplemented on day 1 of stage 5 only. h. Stage 6 (7 days): 2.7 g / 1000 mL sodium bicarbonate containing 1: 200 dilution of ITS-X; 14.5 to achieve a final concentration of 20 mM D-glucose 1 mM D-glucose; 1x GlutaMAX™; 2% FAF-BSA; 10 μL g / mL heparin ("H"); 10 μM ZnSO4; 100 nM LDN-HCl; 1 μM T3; 10 μM ALK5 inhibitor II, and 100 nM gamma secretase inhibitor Aggrewell™ clusters were grown in BLAR001 medium supplemented with antimicrobial agent XX. Processed. i. Stage 7 (6-7 days): Contains 2.7g / 1000mL sodium bicarbonate 1:200 dilution of ITS-X; 1x GlutaMAX™; 2% FAF-BS A; 10 μg / mL heparin ("H"), 10 nM T3 ("low T3"); 1 mM N AC; 0.5 μM ZM"); and the following components comprising Formulation I ("FI"): RPMI vitamin supplement at a 1:200 dilution; MEM non-essential amino acid supplement at a 1:200 dilution agent; 1:2000 dilution of chemically defined lipid concentrate; 1:200 dilution of sodium pyruvate; BLAR0 supplemented with trace element A at a 1:2000 dilution; trace element B at a 1:2000 dilution Aggrewell™ clusters in either 01 or BLAR004 medium The treatment was continued for 7 days. Additional components added during stage 7 included 4 μM AZT, or 1 μM AZT. For clarity, Aggrewel in suspension contained either 1 μM DEZA or 2 μM DEZA. The I™ cluster was grown at the concentrations described above (BLAR001-based medium or BLAR004 Two conditions were used: (i) no ALK5, low T3, ZM , H, NAC; (ii) without ALK5, low T3, ZM, H, NAC, AZT, DEZA Cultured during stage 7.

[0203] For stages 5, 6, and 7, cell cultures were grown in S4D5 Aggrewell (commercially available). By starting with either a target cluster or an S4D3 transition ALI cluster, The S4D5 Aggrewell™ cluster was Harvest from a target 400EX plate at a cell density of 1.5 to 2 million cells / mL, and rotate at a speed of 1.5 to 2.5 times faster. The mixture was transferred to a PBS0.1MAG spinner at 27 rpm (revolutions per minute).

[0204] Quantification and characterization of differentiated cells: Stage 7 Aggrewell™ class Human islets and human pancreatic islets were analyzed by IF, FACS, and qRT-P as described in Examples 2 and 3. Characterized by CR.

[0205] Figure 7A shows the results of (i) growing fresh S4D3 monolayers or S4D3 cryopreserved cells; Assemble into Aggrewell™ clusters, (ii & iii) Stage 5 , 6, and 7, and (iv) S7D7 Aggrew This figure illustrates the procedure for generating Aggrewell™ clusters. During stage 7, the AL (trademark) clusters were observed (Figures 6F-6G). No K5, low T3, ZM, H, NAC, DEZA, AZT, FI BLAR001 S7D7 Aggrewell™ clusters cultured with The NKX6 protein profile was maintained (Figures 7B-7C). 89% of cells expressed NKX6. 1 + CHGA + (left, Fig. 7B), 77.7% were NKX6.1 + NEUROD1 + (right , Figure 7B), and 40.8% were NKX6.1 + insulin + (Left, Figure 7C). The ULIN-positive population (46.4% of all cells) + The majority of the genes were NKX6. 1 + (left, Figure 7C) and was glucagon-positive (3.9% insulin-positive). + Glucagon + ;(right , Figure 7C).

[0206] In addition to the baseline beta cell profile, we also measured the maturation marker MAFA (Figure 7 D), UCN3 (Figure 7E), SLC2A1 (Figure 7F), G6PC2 (Figure 7G), insulin The gene expression of ALK5 (Fig. 7H) and NKX6.1 (Fig. 7I) was significantly higher in stage 7 (ALK5-less) tumors. , Low T3, ZM, H, NAC, DEZA, AZT, FI BLAR001 or BLAR0 Human S7D7 Aggrewell™ cluster conditioned with 04 MAFA (Fig. 3C), G2PC2 (Fig. 3F) were at or above the islet level. , insulin (Figure 3H), and maturation markers such as NKX6.1 (Figure 3L) at S7D7. The expression levels of ALK5 were measured using Aggrewell™ (ALK5-free, low T3, ZM, H, N AC, DEZA, AZT, FI (BLAR001 or BLAR004) The second highest level was in the ALI cluster.

