Cell populations and gene expression associated with in vitro beta cell differentiation

Abstract

Disclosed herein are methods for directing differentiation of stem cells into specific cell types.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 249,546, filed September 28, 2021, the entire teachings of which are incorporated herein by reference. [Background technology]

[0002] Pancreatic beta cells are regulators of blood glucose, and their autoimmune destruction or dysfunction is a cause of type 1 and type 2 diabetes. Recently, in vitro differentiation protocols have been developed to convert pluripotent stem cells into pancreatic beta cells. A challenge in generating any type of cell in vitro is the heterogeneity of cells generated by induced differentiation. At each step of the process, some cells follow a desired pathway while others stray. To improve the yield of the desired cell type, it would be beneficial to guide the differentiation process so that cells follow a specific pathway and become the desired cell type. Summary of the Invention [Means for solving the problem]

[0003] Single-cell RNA sequencing can be used to identify the different cell types produced during the differentiation of pluripotent stem cells into target cell types in vitro. Sequence analysis can provide insight into the sequence of transcriptional changes that underlie the formation of target cells, revealing critical fate decision points and alternative pathways that cells may take along their differentiation path.

[0004] The studies described herein identify markers of cell types produced by in vitro pancreatic cell differentiation. This data can be used to identify genes that are specifically enriched in a single population or a combination of populations. The knowledge that these genes are specific to a given population can be used to further develop in vitro pancreatic β cell differentiation methods and in vitro differentiation methods to other cell types (such as α cells).

[0005] More specifically, these genes can be used as targets for genetic perturbation (e.g., knockout, activation, or inhibition), allowing the creation of "adapted" (non-wild type, non-native) stem cell lines that are gene-edited to prevent misdifferentiation into undesired fates. That is, by controlling gene expression in cells during the differentiation process (at the pluripotent stage and / or at one or more points later in the differentiation process), differentiation pathways can be opened or closed, encouraging or forcing cells toward desirable pathways or away from undesirable pathways.

[0006] Described herein is a method of inducing differentiation of a cell population comprising inhibiting expression of a cell fate regulator in a progenitor cell, where the regulator is selected from Table 1 or Table 2, thereby inducing differentiation of the cell population into SC-β cells.

[0007] In some embodiments, the regulator is selected from the group comprising FBXL14, BCORL1, SHOC2, CCDC6, B3GALT6, HOXA1, DDX3X, CARM1, EXT2, EXT1, DYRK1A, SCAF1, SCAF8, CAND1, NDST1, EYA3, GLCE, DYRK1B, PRDM16, ALG3, CXXC4, SMURF1, PHF21A, SOX4, and TET2. In certain embodiments, the regulator is selected from the group comprising FBXL14, BCORL1, SHOC2, CCDC6, B3GALT6, HOXA1, DDX3X, CARM1, EXT2, and EXT1. In some embodiments, the regulator is selected from the group comprising SOX4, BCORL1, FBXL14, CCDC6, SOX1, CARM1, TNRC18, CAND1, TET2, HOXA1, ASCL1, ARID2, SIRT6, FBXO22, FLVCR1, FOXA1, COPS9, ELAVL1, SSBP3, PROSER1, PROX1, SMURF1, SCAF1, HELLS, and DACH1. In certain embodiments, the regulator is selected from the group comprising SOX4, BCORL1, FBXL14, CCDC6, SOX1, CARM1, TNRC18, CAND1, TET2, and HOXA1.

[0008] Described herein is a method of inducing differentiation of a cell population comprising inhibiting expression of a cell fate control factor in a progenitor cell, where the control factor is selected from Table 3 or Table 4, thereby inducing differentiation of the cell population into an SC-α cell.

[0009] In some embodiments, the modulator is selected from the group comprising PDX1, CCDC6, HES1, PHF21A, PAX4, DYRK1B, DYRK1A, BCORL1, TET2, DDX3X, PROSER1, PBX1, HELLS, CAND1, EYA3, MYT1, AFF4, FBXL14, HOXA1, ZC3H15, SCAF8, PRDM16, HEXIM1, TTC14, ZRANB1, and B3GALT6. In certain embodiments, the modulator is selected from the group comprising PDX1, CCDC6, HES1, PHF21A, PAX4, DYRK1B, DYRK1A, BCORL1, TET2, and DDX3X. In some embodiments, the regulator is selected from the group comprising PAX4, HES1, CCDC6, SOX4, ZBTB10, PHF21A, PBX1, ARID2, TET2, BCORL1, TTC14, CAND1, PROSER1, SOX1, FBXO22, HELLS, DYRK1B, ZRANB1, DYRK1A, ASCL1, ZC3H15, SETBP1, FAM58A, MYT1, and RALGAPB. In certain embodiments, the regulator is selected from the group comprising PAX4, HES1, CCDC6, SOX4, ZBTB10, PHF21A, PBX1, ARID2, TET2, and BCORL1.

[0010] In some embodiments, the expression of the regulator of cell fate is inhibited by knocking down or knocking out the regulator using gene editing technology, such as CRISPR.In some embodiments, gene editing technology, such as CRISPR, is delivered to progenitor cells via retrovirus, such as lentivirus.

