Microenvironmental control improves differentiation of stem cell derived islets

Abstract

Among the various aspects of the present disclosure is the provision of methods for the generation of stem cell-derived beta cells (SC-β), stem cell-derived islets (SC-islets), definitive endoderm cell, pancreatic progenitor cells, and uses thereof. The various cells are generated by contacting human pluripotent stem cells (hPSCs) with an amount of a cytoskeleton depolymerizing agent during initial stem cell differentiation into definitive endoderm cells. A method of treatment of a subject diagnosed with a diabetes condition by transplanting administering a therapeutically effective amount of the stem cell-derived islets (SC-islets) into the subject is also disclosed.

Description

[0001] TITLE OF THE INVENTION

[0002] MICROENVIRONMENTAL CONTROL IMPROVES DIFFERENTIATION OF STEM CELL DERIVED ISLETS

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application is a PCT application that claims the benefit of priority to US Provisional Application No. 63 / 676,328 filed on 26 July 2024, the content of which is incorporated by reference herein in its entirety.

[0005] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0006] This invention was made with government support under DK114233 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] MATERIAL INCORPORATED-BY-REFERENCE

[0008] Not applicable.

[0009] FIELD OF THE INVENTION

[0010] The present disclosure generally relates to methods to enhance stem cell- derived beta cell (SC-beta cell) differentiation.

[0011] BACKGROUND OF THE INVENTION

[0012] Pancreatic [3 cells regulate glucose metabolism throughout the body by secreting insulin into the bloodstream, facilitating glucose uptake by many cell types so that it can be utilized for energy production and fat storage. Within the pancreas, [3 cells are clustered together with other endocrine cell types to form the islets of Langerhans. In type 1 diabetes (T1 D), [3 cells are erroneously targeted by the immune system and selectively eliminated. As a result, T1 D patients must inject exogenous insulin in order to restore proper insulin signaling throughout the body, which is required for survival. Replicating the precise insulin secretion kinetics of [3 cells with insulin injections can be difficult because insulin requirements change dynamically based on factors related to energy metabolism, such as the amount and types of food consumed, the duration and intensity of physical activity, and the levels of circulating stress hormones. Over time, chronically elevated blood glucose levels can lead to severe complications, such as cardiovascular, kidney, and eye disease, emphasizing the importance of keeping blood glucose levels as close to normal as possible.

[0013] In an effort to improve blood glucose control and ease the burden of diabetes management, primary [3 cells from deceased donors have been transplanted into patients with T1 D, demonstrating that replacing this lost [3 cell mass is a safe and effective method to restore glucose homeostasis. However, issues with donor quality, quantity, and immunogenicity have hampered its widespread use. As an alternative, stem cell-derived islets (SC-islets) containing stem cell-derived [3 cells (SC-[3 cells) and other pancreatic endocrine cell types could provide an unlimited source of islets for transplantation. These SC-islets can be derived from a single stem cell source that is extensively characterized, resulting in more reproducible transplant outcomes. Furthermore, these cells can be gene-edited to have desirable characteristics that enhance transplant results, such as decreasing their immunogenicity to circumvent the need for immunosuppressive drugs or making them more resistant to the stresses experienced during transplantation. SC-islets can also be utilized as an effective in vitro model to study different aspects of the disease and develop additional therapeutic strategies.

[0014] To this end, directed differentiation protocols have been developed in recent years to generate SC-islets using a stepwise combination of growth factors and small molecules to drive hPSCs through several intermediate cell types on their way to becoming pancreatic endocrine cells. While there are a number of different iterations and improvements that have been made since the original development of these protocols, all successful SC-islet differentiation methods follow the same basic strategy. Specifically, they attempt to recreate the specific sequence of signaling events that must occur to form the pancreas during embryonic development. This process begins with the specification of the definitive endoderm germ layer from hPSCs, which is driven by a coordination of Activin / Nodal, WNT, and BMP signaling. This definitive endoderm is then specified as the primitive gut tube, which will eventually form the organs of the gastrointestinal and respiratory tracts. The primitive gut tube is progressively segmented into the foregut, midgut, and hindgut through signaling gradients of FGF, BMP, WNT, and retinoic acid (RA) ligands that are secreted from the splanchnic mesodermal tissue surrounding the gut tube, as well as Hedgehog and Notch signaling originating in the gut tube itself. The intensity and timing of these signals induce gene expression patterns that segment the gut tube into the various organ-forming regions. For example, S0X2 expression is restricted to the foregut, while CDX2 expression is localized to the mid- and hindgut. Furthermore, differential expressions of various HOX genes along the gut tube provide a spatial code that helps guide these different regions to develop into their respective organs. Cells of the foregut will subsequently generate organs such as the thyroid, thymus, trachea, lungs, esophagus, stomach, pancreas, and liver, while the midgut forms the small intestine and the hindgut develops into the large intestine. The pancreas arises from the posterior segment of the foregut, where high RA signaling helps induce the expression of PDX1. These multipotent PDX1 + pancreatic progenitors subsequently develop into duct and acinar exocrine cells as well as all the endocrine cell types of the islet.

[0015] SUMMARY OF THE INVENTION

[0016] Among the various aspects of the present disclosure is the provision of methods to enhance SC-beta cell production, functionality, and utility.

[0017] Disclosed herein is a method of generating stem cell-derived islets (SC- islets) containing stem cell-derived [3-cells (SC-[3s). The method includes contacting a plurality of human pluripotent stem cells (hPSCs) with a cytoskeleton depolymerizing agent during initial stem cell differentiation into definitive endoderm cells. In one aspect, the stem cell differentiation process comprises (Stage 1 ) providing a stem cell, providing serum-free media and contacting the stem cell with a TGF[3 / Activin agonist or a glycogen synthase kinase 3 (GSK) inhibitor or WNT agonist for an amount of time sufficient to form a definitive endoderm cell; (Stage 2) contacting the definitive endoderm cell with a FGFR2b agonist for an amount of time sufficient to form a primitive gut tube cell; (Stage 3) contacting the primitive gut tube cell with an RAR agonist, and optionally a rho kinase inhibitor, a smoothened antagonist, a FGFR2b agonist, a protein kinase C activator, or a BMP type 1 receptor inhibitor for an amount of time sufficient to form an early pancreas progenitor cell; (Stage 4) incubating the early pancreas progenitor cell for at least about 3 days and optionally contacting the early pancreas progenitor cell with a rho kinase inhibitor, a TGF-[3 / Activin agonist, a smoothened antagonist, an FGFR2b agonist, or a RAR agonist for an amount of time sufficient to form a pancreatic progenitor cell; (Stage 5) contacting the pancreatic progenitor cell with an Alk5 inhibitor, a gamma secretase inhibitor, SANT1 , Erbbl (EGFR) or Erbb4 agonist, or a RAR agonist for an amount of time sufficient to form an endoderm cell; and (Stage 6) allowing the endoderm cell to mature in serum-free media for an amount of time sufficient to form a beta cell.

[0018] In one aspect, a method of generating at least one stem cell-derived islet (SC-islet) comprising a plurality of stem cell-derived [3-cells (SC-[3s) is disclosed, the method comprising contacting at least one human pluripotent stem cell (hPSC) with an amount of a cytoskeleton depolymerizing agent during initial stem cell differentiation into definitive endoderm cells. In some aspects, the at least one human pluripotent stem cell is contacted with the amount of the cytoskeleton depolymerizing agent during the first 24 hours of the initial stem cell differentiation. In some aspects, the cytoskeleton depolymerizing agent is configured to depolymerize a cytoskeleton component comprising F-actin. In some aspects, the cytoskeleton depolymerizing agent is selected from latrunculin A (latA), latrunculin B, cytochalasin D, and any combination thereof. In some aspects, the at least one human pluripotent stem cell is contacted with the latA at a concentration ranging from about 0.01 pM to about 1 pM. In some aspects, the method produces a stem cell-derived islet (SC-islet) with reduced off-target and undifferentiated cells compared to a corresponding SC-islet produced without a cytoskeleton depolymerizing agent. In some aspects, the method produces a stem cell-derived islet (SC-islet) with reduced formation of tumor cells. In some aspects, the method produces at least 5% more SC-[3s compared to a corresponding number of SC-[3s SC-islet produced without a cytoskeleton depolymerizing agent In some aspects, the method produces a stem cell-derived islet (SC-islet) with enhanced insulin secretion compared to a corresponding SC-islet produced without a cytoskeleton depolymerizing agent. In some aspects, the at least one hPSC contacted with the cytoskeleton depolymerizing agent comprises at least one hPSC selected from a stem cell line selected from HUES8, WS4c, AN1 .1 , and H1 .

[0019] In another aspect, a method of treating a subject is disclosed, the method comprising: contacting a plurality of human pluripotent stem cells (hPSCs) with an amount of a cytoskeleton depolymerizing agent during initial stem cell differentiation into definitive endoderm cells to generate a plurality of stem cell- derived islets (SC-islets) each comprising a plurality of stem cell-derived [3-cells (SC-|3s); and transplanting a therapeutically effective amount of the stem cell- derived islets (SC-islets) into the subject. In some aspects, the subject comprises a patient diagnosed with a diabetes disorder selected from type 1 diabetes mellitus (T1 D), type 2 diabetes mellitus (T2D), gestational diabetes mellitus, latent autoimmune diabetes of adults (LADA), or maturity onset diabetes of the young (MODY). In some aspects, the at least one human pluripotent stem cell is contacted with the amount of the cytoskeleton depolymerizing agent during the first 24 hours of the initial stem cell differentiation. In some aspects, the cytoskeleton depolymerizing agent is configured to depolymerize a cytoskeleton component comprising F-actin. In some aspects, the cytoskeleton depolymerizing agent comprises latrunculin A (latA). In some aspects, the plurality of human pluripotent stem cells is contacted with the latA at a concentration ranging from about 0.01 pM to about 1 pM. In some aspects, the SC-islets comprise enhanced insulin secretion compared to corresponding SC-islets produced without a cytoskeleton depolymerizing agent. In some aspects, the therapeutically effective amount of the stem cell-derived islets (SC-islets) produce insulin sufficient to control a blood glucose level of the subject.

[0020] In another aspect, at least one definitive endoderm cell generated by contacting at least one human pluripotent stem cell (hPSC) with an amount of a cytoskeleton depolymerizing agent during initial stem cell differentiation into definitive endoderm cells is disclosed, wherein the at least one definitive endoderm cell, wherein the at least one definitive endoderm cell comprises higher expression levels of at least one first protein selected from Gata3, Bambi, Id 1 , Id2, Id3, Id4 and any combination thereof and further comprises lower expression levels of at least one second protein selected from Cer1 , Gsc, Leftyl , Lefty2, Nodal, Tagln, Gata6, Otx2 and any combination thereof, the higher and lower expression levels defined relative to corresponding expression levels of at least one comparison definitive endoderm cell generated in an absence of the cytoskeleton depolymerizing agent. In some aspects, the at least one human pluripotent stem cell is contacted with the amount of the cytoskeleton depolymerizing agent during the first 24 hours of the initial stem cell differentiation. In some aspects, the cytoskeleton depolymerizing agent is configured to depolymerize a cytoskeleton component comprising F-actin. In some aspects, the cytoskeleton depolymerizing agent is selected from latrunculin A (latA), latrunculin B, cytochalasin D, and any combination thereof. In some aspects, the at least one human pluripotent stem cell is contacted with the latA at a concentration ranging from about 0.01 pM to about 1 pM. In some aspects, the at least one hPSC contacted with the cytoskeleton depolymerizing agent comprises at least one hPSC selected from a stem cell line selected from HUES8, WS4c, AN1.1 , and H1.

[0021] In another aspect, at least one pancreatic progenitor cell generated by contacting at least one human pluripotent stem cell (hPSC) with an amount of a cytoskeleton depolymerizing agent during initial stem cell differentiation into definitive endoderm cells is disclosed, wherein the at least one pancreatic progenitor cell, comprises higher expression levels of at least one first protein selected from of PTF1a, ONECUT1 , OSR1 , HOXA1 , HOXA3 and any combination, the higher expression levels defined relative to corresponding expression levels of at least one comparison pancreatic progenitor cell generated in an absence of the cytoskeleton depolymerizing agent. In some aspects, the at least one human pluripotent stem cell is contacted with the amount of the cytoskeleton depolymerizing agent during the first 24 hours of the initial stem cell differentiation. In some aspects, the cytoskeleton depolymerizing agent is configured to depolymerize a cytoskeleton component comprising F- actin. In some aspects, the cytoskeleton depolymerizing agent is selected from latrunculin A (latA), latrunculin B, cytochalasin D, and any combination thereof. In some aspects, the at least one human pluripotent stem cell is contacted with the latA at a concentration ranging from about 0.01 pM to about 1 pM. In some aspects, the at least one hPSC contacted with the cytoskeleton depolymerizing agent comprises at least one hPSC selected from a stem cell line selected from HUES8, WS4c, AN1.1 , and H1.

[0022] In another aspect, at least one SC-beta cell generated by contacting at least one human pluripotent stem cell (hPSC) with an amount of a cytoskeleton depolymerizing agent during initial stem cell differentiation into definitive endoderm cells is disclosed, wherein the at least one SC-beta cell comprises lower expression levels of at least one first protein selected from of LRP1 B, SORCS1 , RALYL, HDAC9 and high CACNA1A, DSP, HS6ST3, MY03A, SAMD4A, ASPH and any combination thereof, the lower expression levels defined relative to corresponding expression levels of at least one comparison SC-beta cell generated in an absence of the cytoskeleton depolymerizing agent. In some aspects, the at least one human pluripotent stem cell is contacted with the amount of the cytoskeleton depolymerizing agent during the first 24 hours of the initial stem cell differentiation. In some aspects, the cytoskeleton depolymerizing agent is configured to depolymerize a cytoskeleton component comprising F-actin. In some aspects, the cytoskeleton depolymerizing agent is selected from latrunculin A (latA), latrunculin B, cytochalasin D, and any combination thereof. In some aspects, the at least one human pluripotent stem cell is contacted with the latA at a concentration ranging from about 0.01 pM to about 1 pM. In some aspects, the at least one hPSC contacted with the cytoskeleton depolymerizing agent comprises at least one hPSC selected from a stem cell line selected from HUES8, WS4c, AN1.1 , and H1.

[0023] Other objects and features will be in part apparent and in part pointed out hereinafter.

[0024] DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1 A is a schematic diagram illustrating an experimental protocol for testing cytoskeletal-modulating compounds during a process of SC-islet differentiation from human pluripotent stem cells (hPSCs).

[0026] FIG. 1 B contains a series of microscopic images of cells from the HUES8 stem cell line on stage 6, day 7 of the differentiation process shown illustrated in FIG. 1A, demonstrating that compounds that induce polymerization of the actin cytoskeleton (nocodazole, S1 P) produced non-endocrine cell types, while those compounds that depolymerized the cytoskeleton (latrunculin A, latrunculin B, cytochalasin D) in the same part of the differentiation process was associated with robust endocrine-type cell formation. The added compounds were contacted with differentiating cells for the first 24 hours of differentiation during which cytoskeletal modulation occurred. Scale bar = 500 pm.