[0207] In fact, Figures 7J-7M show that DEZA and AZT are compared with "no ALK5, low T3, ZM, H, By adding BLAR004 to NAC and FI's Stage 7 conditioning, a significant number of non- Lucagon (Figure 7J; bottom row, right) C-peptide cells (in terms of protein abundance at S7D7) co-expressed), PDX1 (Fig. 7J; bottom row, left), NKX6.1 (Fig. 7J; bottom row, center), MAFA (Fig. 7K; lower row, left), UCN3 (Fig. 7K; lower row, center), and SLC2A1 ( Figure 7K; bottom right) demonstrates that IF staining was performed on C-Phe. The peptides are shown as single channels in the upper row. MAFA, SLC2A1, and UC The presence of N3 protein is not restricted to C-peptide positive cells, but is present in at least some of the cell population. Approximately 10% of the genes involved C-peptide, PDX1, NKX6.1, MAFA, SLC2A1, and In contrast, Figures 7L-7M show that the protein is present. In terms of S7D7, a partial aggregation of the maturation marker PDX1 (Fig. 7L; bottom row, left), NK X6.1 (Figure 7L; bottom row, center), MAFA (Figure 7M; bottom row, left), SLC2A1 (Figure 7 M; lower right), but not glucagon (Fig. 7L; lower right) and UCN3 (Figure 7M; bottom, center) BLAR004' specific stage 7 conditioning with low T3, ZM, H, NAC, and FI Aggrewell™ cluster stage 7 "ALK5-free, low BLAR004" conditioning with T3, ZM, H, NAC, DEZA, AZT, and FI. A significant number of mature adult human pancreatic Mature beta cells resembling islets were generated.

[0208] In summary, this example demonstrates that stage 4 cell clusters respond to changes in stage 7 conditioning. demonstrated that the cells could be further differentiated in suspension culture towards functional beta cells by Specifically, Example 4 demonstrates the proper isolation of beta cells from cell clusters in suspension culture. We demonstrate the generation of C-peptide cells that co-express beta cell maturation markers essential for normal function. vinegar.

[0209] Example 5 Glucose-dependent mitochondrial respiration and glucose-stimulated islet function similar to that of mature human islets Suspension culture generation of insulin-secreting endocrine cells The following examples demonstrate the glucose-dependent mitochondrial respiration and glucose regulation in a manner similar to that in human pancreatic islets. Functionally mature beta cells with glucose-stimulated insulin secretion ("GSIS") kinetics This demonstrates production by suspension culture. The culture conditions are the same as in Example 4. EZ8 was produced at 28 passages. Cells of the H1-hESC cell line cultured in DM supplemented with 10 μM Y compound were then cultured in DM. EM-F12, GlutaMAX™ at 1:100 dilution ("1x concentration"), 0.25 mM ascorbic acid, 100ng / mL FGF2, 1ng / mL TGFβ, 1:10 0 dilution of ITS-X, 2% FAF-BSA, 20 ng / mL IGF-1 in medium 0.094 × 10 cells onto a dish coated with ATRIGEL™ at a 1:30 dilution. 6 cells / cm 2 Y compounds were added to the cells as single cells during the first 24 hours after seeding. 48 hours after seeding, cultures were washed in PBS(- / -).