[0011] Also described herein are enriched populations of SC-β cells produced by the methods described herein, in some embodiments, the methods produce cell populations that comprise 70%, or in some aspects 80% SC-β cells.

[0012] Also described herein are SC-islets that comprise an enriched population of SC-β cells as described herein. The patent or application file contains at least one color drawing. Copies of this patent or patent application publication containing color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0013] [Figure 1] Schematic diagram of in vitro human developmental genetic screen. HUES8, human embryonic stem cells, are expanded and plated for differentiation. Cells are differentiated to stage 6, day 15 or more, stained for intracellular markers, and target populations (SC-β cells, SC-α cells, SC-EC cells, and triple negative cells) are sorted and sequenced. All tissue cultures are performed at >100 million cells. [Diagram 2] The sorting strategy is shown, with definition of cell types (SC-β cells, SC-α cells, SC-EC cells, and triple-negative cells). Cells are sorted based on the presence or absence of INS, GCG, and SLC18A1 expression. [Diagram 3] Technical controls in validation experiments demonstrate the expected pattern of depletion in the desired populations. Each vertical plot compares a pair of populations (between SC-β, SC-α, and triple negative). Each dot represents a gene in the secondary screen, and the y-axis shows the effect of disrupting that gene on the likelihood of generating each cell type. Vertical bars show the values ​​for the labeled control genes. [Figure 4]Perturbation targets (Table 1) that increase differentiation of SC-β cells versus triple-negative cells (TN) are shown. Orange dots indicate where perturbations affect 50 genes in Table 1. Highlighted panels show how this group was defined. [Diagram 5] Perturbation targets that increase differentiation of SC-β cells relative to SC-EC cells (Table 2) are shown. Orange dots indicate where perturbations affect 50 genes in Table 2. Highlighted panels show how this group was defined. [Figure 6] Perturbation targets that increase differentiation of SC-α cells versus triple-negative cells (TN) are shown (Table 3). Orange dots indicate where the perturbation effect of 50 genes in Table 3 is located. Highlighted panels show how this group was defined. [Figure 7] Perturbation targets that increase differentiation of SC-α cells versus SC-EC cells (Table 4) are shown. Orange dots indicate where the perturbation effect of 50 genes in Table 4 is located. Highlighted panels show how this group was defined. [Figure 8] The effect of knocking out FBXL14 by lentivirus-mediated CRISPR is shown. The columns indicate the effect of the knockout, and the orange line indicates the degree to which the cell population containing the knockout makes more cells of one type versus the other. The three cell types measured are beta cells (SC-β), alpha cells (SC-α), and enterochromaffin cells (SC-EC). At the bottom of each column, TN indicates "triple negative," meaning the cells are neither alpha, beta, nor EC. All black dots represent other gene knockouts. The same data is displayed in another way, as a circle, with orange crosses representing the location of the knockout. This shows that knocking out FBXL14 produces more beta cells. The histograms are quantified using different guide RNAs (g1, g2, etc.) to achieve the CRISPR knockout. It is shown that knocking out FBXL14 increases the percentage of beta cells (with no significant effect on EC cells). [Figure 9] The effect of knocking out FBXO22 by lentivirus-mediated CRISPR is shown. The columns indicate the effect of the knockout, and the orange line indicates the degree to which the cell population containing the knockout makes more of one type of cell versus the other. The three cell types measured are beta cells (SC-β), alpha cells (SC-α), and enterochromaffin cells (SC-EC). At the bottom of each column, TN indicates "triple negative," meaning the cells are neither alpha, beta, nor EC. All black dots represent other gene knockouts. The same data is displayed in another way, as a circle, with the orange crosses representing the location of the knockout. This shows that knocking out FBXO22 results in the production of fewer EC cells. The histograms are quantified using different guide RNAs (g1, g2, etc.) to achieve the CRISPR knockout. It is shown that knocking out FBXO22 reduces the percentage of EC cells (with no significant effect on beta cells). [Figure 10A] We demonstrate single-cell RNA sequencing of in vitro beta cell differentiation. We provide an overview of cell populations identified by flow cytometry at the end of stages 3-6 of the SC-β protocol of Pagliuca et al.. PDX1 stands for pancreatic transcription factor, NKX6.1 stands for beta cell transcription factor, INS stands for insulin, a beta cell hormone, and CHGA stands for chromogranin A, a pan-endocrine marker. [Figure 10B] We demonstrate single-cell RNA sequencing of in vitro β-cell differentiation. We describe the use of inDrops to sample cells from multiple time points of the same differentiation. [Figure 10C] We demonstrate single-cell RNA sequencing of in vitro beta cell differentiation. We provide expression profiles of developmentally relevant genes and markers across cell types identified during SC-beta differentiation. Shading indicates mean expression (z-normalized tpm) and diameter indicates percentage of expression. [Figure 10D]Demonstrating single-cell RNA sequencing of in vitro beta cell differentiation. Shown are tSNE projections of cells sampled at the end of stages 3-6 of the "x1" protocol. Cells are colored according to their assigned cluster. Horizontal bars indicate the proportion of cell types. [Figure 10E] Demonstrating single-cell RNA sequencing of in vitro beta cell differentiation. Shown are tSNE projections of cells sampled at the end of stages 3-6 of the "x1" protocol. Cells are colored according to their assigned cluster. Horizontal bars indicate the proportion of cell types. [Figure 10F] Demonstrating single-cell RNA sequencing of in vitro beta cell differentiation. Shown are tSNE projections of cells sampled at the end of stages 3-6 of the "x1" protocol. Cells are colored according to their assigned cluster. Horizontal bars indicate the proportion of cell types. [Figure 10G] Demonstrating single-cell RNA sequencing of in vitro beta cell differentiation. Shown are tSNE projections of cells sampled at the end of stages 3-6 of the "x1" protocol. Cells are colored according to their assigned cluster. Horizontal bars indicate the proportion of cell types. [Figure 11A] We demonstrate that SC-β cells maintain identity and acquire maturation marker expression during long-term culture at stage 6. We provide an experimental design to study functional and transcriptional changes during stage 6 of protocol v8. [Figure 11B] We demonstrate that SC-β cells maintain identity and acquire maturation marker expression during long-term culture at stage 6. Glucose-stimulated insulin secretion showing successive low glucose (2.8 