[0027] FIG. 1 C is a bar graph summarizing static GSIS of stage 6 cells from the HUES8 stem cell line treated with cytoskeletal-modulating compounds for the first 24 hours of differentiation (two-way paired t-test comparing insulin secretion between low and high glucose stimulation; n = 4).

[0028] FIG. 1 D contains a series of immunostaining images of F-actin on stage 1 , day 2 of the differentiation process HUES8 stem cells as illustrated in FIG. 1A in response to a 24-hour treatment with either latA, S1 P, or no treatment. Scale bar = 50 pm.

[0029] FIG. 1 E contains a series of immunostaining images of NANOG at the end of stage 1 in response to either latA, S1 P, or no treatment for the first 24 hours of differentiation of HUES8 stem cells. Scale bar = 100 pm.

[0030] FIG. 1 F is a bar graph summarizing the results of qRT-PCR of pluripotency genes of HUES8 stem cells at the end of stage 1 in response to treatment with cytoskeletal-modulating compounds for the first 24 hours of differentiation (Welch’s ANOVA followed by Dunnett’s T3 multiple comparison test compares each condition to control; n = 8).

[0031] FIG. 1 G is a flow chart illustrating an SC-islet differentiation protocol used for all experiments incorporating the latA treatment during the first 24 hours of stage 1 .

[0032] FIG. 1 H is a bar graph summarizing static GSIS of stage 6 cells from HUES8, WS4c, AN1.1 , or H1 stem cell lines treated with latA (0 pM, 0.1 pM, 0.1375 pM, or 0.175 pM) during the first 24 hours of differentiation (two-way paired t-test comparing insulin secretion between low and high glucose stimulation; one-way ANOVA of high glucose between all latA concentrations for each cell line; n = 15-16 for HUES8, n = 6-8 for WS4c, n = 6-7 for AN1 .1 , n = 8 for H1 , n = 11 for human islets from 2 separate donors).

[0033] FIG. 1 1 is a bar graph summarizing insulin secretion in response to 30 mM KCI stimulation in stage 6 cells from HUES8, WS4c, AN1 .1 , or H1 stem cell lines treated with latA (0 pM, 0.1 pM, 0.1375 pM, or 0.175 pM) during the first 24 hours of differentiation (legend in (I); one-way ANOVA of insulin secretion between all latA concentrations for each cell line; n = 4 for all cell lines, n = 11 for human islets from 2 separate donors).

[0034] FIG. 1 J is a bar graph summarizing insulin content of stage 6 cells from HUES8, WS4c, AN1.1 , or H1 stem cell lines treated with latA (0 pM, 0.1 pM, 0.1375 pM, or 0.175 pM) during the first 24 hours of differentiation (one-way ANOVA of insulin content between all latA concentrations for each cell line; n = 4 for HUES8, WS4c, and H1 , n = 3 for AN1.1 , n = 8 for human islets from 2 separate donors).

[0035] FIG. 1 K is a series of graphs summarizing dynamic insulin secretion of HUES8, WS4c, AN1 .1 , or H1 stem cell lines at stage 6 after treatment with latA (0 pM, 0.1375 pM, or 0.175 pM) for the first 24 hours of differentiation (n = 7-10 for HUES8, n = 9 for WS4c, n = 6 forAN1.1, n = 6-8 for H1 , n = 14 for human islets from 2 separate donors). Panels (B-F) were generated with the HUES8 stem cell line. Panels (H-K) were generated with either the HUES8, WS4c, AN 1.1 , or H1 stem cell line, as indicated. All data are represented as the mean, and all error bars represent the SEM. Individual data points are shown for all bar graphs. NS, not significant; *P < 0.05, **P < 0.01 , ***P < 0.001 .

[0036] FIG. 1 L contains graphs summarizing qRT-PCR of pluripotency genes and markers specific to each germ layer after differentiation in either endoderm, mesoderm, or ectoderm media. 0.125 pM latA or 0.3 pM S1 P was added to the media during the first 24 hours of differentiation as indicated (ANOVA followed by Dunnett’s T3 multiple comparison test compares each condition to the control within each differentiation media; n = 3).

[0037] FIG. 1 M contains images with immunostaining of NANOG after the trilineage germ layer differentiation. Scale bars = 100 pm. All data were obtained with the HUES8 cell line. All data are represented as the mean, and all error bars represent the SEM. Individual data points are shown for all bar graphs. NS, not significant; *P < 0.05, **P < 0.01 , ***P < 0.001 .

[0038] FIG. 1 N contains images with immunostaining of lineage specific markers after the tri-lineage germ layer differentiation. Scale bars = 100 pm. All data were obtained with the HUES8 cell line. All data are represented as the mean, and all error bars represent the SEM. Individual data points are shown for all bar graphs. NS, not significant; *P < 0.05, **P < 0.01 , ***P < 0.001 .

[0039] FIG. 2A contains bar graphs summarizing flow cytometry on stage 6, day 7, demonstrating an increase in the percentage of SC-|3 cells (NKX6.1 + / C- peptide+) for all cell lines in response to the stage 1 latA treatment and a decrease in the percentage enterochromaffin cells (SLC18A1 +) for HUES8 and WS4c cell lines (two-way unpaired t-test, n = 3 for HUES8, WS4c, and AN1.1 , n = 4 for H1).

[0040] FIG. 2B contains representative flow cytometry plots for stage 6, day 7 cells generated from HUES8 stained for C-peptide and NKX6.1 .

[0041] FIG. 2C containing immunostaining images of stage 6, day 7 cells generated from HUES8 stained for C-peptide and NKX6.1. Scale bar = 100 pm.

[0042] FIG. 2D contains images of stage 6, day 7 cells generated from HUES8 before and 2 weeks after aggregation into clusters. Scale bar = 500 pm.

[0043] FIG. 2E is a bar graph summarizing qRT-PCR of stage 6, day 7 cells generated from HUES8 (two-way unpaired t-test, n = 3).

[0044] FIG. 2F contains a series of immunostaining images of stage 6, day 7 cells generated from AN1 .1 stained for C-peptide and NKX6.1 . These images show a batch of cells that failed to make endocrine cells with the normal differentiation protocol, while treating this same batch of cells with 0.175 pM latA during stage 1 rescued SC-|3 cell generation. Scale bar = 100 pm.

[0045] FIG. 2G contains a pair of images on stage 6, day 7 showing a batch of AN1 .1 cells that failed to make endocrine cells with the normal differentiation protocol but exhibited the expected endocrine morphology when treated with 0.175 pM latA during stage 1 . Scale bar = 500 pm.

[0046] FIG. 2H is a bar graph summarizing Flow cytometry on stage 6, day 7 of differentiation batches from the AN1 .1 and H1 cell lines in which the control differentiation failed to produce SC-|3 cells, while SC-|3 cell generation was rescued by treating these same batches with latA during stage 1 in a dosedependent manner (one-way ANOVA, n = 3). All data are represented as the mean, and all error bars represent the SEM. Individual data points are shown for all bar graphs. NS, not significant; *P < 0.05, **P < 0.01 , ***P < 0.001 .

[0047] FIG. 2I contains graphs summarizing qRT-PCR of pluripotency genes on stage 2, day 1 cells for all four cell lines, with and without stage 1 latA treatment (two-sided t-tests between control and latA condition for each gene in a given cell line, n = 3).

[0048] FIG.3A contains a series of graphs summarizing fasting blood glucose concentrations over 30 weeks post-transplant of SC-|3 cells (top), and blood glucose concentrations during a glucose tolerance test at week 8 (bottom left) and week 28 (bottom right) post-implantation. Mice were made diabetic with streptozotocin (STZ) injections. Cells were transplanted underneath the kidney capsule using either primary human islets or SC-islets generated with the AN1 .1 stem cell line. These SC-islets were produced either without or with the 0.175 pM latA treatment during stage 1 (untreated control: n = 5 mice; STZ, no transplant: n = 7 mice; AN1 .1 - control: n = 7 mice; AN1 .1 - 0.175 pM latA: n = 8 mice; primary human islets: n = 6 mice). Fasting blood glucose levels demonstrated rapid restoration of blood glucose control with the mice transplanted with the primary human islets and the AN1.1 - 0.175 pM latA SC- islets. These transplanted mice also controlled blood glucose levels during glucose tolerance tests at 8 and 28 weeks. All data are represented as the mean, and all error bars represent the SEM. Individual data points are shown for all bar graphs. NS, not significant; *P < 0.05, **P < 0.01 , ***P < 0.001 .

[0049] FIG.3B contains bar graphs summarizing in vivo GSIS assay results at weeks 6 and 27 after transplantation of SC-islets, demonstrating that the transplanted AN1.1 - 0.175 pM latA SC-islets secreted more insulin 30 minutes after glucose injection, and this insulin secretion was at comparable levels to the transplanted human islets by week 27 (two-way paired t-test between low and high glucose stimulation; two-way unpaired t-test of the 30 minute timepoint between the mice transplanted with AN1.1 - 0.175 pM latA SC-islets and either the AN1 .1 - control SC-islets or human islets). All data are represented as the mean, and all error bars represent the SEM. Individual data points are shown for all bar graphs. NS, not significant; *P < 0.05, **P < 0.01 , ***P < 0.001 .

[0050] FIG.3C contains a series of images of kidneys (left images) and microscopic images of the kidneys and any associated tumors (right images) at week 11 (upper graphs) and week 30 (lower graphs). The mice transplanted with the AN1 .1 - control cells formed large tumors by week 11 , while the mice transplanted with the AN1 .1 - 0.175 pM latA SC-islets did not have any signs of overgrowths during the experiment. Furthermore, the AN1.1 - 0.175 pM latA SC- islet graft was still clearly visible in the kidney after 30 weeks.

[0051] FIG.3D is a series of microscopic images stained for various proteins. Many SC-|3 cells (NKX6.1 + / C-peptide+) were observed in these AN1.1 - 0.175 pM latA SC-islet grafts after 30 weeks in the mice.

[0052] FIG. 4A contains single cell RNA-sequencing UMAPs of cells every day of stage 1 comparing without and with a 0.175 pM latA treatment for the first 24 hours of differentiation. Example nomenclature: s1d1 = stage 1 , day 1.

[0053] FIG. 4B contains a volcano plot of genes differentially expressed at the end of stage 1 with latA treatment.

[0054] FIG. 4C contains a series of graphs summarizing the average gene expression of all cells in a cluster from the single cell RNA-sequencing data at each day of differentiation either without or with latA treatment.

[0055] FIG. 4D contains a series of immunostaining images of several important markers at the end of stage 1 , confirming the expression patterns exhibited at the transcriptional level in the single cell RNA-sequencing data. Scale bar = 100 pm.

[0056] FIG. 4E contains graphs summarizing the results of enzyme-linked immunosorbent assays (ELISAs) measuring the ratio of phosphorylated to total protein of SMADS 1 , 2, and 5 every day of stage 1 without or with latA treatment (two-way unpaired t-test at each time point, n = 4). All assays were performed with the HUES8 cell line. All data are represented as the mean, and all error bars represent the SEM. NS, not significant; *P < 0.05, **P < 0.01 , ***P < 0.001 .

[0057] FIG. 5A contains a series of immunostaining images and a bar graph summarizing flow cytometry analysis (two-way unpaired t-test, n = 3) of the definitive endoderm markers FOXA2 and SOX17 at the end of stage 1 comparing without and with a 0.175 pM latA treatment for the first 24 hours of differentiation. Scale bar = 100 pM.

[0058] FIG. 5B contains a series of immunostaining images and a bar graph summarizing flow cytometry analysis (two-way unpaired t-test, n = 3) of the pancreatic markers PDX1 and NKX6.1 at the end of stage 4 comparing without and with a 0.175 pM latA treatment for the first 24 hours of differentiation. Scale bar = 100 pM.

[0059] FIG. 5C is a bar graph summarizing the quantification of western blots for NKX6.1 protein at the end of stage 4 comparing without and with a 0.175 pM latA treatment for the first 24 hours of differentiation (two-way unpaired t-test, n = 3).

[0060] FIG. 5D is a bar graph summarizing the qRT-PCR results at the end of stage 4 for pancreatic markers (PDX1 , PTF1a, NKX6.1 , S0X9) and markers associate other lineage of the gut tube (CDX2, 0TX2, AFP, HNF4a) (two-way unpaired t-test, n = 3).

[0061] FIG. 5E contains single cell RNA-sequencing UMAPs of cells throughout the SC-islet differentiation process comparing without and with a 0.175 pM latA treatment for the first 24 hours of differentiation. Example nomenclature: s1d1 = stage 1 , day 1 .

[0062] FIG. 5F contains a series of graphs summarizing average gene expression of all cells in a cluster from the single cell RNA-sequencing data at the intermediate stages of differentiation (s2d1 , s3d1 , s4d1 , and s5d1 ) either without or with latA treatment.

[0063] FIG. 5G contains a second series of graphs summarizing average gene expression of all cells in a cluster from the single cell RNA-sequencing data at the intermediate stages of differentiation (s2d1 , s3d1 , s4d1 , and s5d1 ) either without or with latA treatment.

[0064] FIG. 5H is a bar graph summarizing the quantification of western blots for the indicated proteins comparing without and with a 0.175 pM latA treatment for the first 24 hours of differentiation (two-way unpaired t-test, n = 3).

[0065] FIG. 5I is a bar graph summarizing the quantification of western blots for the indicated proteins comparing without and with a 0.175 pM latA treatment for the first 24 hours of differentiation (two-way unpaired t-test, n = 3).

[0066] FIG. 5J contains single cell RNA-sequencing UMAPs of cells on stage 6, day 7 of differentiation, demonstrating differences in the percentage of cell types in latA-treated cells. All assays were performed with the HUES8 cell line. All data are represented as the mean, and all error bars represent the SEM. Individual data points are shown for all bar graphs. NS, not significant; *P < 0.05, **P < 0.01 , ***P < 0.001.

[0067] FIG. 6A contains single-nuclei multi-omic sequencing UMAPs showing cell clustering based on either gene expression, chromatin accessibility, or both combined from each individual cell. Cells were processed on stage 6, day 15, which was 8 days after aggregation into islet-like clusters. Treating cells with 0.175 pM latA during the first 24 hours of stage 1 influenced the percentages of the different cell types produced by the end of the protocol.

[0068] FIG. 6B is a gene expression heatmap of markers associated with different endocrine cell types observed in the multi-omic sequencing.

[0069] FIG. 6C is a heatmap of the chromatin accessibility for the promoters of these gene markers.

[0070] FIG. 6D contains volcano plots within the [3 cell cluster showing differences in gene expression, chromatin accessibility for different gene promoters, and the chromatin accessibility of transcription factor binding motifs in SC-islets generated without and with 0.175 pM latA for the first 24 hours of differentiation. All multi-omic sequencing was performed with the HUES8 cell line.