[0210] For Figures 8A-8I, the cultures were differentiated using the following protocol. During stages 1–4, cultures were maintained on planar adherent cultures. S4D3 monolayers or cryopreserved cultures were used. Aggrewell™ cell clusters were prepared starting from cultured S4D3 cells. The cells were cultured for an additional 48 hours (stage 4, day 5, or S4D5). During stages 5, 6, and 7, Aggrewell ( ™) clusters were grown in suspension culture as described in Example 3. Figures 8E-8 The ALI cluster shown in F was cultured as described in Example 2. Briefly, a. Stage 1 (3 days): Cells were cultured in the following Stage 1 medium: 2.7g / 10 Contains 00 mL sodium bicarbonate, 0.5% FAF-BSA, 1:100 dilution (1 GlutaMAX™ (4x concentration) to obtain a 10 mM D-glucose concentration 0.5 mM D-glucose, 100 ng / mL GDF8, and 1.5 μM MCX compound The cells were cultured for one day in MCDB-131 medium supplemented with 2.7g / 1000mL of PEG-400. Contains sodium bicarbonate, 0.5% FAF-BSA, 1x GlutaMAX ( trademark), 4.5 mM D-glucose to obtain a concentration of 10 mM D-glucose, 10 MCDB- supplemented with 0 ng / mL GDF8 and 0.1 μM GSK-3β inhibitor The cells were cultured for another day in 131 medium. Then, 2.7 g / 1000 mL sodium bicarbonate was added. 0.5% FAF-BSA, 1x GlutaMAX™, 4.5 mM D-glucose to obtain 10 mM D-glucose, and 100 ng / Cells were cultured for an additional day in MCDB-131 supplemented with mL GDF8. b. Stage 2 (2 days): Contains 2.7 g / 1000 mL sodium bicarbonate and 0. 5% FAF-BSA, 1x GlutaMAX™, 10 mM D-glucose 4.5 mM D-glucose, 0.25 mM ascorbic acid, and 50 ng Cells were treated with MCDB-131 medium supplemented with 1 / mL FGF7 for 2 days. c. Stage 3 (2 days): Contains 3.6 g / 1000 mL sodium bicarbonate and 1: 200% dilution of ITS-X; 4.5 mM D-glucose to obtain a concentration of 10 mM Glucose; 1x GlutaMAX™; 1% FAF-BSA; 25 ng / mL FGF7, 0.25 μM SANT-1, 1 μM RA; 0.25 mM ascorbic acid, BLAR001 custom supplemented with 300 nM TPB and LDN-HCl for 2 days The cells were treated with 100% LDN-HCl for 2 days. The concentration of LDN-HCl used on day 1 of stage 3 was 100 nM and for day 2 of stage 3 it was 10 nM. d. Stage 4 (3 days): 3.6 g / 1000 mL sodium bicarbonate containing 1: 200% dilution of ITS-X; 4.5 mM D-glucose to obtain a concentration of 10 mM Glucose; 1x GlutaMAX™; 1% FAF-BSA; 0.25 μL M SANT-1, 50nM RA; 2ng / mL FGF7, 70nM LDN-HC l; BLAR001 supplemented with 0.25 mM ascorbic acid; and 200 nM TPB Cells were treated with the medium for 3 days. e. Cryopreservation of stage 4 day 3 monolayer: Cryopreserved monolayer from S4D3 monolayer A cell bank was established using the procedures outlined in Example 3 and Table VII. f. Thawing of cryopreserved S4D3 monolayer cells: 5.0 x 10 6 S4D3 monolayer thin films Frozen 5 mL vials containing cells were thawed as described in Example 3. The same procedure was followed. Applicable for migration of either ALI or Aggrewell™ clusters Cut. g. Aggrewell™ Cluster Migration or S4D3 ALI Cluster Migration Stage 4 (2 days) for: Aggrewell™ clusters were prepared as described in Example 3. Stage 4 day 3 monolayer cells or thawed cryopreserved stage 4 cells were cultured as described. ALI clusters were generated from day 3 cells of the 4. The cells were generated on day 3 of Di4. As in Example 4, for stages 5, 6, and 7, cell cultures were cultured with S4D5 Agg Starting with either a rewell™ cluster or an S4D3 transition ALI cluster The S4D5 Aggrewell™ cluster was conditioned by Rewell™ 400EX plate was harvested and the cell density was 1.5-2 million cells / mL, and transferred to a PBS0.1MAG spinner at a rotation speed of 27 rpm (revolutions per minute). h. Stage 5 (3 days): 2.7 g / 1000 mL sodium bicarbonate containing 1: 200 dilution of ITS-X; 14.5 to achieve a final concentration of 20 mM D-glucose 1 mM D-glucose; 1x GlutaMAX™; 2% FAF-BSA; 10 μL g / mL heparin ("H"); 10 μM ZnSO4; 0.25 μM SANT-1; 50 nM RA; 100 nM LDN-HCl; 1 μM T3; and 10 μM ALK5 BLAR001 medium supplemented with inhibitor II, Aggrewell™ or ALI Clusters were treated for 3 days. 4 kU / mL DNase I and 5 μM Y compound were added to A The ggrewell™ clusters were replenished only on day 1 of stage 5. i. Stage 6 (7 days): 2.7 g / 1000 mL sodium bicarbonate containing 1: 200 dilution of ITS-X; 14.5 to achieve a final concentration of 20 mM D-glucose 1 mM D-glucose; 1x GlutaMAX™; 2% FAF-BSA; 10 μL g / mL heparin ("H"); 10 μM ZnSO4; 100 nM LDN-HCl; 1 μM T3; 10 μM ALK5 inhibitor II, and 100 nM gamma secretase X Aggrewell™ or ALI class 1 in BLAR001 medium supplemented with X. The target was processed. j. Stage 7 (7-14 days): 2.7g / 1000mL sodium bicarbonate 1:200 dilution of ITS-X; 1x GlutaMAX™; 2% FAF-B SA; 10 μg / mL heparin ("H"), 10 nM T3 ("low T3"); 1 mM NAC; 0.5 μM ZM); and the following ingredients comprising Formulation I ("FI"): 1:200 dilution of RPMI vitamin supplement; 1:200 dilution of MEM non-essential amino acids Supplement: 1:2000 dilution of chemically defined lipid concentrate; 1:200 dilution of sodium pyruvate BLA supplemented with trace element A at a 1:2000 dilution; trace element B at a 1:2000 dilution Aggrewell™ class, either R001 medium or BLAR004 medium The mice were treated for 7 days. Additional components added during stage 7 included 4 μM AZT; For clarity, Aggrew in suspension contained either 1 μM DEZA or 1 μM DEZA. ell™ or ALI clusters were grown at the concentrations described above (BLAR001-based medium or Two conditions were used: (i) without ALK5 and (ii) with BLAR004-based medium. , low T3, ZM, H, NAC; (ii) no ALK5, low T3, ZM, H, NAC, AZ T, DEZA during stage 7. Figures 8A, 8B, 8H, and 8I: During stage 7. Aggrewell clusters in suspension were analyzed under two conditions: (i) ALK5 and (ii) S7D, low T3, 0.5 μM ZM, 10 μg / mL H, 1 mM NAC; 1~S7D5-ALK5 no, low T3, 0.5 μM ZM, 10 μg / mL H, 1 mM NAC;5μM AZT;1μM DEZA;S7D6~S7D13 or S7D14- No ALK5, low T3, 0.5 μM ZM, 10 μg / mL H, 1 mM NAC, S The cells were cultured to 7D13 or S7D14.