mM) and high glucose (20 mM) challenges for three independent differentiations over 5 weeks is shown. [Figure 11C] We demonstrate that SC-β cells maintain identity and acquire maturation marker expression during long-term culture at stage 6. Stimulation index (insulin released at 20 mM glucose / insulin released at 2 mM) is shown for the data in FIG. 11B. [Figure 11D]We demonstrate that SC-β cells maintain identity and acquire maturation marker expression during long-term culture at stage 6. tSNE projections of 38,494 cells from six time points over a 5-week period at stage 6 are shown. Cells are color-coded according to their assigned type. Vertical bars indicate the population ratios for each week. [Figure 11E] We demonstrate that SC-β cells maintain identity and acquire maturation marker expression during long-term culture at stage 6. Expression of endocrine marker genes is shown. [Figure 11F] We demonstrate that SC-β cells maintain identity and acquire maturation marker expression during long-term culture at stage 6. Correlation of expression profiles for each major cell type is shown separated by week. Color coding of cell types corresponds to that in FIG. 11D. [Figure 11G] We demonstrate that SC-β cells maintain identity and acquire maturation marker expression during long-term culture at stage 6. Shown are the pseudo-chronological order of SC-β cells displayed on tSNE (top panel) and the distribution of SC-β pseudo-chronological order stratified by sampling week (bottom panel). [Figure 11H] We demonstrate that SC-β cells maintain identity and acquire maturation marker expression during long-term culture at stage 6. We provide identification of dynamic genes along SC-β pseudotime. Fold changes compare the beginning and end of the pseudotime trajectory. q-values ​​are FDR-adjusted (α=0.001) p-values ​​from likelihood ratio tests comparing full and reduced models (see Methods). [Figure 11I] We demonstrate that SC-β cells maintain identity and acquire maturation marker expression during long-term culture at stage 6. We provide the expression of selected genes indicated along SC-β pseudotime. Each dot represents the expression of a cell, sorted and shaded as in FIG. 11G. Lines show the results of pseudotime regression. [Figure 12A] Characterization of stem cell-derived enterochromaffin cells (SC-EC cells) is provided. Comparison of gene expression profiles of SC-β and SC-EC is provided. Genes in blue are required for serotonin synthesis or enterochromaffin markers. [Figure 12B]1 provides characterization of stem cell-derived enterochromaffin cells (SC-EC cells), showing expression levels of SC-EC enriched genes across in vitro populations (top panel) and human pancreatic endocrine cells (bottom panel). [Figure 12C] Characterization of stem cell-derived enterochromaffin cells (SC-EC cells) is provided. Immunofluorescence staining of SC-EC cell markers showing co-localization with serotonin (5-HT) in the v8 protocol is shown. Dimension bar is 100 μm. [Figure 12D] Characterization of stem cell-derived enterochromaffin cells (SC-EC cells) is provided. Immunofluorescence staining of SC-EC cell markers showing co-localization with serotonin (5-HT) in the v8 protocol is shown. Dimension bar is 100 μm. [Figure 12E] Characterization of stem cell-derived enterochromaffin cells (SC-EC cells) is provided. (v4) Immunofluorescence staining of graft tissue harvested 8 weeks after transplantation of SC-islet clusters is shown. [Figure 13A] 14A-14D demonstrates the purification of SC-β cells by ITGA1 / CD49a. Time course data showing ITGA1 / CD49a expression at stage 6. [Figure 13B] Figure 1. Demonstration of purification of SC-β cells by ITGA1 / CD49a. Immunofluorescence of native, unsorted reaggregated, and CD49a+ sorted reaggregated clusters for SC-β markers (top) and endocrine markers (bottom). Dimensions bar is 100 μm. [Figure 13C] We demonstrate purification of SC-β cells by ITGA1 / CD49a. Flow cytometry quantification of SC-β (C-pep+ / NKX6.1+) and SC-EC (SLC18A1+) cell fractions in three matched conditions for five biologically independent v8 differentiations is provided. Symbols indicate the mean and error bars (where indicated) correspond to the standard error across three independently reaggregated biological replicates. P values ​​are from a (two-tailed) dependent t-test. [Figure 13D]Figure 1 demonstrates the purification of SC-β cells by ITGA1 / CD49a. Stimulation indices for the same differentiation are shown. Symbols indicate the mean and error bars (when indicated) correspond to the standard error across three independently reaggregated biological replicates. P values ​​are from a (two-tailed) dependent t-test. [Figure 14A] We provide a high-resolution map of in vitro endocrine induction. tSNE projections of 51,274 cells are shown and colored according to sampling time within stage 5. [Figure 14B] We provide a high-resolution map of in vitro endocrine induction. tSNE projections of 51,274 cells are shown and colored according to NEUROG3 expression. [Figure 14C] We provide a high-resolution map of in vitro endocrine induction. tSNE projections of 51,274 cells are shown and colored according to assigned cell type. Arrows indicate divergence of key lineages. [Figure 14D] A high-resolution map of in vitro endocrine induction is provided. The percentage of cells from each cluster in FIG. 14C is shown for each day of both independent differentiations. [Figure 14E] A high-resolution map of in vitro endocrine induction is provided, showing colored tSNE of branch assignments and pseudotime values ​​for each cell on the pathway from NKX6.1+ progenitors to SC-β and SC-EC cells. [Figure 14F] We provide a high-resolution map of in vitro endocrine induction. Expression of selected marker genes along the pseudo-time sequence from Figure 14E is shown. Dots indicate expression in single cells, sorted and colored according to the pseudo-time sequence. Lines indicate the regression of each branch against pseudo-time (blue: SC-EC, purple: SC-β). [Figure 14G] We provide a high-resolution map of in vitro endocrine induction. Genes with significant branching-specific expression patterns are shown. q-values ​​are FDR-adjusted (α=0.001) p-values ​​obtained from likelihood ratio tests comparing branching and non-branching models (see Methods). [Figure 14H]14C-14D provide a high-resolution map of endocrine induction in vitro. Mean expression values ​​of transcription factors for the clusters shown in Figures 14C-14D are shown. Shading indicates mean expression (z-normalized tpm) and diameter indicates percentage of expression. [Figure 14I] We provide a high-resolution map of in vitro endocrine induction and a proposed developmental model for the key cell types produced by the SC-β protocol. [Figure 15A] A comparison of the two SC-β protocol variants and the resulting cell types is provided. Immunofluorescence images of differentiated (v8, stage 6, day 13) SC-islets stained for relevant markers. Typically peripherally located SC-β cells are shown to be positive for both NKX6.1 and C-peptide (a fragment of proinsulin). [Figure 15B] A comparison of the