[0071] FIG. 7A contains immunostaining images of F-actin on stage 1 , day 2 in response to a 24-hour treatment with compounds that either induce actin polymerization (nocodazole, S1 P) or that depolymerize actin (latrunculin A, latrunculin B, cytochalasin D). Scale bar = 100 pm for the two most left columns; The far-right column is a zoomed in area of the image in the center column, scale bar = 50 pm.

[0072] FIG. 7B contains immunostaining images of FOXA2 and SOX17 at the end of stage 1 in response to either latA, S1 P, or no treatment for the first 24 hours of differentiation. Scale bar = 100 pm.

[0073] FIG. 7C is a graph summarizing qRT-PCR analysis of cells on stage 6, day 7 that were treated for the first 24 hours of differentiation with the indicated cytoskeletal-modulating compounds. Compounds that polymerized actin decreased expression of pancreatic endocrine genes (Dunnett’s test compares each condition to the control, n = 3). All data were generated with the HUES8 cell line. All data are represented as the mean, and all error bars represent the SEM. Individual data points are shown for all bar graphs. NS, not significant; *P < 0.05, **P < 0.01 , ***P < 0.001 .

[0074] FIG 7D is a graph summarizing additional qRT-PCR analysis of cells on stage 6, day 7 that were treated for the first 24 hours of differentiation with the indicated cytoskeletal-modulating compounds. Compounds that polymerized actin decreased expression of pancreatic endocrine genes (Dunnett’s test compares each condition to the control, n = 3). All data were generated with the HUES8 cell line. All data are represented as the mean, and all error bars represent the SEM. Individual data points are shown for all bar graphs. NS, not significant; *P < 0.05, **P < 0.01 , ***P < 0.001 .

[0075] FIG. 8A contains images of cells on stage 1 , day 2 after a 24-hour treatment with latA (0 pM, 0.1 pM, 0.1375 pM, or 0.175 pM). Scale bar = 100 pm.

[0076] FIG. 8B contains immunostaining images of F-actin on stage 1 , day 2 without and with a 24-hour treatment of 0.175 pM latA. Scale bar = 50 pm.

[0077] FIG. 8C contains images of cell morphology every day of stage 1 either without or with a 0.175 pM latA treatment for the first 24 hours of this stage. Notably, the latA treatment caused cells to round up, but they flattened again once latA was removed. Importantly, while increased cell death is observed throughout stage 1 with latA treatment, there should always be a confluent monolayer of cells still attached to the plate. Scale bar = 500 pm.

[0078] FIG. 8D contains images of cell morphology every day of stage 1 either without or with a 0.175 pM latA treatment for the first 24 hours of this stage. Notably, the latA treatment caused cells to round up, but they flattened again once latA was removed. Importantly, while increased cell death is observed throughout stage 1 with latA treatment, there should always be a confluent monolayer of cells still attached to the plate, scale bar = 100 pm.

[0079] FIG. 8E contains images of cell morphology on stage 2, day 1 and stage 6, day 7, demonstrating that treating the cells with too high of a latA concentration (0.25 pM) in stage 1 resulted in decreased cell density by stage 2, day 1 and non-endocrine cell types by the end of the protocol. In contrast, cells treated with an optimized latA concentration for a given cell line (0.175 pM for HUES8) produced cells with an endocrine morphology similar to the control. All images are of differentiations with the HUES8 cell line.

[0080] FIG 9A is a graph summarizing insulin secretion by SC-islets generated with and without a 0.175 pM latA treatment during the first 24 hours of differentiation. SC-islets generated with the 0.175 pM latA treatment secreted more insulin in response to various secretagogues (two- way paired t-test between low and high glucose; unpaired two-way t-test between control and latA with each secretagogue at high glucose, n = 8). All data are represented as the mean, and all error bars represent the SEM. Individual data points are shown for all bar graphs. NS, not significant; *P < 0.05, **P < 0.01 , ***P < 0.001 .

[0081] FIG. 9B is a graph summarizing the proinsulin content to insulin content ratio in stage 6 in response to latA dosing in stage 1 (one-way ANOVA, n = 4 for HUES8, WS4c, and H1 , n = 3 for AN1.1 , n = 8 for human islets from 2 separate donors).

[0082] FIG. 9C is a graph summarizing flow cytometry quantification of endocrine marker expression in stage 6 in response to increasing latA concentration in stage 1 (one-way ANOVA, n = 3).

[0083] FIG. 9D is a graph quantifying the insulin secretion summarized in FIG.

[0084] 1 H in which the insulin secretion is normalized to the number of SC-|3 cells rather than the total number of cells (two-way paired t-test comparing insulin secretion between low and high glucose stimulation; two-way unpaired t-test between high glucose for each cell line; n = 15-16 for HUES8, n = 6-8 for WS4c, n = 6-7 for AN1 .1 , n = 8 for H1 , n = 11 for human islets from 2 separate donors)

[0085] FIG. 9E contains Immunostaining images of stage 6, day 7 cells generated from the WS4c cell line stained for C-peptide and NKX6.1 . Scale bar = 100 pm.

[0086] FIG, 9F contains qRT-PCR measurements of stage 6, day 7 cells generated from the WS4c cell line (two-way unpaired t-test, n = 3 for WS4c).

[0087] FIG. 9G contains Immunostaining images of stage 6, day 7 cells generated from the AN1.1 cell line stained for C-peptide and NKX6.1. Scale bar = 100 pm.

[0088] FIG, 9H contains qRT-PCR measurements of stage 6, day 7 cells generated from the AN 1.1 cell line (two-way unpaired t-test, n = 3 for AN 1.1 ).

[0089] FIG. 9I contains Immunostaining images of stage 6, day 7 cells generated from the H1 cell line stained for C-peptide and NKX6.1. Scale bar = 100 pm.

[0090] FIG, 9J contains qRT-PCR measurements of stage 6, day 7 cells generated from the H1 cell line (two-way unpaired t-test, n = 4 for H1 ). FIG. 10A contains images of the cells at each stage of the differentiation protocol to compare cell morphology between without or with a 0.175 pM latA treatment during the first 24 hours of the protocol. Notably, the latA-treated cells appeared more homogenous throughout the differentiation process. Scale bar = 500 pm.

[0091] FIG. 10B contains images of the cells at each stage of the differentiation protocol to compare cell morphology between without or with a 0.175 pM latA treatment during the first 24 hours of the protocol. Notably, the latA-treated cells appeared more homogenous throughout the differentiation process. Scale bar = 100 pm.

[0092] FIG, 11 A contains morphology images in stage 6 of successful differentiations from the WS4c, AN1.1 , and H1 cell lines before aggregation into islet-like clusters. Differentiations were performed either without or with a latA treatment during the first 24 hours of the protocol (0.175 pM for WS4c and AN 1 .1 , 0.1375 pM for H 1 ). Scale bars = 500 pm .

[0093] FIG, 11 B contains morphology images in stage 6 of successful differentiations from the WS4c, AN1 .1 , and H1 cell lines after aggregation into islet-like clusters. Scale bars = 500 pm.

[0094] FIG. 11 C is a graph summarizing viable cell counts during the aggregation step on stage 6, day 7 for differentiations performed with the WS4c cell line with a 0.175 pM latA treatment in culture flasks with a surface area of 182.5 cm2.

[0095] FIG. 12A contains immunostaining images of stage 4, day 1 cells generated from the AN1 .1 line stained for PDX1 . These images show a batch of cells that failed to make pancreatic progenitors with the normal differentiation protocol, while treating this same batch of cells with 0.175 pM latA during stage 1 rescued induction of PDX1 + pancreatic progenitors. Scale bar = 100 pm.

[0096] FIG. 12B contains immunostaining images of stage 6, day 7 cells generated from the H1 line stained for C-peptide and NKX6.1 . These images show a batch of cells that failed to make endocrine cells with the normal differentiation protocol, while treating this same batch of cells with 0.1375 pM latA during stage 1 rescued SC-|3 cell generation. Scale bar = 100 pm. FIG. 12C contains morphology images of cells generated with the AN1.1 and H1 cell lines on stage 6, day 7. The control differentiations for these cell lines would often generate non-endocrine cell types (left), while treating the cells with latA during the first 24 hours of stage 1 would rescue endocrine formation in these same batches of cells (right).

[0097] FIG 12D contains graphs summarizing qRT-PCR results of stage 6, day 7 cells differentiated from the AN1.1 (top graph) and H1 (bottom graph) cell lines from batches of cells in which the control differentiation failed. Increasing latA concentration in stage 1 progressively increased expression of pancreatic endocrine genes by stage 6, day 7 (one-way ANOVA for each gene, n = 3). All data are represented as the mean, and all error bars represent the SEM. Individual data points are shown for all bar graphs. NS, not significant; *P < 0.05, **P < 0.01 , ***P < 0.001.

[0098] FIG. 13A is a graph summarizing random blood glucose measurements of diabetic mice that had cells transplanted underneath the kidney capsule using either primary human islets or SC-islets generated with the AN1 .1 stem cell line. These SC-islets were produced either without or with the 0.175 pM latA treatment during stage 1 (untreated control: n = 5 mice; STZ, no transplant: n = 7 mice; AN1 .1 - control: n = 7 mice; AN1 .1 - 0.175 pM latA: n = 8 mice; primary human islets: n = 6 mice).

[0099] FIG. 13B contains immunostaining images of histology sections taken from mouse kidneys transplanted with primary human islets, showing the presence of human [3 cells (C-peptide+ / NKX6.1 +) 30 weeks after transplantation. Scale bar = 100 pm.

[0100] FIG. 14A contains volcano plots from single cell RNA-sequencing of genes differentially expressed throughout stage 1 with latA treatment, corresponding to the single cell-RNA sequencing data presented in FIG. 4C.

[0101] FIG. 14B contains a graph of the average mRNA expression of the whole cell population and the corresponding violin plots for ACTB at each day of stage 1 , , corresponding to the single cell-RNA sequencing data presented in FIG. 4C.

[0102] FIG. 14C contains violin plots from single cell RNA-sequencing of genes differentially expressed throughout stage 1 with latA treatment, , corresponding to the single cell-RNA sequencing data presented in FIG. 4C. FIG. 14D contains graphs summarizing qRT-PCR of genes differentially expressed at the end of stage 1 , demonstrating similar trends in the expression of these specific genes in response to latA treatment in all 4 cell lines (two-way unpaired t-test; n = 3 for HUES8 and WS4c, n = 4 for AN1.1 , n = 6 for H1 ). All data are represented as the mean, and all error bars represent the SEM. Individual data points are shown for all bar graphs. NS, not significant; *P< 0.05, **P< 0.01 , ***P< 0.001.

[0103] FIG, 15A contains single-cell RNA-sequencing LIMAP on stage 2, day 1 of cells treated without and with 0.175 pM latA for the first 24 hours of differentiation.

[0104] FIG. 15B contains feature plots for select genes on stage 2, day 1 . All sequencing data was performed with HUES8 and corresponds to the single cell- RNA sequencing data presented in FIG. 4C.

[0105] FIG. 16A contains single cell RNA-sequencing LIMAP (top) and violin plots (bottom) of HUES8 stem cells treated with either 0 pM, 0.175 pM, or 1.5 pM latA for 24 hours in mTeSRI .

[0106] FIG. 16B contains volcano plots showing genes differentially expressed between these latA treatments.

[0107] FIG. 16C contains morphology images of stem cells treated with either 0 pM, 0.175 pM, or 1 .5 pM latA for 24 hours in mTeSRI . A much higher latA concentration (1.5 pM) was required to induce a round morphology in mTeSRI when compared to cells cultured in stage 1 media (0.175 pM), regardless of the presence of AA and CHIR. Scale bars = 500 pm (top row) and 200 pM (bottom row).

[0108] FIG. 17A contains single nuclei ATAC-sequencing LIAMP (top) at the end of stage 1 of HUES8 cells treated without and with 0.175 pM latA for the first 24 hours of differentiation.

[0109] FIG. 17B contains volcano plots showing differences in chromatin accessibility for gene promoters and the chromatin accessibility of transcription factor binding motifs at the end of stage 1 .

[0110] FIG. 17C contains motif enrichment analysis in latA-treated cells at the end of stage 1 . FIG. 17D contains an ATAC plot showing the chromatin accessibility around the NODAL genomic region.

[0111] FIG. 18A contains immunostaining images of cells each day of stage 1 treated without or with 0.175 pM latA for the first 24 hours of differentiation and stained for NODAL. The images on stage 2, day 1 shown here are the same as in FIG. 4D to show comparison to the other days in stage 1 . Scale bar = 100 pm

[0112] FIG. 18B contains immunostaining images of cells each day of stage 1 treated without or with 0.175 pM latA for the first 24 hours of differentiation and stained for LEFTY 1 / 2. The images on stage 2, day 1 shown here are the same as in FIG. 4D to show comparison to the other days in stage 1 . Scale bar = 100 pm

[0113] FIG. 18C contains immunostaining images of cells each day of stage 1 treated without or with 0.175 pM latA for the first 24 hours of differentiation and stained for DAPI. The images on stage 2, day 1 shown here are the same as in Fig. 4D to show comparison to the other days in stage 1 . Scale bar = 100 pm.

[0114] FIG. 19A contains immunostaining images of cells each day of stage 1 treated without or with 0.175 pM latA for the first 24 hours of differentiation and stained for NANOG. The images on stage 2, day 1 shown here are the same as in FIG. 4D to show comparison to the other days in stage 1 . Scale bar = 100 pm.

[0115] FIG. 19B contains immunostaining images of cells each day of stage 1 treated without or with 0.175 pM latA for the first 24 hours of differentiation and stained for c- Jun. The images on stage 2, day 1 shown here are the same as in FIG. 4D to show comparison to the other days in stage 1 . Scale bar = 100 pm.

[0116] FIG. 19C contains immunostaining images of cells each day of stage 1 treated without or with 0.175 pM latA for the first 24 hours of differentiation and stained for DAPI. The images on stage 2, day 1 shown here are the same as in FIG. 4D to show comparison to the other days in stage 1 . Scale bar = 100 pm.

[0117] FIG. 20A contains Immunostaining images of cells each day of stage 1 treated without or with 0.175 pM latA for the first 24 hours of differentiation and stained for F-actin. The images on stage 2, day 1 shown here are the same as in FIG. 4D to show comparison to the other days in stage 1 . Scale bar = 100 pm.

[0118] FIG. 20B contains Immunostaining images of cells each day of stage 1 treated without or with 0.175 pM latA for the first 24 hours of differentiation and stained for TAGLN. The images on stage 2, day 1 shown here are the same as in FIG. 4D to show comparison to the other days in stage 1 . Scale bar = 100 pm.

[0119] FIG. 20C contains Immunostaining images of cells each day of stage 1 treated without or with 0.175 pM latA for the first 24 hours of differentiation and stained for DAPI. The images on stage 2, day 1 shown here are the same as in FIG. 4D to show comparison to the other days in stage 1 . Scale bar = 100 pm.