[0211] Quantification and characterization of differentiated cells: glucose dependence of S6D7 ALI clusters Stage 7 Aggrewell™ cluster for quantification of mitochondrial activity Human islet cells and mature human islet cells were cultured in Seahorse XF as described in Example 2.e 24 days ALI / Aggrewell™ clusters or human pancreatic Islets were harvested and their oxygen consumption rate ("OCR") was measured after an injection of 20 mM D-glucose. Before and after XF e 24 Measured on an Extracellular Flux Analyzer ALI / Aggrewell™ clusters or human islets were analyzed to determine their status. Remove from conditioning on day 7 and maintain a 37 °C non-CO2 environment to achieve baseline OCR. The cells were incubated for 2 hours in both the pre-incubation medium and the designed medium. The medium was XF base medium containing 1 mM D-glucose, 1 mM L-glutamine, and 1 mM After preincubation, ALI / Aggrew The ell™ clusters or human islets were loaded into the Seahorse machine, where The following measurements were taken: (i) baseline OCR three times (before D-glucose injection); i) D-glucose injection was performed 5 times (incubation time after injection: 72 minutes). OCR measurements were normalized to the DNA content of each individual sample. The DNA content was determined by the Aamp DNA MicroKit and analyzed by NanoDro The measurements were carried out by a p 8000 UV-Vis Spectrophotometer.

[0212] For quantification of glucose-dependent insulin secretion, stage 7 ALI cluster, A The Grewell™ clusters and mature human islets were subjected to cell perfusion. usion) system (BioRep Technologies, Miami, Florida) da, Catalog No. PERI4-02). Briefly, insulin secretion was To normalize to the baseline, the ALI / Aggrewell™ class Mouse or human islets were transferred to warm (37°C) Krebs buffer (Table IX). Chamber (BioRep Technologies, Cat. No. PERI-CHA MBER) and the following: (i) 3 mM D-glucose; (ii) 16 0.7 mM D-glucose ± 100 ng / mL exendin-4 ("Ex4"); and ( iii) four supplemented with either 25 mM KCl containing 3 mM D-glucose; The cells were continuously perfused with successive Krebs buffer solutions (flow rate 100 μL / min). The washout protocol is shown in each figure. Perfusate samples were collected every minute to measure C-peptide protein. Protein levels (unit: ng / mL) were measured using C-PEPTIDE ELISA (Mercodia, Uppsala, Sweden, Cat. No. 10-1136-01) .

[0213] [Table 12]

[0214] Figures 8A and 8B show the effect of S7D13 Agg in suspension on 20 mM D-glucose. Figures 8A and 8B show the mitochondrial response of OC Rewell™ clusters. R > 123.3% ± 12.92% of baseline at 15 minutes ("min") post-injection ("ip"). Human pancreatic islets (black circle line) rapidly respond to high D-glucose, as demonstrated by the The subjects responded well and maintained a high OCR (131.5% ± 11.32; 72 min after injection) over time. Conversely, the S6D7 ALI cluster enriched for immature C-peptide positive cells. Star (gray triangular line) lacks a rapid OCR response to high D-glucose (1 04.4% ± 3.37; 15 min after injection), exhibiting a relatively weak OCR response over time (11 3.3% ± 4.5%; 72 min after injection). Glucose-dependent mitochondria similar to those in human pancreatic islets Rear respiratory dynamics in relation to Aggrewell™ clusters in suspension S7D1 Observed in the three "no ALK5, low T3, ZM, H, NAC, FI, BLAR004" conditions (110.7% ± 2.46 - 15 min after injection; 125.9% ± 2.27 - 72 min after injection) ) (gray square line) (Figure 8A). In contrast, "no ALK5, low T3, ZM, H, NA" C, FI BLAR001 or BLAR004' S7D7 ALI cluster in the human pancreas The islet-level glucose-dependent mitochondrial respiration dynamics were not observed (Figures 5C and 5E). In addition, "ALK5-free, low T3, ZM, H, NAC, AZT, DEZA, FI's BLAR004 Aggrewell™ cluster provides high glucose stimulation In response to the α-glucan, the islets consumed oxygen at levels far exceeding those of human islets (162.0% ± 11.5%). 1 - 15 min after injection; 177.1% ± 0.99 - 72 min after injection) (gray square line) (Figure 8 B). DEZA and AZT were administered only during the first 4 days of stage 7 conditioning. Functional mitochondrial response induced by T and DEZA was observed in stage 7 Ag It was shown to be stable for at least 10 days in grownell™ clusters. was suggested.