two SC-β protocol variants and the resulting cell types is provided. Immunofluorescence images of differentiated (v8, stage 6, day 13) SC-islets stained for relevant markers. SC-EC cells are shown to be positive for SLC18A1, an enterochromaffin cell marker. These cells are also present in the periphery. [Figure 15C] A comparison of the two SC-β protocol variants and the resulting cell types is provided. Immunofluorescence images of differentiated (v8, stage 6, day 13) SC-islets stained for relevant markers. Non-endocrine cells, indicated by SOX9, are most commonly found near the center of the SC-islets. Dimension bars are 100 μm. [Figure 15D] A comparison of the two SC-β protocol variants and the resulting cell types is provided. An overview of the changes in stages 3 and 4 in protocol x1 is shown, as well as representative flow cytometry results at the end of stages 4 and 6. [Figure 15E] A comparison of the two SC-β protocol variants and the resulting cell types is provided. An overview of the changes in stages 3 and 4 in protocol x2 is shown, as well as representative flow cytometry results at the end of stages 4 and 6. [Figure 15F]We provide a comparison of the two SC-β protocol variants and the resulting cell types. We provide tSNE projections of cells sampled from the end time points of stages 3-6 of protocol x2. Cells are colored according to their assigned clusters. Horizontal bars indicate the proportion of cell types. (Related to Figures 10D-10G). [Figure 15G] We provide a comparison of the two SC-β protocol variants and the resulting cell types. We provide tSNE projections of cells sampled from the end time points of stages 3-6 of protocol x2. Cells are colored according to their assigned clusters. Horizontal bars indicate the proportion of cell types. (Related to Figures 10D-10G). [Figure 15H] We provide a comparison of the two SC-β protocol variants and the resulting cell types. We provide tSNE projections of cells sampled from the end time points of stages 3-6 of protocol x2. Cells are colored according to their assigned clusters. Horizontal bars indicate the proportion of cell types. (Related to Figures 10D-10G). [Figure 15I] We provide a comparison of the two SC-β protocol variants and the resulting cell types. We provide tSNE projections of cells sampled from the end time points of stages 3-6 of protocol x2. Cells are colored according to their assigned clusters. Horizontal bars indicate the proportion of cell types. (Related to Figures 10D-10G). [Figure 15J] A comparison of the two SC-β protocol variants and the resulting cell types is provided. A comparison of the cell populations of protocols x1 and x2 is provided. Correlations are calculated using the z-scores of the average tpm values ​​(for each cluster) of 2000 highly variable genes. Rows and columns are ordered using hierarchical clustering. Cells are labeled as in Figures 15F-15I and Figures 10D-10G. [Figure 15K] We provide a comparison of the two SC-β protocol variants and the resulting cell types. We provide tSNE projections of stage 6 from the three differentiations, color-coded by cell type. [Figure 15L]We provide a comparison of the two SC-β protocol variants and the resulting cell types. We provide tSNE projections of stage 6 from the three differentiations, color-coded by differentiation. [Figure 15M] A comparison of the two SC-β protocol variants and the resulting cell types is provided. Correlations are shown for cell populations derived from HUES8 (ES cells, v4 and x3) and iPS1016 / 31 (iPS cells, v4). Colors are the same as in Figure 15K. Correlations are calculated as in Figure 15J. [Figure 16A] A functional assay of glucose-stimulated insulin secretion (GSIS) during the time course of stage 6 is provided. A sequential GSIS assay design is provided. [Figure 16B] A functional assay of glucose-stimulated insulin secretion (GSIS) over a time course of stage 6 is provided. Complete data is provided for three independent flasks assayed over several weeks. Circles are individual technical triplicates and bars indicate the average of those triplicates. [Figure 16C] A functional assay of glucose stimulated insulin secretion (GSIS) is provided over a time course of stage 6. Full data from 7 donors of cadaveric human islets are provided, run in parallel with samples from FIG. 16B. [Figure 17A] We demonstrate that stage 6 SC-β cells express characteristic β cell markers. We provide tSNE projections of stage 6 time course data shaded by sampling time. Expression is normalized to maximum and smoothed to adjacent cells. [Figure 17B] We demonstrate that stage 6 SC-β cells express characteristic β cell markers. We provide tSNE projections of stage 6 time course data colored by representative marker genes. Expression is normalized to maximum and smoothed to adjacent cells. [Figure 17C] We demonstrate that stage 6 SC-β cells express characteristic β cell markers. We provide an expression profile of key genes required for β cell function. Shading indicates mean expression (tpm, log scale) and diameter indicates percentage of expression. [Figure 17D] We demonstrate that stage 6 SC-β cells express characteristic β-cell markers. We provide a comparison of global expression between human cadaveric islet-derived β-cells and in vitro progenitor cells. Note the shift in gene expression from progenitor cells to SC-β-cells. All genes shown in all panels of Figure 17C are circled in red. [Figure 17E] We demonstrate that stage 6 SC-β cells express characteristic β-cell markers. We provide a comparison of global expression between human cadaveric islet-derived β-cells and SC-β-cells. Note the shift in gene expression from progenitors to SC-β-cells. All genes shown in all panels of FIG. 17C are circled in red. [Figure 17F] We demonstrate that stage 6 SC-β cells express characteristic β-cell markers. We provide the results of gene set enrichment analysis (GSEA). We show that the gene sets from Figure 17C are significantly upregulated during differentiation. Values ​​plotted are -log10 of the FDR q-values ​​(capped at 10) reported in GSEA, and the sign indicates the direction of the effect (i.e., positive values ​​in purple are upregulated in SC-β cells compared to NKX6.1 progenitors). [Figure 18A] Comparison of SC-β and SC-α cells with each other and with their islet counterparts is provided. Insulin and glucagon expression in SC-β cells (purple distribution) and SC-α cells (red distribution) during several weeks of stage 6 is shown as violin plots of SC-β or SC-α cells from that particular time point. Connecting lines connect the medians of each population at each time point. [Figure 18B] We provide a comparison of SC-β and SC-α cells to each other and to their islet counterparts. We show the identification of genes enriched in cadaveric islet α and islet β cells from data from Baron et al. 2016. [Figure 18C]18B provides a comparison of SC-β and SC-α cells with each other and with their islet counterparts.18C provides a heat map of expression levels of genes from FIG. 18B shown for islet α cells, SC-α cells, SC-β cells, and islet β cells. [Figure 18D] Comparison of SC-β