[0120] FIG. 21 A contains immunostaining images of cells each day of stage 1 treated without or with 0.175 pM latA for the first 24 hours of differentiation and stained for [3-catenin. Scale bar = 100 pm.

[0121] FIG. 21 B contains immunostaining images of cells each day of stage 1 treated without or with 0.175 pM latA for the first 24 hours of differentiation and stained for DAPI. Scale bar = 100 pm.

[0122] FIG. 22A contains immunostaining images of the HUES8, WS4c, AN1 .1 , and H1 hPSC lines stained for the pluripotency markers OCT4 and NANOG. Scale bar = 100 pm.

[0123] FIG. 22B contains immunostaining images of the HUES8, WS4c, AN1.1 , and H1 hPSC lines stained for c-Jun and F-actin. Scale bar = 100 pm.

[0124] FIG. 23A contains graphs summarizing qRT-PCR of cells on stage 5, day 1 for pancreatic markers (left side) and markers of other endodermal lineages (right side). Cells were treated without or with 0.175 pM latA for the first 24 hours of differentiation. The BMP inhibitor LDN193189 was also added to the specified conditions starting on stage 1 , day 2 through the end of stage 1 (two-way unpaired t-test for each gene, n = 4).

[0125] FIG. 23B contains immunostaining images of the conditions specified in FIG. 23A on stage 5, day 1 stained for PDX1 and NKX6.1 .

[0126] FIG. 23C contains graphs summarizing qRT-PCR each day of stage 1 of genes associated with WNT signaling in cells treated without or with 0.175 pM latA.

[0127] FIG. 23D contains graphs summarizing ELISAs measuring the ratio of phosphorylated to total GSK-3[3 protein as well as total [3-catenin protein every day of stage 1 without or with latA treatment (two-way unpaired t-test at each time point, n = 4).

[0128] FIG. 24A is a schematic diagram illustrating gradients of signaling factors patterning the gut tube into different organ-forming regions during development. The SC-islet differentiation protocol shown in FIG. 2A attempts to replicate these signaling dynamics to progressively specify the gut tube, then the pancreatic region, and finally the endocrine portion of the pancreas.

[0129] FIG. 24B contains violin plots from the single cell RNA-sequencing data presented in FIG. 5 at the intermediate stages of differentiation (s2d1 , s3d1 , s4d1 , and s5d1 ) either without or with latA treatment.

[0130] FIG. 24C contains violin plots from the single cell RNA-sequencing data presented in FIG. 5 at the intermediate stages of differentiation (s2d1 , s3d1 , s4d1 , and s5d1 ) either without or with latA treatment.

[0131] FIG. 24D contains violin plots from the beta cell and enterochromaffin cell clusters on stage 6, day 7 from the single cell RNA-sequencing data presented in FIG. 5.

[0132] FIG. 25A contains immunostaining images of cells either without or with latA treatment at the intermediate stages of differentiation (s2d1 , s3d1 , and s4d1 ) stained for 0TX2 and PDX1 . Scale bar = 100 pm.

[0133] FIG. 25B contains Western blot images for markers in cells either without or with latA treatment at the intermediate stages of differentiation (s2d1 , s3d1 , and s4d1 ) (n = 3). Blots for each marker were stripped and re-probed for GAPDH, which was used as the loading control. Unprocessed western blot scans are shown in Figure 27.

[0134] FIG. 25C contains immunostaining images of cells either without or with latA treatment at stage 4, day 1 stained for ONECUT 1 . Scale bar = 100 pm. All assays were performed with the HUES8 cell line.

[0135] FIG. 26A contains single-nuclei multi-omic sequencing UMAP data on stage 6, day 15 combining both RNA expression and ATAC chromatin accessibility for each cell.

[0136] FIG. 26B contains a heatmap of the mRNA expression for these gene promoters in each cluster. FIG. 26C contains a heatmap of the chromatin accessibility for these gene promoters in each cluster.

[0137] FIG. 27A unprocessed scans of western blots for NKX6-1 on s5d1. Blots for each marker were stripped and re-probed for GAPDH, which was used as the loading control. The relevant blots in each image are labeled.

[0138] FIG. 27B contains unprocessed scans of western blots for 0TX2 on s2d1 , s3d1 , and s4d1 . Blots for each marker were stripped and re-probed for GAPDH, which was used as the loading control. The relevant blots in each image are labeled.

[0139] FIG. 27C contains unprocessed scans of western blots for PDX1 on s2d1 , s3d1 , and s4d1 . Blots for each marker were stripped and re-probed for GAPDH, which was used as the loading control. The relevant blots in each image are labeled.

[0140] FIG. 27D contains unprocessed scans of western blots for CYP26A1 and CRABP2 on s2d1 . Blots for each marker were stripped and re-probed for GAPDH, which was used as the loading control. The relevant blots in each image are labeled.

[0141] FIG. 28 contains a series of microscopic images of pancreatic progenitor cells contacted with latrunculin A at different concentrations on the first day of the development of human pluripotent stem cell derived [3-cells from cells at pancreatic progenitor stage 1 cells. Higher concentrations of latA were associated with more homogeneous cell layers.

[0142] FIG. 29 contains bar graphs summarizing the insulin secretion of human pluripotent stem cell (HUES8) derived [3-cells produced from contacting cells at pancreatic progenitor stage 1 with various concentrations of actin depolymerizing compounds, including cytochalasin D, latrunculin B, and latrunculin A at different concentrations on the first day of stage 1 of the pancreatic progenitor cells. The effects of cytochalasin D and latrunculin B exhibited similar effects to the effects of latrunculin A at improving the function of SC-|3 cells when added for the first 24 hours of stage 1 of pancreatic progenitor cells

[0143] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0144] DETAILED DESCRIPTION OF THE INVENTION

[0145] The present disclosure is based, at least in part, on the discovery that the generation of insulin-producing islets from human pluripotent stem cells (hPSCs) is improved by treatment with latrunculin A (latA).

[0146] One aspect of the present disclosure provides methods to improving generation of insulin-producing islets from human pluripotent stem cells (hPSCs). Here we demonstrated that cytoskeletal state at the onset of differentiation is critical for the exit of hPSCs from pluripotency and subsequent definitive endoderm formation. Specifically, depolymerizing the actin cytoskeleton with latA during the first 24 hours of differentiation facilitated a more robust exit from pluripotency and altered signaling dynamics during definitive endoderm formation, modifying the temporal activation of the Activin / Nodal, BMP, JNK-JUN, and WNT pathways. These changes in stage 1 signaling influenced downstream patterning of the gut tube, including modified expression of components of the RA signaling pathway and altered HOX gene expression. Subsequently, latA treated cells exhibited improved pancreatic identity by s4d1 and decreased expression of genes associated with other endodermal lineages along the gut tube, such as AFP (liver) and CDX2 (intestine). This improved pancreatic specification led to an increased percentage of PDX1 + / NKX6.1 + pancreatic progenitors by the end of stage 4 and ultimately generated SC-islets with a higher percentage of [3 cells and lower percentage of enterochromaffin cells by the end of the protocol. These SC-islets contained [3 cells with a more mature identity and greatly improved insulin secretion in response to glucose stimulation. Furthermore, this latA treatment could rescue differentiations from inconsistent hPSC lines in batches that otherwise failed to produce endocrine cells.

[0147] In various aspects the disclosed method comprises contacting the stem cells with a cytoskeleton depolymerizing agent including, but not limited to, latrunculin A (latA), latrunculin B, cytochalasin D, and any combination thereof, during a process of initial stem cell differentiation into definitive endoderm cells. In some aspects, the stem cells are contacted with latA during the initial differentiation process. A detailed description of a suitable process for producing stem cell- derived stem cell derived [3-cells and islets from pluripotent stem cells is described in Hogrebe, N. J. et al., Nat. Protoc. 16, 4109-4143 (2021), the content of which is incorporated by reference herein in its entirety.

[0148] In some aspects, the at least one human pluripotent stem cell is contacted with the latA at a concentration ranging from about 0.01 pM to about 1 pM. In other aspects, the at least one human pluripotent stem cell is contacted with the latA at a concentration ranging from about 0.01 pM to about 0.03 pM, from about 0.02 pM to about 0.04 pM, from about 0.03 pM to about 0.05 pM, from about

[0149] 0.04 pM to about 0.06 pM, from about 0.05 pM to about 0.07 pM, from about

[0150] 0.06 pM to about 0.08 pM, from about 0.07 pM to about 0.09 pM, from about

[0151] 0.08 pM to about 0.10 pM, from about 0.09 pM to about 0.11 pM, from about 0.1 pM to about 0.3 pM, from about 0.2 pM to about 0.4 pM, from about 0.3 pM to about 0.5 pM, from about 0.4 pM to about 0.6 pM, from about 0.5 pM to about 0.7 pM, from about 0.6 pM to about 0.8 pM, from about 0.7 pM to about 0.9 pM, and from about 0.8 pM to about 1 pM.

[0152] The disclosed results show how refining earlier stages of a differentiation protocol, particularly in the context of modifying cytoskeletal state, can improve the final cell product. Directed hPSC differentiations are very dependent on factor concentration and timing, and thus small changes early on can have large effects on the final cell populations. In this case, we altered the exit of hPSCs from pluripotency and subsequent definitive endoderm formation by chemically modulating the actin cytoskeleton, which influenced downstream patterning of the gut tube. While the differences in these cell populations were subtle at certain stages, there were several critical signaling changes that allowed the latA-treated cells to be better specified as pancreatic progenitors, which then gave rise to better SC-islets. Furthermore, our results demonstrate that refining pancreatic progenitor generation influenced the decision between an enterochromaffin or SC-|3 cell fate. Hence, revisiting early stages of other hPSC differentiation protocols to refine the exit of hPSCs from pluripotency and subsequent generation of progenitor populations through cytoskeletal modulation may improve specification of the final desired cell types. SCREENING

[0153] Also provided are methods for screening. The screening method can comprise providing a generated cell by any of the methods described herein and introducing a compound or composition (e.g., a secretagogue) to the cell. For example, the screening method can be used for drug screening or toxicity screening on any cell of endodermal lineage or beta cell provided herein.

[0154] The subject methods find use in the screening of a variety of different candidate molecules (e.g., potentially therapeutic candidate molecules). Candidate substances for screening according to the methods described herein include, but are not limited to, fractions of tissues or cells, nucleic acids, polypeptides, siRNAs, antisense molecules, aptamers, ribozymes, triple helix compounds, antibodies, and small (e.g., less than about 2000 mw, or less than about 1000 mw, or less than about 800 mw) organic molecules or inorganic molecules including but not limited to salts or metals.

[0155] Candidate molecules encompass numerous chemical classes, for example, organic molecules, such as small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons. Candidate molecules can comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, and usually at least two of the functional chemical groups. The candidate molecules can comprise cyclical carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups.

[0156] A candidate molecule can be a compound in a library database of compounds. One of skill in the art will be generally familiar with, for example, numerous databases for commercially available compounds for screening (see e.g., ZINC database, LICSF, with 2.7 million compounds over 12 distinct subsets of molecules; Irwin and Shoichet (2005) J Chem Inf Model 45, 177-182). One of skill in the art will also be familiar with a variety of search engines to identify commercial sources or desirable compounds and classes of compounds for further testing (see e.g., ZINC database; eMolecules.com; and electronic libraries of commercial compounds provided by vendors, for example: ChemBridge, Princeton BioMolecular, Ambinter SARL, Enamine, ASDI, Life Chemicals etc.).

[0157] Candidate molecules for screening according to the methods described herein include both lead-like compounds and drug-like compounds. A lead-like compound is generally understood to have a relatively smaller scaffold-like structure (e.g., molecular weight of about 150 to about 350 kD) with relatively fewer features (e.g., less than about 3 hydrogen donors and / or less than about 6 hydrogen acceptors; hydrophobicity character xlogP of about -2 to about 4) (see e.g., Angewante (1999) Chemie Int. ed. Engl. 24, 3943-3948). In contrast, a drug-like compound is generally understood to have a relatively larger scaffold (e.g., molecular weight of about 150 to about 500 kD) with relatively more numerous features (e.g., less than about 10 hydrogen acceptors and / or less than about 8 rotatable bonds; hydrophobicity character xlogP of less than about 5) (see e.g., Lipinski (2000) J. Pharm. Tox. Methods 44, 235-249). Initial screening can be performed with lead-like compounds.

[0158] When designing a lead from spatial orientation data, it can be useful to understand that certain molecular structures are characterized as being “druglike”. Such characterization can be based on a set of empirically recognized qualities derived by comparing similarities across the breadth of known drugs within the pharmacopoeia. While it is not required for drugs to meet all, or even any, of these characterizations, it is far more likely for a drug candidate to meet with clinical successful if it is drug-like.

[0159] Several of these “drug-like” characteristics have been summarized into the four rules of Lipinski (generally known as the “rules of fives” because of the prevalence of the number 5 among them). While these rules generally relate to oral absorption and are used to predict bioavailability of compound during lead optimization, they can serve as effective guidelines for constructing a lead molecule during rational drug design efforts such as may be accomplished by using the methods of the present disclosure.

[0160] The four “rules of five” state that a candidate drug-like compound should have at least three of the following characteristics: (i) a weight less than 500 Daltons; (ii) a log of P less than 5; (iii) no more than 5 hydrogen bond donors (expressed as the sum of OH and NH groups); and (iv) no more than 10 hydrogen bond acceptors (the sum of N and O atoms). Also, drug-like molecules typically have a span (breadth) of between about 8A to about 15A.

[0161] FORMULATION

[0162] The agents and compositions described herein can be formulated by any conventional manner using one or more pharmaceutically acceptable carriers or excipients as described in, for example, Remington’s Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005), incorporated herein by reference in its entirety. Such formulations will contain a therapeutically effective amount of a biologically active agent described herein, which can be in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.

[0163] The term "formulation" refers to preparing a drug in a form suitable for administration to a subject, such as a human. Thus, a "formulation" can include pharmaceutically acceptable excipients, including diluents or carriers.

[0164] The term "pharmaceutically acceptable" as used herein can describe substances or components that do not cause unacceptable losses of pharmacological activity or unacceptable adverse side effects. Examples of pharmaceutically acceptable ingredients can be those having monographs in United States Pharmacopeia (USP 29) and National Formulary (NF 24), United States Pharmacopeial Convention, Inc, Rockville, Maryland, 2005 ("USP / NF"), or a more recent edition, and the components listed in the continuously updated Inactive Ingredient Search online database of the FDA. Other useful components that are not described in the USP / NF, etc. may also be used.

[0165] The term “pharmaceutically acceptable excipient,” as used herein, can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic, or absorption delaying agents. The use of such media and agents for pharmaceutical active substances is well known in the art (see generally Remington’s Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005)). Except insofar as any conventional media or agent is incompatible with an active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0166] A "stable" formulation or composition can refer to a composition having sufficient stability to allow storage at a convenient temperature, such as between about 0 °C and about 60 °C, for a commercially reasonable period of time, such as at least about one day, at least about one week, at least about one month, at least about three months, at least about six months, at least about one year, or at least about two years.