[0215] Figure 8C shows that mature beta cells in human pancreatic islets respond to glucose stimulation with rapid biphasic induction. This demonstrates that the second biphasic GSIS response exhibits the ability to induce multiple rounds of insulin secretion. The response was slower than the first response (about 7-8 times the first phase of the first GSIS response). In addition, human pancreatic islets have the ability to perform multiple rounds of "on-off" switching of insulin secretion, and and the ability to release large amounts of insulin granules upon membrane depolarization mediated by KCl. All conditions tested demonstrated a strong insulin secretory response to KCl. The addition of exendin-4 ("Ex4") to the human islets shown in Figures 8C-8I. did not increase the magnitude of the GSIS response in ALI or Aggrewell (commercial BLAR001 (Figure 8D) was included for comparison to the GSIS profile. ALK5-free, low T3, ZM, H , NAC, FI" during stage 7 S7D8~S7D10-AL Cluster I showed a single biphasic GSIS response (approximately 4-10 times the first GSIS The first phase of the response), no second biphasic GSIS response, and relatively slow biphasic GSIS Furthermore, the ALI clusters were significantly increased after reperfusion with 3 mM D-glucose after stimulation. In contrast, ALK5-free mice lacked the ability to block the second phase of insulin secretion. Conditioned during stage 7 with BLAR004, low T3, ZM, H, NAC, FI The 7D14 Aggrewell™ cluster induces a potent first biphasic GSIS (approximately 5-fold increase in the first phase of the GSIS response), followed by the ability to completely block GSIS, and The patient presented with a second monophasic response (Fig. 8G). ZM, H, NAC, FI, BLAR004" by adding to conditioning S7D14 Aggrewell™ clusters are biphasic clusters similar to multi-round human pancreatic islets. between GSIS (approximately 5-7 times the first phase of the GSIS response) and the high glucose pulse. ALK demonstrated the ability to completely block GSIS (Fig. 8H-8I). 5 None, Low T3, ZM, H, NAC, DEZA, AZT BLAR004" S7D14 Two biological replicates of Aggrewell™ cluster conditions are shown. In this study, DEZA and AZT were administered only for the first 4 days of stage 7 conditioning. The robust biphasic GSIS response induced by T and DEZA was observed in stage 7 Aggr The results suggest that the compound was stable for at least 10 days in the ewell™ cluster. It was.

[0216] In summary, Example 5 demonstrates glucose-dependent mitochondrial respiration and mitochondrial function similar to that of human pancreatic islets. hESC-derived functional beta cells by cell aggregation and suspension culture with GSIS kinetics Demonstrate generation (stage 7).

[0217] [Table 13-1]

[0218] [Table 13-2]

[0219] [Table 13-3]

[0220] [Table 14-1]

[0221] [Table 14-2]

[0222] [Table 14-3]

[0223] [Table 14-4]

[0224] [Table 14-5]

[0225] [Table 14-6]

[0226] [Table 14-7] ...

Claims

1. Pancreatic endoderm cells were cultured using a culture medium expressing PDX1, NKX6.1, MAFA, UCN3, and SLC2A1. A method for differentiating pancreatic endocrine cells into functional beta cells expressing UNC063 8, UNC0642, UCN0646, TC-E5003, A366, PF038147 35, ZM447439, SB747651A, PFI1, LY303511, MS43 6, AZT, DEZA, pyroxamide, CI9994, or MC1568 The method comprises culturing the cells in a medium supplemented with two or more of:

2. The culture medium may contain heparin, N-acetylcysteine, formulation I, and T3, T4, or 10. The method of claim 1, further supplemented with one or more of its analogs.

3. The method of claim 1 or 2, wherein the culture medium lacks an ALK5 inhibitor.

4. 3. The method of claim 1 or 2, wherein the culture medium is supplemented with an ALK5 inhibitor.

5. The medium contained ZM447439, heparin, N-acetylcysteine, and T3, T4 or one or more of its analogs, wherein the medium contains an ALK5 inhibitor. The method of claim 1 , wherein the method does not have

6. 6. The method of claim 5, wherein the medium is supplemented with T3.

7. The method of claim 6, wherein the medium is supplemented with AZT.

8. The method of claim 7, wherein the medium is supplemented with DEZA.

9. The functional beta cells may be cells that express markers characteristic of definitive endoderm, early intestinal cells, cells expressing markers characteristic of foregut endoderm cells, and cells expressing markers characteristic of foregut endoderm cells. cells, cells expressing markers characteristic of pancreatic endoderm cells, and cells expressing markers characteristic of pancreatic endocrine progenitor cells. cells expressing CAR, cells expressing markers characteristic of immature beta cells, and functional The stepwise differentiation of one or more cells expressing markers characteristic of beta cells. The method according to claim 1, obtained thereby.

10. The method of claim 1, wherein the method comprises culturing the cells at an air-liquid interface. 。

11. 10. The method of claim 1, wherein the method comprises culturing the cells in suspension clusters. Law.

12. Single hormone insulin, PDX1, obtained by in vitro differentiation of pancreatic endocrine cells In vitro populations of functional beta cells expressing NKX6.1, NKX6.1, and MAFA.

13. The population of claim 12, wherein the cells also express UCN3 and SLC2A1.

14. The cells are capable of regulating glucose-stimulated insulin secretion and glucose-dependent mitochondrial respiration.

13. The population of claim 12, exhibiting asthma.

15. The glucose-stimulated insulin secretion and glucose-dependent mitochondrial respiration are 15. The population of claim 14, wherein the population is similar to that of human pancreatic islet cells.

16. UNC0638, UNC0642, UCN0646, TC-E5003, A366, P F03814735, ZM447439, SB747651A, PFI1, LY3035 11, MS436, AZT, DEZA, pyroxamide, CI9994, or MC1568 The present invention relates to a method for producing pancreatic endocrine cells, the method comprising culturing pancreatic endocrine cells in a medium supplemented with one or more of the following: The pancreas expresses the single hormones insulin, PDX1, NKX6.1, and MAFA. In vitro populations of pancreatic beta cells.