and SC-α cells with each other and with their islet counterparts is provided. Genes enriched in islet β cells are upregulated in SC-β cells, and genes enriched in α cells are upregulated in SC-α cells. The p-values ​​shown were calculated using a (two-tailed) Wilcoxon rank sum test. In the box plots, the box extends from the 1st to the 3rd quartile, the whiskers extend from the 5th to the 95th percentile, the middle line indicates the median, and the notches in the box indicate the 95th percentile confidence interval for the median. [Figure 19A] We demonstrate that SC-EC cells secrete serotonin, as is present in other protocols. We provide a schematic of serotonin synthesis from tryptophan. Enterochromaffin cells use TPH1, whereas serotonergic neurons use TPH2 for the first, rate-limiting synthesis step. [Figure 19B] We demonstrate that SC-EC cells secrete serotonin and that it is present in other protocols. Serotonin release during successive challenges of low and high glucose followed by KCl depolarization is shown. The top panel shows clusters from three independent SC-β differentiations, the bottom panel shows human cadaveric islets from two donors. Symbols indicate individual replicates for each sample (different clusters from the same sample were split and measured separately). p-values ​​were calculated using a (two-tailed) Wilcoxon rank sum test (ns means not significant at p>0.05). [Figure 19C]We demonstrate that SC-EC cells secrete serotonin and are present in other protocols. Expression of EC marker genes (shown in blue) was detectable by bulk RNA sequencing (from Gupta et al.) and enriched by sorting of NKX6.1(GFP)+ cells, as shown by fold change, mean expression and differential expression q-values. Positive fold change indicates high expression in NKX6.1(GFP)+ cells. Enrichment of SC-EC markers is comparable to β-cell markers (shown in purple) and opposite to α-cell markers (shown in red). All values ​​shown are directly reproduced from results calculated and deposited by Gupta et al. 2018. [Figure 19D] We demonstrate that SC-EC cells secrete serotonin and are present in other protocols. Expression of EC marker genes (shown in blue) was detectable by bulk RNA sequencing (from Gupta et al.) and enriched by sorting of NKX6.1(GFP)+ cells, as shown by fold change, mean expression and differential expression q-values. Positive fold change indicates high expression in NKX6.1(GFP)+ cells. Enrichment of SC-EC markers is comparable to β-cell markers (shown in purple) and opposite to α-cell markers (shown in red). All values ​​shown are directly reproduced from results calculated and deposited by Gupta et al. 2018. [Figure 19E] Demonstrate that SC-EC cells secrete serotonin and are present in other protocols.Provide flow cytometry showing that SLC18A1 is co-expressed with NKX6.1+ in SC-EC cells differentiated by V8 SC-β protocol.This example is representative of more than 100 independent differentiations. [Figure 19F] We demonstrate that SC-EC cells secrete serotonin and are present in other protocols. We provide a comparison of gene expression between WT mouse islets and mouse islets 25 weeks after beta cell-specific PRC2 ablation with EED knockout. Genes in purple are examples of downregulated beta cell identity genes, while genes in blue represent serotonin / EC signatures. q values ​​are FDR-corrected (α=0.05) p-values ​​from Limma differential expression analysis. [Figure 20A] Characterization of non-endocrine cells from a stage 6 time course is provided. tSNE projections of non-endocrine cells from a stage 6 time course are shown shaded by collection day. Expression is normalized to maximum and smoothed to adjacent cells. [Figure 20B] We provide a characterization of non-endocrine cells from a time course of stage 6. tSNE projections of non-endocrine cells from a time course of stage 6 are shown, colored by genes associated with cell identity. Expression is normalized to maximum and smoothed relative to adjacent cells. [Figure 20C] We provide characterization of non-endocrine cells from a time course of stage 6. We provide tSNE projections colored by assigned cluster and bar graphs of cell fractions in each cluster by week of differentiation. [Figure 20D] Characterization of non-endocrine cells from a time course of stage 6 is provided. Gene expression of population-specific markers for each subpopulation of non-endocrine cells is shown. Shading indicates mean expression (z-normalized tpm) and diameter indicates percentage of expression. [Figure 21A] We demonstrate that reaggregation is a scalable, functionally-retentive method for enriching endocrine cells. We provide a schematic of the reaggregation procedure to remove non-endocrine cells. Cells are enzymatically dissociated and reaggregated during continued suspension culture. Non-endocrine cells fail to adhere and are removed by filtration. [Figure 21B] We demonstrate that reaggregation is a scalable, function-preserving method for enriching endocrine cells. We provide a schematic of the CD49a enrichment procedure to produce SC-β enriched clusters. Dissociated cells are stained with anti-CD49a PE-labeled antibody, cultured with anti-PE magnetic microbeads, and magnetically separated. Enriched cells are reaggregated into 6-well plates on a rocker. [Figure 21C]We demonstrate that reaggregation is a scalable, function-preserving method for enriching endocrine cells. tSNE projections of sequenced cells from native and reaggregated clusters derived from a single differentiation are provided, showing strong depletion of non-endocrine populations. Cells in both panels were differentiated with protocol v8. [Figure 21D] We demonstrate that reaggregation is a scalable, function-preserving method to enrich for endocrine cells. Immunofluorescence staining for C-peptide, GCG and SLC18A1 is shown, demonstrating distinct regions in the reaggregated clusters (protocol v8). Images shown are maximum intensity projections from z-stacks. Each panel shows a separate representative cluster stained with all markers. Dimension bars are 100 μm. [Figure 21E] We demonstrate that reaggregation is a scalable, functional method for enriching endocrine cells. Representative flow cytometry analysis of endocrine cell mass (from protocol v8) before and after reaggregation is shown. Endocrine cells express CHGA. [Figure 21F] We demonstrate that reaggregation is a scalable, functional method for enriching endocrine cells. Representative flow cytometry analysis of endocrine cell mass (from protocol v8) before and after reaggregation is shown. Endocrine cells express CHGA. [Figure 21G] We demonstrate that reaggregation is a scalable, function-preserving method to enrich for endocrine cells. An overview of the population composition (assayed by flow cytometry) in 60 reaggregations (RA) and 41 native independent differentiations performed with protocol v8 is shown. Reaggregations were performed in spinner flasks. p-values ​​were calculated using a (two-tailed) Wilcoxon rank sum test. In the box plots, the boxes extend from the 1st to the 3rd quartile, the whiskers extend from the 5th to the 95th