[0167] The formulation should suit the mode of administration. The agents of use with the current disclosure can be formulated by known methods for administration to a subject using several routes which include, but are not limited to, parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal. The individual agents may also be administered in combination with one or more additional agents or together with other biologically active or biologically inert agents. Such biologically active or inert agents may be in fluid or mechanical communication with the agent(s) or attached to the agent(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophilic or other physical forces.

[0168] Controlled-release (or sustained-release) preparations may be formulated to extend the activity of the agent(s) and reduce dosage frequency. Controlled- release preparations can also be used to affect the time of onset of action or other characteristics, such as blood levels of the agent, and consequently affect the occurrence of side effects. Controlled-release preparations may be designed to initially release an amount of an agent(s) that produces the desired therapeutic effect, and gradually and continually release other amounts of the agent to maintain the level of therapeutic effect over an extended period of time. In order to maintain a near-constant level of an agent in the body, the agent can be released from the dosage form at a rate that will replace the amount of agent being metabolized or excreted from the body. The controlled release of an agent may be stimulated by various inducers, e.g., change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules.

[0169] Agents or compositions described herein can also be used in combination with other therapeutic modalities, as described further below. Thus, in addition to the therapies described herein, one may also provide to the subject other therapies known to be efficacious for treatment of the disease, disorder, or condition.

[0170] THERAPEUTIC METHODS

[0171] Also provided is a process of using generated cells for cell replacement therapies or stem cell transplant. For example, the disclosed compositions and methods can be used to treat diabetes or other disease associated with dysfunctional endodermal cells in a subject in need of administration of a therapeutically effective number of cells of endodermal lineage or beta cells, so as to induce insulin secretion.

[0172] As defined herein, the term “diabetes” refers to any form of diabetes including, but not limited to, type 1 diabetes mellitus (T1 D), type 2 diabetes mellitus (T2D), gestational diabetes mellitus, latent autoimmune diabetes of adults (LADA), and maturity onset diabetes of the young (MODY). Although the compositions and methods are disclosed herein in the context of T1 D, it is to be understood that the disclosed compositions and methods may be used with minimal modification for the treatment of any of the forms of diabetes described above.

[0173] Methods described herein are generally performed on a subject in need thereof. A subject in need of the therapeutic methods described herein can be a subject having diagnosed with, suspected of having, or at risk for developing a diabetes or other disease associated with dysfunctional endodermal cells. A determination of the need for treatment will typically be assessed by a history and physical exam consistent with the disease or condition at issue. Diagnosis of the various conditions treatable by the methods described herein is within the skill of the art. The subject can be an animal subject, including a mammal, such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and chickens, and humans. For example, the subject can be a human subject.

[0174] Generally, a safe and effective number of cells of endodermal lineage (e.g., insulin-expressing cells (e.g., [3 cells, SC-|3 cells)), is, for example, an amount that would cause the desired therapeutic effect in a subject while minimizing undesired side effects. In various embodiments, an effective amount of endodermal lineage or beta cells described herein can respond to glucose by secretion of insulin. In various embodiments, an effective number of cells described herein can treat diabetes or other disease associated with dysfunctional endodermal cells, substantially inhibit diabetes or other disease associated with dysfunctional endodermal cells, slow the progress of diabetes or other disease associated with dysfunctional endodermal cells, or limit the development of diabetes or other disease associated with dysfunctional endodermal cells.

[0175] According to the methods described herein, administration can be a cell transplantation, cell implantation, parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.

[0176] When used in the treatments described herein, a therapeutically effective amount of beta cells or cells of endodermal lineage can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form and with or without a pharmaceutically acceptable excipient. For example, the compounds of the present disclosure can be administered, at a reasonable benefit / risk ratio applicable to any medical treatment, in a sufficient amount to induce insulin secretion.

[0177] The amount of a composition described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of agent contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses.

[0178] Toxicity and therapeutic efficacy of compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals for determining the LD50 (the dose lethal to 50% of the population) and the ED50, (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio LD50 / ED50, where larger therapeutic indices are generally understood in the art to be optimal. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the seventy of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e.g., Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics, 4thed., Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw-Hill / Appleton & Lange, ISBN 0071375503). For example, it is well within the skill of the art to start doses of the composition at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration. Consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. It will be understood, however, that the total daily usage of the compounds and compositions of the present disclosure will be decided by an attending physician within the scope of sound medical judgment.

[0179] Again, each of the states, diseases, disorders, and conditions, described herein, as well as others, can benefit from compositions and methods described herein. Generally, treating a state, disease, disorder, or condition includes preventing or delaying the appearance of clinical symptoms in a mammal that may be afflicted with or predisposed to the state, disease, disorder, or condition but does not yet experience or display clinical or subclinical symptoms thereof. Treating can also include inhibiting the state, disease, disorder, or condition, e.g., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof. Furthermore, treating can include relieving the disease, e.g., causing regression of the state, disease, disorder, or condition or at least one of its clinical or subclinical symptoms. A benefit to a subject to be treated can be either statistically significant or at least perceptible to the subject or to a physician. Administration of cells of endodermal lineage or beta cells can occur as a single event or over a time course of treatment. For example, cells of endodermal lineage or beta cells can be administered daily, weekly, bi-weekly, or monthly. For treatment of acute conditions, the time course of treatment will usually be at least several days. Certain conditions could extend treatment from several days to several weeks. For example, treatment could extend over one week, two weeks, or three weeks. For more chronic conditions, treatment could extend from several weeks to several months or even a year or more.

[0180] Treatment in accord with the methods described herein can be performed prior to, concurrent with, or after conventional treatment modalities for diabetes or other disease associated with dysfunctional endodermal cells.

[0181] KITS

[0182] Also provided are kits. Such kits can include an agent or composition described herein and, in certain embodiments, instructions for administration. Such kits can facilitate performance of the methods described herein. When supplied as a kit, the different components of the composition can be packaged in separate containers and admixed immediately before use. Components include, but are not limited to stem cells, media, and factors as described herein, including, but not limited to, the cytoskeleton depolymerizing agent to be contacted with the stem cells during initial stem cell differentiation into definitive endoderm cells. Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil such as a blister pack. Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components.

[0183] Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately. For example, sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline each of which has been packaged under a neutral non-reacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules, and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix. Removable membranes may be glass, plastic, rubber, and the like.

[0184] In certain embodiments, kits can be supplied with instructional materials. Instructions may be printed on paper or other substrate, and / or may be supplied as an electronic-readable medium or video. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit.

[0185] A control sample or a reference sample as described herein can be a sample from a healthy subject. A reference value can be used in place of a control or reference sample, which was previously obtained from a healthy subject or a group of healthy subjects. A control sample or a reference sample can also be a sample with a known amount of a detectable compound or a spiked sample.

[0186] Compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001 ) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005) Protein Expr Purif. 41 (1 ), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253). Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0187] In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.” In some embodiments, the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. The recitation of discrete values is understood to include ranges between each value.

[0188] In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0189] The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

[0190] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0191] Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0192] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

[0193] Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.

[0194] EXAMPLES

[0195] The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

[0196] EXAMPLE 1: DEPOLYMERIZING F-ACTIN ACCELERATES THE EXIT FROM PLURIPOTENCY TO ENHANCE STEM CELL-DERIVED ISLET DIFFERENTIATION

[0197] Pancreatic [3 cells regulate glucose metabolism throughout the body by secreting insulin into the bloodstream, facilitating glucose uptake by many cell types so that it can be utilized for energy production and fat storage. Within the pancreas, [3 cells are clustered together with other endocrine cell types to form the islets of Langerhans. In type 1 diabetes (T1 D), [3 cells are erroneously targeted by the immune system and selectively eliminated. As a result, T1 D patients must inject exogenous insulin in order to restore proper insulin signaling throughout the body, which is required for survival. Replicating the precise insulin secretion kinetics of [3 cells with insulin injections can be difficult because insulin requirements change dynamically based on factors related to energy metabolism, such as the amount and types of food consumed, the duration and intensity of physical activity, and the levels of circulating stress hormones. Over time, chronically elevated blood glucose levels can lead to severe complications, such as cardiovascular, kidney, and eye disease, emphasizing the importance of keeping blood glucose levels as close to normal as possible.

[0198] In an effort to improve blood glucose control and ease the burden of diabetes management, primary [3 cells from deceased donors have been transplanted into patients with T1 D, demonstrating that replacing this lost [3 cell mass is a safe and effective method to restore glucose homeostasis. However, issues with donor quality, quantity, and immunogenicity have hampered its widespread use. As an alternative, stem cell-derived islets (SC-islets) containing stem cell-derived [3 cells (SC-[3 cells) and other pancreatic endocrine cell types could provide an unlimited source of islets for transplantation. These SC-islets can be derived from a single stem cell source that is extensively characterized, resulting in more reproducible transplant outcomes. Furthermore, these cells can be gene-edited to have desirable characteristics that enhance transplant results, such as decreasing their immunogenicity to circumvent the need for immunosuppressive drugs or making them more resistant to the stresses experienced during transplantation. SC-islets can also be utilized as an effective in vitro model to study different aspects of the disease and develop additional therapeutic strategies.

[0199] To this end, directed differentiation protocols have been developed in recent years to generate SC-islets using a stepwise combination of growth factors and small molecules to drive hPSCs through several intermediate cell types on their way to becoming pancreatic endocrine cells. While there are a number of different iterations and improvements that have been made since the original development of these protocols, all successful SC-islet differentiation methods follow the same basic strategy. Specifically, they attempt to recreate the specific sequence of signaling events that must occur to form the pancreas during embryonic development. This process begins with the specification of the definitive endoderm germ layer from hPSCs, which is driven by a coordination of Activin / Nodal, WNT, and BMP signaling. This definitive endoderm is then specified as the primitive gut tube, which will eventually form the organs of the gastrointestinal and respiratory tracts. The primitive gut tube is progressively segmented into the foregut, midgut, and hindgut through signaling gradients of FGF, BMP, WNT, and retinoic acid (RA) ligands that are secreted from the splanchnic mesodermal tissue surrounding the gut tube, as well as Hedgehog and Notch signaling originating in the gut tube itself. The intensity and timing of these signals induce gene expression patterns that segment the gut tube into the various organ-forming regions. For example, S0X2 expression is restricted to the foregut, while CDX2 expression is localized to the mid- and hindgut. Furthermore, differential expressions of various HOX genes along the gut tube provide a spatial code that helps guide these different regions to develop into their respective organs. Cells of the foregut will subsequently generate organs such as the thyroid, thymus, trachea, lungs, esophagus, stomach, pancreas, and liver, while the midgut forms the small intestine and the hindgut develops into the large intestine. The pancreas arises from the posterior segment of the foregut, where high RA signaling helps induce the expression of PDX1. These multipotent PDX1 + pancreatic progenitors subsequently develop into duct and acinar exocrine cells as well as all the endocrine cell types of the islet.

[0200] In this study, we modulated the state of the actin cytoskeleton to alter signaling dynamics during the exit of hPSCs from pluripotency and subsequent definitive endoderm formation, which influenced downstream patterning of the gut tube to improve SC-islet differentiation outcomes. Specifically, depolymerizing the actin cytoskeleton with the compound latA during the first 24 hours of differentiation facilitated a more robust exit from pluripotency and induced a unique pattern of Activin / Nodal, BMP, JNK-JUN, and WNT signaling during definitive endoderm formation that biased these cells toward a pancreatic fate. LatA-treated cells subsequently differentiated into SC-islets with a higher percentage of SC-|3 cells as well as fewer enterochromaffin cells, which are a type of intestinal endocrine cell that is typically generated during these SC-islet differentiation protocols. Importantly, depolymerizing the actin cytoskeleton with latA during the first 24 hours of differentiation drastically improved multiple components of SC-|3 cell function by the end of the protocol 5 weeks later, including higher insulin content and increased insulin secretion in response to glucose stimulation. Not only did this latA treatment improve insulin secretion of two stem cell lines that already generated functional SC-islets reliably, but it also rescued two hPSC lines that failed to consistently produce SC-islets. Overall, this work demonstrates the importance of cytoskeletal state during the exit of hPSCs from pluripotency as well as highlights how early interventions that refine patterning of the gut tube to better specify pancreatic progenitors can improve SC-islet generation.

[0201] RESULTS Depolymerizing the actin cytoskeleton during the first 24 hours of differentiation improved the specification to and function of SC-islets

[0202] Previously, we demonstrated that the polymerization state of the actin cytoskeleton was critical for the conversion of pancreatic progenitors to pancreatic endocrine and exocrine cells. Specifically, a highly polymerized actin cytoskeleton induced by culture on stiff polystyrene plasticware blocked NEUR0G3-induced endocrine formation, keeping the cells in a pancreatic progenitor state despite the presence of endocrine-inducing factors. Depolymerizing F-actin at the onset of endocrine induction with the compound latA, however, facilitated the expression of NEUR0G3 and the initiation of the endocrine program even when the cells continued to be grown on the stiff tissue culture polystyrene substrate.

[0203] Since the cytoskeleton is important to tissue morphogenesis throughout development, we wanted to investigate if cytoskeletal state was also important to other stages of the in vitro directed differentiation of hPSCs to SC-islets. While chemically modulating the cytoskeleton at various other stages most often negatively influenced differentiation outcomes, we observed a more diverse response to cytoskeletal manipulation during definitive endoderm formation (Fig. 1A-B, Fig. 7A). In particular, adding compounds that depolymerized F-actin during the first 24 hours of stage 1 (Fig. 7A) resulted in SC-islets that secreted more insulin in response to glucose stimulation by the end of the 5-week protocol (Fig. 1 C). In contrast, compounds that increased actin polymerization during this initial 24-hour period generated non-endocrine cell types (Fig. 1 B, Fig. 7C-E) and decreased insulin secretion of the final cell clusters (Fig. 1 C).

[0204] In agreement with the improved SC-islet differentiation, cells receiving compounds that depolymerized F-actin (Fig. 1 D, Fig. 7A) displayed reduced expression of pluripotency markers 0CT4, NANOG, S0X2) by the end of stage 1 (Fig. 1 E-F), indicating that these cells experienced a more robust exit from pluripotency. In contrast, compounds that induced actin polymerization (Fig. 1 D, Fig. 7A) exhibited much higher levels of these pluripotency genes (Fig. 1 E-F), the extent of which was dependent upon the compound and degree of actin polymerization. While we initially tried to polymerize actin directly with jasplakinolide, we observed extensive cell death before reaching concentrations that induced visible actin polymerization. Thus, we utilized compounds that induced actin polymerization indirectly through other mechanisms. Specifically, nocodazole depolymerizes microtubules, which have been shown to induce actin hypercontractility and polymerization. Sphingosine-1 -phosphate (S1 P), on the other hand, is a sphingolipid that has been shown to increase actin polymerization via activation of the Rho family of GTPases. Nocodazole specifically led to increased cortical F-actin as the cells rounded up after microtubule depolymerization, while S1 P led to a more robust overall increase in actin polymerization (Fig. 1 D, Fig. 7A). Consequently, S1 P-treated cells displayed the highest expression of pluripotency genes at the end of stage 1 (Fig. 1 E-F), the most difficulty in forming FOXA2+ / SOX17+ definitive endoderm (Fig. 7B), and subsequently the fewest number of endocrine cells by the end of the protocol (Fig. 1 B-C, Fig. 7C-D). Thus, a polymerized actin cytoskeleton appeared to present a barrier to hPSC exit from pluripotency and subsequent differentiation to definitive endoderm and SC-islets, which was overcome with chemical depolymerization at the onset of differentiation.