17. The medium may contain heparin, N-acetylcysteine, Preparation I, and T3, T4, or its derivatives.

17. The population of claim 16, further supplemented with one or more of the analogs.

18. 18. The population of claim 16 or 17, wherein the culture medium lacks an ALK5 inhibitor.

19. 18. The population of claim 16 or 17, wherein the culture medium is supplemented with T3.

20. The medium contained ZM447439, heparin, N-acetylcysteine, formulation I, and T. and the medium is supplemented with one or more of ALK5 inhibitors.

17. The population of claim 16, which is free of harmful agents.

21. 17. The population of claim 16, wherein the medium is supplemented with T3.

22. 22. The population of claim 21, wherein the medium is supplemented with AZT.

23. 23. The population of claim 22, wherein the medium is supplemented with DEZA.

24. The population of claim 16, wherein the cells also express UCN3 and SLC2A1.

25. The cells are capable of regulating glucose-stimulated insulin secretion and glucose-dependent mitochondrial respiration.

17. The population of claim 16, exhibiting asthma.

26. The glucose-stimulated insulin secretion and glucose-dependent mitochondrial respiration are The population of claim 16, which is similar to that of islet cells.

27. Pluripotent stem cells were cultured to express PDX1, NKX6.1, MAFA, UCN3, and SLC2A.

1. A method for differentiating a beta cell into a functional beta cell that expresses the beta cell, comprising: a. Differentiating pluripotent stem cells into immature beta cells; and b. UNC0638, UNC0642, UCN0646, TC-E5003, A366 , PF03814735, ZM447439, SB747651A, PFI1, LY30 3511, MS436, AZT, DEZA, pyroxamide, CI9994, or MC15 and culturing the immature beta cells in a medium supplemented with one or more of the following: and the immature beta cells are then cultured to express PDX1, NKX6.1, MAFA, UCN3, and differentiating the cells into functional beta cells that express SLC2A1.

28. The functional beta cells contain glucose-dependent mitochondria similar to human pancreatic islet cells.

28. The method of claim 27, comprising:

29. The functional beta cells are capable of glucose-stimulated insulin secretion similar to human pancreatic islet cells.

28. The method of claim 27, wherein the secretion is

30. 28. The method of claim 27, wherein the functional beta cells secrete insulin in multiple phases. How to do it.

31. The culture medium may contain heparin, N-acetylcysteine, formulation I, and T3, T4, or 28. The method of claim 27, further supplemented with one or more of its analogs.

32. 32. The method of claim 27 or 31, wherein the culture medium lacks an ALK5 inhibitor.

33. 32. The method of claim 27 or 31, wherein the culture medium is supplemented with T3.

34. The medium contained ZM447439, heparin, N-acetylcysteine, and T3, T4 or one or more of its analogs, wherein the medium contains an ALK5 inhibitor.

28. The method of claim 27, wherein the

35. 35. The method of claim 34, wherein the medium is supplemented with T3.

36. 36. The method of claim 35, wherein the medium is supplemented with AZT.

37. 37. The method of claim 36, wherein the medium is supplemented with DEZA.

38. 3. The method of claim 2, wherein the method comprises culturing the immature beta cells at an air-liquid interface.

7. The method according to claim 7.

39. 10. The method of claim 1, wherein the method comprises culturing the immature beta cells in suspension clusters.

27. The method according to claim 27.

40. The step of differentiating pluripotent stem cells comprises: a. The pluripotent stem cells are cultured to form cells expressing markers characteristic of definitive endoderm ("stage and differentiating the cells into "1-cell" ("1-cell"). b. The stage 1 cells are classified as cells expressing markers characteristic of early intestinal cells ("stage 1 cells"). and differentiating the cells into "age 2 cells" ("age 2 cells"). c. The stage 2 cells are classified as cells expressing markers characteristic of foregut endoderm cells ("Stage 2 cells"). Differentiation of the cells into stage 3 cells; d. The stage 3 cells are classified as cells expressing markers characteristic of pancreatic endoderm cells ("stage 3 cells"). and differentiating the cells into "age 4 cells" ("age 4 cells"). e. Differentiating the Stage 4 cells into pancreatic endocrine precursor cells ("Stage 5 cells"). The process and f. differentiating the stage 5 cells into immature beta cells.

27. The method according to claim 27.

41. 41. The method of claim 40, wherein steps e. and f. comprise culturing at the air-liquid interface. How to post.

42. 10. The method of claim 9, wherein steps e. and f. comprise culturing the cells in suspension clusters.

40. The method according to claim 40.

43. The method further comprises culturing the pluripotent stem cells in a medium supplemented with an MCX compound and GDF-8. The method of claim 1 further comprises differentiating pluripotent stem cells into stage 1 cells by culturing the pluripotent stem cells.

40. The method according to claim 40.

44. The method comprises culturing the Stage 1 cells in a medium supplemented with FGF7 and ascorbic acid. and differentiating the stage 1 cells into stage 2 cells by culturing the cells.

41. The method of claim 40.

45. The method further comprises treating the stage 2 cells with FGF7, retinoic acid, SANT-1, PKC activity, by culturing in a medium supplemented with an anti-inflammatory agent, a BMP inhibitor, and ascorbic acid.