percentile, the middle line indicates the median, and the box notches indicate the 95th percentile confidence interval for the median. [Fig. 21H]We demonstrate that reaggregation is a scalable, function-preserving method to enrich for endocrine cells. In the box plots, the box extends from the 1st to the 3rd quartile, the whiskers extend from the 5th to the 95th percentile, the middle line indicates the median, and the box notches indicate the 95th percentile confidence interval for the median. Stimulation indices (insulin released at 20 mM glucose / insulin released at 2 mM) of 52 independent protocol v8 differentiations are provided in paired comparisons of native vs. redifferentiated. p values ​​were calculated using a (two-tailed) Wilcoxon signed rank test. [Figure 21I] We demonstrate that reaggregation is a scalable, functionally-preserving method for enriching endocrine cells. Complete data are provided for static glucose-stimulated insulin secretion assays performed as in Figure 16, corresponding to the stimulation index shown in Figure 13D. Circles are individual technical triplicates and bars represent the average of those triplicates. [Figure 21J] We demonstrate that reaggregation is a scalable, function-preserving method for enriching endocrine cells. Dynamic washout assays of glucose-responsive insulin secretion of human islets, native SC-β clusters (stage 6, day 22, v8), and matched CD49a magnetic sorting enriched SC-β islets are shown. Each point is the mean of three technical replicates, and the vertical bars indicate the standard error across those triplicates. [Figure 21K] We demonstrate that reaggregation is a scalable, functionally preserving method for enriching endocrine cells. Areas under the curve comparing initial low glucose stimulation with high glucose stimulation, normalized to equal effective times for each treatment, are shown. [Figure 22A] Demonstrating stage 5 time course markers and progenitor population heterogeneity. tSNE projections of stage 5 time course data are shown shaded by collection date. Expression is normalized to maximum and smoothed to adjacent cells. [Figure 22B] Demonstrating stage 5 time course markers and progenitor population heterogeneity. tSNE projections of stage 5 time course data colored by population marker genes are shown. Expression is normalized to maximum and smoothed to adjacent cells. [Figure 22C] Demonstrating stage 5 time course markers and heterogeneity of progenitor populations. Pseudotime analysis of progenitor cells at day 0 (top) and day 1 (bottom) is shown. Coloring on each tSNE indicates the pseudotime value assigned to each cell. [Figure 22D] Demonstrating stage 5 time course markers and progenitor population heterogeneity. Pseudo-time ordering of progenitor cells at stage 5 day 0 (top) and day 1 (bottom) is shown, demonstrating population heterogeneity among early progenitors. Individual cells are shown as colored dots as in FIG. 22C. Overlaid lines show predicted gene expression from pseudo-time regressions. [Figure 22E] Demonstrating heterogeneity of stage 5 time course markers and progenitor populations. Providing an overview of heterogeneity at stage 5 day 0 captured by pseudotime analysis. Fold change between the start and end of the pseudotime sequence. q-values ​​from likelihood ratio tests of models with and without pseudotime. [Figure 22F] Demonstrating stage 5 time course markers and progenitor population heterogeneity. Heatmap of dynamically expressed receptors, ligands, and signaling effectors across stage 5 populations. Shading indicates mean expression (z-normalized tpm) and diameter indicates percentage of expression. [Diagram 23] Expression of key marker genes across the entire population from time course datasets and cadaveric islets is shown. The left column shows the original dataset. Shading indicates mean expression (z-normalized tpm) and diameter indicates percentage of expression. [Figure 24] Figure 1. Expression of enteroendocrine marker genes across the entire population from a time course dataset. The left column shows the original dataset. Shading indicates mean expression (z-normalized tpm) and diameter indicates percentage of expression. [Diagram 25]A-D demonstrate an example of a flow cytometry gating strategy. A-C show stage 6 time course differentiation 1 (internal ID: DA-089) at stage 6, day 13 of the v8 protocol. A shows a secondary only control. B shows identification of SC-β cells by staining for C-peptide and NKX6.1. C shows identification of endocrine cells by staining for CHGA and NKX6.1. Results are representative across more than 100 v8 differentiations with a typical SC-β percentage of 25-45%. D provides an example of CD49a+ magnetic purification. The left panel shows the distribution of CD49a+ before sorting and the right panel shows the distribution after one round of magnetic separation (see methods). Results are representative across more than 10 enrichment experiments. [Figure 26] Provide specifications for the differentiation protocol used in this study. Overview of the different versions of the SC-β protocol used throughout this study. [Figure 27] We provide an overview of all cell populations identified in this study. For each population, we describe the markers important for their identification, the dataset in which they were identified, and, for rare populations, a description of their relationship to other populations. [Figure 28] We provide an overview of the single-cell RNA sequencing datasets generated in this study. The table shows the protocol, cell line, number of inDrops libraries, source of inDrops reagents, and number of cells sequenced for each dataset in this study, as well as the corresponding figures. [Figure 29] Demonstrating that homozygous deletion of FBXL14 increases the percentage of SC-β cells. This figure provides a comparison of HUES8 ESC monoclonal lines for endocrine cell formation potential. Three genotypes (WT: wild type, FBXL14 KO: homozygous deletion of FBXL14, NEUROG3 KO: homozygous deletion of NEUROG3) are compared, each point is an independent differentiation. FBXL14 increases the percentage of SC-β cells by 50% relative to WT. NEUROG3 KO is the control showing a complete loss of endocrine cell formation. [Diagram 30]We demonstrate that overexpression of screen hit transcription factors (TFs) alters the proportion of endocrine cells. Overexpression of ISL1 increases SC-β and SC-α cells while decreasing the formation of SC-EC cells. Lentiviruses constitutively expressing GFP and specific TFs were generated and administered separately throughout the cluster. 1-5% of the outer layer of cells were transduced. At S6d1, SC-islands were dissociated, fixed, and stained for flow cytometry. GFP+ fractions were compared specifically between target genes and neutral controls (transduced with LUC2) and log2 fold changes were calculated. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Aspects of the present disclosure relate to methods for directing differentiation of cells having multiple potential differentiation outcomes toward or away from a particular differentiation outcome.