[0205] Because the polymerization state of the actin cytoskeleton appeared to alter the exit of stem cells from pluripotency during directed differentiation to definitive endoderm, we tested its influence on the specification to the other germ layers using their respective differentiation protocols. Using a commercially available tri-lineage differentiation kit, we first replicated these results for the differentiation of stem cells to definitive endoderm. Specifically, depolymerizing F-actin with latA for the first 24 hours of differentiation decreased expression of pluripotency genes by the end of this definitive endoderm protocol, while increasing actin polymerization with S1 P both maintained expression of pluripotency genes and reduced expression of the endoderm marker F0XA2 (Fig. 1 L, upper right). We observed similar results with the directed differentiation to mesoderm, where latA treatment reduced pluripotency gene expression while simultaneously increased expression of the important mesoderm marker TBXT (Fig. 1 L, lower left). In contrast, inducing actin polymerization with S1 P during a directed ectoderm protocol greatly increased the expression of the key ectoderm marker PAX6 and reduced pluripotency gene expression (Fig. 1 L, lower right). Thus, controlling the polymerization state of the actin cytoskeleton during the first 24 hours of differentiation influenced the exit of stem cells from pluripotency and could drastically improve key marker expression of each germ layer, but these effects were context dependent. Specifically, a depolymerized actin cytoskeleton at the onset of differentiation promoted mesendoderm lineages, while a polymerized actin cytoskeleton facilitated directed ectoderm differentiation.

[0206] To further explore the benefits of depolymerizing the actin cytoskeleton during the onset of differentiation, we tested a range of latA concentrations for the first 24 hours of stage 1 and continued the remainder of the SC-islet protocol as normal (Fig. 1 G). Increasing latA concentration progressively induced a round morphology (Fig. 8A) and disrupted actin polymerization during this initial 24- hour period (Fig. 8B). Once latA was removed, the cells flattened out again for the rest of stage 1 (Fig. 8C-D), as they were once again able to polymerize their actin cytoskeleton to pull against the tissue culture substrate. Interestingly, there was consistently more cell death throughout the rest of stage 1 even after latA was removed, particularly on days 3 and 4. However, a monolayer of cells remained attached to the plate throughout stage 1 at these latA concentrations (Fig. 8C-D). If the latA concentration was too high, however, cell density ended up being too low by the end of stage 1 , and these cells were unable to make endocrine cells by the end of the protocol (Fig. 8E).

[0207] We evaluated 4 hPSC lines with this latA treatment. Based on our prior work, the HUES8 and WS4c lines reliably generate high-functioning SC-islets, while differentiation from the AN1 .1 and H1 lines is less efficient with our previous protocol. In this study, we found that 0.175 pM seemed to be the highest latA concentration that consistently resulted in high quality SC-islets by the end of the protocol for the HUES8, WS4c, and AN1.1 stem cell lines. The H1 stem cell line was more sensitive to this stage 1 latA treatment, and thus a maximum of 0.1375 pM latA was used for all H1 experiments. Strikingly, this latA treatment during the first 24 hours of the differentiation led to a dose-dependent increase in the function of SC-islets generated from these 4 hPSC lines by the end of the 5-week protocol. Specifically, this treatment drastically increased insulin secretion in both static (Fig. 1 H) and dynamic (Fig. 1 K) glucose- stimulated insulin secretion (GSIS) assays as well as in response to various secretagogues (Fig. 11, Fig. 9A). These SC-islets also exhibited much higher insulin content (Fig. 1 J) while retaining a similar proinsulin to insulin content ratio (Fig. 9B). With these improvements, the SC-islets approached the insulin secretion levels of primary human islets in response to glucose stimulation, particularly with the HUES8 and WS4c lines, and exceeded it in response to KCI stimulation for all 4 cell lines at the highest latA concentration. Similarly, SC- islets from all 4 cell lines had higher insulin content on a per cell basis than primary human islets at the highest latA concentration.

[0208] Latrunculin A treatment at the onset of definitive endoderm formation improved SC-islet composition by the end of the directed differentiation protocol. Upon further characterization, we observed that this stage 1 latA treatment consistently increased the number of SC-|3 cells by approximately 5-10% for any given batch (Fig. 2A-B), and this effect was dose-dependent (Fig. 9C). For the HUES8, WS4c, and AN1 .1 cell lines, the total number of CHGA+ endocrine cells did not change with the latA treatment, while the number of SCL18A1 + enterochromaffin cells decreased by approximately 5-15% (Fig. 2A). Thus, the stage 1 latA treatment improved the ratio of SC-|3 cells and the off-target enterochromaffin cells with these cell lines. Furthermore, the expression of genes associated with [3 cells (e.g, / / VS, IAPP) increased, while those associated with off-target lineages (e.g., TPH1 for enterochromaffin cells, CDX2 for intestinal cells, AFP for liver cells) decreased (Fig. 2E, Figs. 9F, 9H, and 9J). In the H1 line, the latA treatment appeared to also increase overall endocrine differentiation efficiency as indicated by an increase in the total number of CHGA+ cells, and thus there were also more SLC18A1 + cells in addition to the increase in SC-|3 cells (Fig. 2A). Re-normalizing the GSIS results to the number of SC-p cells rather than the total number of cells in the SC-islets (Fig. 9D) revealed that latA-treated cells still exhibited higher insulin secretion, demonstrating that the stage 1 latA treatment not only increased the number of SC-p cells but also improved the insulin secretion per SC-p cell.

[0209] Stage 1 latA treatment rescued differentiation to SC-islets from inconsistent hPSC lines

[0210] Throughout the protocol, the morphology of the latA-treated cells appeared more homogenous than the control (Fig. 10A-B), though both conditions for all 4 hPSC lines could generate high quality endocrine cells that stained for SC-p cell markers (Fig. 2C, Fig. 9E,G, I) and could be aggregated into islet-like clusters (Fig. 2D, Fig. 11A-B). SC-islet yields with this stage 1 latA treatment were similar to our previously reported values for this differentiation process, typically generating between 0.5 and 0.75 million total cells per cm2(Figure 11 C). The HUES8 and WS4c lines always yielded successful differentiations both with and without the stage 1 latA treatment, consistent with our previous publications demonstrating that these lines reliably generate high quality SC-islets that can cure severe pre-existing diabetes in mice without any signs of tumor formation. Differentiations of the AN1 .1 and H1 lines without the stage 1 latA treatment were inconsistent, however, often generating a mix of off- target and endocrine cell types or occasionally not producing any endocrine cells at all (Fig. 2F-G, Fig. 12A-C). The H1 line has also been noted by others for being inefficient at differentiating to SC-islets. Strikingly, the stage 1 latA treatment rescued batches of cells in which the control differentiation failed to generate endocrine cells (Fig. 2F-G, Figure 12A-C). This effect was dosedependent, with the highest latA concentrations restoring the typical percentage (~30-50%) of SC-[3 cells generated by the end of the protocol (Fig. 2H, Fig. 12E). Interestingly, the control differentiations for the AN1.1 and H1 lines retained much higher expression of pluripotency genes by the end of stage 1 (Fig. 2I, Fig. 12D), mimicking the phenotype observed after treatment with actin-polymerizing compounds (Fig. 1 F). These data suggest that the AN 1.1 and H1 hPSC lines normally have trouble exiting pluripotency at the onset of differentiation, resulting in inefficiencies in pancreatic specification later on. In contrast, the latA treatment drastically decreased expression of OCT4, NANOG, and S0X2 by the end of stage 1 in all four hPSC lines, further suggesting that the latA treatment helps hPSCs exit pluripotency more robustly (Fig. 2I, Fig. 12D).

[0211] Off-target or residual undifferentiated cells produced during SC-islet differentiations as a result of inefficient exit from the pluripotent state are an important safety concern for a cell-based therapy, as they could form tumors upon transplantation.

[0212] Latrunculin A treatment at the onset of definitive endoderm formation generated SC-islets from the AN1 .1 cell line that rapidly cured severe preexisting diabetes in mice. To further demonstrate the drastic improvement that this stage 1 latA treatment could have on SC-islet generation, function, and safety, we transplanted SC-islets generated both with and without the stage 1 latA treatment using the AN1 .1 hPSC line under the kidney capsule of severely diabetic mice. SC-islets generated with this induced pluripotent stem cell (iPSC) line have not been previously transplanted into mice. The fasting blood glucose levels of the mice receiving the SC-islets generated without the latA treatment (AN1 .1 - control) came down gradually, finally dipping below 200 mg / dL by week 11 (Fig. 3A). In contrast, the fasting blood glucose of the mice transplanted with the latA-treated SC-islets (AN1 .1 - 0.175 pM latA) fell below 200 mg / dL between weeks 1 and 2 (Fig. 3A). By week 5, the fasting blood glucose of these mice was approximately 80 mg / dL, matching the blood glucose levels of mice transplanted with primary human islets (Fig. 3A). The fasting blood glucose of the mice transplanted with the AN1 .1 - 0.175 pM latA SC-islets remained between approximately 70-80 mg / dL for the remainder of the experiment (30 weeks total), mimicking the fasting blood glucose set point in humans. Similarly, random blood glucose measurements of these mice averaged 146 mg / dL by week 6 and came down further overtime (Fig. 13A). A glucose tolerance test at week 8 demonstrated that the AN 1.1 - 0.175 pM latA SC-islets were much better at controlling blood glucose levels than the control cells (Fig. 3A). Furthermore, they secreted much more insulin and were glucose responsive in vivo (Fig. 3B), mirroring the large difference observed during in vitro GSIS (Fig. 1 H). As they matured overtime in vivo, the AN1.1 - 0.175 pM latA SC-islets secreted more insulin (Fig. 3B), became more glucose responsive (Fig. 3B), and improved their ability to control blood glucose to a level similar as primary human islets (Fig. 3A). The kidneys containing the AN1.1 - 0.175 pM latA SC-islets and primary human islets were removed 30 weeks after transplantation, resulting in a rapid reversal to a diabetic state in these mice (Fig. 3A). These grafts looked healthy with no overgrowths and contained many C-peptide+ / NKX6.1 + SC-|3 cells (Figs. 3C and 3D), similar to the primary human islet grafts (Fig. 13B). In contrast, the AN1 .1 - control SC-islets formed tumors by week 11 (Fig. 3C), despite transplanting batches of cells that successfully generated SC-islets. This tumor formation is unsurprising, however, given the observed off-targets that were often generated with the AN 1.1 line without latA treatment (Fig. 12C). Taken together, these data demonstrate that treating the otherwise inconsistent AN1 .1 hPSC line with latA during the first 24 hours of stage 1 generated SC-islets with drastically improved function that were able to rapidly cure severely diabetic mice to a similar degree as in our previous publications using the HUES8 and WS4c lines and without tumor formation. Stage 1 I at A treatment altered the signaling dynamics of major pathways during the specification of definitive endoderm

[0213] Collectively, these data suggest that cytoskeletal state is critically important for the exit of hPSCs from pluripotency and during definitive endoderm specification, ultimately influencing the subsequent identity and function of differentiated SC-islets. Latrunculin A treatment altered the signaling dynamics of key pathways during endoderm formation. To further elucidate the mechanism by which latA influenced this process, we performed single cell RNA-sequencing every day of stage 1 on HUES8 cells treated with or without 0.175 pM latA for the first 24 hours of differentiation (Fig. 4A). After these initial 24 hours, the latA- treated cells had decreased expression of multiple genes encoding metallothionein proteins (MT1H, MT1G, MT2A, MT1E, MT1F) as well as increased expression of several genes related to histone structure (HIST1H1B, HIST1H1D), inhibition of canonical WNT signaling (DKK1, DKK4), and stress mediation (TXNIP) (Fig. 14A). Expression of actin (ACTB) itself also decreased in response to the latA treatment (Fig. 14A-B). Despite differential expression of these few genes, the control and latA-treated cells clustered similarly for the first several days of stage 1 (Fig. 4A). As stage 1 progressed, however, the latA- treated cells became more distinct, separating from the untreated cells after stage 1 , day 3 (s1d3). By the end of stage 1 , the latA-treated cells formed their own distinct cluster with notable differences in the expression of genes known to be involved in definitive endoderm formation, while the control cells at s2d1 clustered more similarly to the previous day (Fig. 4A-B).

[0214] We compared the gene expression of the control and latA-treated cells throughout stage 1 and observed decreases in the average expression of the pluripotency markers OCT4 (POU5F1), NANOG, and S0X2, as expected (Fig. 4C, Fig. 14C). The mesendoderm markers TBXT and MIXL1 peaked on s1d2 but were downregulated on subsequent days as the cells differentiated into FOXA2+ / SOX17+ definitive endoderm (Fig. 4C, Fig. 14C). While the expression of F0XA2 and S0X17 progressed similarly between the control and latA-treated cells throughout stage 1 , other markers of definitive endoderm exhibited unique expression patterns. Specifically, while CER1 and GSC expressions peaked on s1 d3 in both conditions, their expression was strongly downregulated by the end of stage 1 in the latA-treated cells but maintained in the control cells (Fig. 40, Fig. 14C). Similarly, LHX1, EOMES, and JUN were more downregulated by the end of stage 1 in the latA-treated cells (Fig. 4C, Fig. 14C).

[0215] Identifying some of the most differentially expressed genes at s2d1 revealed further interesting trends of genes related to several major signaling pathways known to be involved in the specification of definitive endoderm (Fig. 4B). Specifically, while the expression levels of the TGF-[3 superfamily members NODAL and its antagonists LEFTY1 and LEFTY2 initially rose in both conditions, they were strongly downregulated in the latA-treated cells by the end of stage 1 (Fig. 4C, Fig. 14C). TAGLN, which encodes the actin-binding protein transgelin and is downstream of TGF-[3 signaling, concurrently rose in the control cells (Fig. 4C, Fig. 14C). Conversely, the expression of the TGF-[3 inhibitor BAMBI and the BMP downstream targets ID1, ID2, ID3, and ID4 all increased drastically during the second half of stage 1 (Fig. 4C, Fig. 14C). The expression of several other genes further distinguished the latA-treated population by the end of stage 1 , including increased GATA3 expression and downregulated GATA6 and FGF17 expression (Fig. 4C, Fig. 14C). Re-clustering of the cells only at s2d1 highlighted the distinct population generated by the latA-treated cells by the end of stage 1 when compared to the control cells (Fig. 15A). Despite most cells expressing the definitive endoderm markers F0XA2 and S0X17 in both conditions, the expression of many of these key genes were constrained to either population (Fig. 15B). qRT-PCR revealed similar expression patterns for these genes in all 4 hPSC lines in response to the stage 1 latA treatment (Fig. 15D). In a parallel experiment, treating hPSCs with latA in mTeSRI altered expression of many of these same genes (e.g., NODAL, LEFTY1, LEFTY2, JUN, TAGLN, ID1, and multiple metallothionein genes), highlighting the strong effect latA has on the expression of these specific genes (Fig. 16A-B). Interestingly, however, a much higher latA concentration was required in mTeSRI to see the same rounding effect on cell morphology when compared to latA added in stage 1 media, both with and without Activin A and CHIR99021 (Fig. 16C).