41. The method of claim 40, comprising differentiating the stage 2 cells into stage 3 cells. Law.

46. The method further comprises treating the stage 3 cells with FGF7, retinoic acid, SANT-1, PKC activity, by culturing in a medium supplemented with an anti-inflammatory agent, a BMP inhibitor, and ascorbic acid.

41. The method of claim 40, comprising differentiating the stage 3 cells into stage 4 cells. Law.

47. The method further comprises treating the stage 4 cells with SANT-1, a PKC activator, a BMP inhibitor, and The stage 4 cells were cultured in medium supplemented with ascorbic acid.

41. The method of claim 40, comprising differentiating the cells into Age 5 cells.

48. The medium is further supplemented with one or more of T3, T4, or analogs thereof.

48. The method of claim 47.

49. The method further comprises treating the stage 5 cells with a BMP inhibitor, ascorbic acid, T3, T4, or by culturing in a medium supplemented with one or more of the analogs thereof.

41. The method of claim 40, comprising culturing stage 5 cells into immature beta cells.

50. 5. The method of claim 4, wherein the medium is further supplemented with an ALK5 inhibitor or a gamma secretase inhibitor.

9. The method according to claim 9.

51. The method further comprises depriving the immature beta cells of an ALK5 inhibitor, and administering ZM447439. AZT, N-acetylcysteine, DEZA, Formulation I, and T3, T4, or analogs thereof The immature base is cultured in a medium supplemented with one or more of the following: T cells expressing PDX1, NKX6.1, MAFA, UCN3, and SLC2A 28. The method of claim 27, comprising differentiating into functional beta cells.

52. The oxygen consumption rate response of the glucose-dependent mitochondrial respiration after glucose stimulation is Claims 14, 25, and 28, which are in the range of about 20% to about 80% above the basal oxygen consumption rate The method described below.

53. 53. The method of claim 52, wherein the oxygen consumption rate response occurs at least 15 minutes after glucose stimulation. The method described below.

54. The glucose-stimulated insulin secretion is a rapid, two-phase insulin secretion in response to glucose stimulation.

30. The method of claims 14, 25, and 28, comprising phosphorus secretion.

55. The first phase of the biphasic insulin secretion is increased by at least 4 to at least 8 times the basal secretion. and the second phase is increased by at least two to at least four times the basal secretion. The method described below.

56. the insulin secretion occurs at least 5 minutes to at least 10 minutes after the glucose stimulation The method of claim 54, wherein

57. The functional beta cells have glucose-dependent mitochondria similar to human pancreatic islet cells 52. The method of claim 51, comprising breathing.

58. The functional beta cells have glucose-stimulated insulin secretion similar to pancreatic islet cells. The method of claim 51 .

59. The oxygen consumption rate response of the glucose-dependent mitochondrial respiration after glucose stimulation is 58. The method of claim 57, wherein the oxygen consumption rate is in the range of about 20% to about 80% above the basal oxygen consumption rate.

60. 59. The method of claim 59, wherein the oxygen consumption rate response occurs at least 15 minutes after glucose stimulation. The method described below.

61. The glucose-stimulated insulin secretion is a rapid biphasic insulin secretion in response to glucose stimulation.

59. The method of claim 58, comprising secretion of sulin.

62. The first phase of the biphasic insulin secretion is increased by at least 4 to at least 8 times the basal secretion. and the second phase is increased by at least two to at least four times the basal secretion. The method described below.

63. the insulin secretion occurs at least 5 minutes to at least 10 minutes after the glucose stimulation The method of claim 61, wherein

64. 41. The method of claim 40, wherein the stage 4 cells are cryopreserved.

65. 41. The method of claim 40, wherein step e comprises culturing stage 4 cryopreserved cells. Law.

66. The T3 of claims 6, 21, 35, and 51 is in the range of 1 nM to 100 nM. How to do it.

67. Claims 48 to 5, wherein the medium is supplemented with T3, and the T3 is in the range of 1 nM to 1 μM. The method according to claim 0.

68. Pluripotent stem cells were cultured to express PDX1, NKX6.1, MAFA, UCN3, and SLC2A.

1. A method for differentiating a beta cell into a functional beta cell that expresses the beta cell, comprising: a. Incubating the pluripotent stem cells in a culture medium supplemented with activin A and WNT3A. Cells expressing markers characteristic of definitive endoderm obtained by culturing in a medium containing Differentiate into b. Differentiating the cells expressing markers characteristic of the definitive endoderm into immature beta cells and c. UNC0638, UNC0642, UCN0646, TC-E5003, A366 , PF03814735, ZM447439, SB747651A, PFI1, LY30 3511, MS436, AZT, DEZA, pyroxamide, CI9994, or MC15 and culturing the immature beta cells in a medium supplemented with one or more of the following: and the immature beta cells are then cultured to express PDX1, NKX6.1, MAFA, UCN3, and differentiating the cells into functional beta cells that express SLC2A1.

69. The functional beta cells have glucose-dependent mitochondria similar to human pancreatic islet cells 69. The method of claim 68, comprising breathing.