[0015] definition For convenience, certain terms used in the specification, examples, and appended claims are collected here. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0016] The term "differentiated cell" refers to any primary cell that is not pluripotent in its native form, as defined herein. In other words, the term "differentiated cell" refers to a more specialized cell type derived from a less specialized cell type (e.g., stem cell, such as induced pluripotent stem cell) during the cell differentiation process. Without wishing to be limited by theory, pluripotent stem cells can differentiate during normal development first into endodermal cells that can form pancreatic cells and other endodermal cell types. Further differentiation of endodermal cells leads to the pancreatic pathway, where approximately 98% of cells become exocrine, ductal, or matrix cells, and approximately 2% become endocrine cells.

[0017] As used herein, the term "somatic cell" refers to any cell that forms the body of an organism, as opposed to a germ cell. In mammals, germ cells (also called "gametes") are sperm and eggs that fuse during fertilization to produce a cell called a zygote, from which the entire mammalian embryo develops. Apart from sperm and eggs, the cells from which they arise (gamete cells), and undifferentiated stem cells, all other cell types in a mammal's body are somatic cells: internal organs, skin, bones, blood, and connective tissues are all made up of somatic cells. In some embodiments, the somatic cell is a "non-embryonic somatic cell," which refers to a somatic cell that is not present in or obtained from an embryo, and not by propagation of such a cell in vitro. In some embodiments, the somatic cell is an "adult somatic cell," which refers to a cell that is present in or obtained from an organism other than an embryo or fetus, or by propagation of such a cell in vitro. Unless otherwise indicated, the methods described herein can be performed in vivo or in vitro.