[0216] In agreement with the gene expression data, single-nuclei ATAC sequencing at the end of stage 1 demonstrated that the control cells had higher promoter chromatin accessibility for NODAL (Figs. 17B and 17D). Furthermore, the control cells had higher motif chromatin accessibility for the SMAD2::SMAD3::SMAD4 and FOS::JUN binding domains, indicative of increased signaling through the Activin / NODAL and JNK-JUN pathways, respectively (Fig. 17B-C). Conversely, latA-treated cells had greater motif accessibility for HNF1A and HNF1B (Fig. 17B-C), which are important during the specification of the gut tube that occurs after definitive endoderm formation. Taken together, these data indicate that while the latA treated cells generated S0X17+I F0XA2+ definitive endoderm in a similar proportion as the untreated control cells, they formed a unique endodermal state that was better at generating functional SC-|3 cells (Figs. 1 and 2). This unique endodermal state appears to be the result of drastic changes in the temporal dynamics of several key signaling pathways in the latA-treated cells, including increased BMP signaling and decreased Activin / Nodal signaling during the second half of stage 1.

[0217] To confirm these signaling changes at the protein level, we immunostained for a number of these markers throughout stage 1 . Mirroring the gene expression patterns, LEFTY1 / 2 and NODAL initially increased in both conditions but were downregulated during the second half of stage 1 in latA- treated cells (Fig. 4D, Fig. 18A-C). C-Jun stained much more intensely on s1d2 in the latA-treated cells but was strongly downregulated by the end of stage 1 in these cells, while the control cells retained c-Jun protein throughout stage 1 (Fig. 4D, Fig. 19B-C). Furthermore, latA-treated cells had essentially eliminated NANOG and 0CT4 protein by the end of stage 1 , while heterogeneous NANOG protein expression and a few Oct4 positive cells persisted in the control (Fig. 4D, Figs. 19A and 19C). Similar to the trends observed in gene expression, the amount of transgelin protein increased as stage 1 progressed in the control but not in latA-treated cells (Fig. 4D, Fig. 20B-C). While latA-treated cells recovered their ability to polymerize actin after latA removal, differences in F-actin staining persisted throughout stage 1 (Fig. 4D, Figs. 19A and 19C). Mimicking the pattern of F-actin staining, [3-catenin became much more localized to the cell membrane in latA-treated cells, suggesting a difference in canonical WNT signaling (Fig. 4D, Fig. 21A-B). Another interesting observation from these immunostaining studies was that, despite all hPSC lines initially staining homogenously for 0CT4 and NANOG to demonstrate pluripotency (Fig. 22A), there was heterogeneity in initial c-Jun and F-actin staining between hPSC lines. Specifically, H1 and AN1.1 hPSCs appeared to have lower overall c-Jun staining than either HUES8 or WS4c, and AN1.1 hPSCs in particular had heterogenous F-actin staining (Fig. 22B).

[0218] As further evidence for these changes in stage 1 signaling dynamics, the downstream Activin / Nodal signaling effector SMAD2 had a decreased percentage of proteins in their phosphorylated state with the latA treatment during the second half of stage 1 , indicating decreased Activin / Nodal signaling during this time (Fig. 4E). Conversely, the BMP signaling effectors SMAD1 and SMAD5 had an increased percentage of phosphorylated proteins during this time period in latA-treated cells, suggesting increased BMP signaling in the second half of stage 1 (Fig. 4E). In support of the notion that BMP signaling is important to definitive endoderm formation and downstream specification of the pancreas, application of the BMP inhibitor LDN193189 from s1 d2 through the end of stage 1 prevented the formation of PDX1 + / NKX6.1 + pancreatic progenitors by s5d1 , even in latA-treated cells (Fig. 23A-B). The dynamics of WNT signaling throughout stage 1 also seemed to be different in latA-treated cells. For example, while the ratio of phosphorylated GSK-3[3 to the total amount of GSK- 3|3 protein was lower in latA-treated cells for most of stage 1 , it increased sharply during the last day, indicating changes in canonical WNT signaling during this time (Fig. 23D). Total [3-catenin protein followed a similar trend (Fig. 23D), and gene expression of WNT signaling components changed during this time as well (Fig. 23C). Collectively, these data demonstrate that treating cells with latA during the first 24 hours of differentiation influenced the signaling dynamics throughout stage 1 of major pathways known to be involved in the specification of definitive endoderm, generating a unique definitive endoderm population by the end of stage 1 .

[0219] Stage 1 latA treatment altered downstream patterning of the gut tube and improved pancreatic progenitor specification

[0220] Latrunculin A treatment at the onset of definitive endoderm formation altered the expression of lineage-specific genes as well as those associated with RA signaling during subsequent gut tube patterning. Despite these differences in major signaling pathways during stage 1 , both conditions yielded close to 100% FOXA2+ / SOX17+ definitive endoderm by the end of stage 1 (Fig. 5A). By s5d1 , however, the latA-treated cells had a higher percentage of PDX1 + / NKX6.1 + cells (Fig. 5B-C, Fig. 23E), indicating better specification to pancreatic progenitors. Furthermore, the latA-treated cells demonstrated higher expression of pancreatic progenitor genes (PDX1, NKX6. 1, PTF1a, S0X9) as well as decreased expression of genes associated with other endodermal lineages (CDX2, 0TX2, AFP, HNF4a) (Fig. 5D). To further elucidate the changes in signaling induced by the latA treatment that led to improved pancreatic progenitor specification and subsequent SC-|3 cell formation, we performed single cell RNA-sequencing at each stage of differentiation (Fig. 5E). Overall, the control and latA-treated cells clustered similarly at most stages, with the end of stage 1 (s2d1 ) still exhibiting the most distinct populations between the two conditions. Despite these similarities, the two conditions still demonstrated differences in the expression of important genes at various stages (Fig. 5F). Specifically, markers of anterior foregut (07X2, LHX1, FZD8, SISHA2, SIX3, HESX1) were elevated in the control cells at s2d1 and s3d1 (Fig. 5F, Fig. 24B). By s4d1 , however, the control cells demonstrated elevated levels of posterior markers (AFP, TTR), while the latA-treated cells exhibited transient upregulation of more anterior markers (S0X2, 0SR1) (Fig. 5F, Fig. 24B). By s5d1 , importantly, the latA-treated cells demonstrated higher expression of key pancreatic markers (0NECUT1, PTF1a, PDX1, NKX6.1) (Fig. 5F, Fig. 24B). Confirming some of these changes at the protein level, we observed that the amount of the anterior marker 0TX2 was initially higher at s2d1 in the control (Fig. 5H, Figs. 25A-B). Conversely, the latA- treated cells had higher protein levels of PDX1 and 0NECUT1 by s4d1 (Fig. 5H, Figs. 25A-C) as well as NKX6.1 by s5d1 (Fig. 5C). Re-clustering of the sequencing data at s6d7 demonstrated that the latA-treated cells generated an increased percentage of [3 cells and a lower percentage of enterochromaffin cells in the final populations (Fig. 5J), further confirming that these improvements in pancreatic progenitor specification resulted in improved [3 cell differentiation. Furthermore, the [3 cell population demonstrated higher expression of [3 cell genes (e.g., INS, IAPP, ER01B) and lower expression of other hormones (GCG) as well as genes not normally found in [3 cells (LDHA, DDC) (Fig. 24D).

[0221] Other genes that stood out in this single cell RNA-sequencing dataset for exhibiting differences in the latA-treated cells during differentiation were those relating to components of the retinoic acid (RA) signaling pathway as well as its downstream targets. In particular, CYP26A1, which degrades RA, was strongly elevated in the control cells at s2d1 (Fig. 5G, Fig. 24C). In contrast, CRABP2, which helps shuttle RA to the nucleus and promote RA signaling, was elevated at this time in latA-treated cells (Fig. 5G, Fig. 24C). These differences were confirmed at the protein level (Fig. 5I, Fig. 23B) and suggest that latA-treated cells may be better primed for RA signaling in subsequent stages. During stage 3, a high dose of RA is used to induce specification of the posterior portion of the foregut and expression of the key pancreatic transcription factor PDX1 (Fig. 24A). Expectedly, the nuclear RA receptors RARA and RARB peaked at the end of this stage (s4d1 ) (Fig. 5G, Fig. 24C). Interestingly, the latA-treated cells exhibited higher expression of many RA target genes at s4d1 (RBP1, NR2F1, MEIS1, MLLT3, PBX1, DHRS3, ZNF703, H0XA1, H0XB2, H0XA3), indicating increased RA signaling during stage 3 (Fig. 5G, Fig. 24C). These included several of the HOX genes, which are important for segmenting the gut tube into different organ-forming regions. Correspondingly, the latA-treated cells had increased PDX1 protein expression at s4d1 (Fig. 5H, Fig. 25A-B), indicating better specification of the pancreatic portion of the gut tube. These peaks in expression also coincided with the peaks of other important gut tube genes mentioned earlier (Fig. 5F-G, Fig. 24B-C). Conversely, the control cells demonstrated increased expression of FGF8 at this timepoint (Fig. 5G, Fig. 24C). Because RA signaling is known to repress FGF8 expression, this further suggests that the control cells experienced decreased RA signaling during stage 3. Taken together, these data indicate the latA treatment in stage 1 influenced the temporal expression of key genes in subsequent stages that altered gut tube patterning, resulting in better specification of pancreatic progenitors by s5d1 and improved SC-|3 cell differentiation by the end of the protocol. These effects appear to be at least partially mediated by altered RA signaling dynamics during the specification of the posterior foregut.

[0222] Latrunculin A treatment during the first 24 hours of differentiation generated SC-islets containing cells with more mature cell identities. To further characterize the SC-islets generated with the stage 1 latA treatment, we performed single-nucleus multi-omic sequencing (mRNA and ATAC) on s6d15 after the cells had been aggregated into islet-like clusters for over one week (Fig. 6A, Fig. 26A-C). Similar to the observations made with the single cell RNA- sequencing and flow cytometry analysis on s6d7, the combined RNA and ATAC characterization demonstrated that the SC-islet clusters generated with the stage 1 latA treatment had a higher percentage of [3 cells and a lower percentage of enterochromaffin cells (Fig. 6A). Furthermore, each cell type in the SC-islets generated with the stage 1 latA treatment generally demonstrated a more mature identity for that cell type in terms of both mRNA expression (Fig. 6B) as well as promoter accessibility for those genes (Fig. 6C). Looking specifically at the [3 cell population, the latA-treated cells had higher mRNA expression of genes such as / / VS and those involved in calcium regulation (CACNA1A, ASPH) (Fig. 6D). They also exhibited increased promoter accessibility for [3 cell genes (INS, ISL1, 0NECUT1) as well as multiple HOX genes (Fig. 6D). Furthermore, the CTCF binding motif was more accessible in the control, which we previously demonstrated to promote an enterochromaffin lineage (Fig. 6D). In summary, treating the cells with latA for the first 24 hours of differentiation altered the signaling dynamics during definitive endoderm formation, leading to altered gut tube patterning and better pancreatic progenitor specification. These cells ultimately generated SC-islets that had more [3 cells as well as a decreased number of enterochromaffin cells. Importantly, these [3 cells exhibited a more mature identity and secreted more insulin in response to glucose stimulation.

[0223] DISCUSSION

[0224] In this study, we demonstrated that cytoskeletal state at the onset of differentiation is critical for the exit of hPSCs from pluripotency and subsequent definitive endoderm formation. Specifically, depolymerizing the actin cytoskeleton with latA during the first 24 hours of differentiation facilitated a more robust exit from pluripotency and altered signaling dynamics during definitive endoderm formation, modifying the temporal activation of the Activin / Nodal, BMP, JNK-JUN, and WNT pathways. These changes in stage 1 signaling influenced downstream patterning of the gut tube, including modified expression of components of the RA signaling pathway and altered HOX gene expression. Subsequently, latA treated cells exhibited improved pancreatic identity by s4d1 and decreased expression of genes associated with other endodermal lineages along the gut tube, such as AFP (liver) and CDX2 (intestine). This improved pancreatic specification led to an increased percentage of PDX1 + / NKX6.1 + pancreatic progenitors by the end of stage 4 and ultimately generated SC-islets with a higher percentage of [3 cells and lower percentage of enterochromaffin cells by the end of the protocol. These SC-islets contained [3 cells with a more mature identity and greatly improved insulin secretion in response to glucose stimulation. Furthermore, this latA treatment could rescue differentiations from inconsistent hPSC lines in batches that otherwise failed to produce endocrine cells.

[0225] While the exact mechanistic link between actin polymerization state and these specific changes in signaling dynamics remains to be fully elucidated, the data suggest that depolymerizing the actin cytoskeleton with latA helped transition the hPSCs out of pluripotency. For example, compounds that increased actin polymerization caused these cells to retain higher expression of pluripotency genes by the end of stage 1 (Fig. 1 F), and consequently these cells had difficulty differentiating into SC-islets (Fig. 1 B-C). Similarly, the AN1.1 and H1 hPSC lines retained much higher expression of these pluripotency genes at the end of stage 1 in the absence of the latA treatment, which correlated with the inconsistent differentiations normally observed with these hPSC lines. Even with the HUES8 hPSC line, the control cells demonstrated heterogenous NANOG protein expression and some 0CT4 positive cells by the end of stage 1 , while these proteins were essentially gone in the latA-treated cells (Fig. 4D, Figs 19A and 19C). Additionally, the HUES8 control cells consistently appeared to lag behind the latA-treated cells throughout the differentiation. For instance, the anterior marker 0TX2 was turned off more slowly in the control during stages 2 and 3, while the master pancreatic regulator PDX1 turned on more strongly in the latA-treated cells in stage 3 (Fig. 5H, Fig. 25A-B). These results support emerging evidence for the importance of cytoskeletal dynamics in pluripotent stem cells. Interestingly, some of the only genes strongly downregulated immediately after the 24-hour latA treatment were those encoding metallothionein proteins, which play an important role in the homeostasis of metal ions within a cell (Fig. 14A, 16A). In agreement with this observation, it has been demonstrated that metallothionein genes must be downregulated in order for cells to differentiate into definitive endoderm. Furthermore, differences in metallothionein expression may contribute to variation in pluripotency between stem cell lines. Similarly, JNK-JUN signaling has also been shown inhibit exit from pluripotency and impede differentiation to definitive endoderm. While latA- treated cells exhibited a strong initial increase in nuclear accumulation of c-Jun, c-Jun was quickly eliminated in the subsequent days from latA-treated cells as they regained their ability to polymerize actin (Fig. 4C-D, Fig. 19B-C). In agreement with this observation, depolymerizing actin has been shown to strongly increase c-Jun expression. Thus, depolymerization of F-actin at the onset of differentiation appears to affect the timing and intensity of JNK-JUN signaling, which may subsequently influence how cells exit pluripotency and respond to differentiation factors. Interestingly, we observed differences in initial c-Jun staining in the H1 and AN1 .1 hPSC lines as well as a heterogenous F- actin structure in AN1 .1 (Fig. 25B). These observations suggest that differences in endogenous signaling involving actin and c-Jun may contribute to the difficulties these hPSC lines had in exiting pluripotency and subsequently differentiating into SC-islets without latA treatment.