70. The functional beta cells resemble human pancreatic islet cells in glucose-stimulated insulin secretion 69. The method of claim 68, comprising:

71. 69. The method of claim 68, wherein the functional beta cells secrete insulin in multiple phases. How to do it.

72. The culture medium may contain heparin, N-acetylcysteine, formulation I, and T3, T4, or 69. The method of claim 68, wherein one or more of said analogs are further replenished.

73. 73. The method of claim 68 or 72, wherein the culture medium lacks an ALK5 inhibitor.

74. 73. The method of claim 68 or 72, wherein the culture medium is supplemented with T3.

75. The medium contained ZM447439, heparin, N-acetylcysteine, and T3, T4 or an analog thereof, and the medium is supplemented with an ALK5 inhibitor 69. The method of claim 68, wherein the method does not contain

76. 76. The method of claim 75, wherein the medium is supplemented with T3.

77. 77. The method of claim 76, wherein the medium is supplemented with AZT.

78. 78. The method of claim 77, wherein the medium is supplemented with DEZA.

79. The method comprises: at the air-liquid interface, in suspended clusters, in a roller bottle; or 69. The method of claim 68, comprising culturing the immature beta cells on microcarriers. method.

80. 7. The method of claim 6, wherein the method comprises culturing the immature beta cells in a roller bottle.

8. The method according to claim 8.

81. The method comprises culturing the immature beta cells in roller bottles on microcarriers.

81. The method of claim 80, comprising:

82. The step of differentiating pluripotent stem cells comprises: a. Culturing the pluripotent stem cells in a medium supplemented with activin A and WNT3A; By this, the pluripotent stem cells are differentiated into cells expressing markers characteristic of the definitive endoderm (" differentiating the cells into "stage 1 cells"); b. The stage 1 cells are classified as cells expressing markers characteristic of early intestinal cells ("stage 1 cells"). and differentiating the cells into "age 2 cells" ("age 2 cells"). c. The stage 2 cells are classified as cells expressing markers characteristic of foregut endoderm cells ("Stage 2 cells"). Differentiation of the cells into stage 3 cells; d. The stage 3 cells are classified as cells expressing markers characteristic of pancreatic endoderm cells ("stage 3 cells"). and differentiating the cells into "age 4 cells" ("age 4 cells"). e. Differentiating the stage 4 cells into pancreatic endocrine precursor cells ("stage 5 cells"). and f. differentiating the stage 5 cells into immature beta cells.

68. The method according to claim 68.

83. Steps e. and f. are performed by using a roller in the suspended cluster at the air-liquid interface.

83. The method of claim 82, comprising culturing in bottles or on microcarriers.

84. 9. The method of claim 8, wherein steps e. and f. comprise culturing the cells in roller bottles.

2. The method according to claim 2.

85. Steps e. and f. are performed by culturing the immature beta cells in a roller bottle on microcarriers.

83. The method of claim 82, comprising culturing the cells.

86. The method comprises culturing the Stage 1 cells in a medium supplemented with FGF7 and ascorbic acid. and differentiating the stage 1 cells into stage 2 cells by culturing the cells.

83. The method of claim 82.

87. The method further comprises treating the stage 2 cells with FGF7, retinoic acid, SANT-1, PKC activity, The stage 2 strain was cultured in a medium supplemented with an antimicrobial agent and ascorbic acid.

83. The method of claim 82, comprising differentiating the cells into stage 3 cells.

88. 88. The method of claim 87, wherein the medium lacks a BMP inhibitor.

89. The method further comprises treating the stage 3 cells with FGF7, retinoic acid, SANT-1, PKC activity, The stage 3 strain was cultured in a medium supplemented with an antimicrobial agent and ascorbic acid.

83. The method of claim 82, comprising differentiating the cells into stage 4 cells.

90. 90. The method of claim 89, wherein the medium lacks a BMP inhibitor.

91. The method further comprises treating the stage 4 cells with SANT-1, a PKC activator, a BMP inhibitor, and The stage 4 cells were cultured in medium supplemented with ascorbic acid.

83. The method of claim 82, comprising differentiating the cells into a Stage 5 cell.

92. The medium is further supplemented with one or more of T3, T4, or analogs thereof.

92. The method of claim 91 .

93. The method further comprises treating the stage 5 cells with a BMP inhibitor, ascorbic acid, T3, T4, or by culturing in a medium supplemented with one or more of the analogs thereof.

83. The method of claim 82, comprising culturing stage 5 cells into immature beta cells.

94. 10. The method of claim 9, wherein the medium is further supplemented with an ALK5 inhibitor or a gamma secretase inhibitor.

3. The method according to claim 3.

95. The method further comprises treating the immature beta cells with an ALK5 inhibitor and administering ZM447439. , AZT, N-acetylcysteine, DEZA, Preparation I, and T3, T4, or the like The immature vectors are cultured in a medium supplemented with one or more of the following: The cells were cultured to express PDX1, NKX6.1, MAFA, UCN3, and SLC2A.

69. The method of claim 68, comprising differentiating the cells into functional beta cells.

96. 28. The method of claim 27, wherein the pluripotent stem cells are human H1 or H9 cells.

97. 69. The method of claim 68, wherein the pluripotent stem cells are human CyT49 cells.

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