[0226] Because F-actin depolymerization influenced hPSC exit from pluripotency, the signaling dynamics changed during stage 1 , leading to a unique definitive endoderm population by the end of this stage. Many of these signaling differences centered around the Activin / Nodal and BMP signaling pathways, which are critical during early embryogenesis. Specifically, the latA-treated cells sharply downregulated expression of LEFTY1, LEFTY2, and NODAL (Figs. 4C and 4D) and decreased phosphorylation of SMAD2 during the second half of stage 1 (Fig. 4E), indicating decreased Activin / Nodal signaling. Conversely, BMP signaling targets such as ID1 increased (Fig. 4C) in conjunction with an increase in phosphorylation of the BMP effectors SMAD1 and SMAD5 during this same time period (Fig. 4E). Intriguingly, a recent study utilizing a CRISPR interference screen during definitive endoderm formation demonstrated that inhibition of SMAD2 and SMAD4 generated a definitive endoderm population with very similar gene characteristics to the latA-treated cells. Specifically, this cluster expressed high levels of F0XA2, S0X17, and ID1 but low levels LEFTY1, NODAL, CER1, and GATA6, further suggesting that our latA treatment is causing inhibition of SMAD2 signaling during the second half of stage 1 . However, it was concluded that the population generated with SMAD2 / SMAD4 inhibition was not proper definitive endoderm, and no further characterization or differentiation of this population was conducted. Our results, however, indicate that a definitive endoderm population with these unique gene signatures is in fact better suited to make SC-islets. In particular, the latA-treated cells appeared to be more receptive to RA signaling during the subsequent patterning of the gut tube, leading to better specification of pancreatic progenitors.

[0227] The improvements in SC-islet generation described here have noteworthy implications for their performance and safety as a cell therapy for treating diabetes. Prior studies have demonstrated that SC-islets produced from hPSCs are immature in vitro in terms of both function and identity. Here, we were able to significantly improve insulin secretion, insulin content, and cell identity of SC- islets with multiple cell lines. This new process increased the proportion of SC-|3 cells and lowered the percentage of enterochromaffin cells in SC-islets, demonstrating improved pancreatic specification and reduction of unwanted off- target cell types. Furthermore, this stage 1 latA treatment increased differentiation consistency and reduced line-to-line variability, particularly with the H1 and AN1 .1 hPSC lines. For example, while we have never observed tumor formation when transplanting SC-islets generated with the HUES8 and WS4c lines with our recent protocols, residual off-target cells in the control AN1 .1 differentiations proliferated and caused tumors in mice by week 11 after transplantation (Fig. 3C). In contrast, the stage 1 latA treatment of AN1.1 cells generated SC-islets that both rapidly cured severely diabetic mice and prevented tumor formation (Fig. 3A-D). This reversal of hyperglycemia was maintained long-term in mice without evidence of hypoglycemia, despite differences in the in vitro insulin secretion kinetics between SC-islets and primary huma islets. Thus, this new version of the SC-islet differentiation protocol can help improve the performance of SC-islets while simultaneously preventing the formation of troublesome off-target cell types, particularly when utilizing new cell lines. The ability to seamlessly adapt SC-islet differentiation protocols to clinical grade cell lines is crucial for the safety, efficacy, and speed of translation of this cell-based therapy. Furthermore, this new differentiation strategy could be combined with other advances in transplantation to develop novel cell therapy products.

[0228] In terms of the practicality of using this new method, latA can simply be added for the first 24 hours of our previously published SC-islet differentiation protocol. The latA concentrations for the cell lines shown here can be used as written. However, when starting with a new cell line, we recommend testing a range of concentrations, with the 0.1 pM to 0.175 pM range being a good place to start. The concentration of latA needed is dependent upon the particular cell line and starting cell density. The goal should be to get the cells as round as possible after 24 hours without too many of them falling off the plate (Fig. 1 D, Fig. 8A-B), as the protocol will not work if cell density becomes too low (Fig. 8C). If the cells are very round on the second day of differentiation, the media can be changed several hours early to prevent too many cells from detaching from the plate. Of particular note, there will be more cell death throughout stage 1 than without the latA treatment, especially on s1d3 and s1d4 when the media can be cloudy with floating cells. As long as there is a confluent layer of cells attached to the flask, however, the differentiation should be successful (Fig. 8A-C). While this latA treatment improved differentiation to SC-islets with the 4 hPSC lines presented here, it is possible that it will not work for all hPSC lines, particularly if those lines have variations in endogenous signaling of some of the pathways influenced by the latA treatment. For cell lines that are very sensitive to latA, latrunculin B or cytochalasin D can be tried as alternatives, as cells treated with these compounds also generated SC-islets with improved function. It is important to note that this stage 1 latA treatment is in addition to the latA treatment on s5d1 , and the alteration of cytoskeletal state seems to be influencing different pathways at their respective points in the differentiation. Interestingly, the latA concentration used during stage 1 had to be much lower than the concentration used later on in the protocol during stage 5. Furthermore, a much higher concentration of latA was needed in mTeSRI than in stage 1 media in order to induce the same rounding effect on hPSC morphology (Fig. 16C), emphasizing the context-dependent effect of latA on cytoskeletal signaling.

[0229] Overall, our results highlight how refining earlier stages of a differentiation protocol, particularly in the context of modifying cytoskeletal state, can improve the final cell product. Directed hPSC differentiations are very dependent on factor concentration and timing, and thus small changes early on can have large effects on the final cell populations. In this case, we altered the exit of hPSCs from pluripotency and subsequent definitive endoderm formation by chemically modulating the actin cytoskeleton, which influenced downstream patterning of the gut tube. While the differences in these cell populations were subtle at certain stages, there were several critical signaling changes that allowed the latA-treated cells to be better specified as pancreatic progenitors, which then gave rise to better SC-islets. Furthermore, our results demonstrate that refining pancreatic progenitor generation influenced the decision between an enterochromaffin or SC-|3 cell fate. Hence, revisiting early stages of other hPSC differentiation protocols to refine the exit of hPSCs from pluripotency and subsequent generation of progenitor populations through cytoskeletal modulation may improve specification of the final desired cell types.

Claims

CLAIMSWhat is claimed is:1 . A method of generating at least one stem cell-derived islet (SC-islet) comprising a plurality of stem cell-derived [3-cells (SC-[3s), the method comprising contacting at least one human pluripotent stem cell (hPSC) with an amount of a cytoskeleton depolymerizing agent during initial stem cell differentiation into definitive endoderm cells.

2. The method of claim 1 , wherein the at least one human pluripotent stem cell is contacted with the amount of the cytoskeleton depolymerizing agent during the first 24 hours of the initial stem cell differentiation.

3. The method of claim 1 , wherein the cytoskeleton depolymerizing agent is configured to depolymerize a cytoskeleton component comprising F-actin.

4. The method of claim 1 , wherein the cytoskeleton depolymerizing agent is selected from latrunculin A (latA), latrunculin B, cytochalasin D, and any combination thereof.

5. The method of claim 4, wherein the at least one human pluripotent stem cell is contacted with the latA at a concentration ranging from about 0.01 pM to about 1 pM.

6. The method of claim 1 , wherein the method produces a stem cell-derived islet (SC-islet) with reduced off-target and undifferentiated cells compared to a corresponding SC-islet produced without a cytoskeleton depolymerizing agent.

7. The method of claim 6, wherein the method produces a stem cell-derived islet (SC-islet) with reduced formation of tumor cells.

8. The method of claim 1 , wherein the method produces at least 5% more SC-[3s compared to a corresponding number of SC-[3s SC-islet produced without a cytoskeleton depolymerizing agent.

9. The method of claim 1 , wherein the method produces a stem cell-derived islet (SC-islet) with enhanced insulin secretion compared to a corresponding SC-islet produced without a cytoskeleton depolymerizing agent.

10. The method of claim 5, wherein the at least one hPSC contacted with the cytoskeleton depolymerizing agent comprises at least one hPSC selected from a stem cell line selected from HUES8, WS4c, AN1.1 , and H1.11 . A method of treating a subject , the method comprising: i. contacting a plurality of human pluripotent stem cells (hPSCs) with an amount of a cytoskeleton depolymerizing agent during initial stem cell differentiation into definitive endoderm cells to generate a plurality of stem cell-derived islets (SC-islets) each comprising a plurality of stem cell-derived [3-cells (SC-|3s); and ii. transplanting a therapeutically effective amount of the stem cell- derived islets (SC-islets) into the subject.

12. The method of claim 11 , wherein the subject comprises a patient diagnosed with a diabetes disorder selected from type 1 diabetes mellitus (T1 D), type 2 diabetes mellitus (T2D), gestational diabetes mellitus, latent autoimmune diabetes of adults (LADA), or maturity onset diabetes of the young (MODY).

13. The method of claim 11 , wherein the at least one human pluripotent stem cell is contacted with the amount of the cytoskeleton depolymerizing agent during the first 24 hours of the initial stem cell differentiation.

14. The method of claim 11 , wherein the cytoskeleton depolymerizing agent is configured to depolymerize a cytoskeleton component comprising F-actin.

15. The method of claim 11 , wherein the cytoskeleton depolymerizing agent comprises latrunculin A (latA).

16. The method of claim 15, wherein the plurality of human pluripotent stem cells is contacted with the latA at a concentration ranging from about 0.01 pM to about 1 pM.

17. The method of claim 16, wherein the SC-islets comprise enhanced insulin secretion compared to corresponding SC-islets produced without a cytoskeleton depolymerizing agent.

18. The method of claim 17, wherein the therapeutically effective amount of the stem cell-derived islets (SC-islets) produce insulin sufficient to control a blood glucose level of the subject.

19. At least one definitive endoderm cell generated by contacting at least one human pluripotent stem cell (hPSC) with an amount of a cytoskeleton depolymerizing agent during initial stem cell differentiation into definitive endoderm cells, wherein the at least one definitive endoderm cell, wherein the at least one definitive endoderm cell comprises higher expression levels of at least one first protein selected from Gata3, Bambi, Id 1 , Id2, Id3, Id4 and any combination thereof and further comprises lower expression levels of at least one second protein selected from Cer1 , Gsc, Leftyl , Lefty2, Nodal, Tagln, Gata6, Otx2 and any combination thereof, the higher and lower expression levels defined relative to corresponding expression levels of at least one comparison definitive endoderm cell generated in an absence of the cytoskeleton depolymerizing agent.

20. The cell of claim 19, wherein the at least one human pluripotent stem cell is contacted with the amount of the cytoskeleton depolymerizing agent during the first 24 hours of the initial stem cell differentiation.

21. The cell of claim 19, wherein the cytoskeleton depolymerizing agent is configured to depolymerize a cytoskeleton component comprising F-actin.

22. The cell of claim 19, wherein the cytoskeleton depolymerizing agent is selected from latrunculin A (latA), latrunculin B, cytochalasin D, and any combination thereof.

23. The cell of claim 22, wherein the at least one human pluripotent stem cell is contacted with the latA at a concentration ranging from about 0.01 pM to about 1 pM.

24. The cell of claim 19, wherein the at least one hPSC contacted with the cytoskeleton depolymerizing agent comprises at least one hPSC selected from a stem cell line selected from HUES8, WS4c, AN1.1 , and H1.

25. At least one pancreatic progenitor cell generated by contacting at least one human pluripotent stem cell (hPSC) with an amount of a cytoskeleton depolymerizing agent during initial stem cell differentiation into definitive endoderm cells, wherein the at least one pancreatic progenitor cell, comprises higher expression levels of at least one first protein selected from of PTF1a, ONECUT1 , OSR1 , HOXA1 , HOXA3 and any combination, the higher expression levels defined relative to corresponding expression levels of at least one comparison pancreatic progenitor cell generated in an absence of the cytoskeleton depolymerizing agent.

26. The cell of claim 25, wherein the at least one human pluripotent stem cell is contacted with the amount of the cytoskeleton depolymerizing agent during the first 24 hours of the initial stem cell differentiation.

27. The cell of claim 25, wherein the cytoskeleton depolymerizing agent is configured to depolymerize a cytoskeleton component comprising F-actin.

28. The cell of claim 25, wherein the cytoskeleton depolymerizing agent is selected from latrunculin A (latA), latrunculin B, cytochalasin D, and any combination thereof.

29. The cell of claim 28, wherein the at least one human pluripotent stem cell is contacted with the latA at a concentration ranging from about 0.01 pM to about 1 pM.

30. The method of claim 25, wherein the at least one hPSC contacted with the cytoskeleton depolymerizing agent comprises at least one hPSC selected from a stem cell line selected from HUES8, WS4c, AN1.1 , and H1.31 .At least one SC-beta cell generated by contacting at least one human pluripotent stem cell (hPSC) with an amount of a cytoskeleton depolymerizing agent during initial stem cell differentiation into definitive endoderm cells, wherein the at least one SC-beta cell comprises lower expression levels of at least one first protein selected from of LRP1 B, SORCS1 , RALYL, HDAC9 and high CACNA1A, DSP, HS6ST3, MY03A, SAMD4A, ASPH and any combination thereof, the lower expression levels defined relative to corresponding expression levels of at least one comparison SC-beta cell generated in an absence of the cytoskeleton depolymerizing agent.

32. The cell of claim 31 , wherein the at least one human pluripotent stem cell is contacted with the amount of the cytoskeleton depolymerizing agent during the first 24 hours of the initial stem cell differentiation.

33. The cell of claim 31 , wherein the cytoskeleton depolymerizing agent is configured to depolymerize a cytoskeleton component comprising F-actin.

34. The cell of claim 31 , wherein the cytoskeleton depolymerizing agent is selected from latrunculin A (latA), latrunculin B, cytochalasin D, and any combination thereof.

35. The cell of claim 34, wherein the at least one human pluripotent stem cell is contacted with the latA at a concentration ranging from about 0.01 pM to about 1 pM.

6. The method of claim 31 , wherein the at least one hPSC contacted with the cytoskeleton depolymerizing agent comprises at least one hPSC selected from a stem cell line selected from HUES8, WS4c, AN1.1 , and H1.

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