[0018] As used herein, the term "adult cell" refers to cells found throughout the body after embryonic development.

[0019] As used herein, the term "endodermal cells" refers to cells derived from one of the three major germ cell layers in the very early embryo (the other two are mesoderm and ectoderm). The endoderm is the innermost of the three layers. Endoderm cells differentiate to give rise first to the embryonic gut, then to the lining of the respiratory and digestive tracts (such as the intestine), the liver, and the pancreas.

[0020] As used herein, the term "endodermal derived cells" refers to any cell that develops or differentiates from an endoderm cell. For example, endoderm derived cells include liver, lung, pancreas, thymus, intestine, stomach and thyroid cells. Without wishing to be limited by theory, liver and pancreatic progenitor cells (also called pancreatic progenitor cells) develop from endoderm cells in the embryonic foregut. Shortly after their differentiation, liver and pancreatic progenitor cells rapidly acquire strikingly different cellular functions and regenerative capabilities. These changes are driven by inductive signals and genetic regulators that are highly conserved among vertebrates.

[0021] The term "pancreatic progenitor cell" or "pancreatic precursor" is used interchangeably herein and refers to a stem cell that can form either pancreatic endocrine cell, pancreatic exocrine cell, or pancreatic duct cell. As used herein, the term "Pdx1 positive pancreatic progenitor cell" or "Pdx1+ pancreatic progenitor cell" refers to a cell that is a pancreatic endoderm (PE) cell. Pdx1 positive pancreatic progenitor cell expresses the marker Pdx1. Other markers include, but are not limited to, Cdcp1, or Ptf1a, or HNF6, or NRx2.2. The expression of Pdx1 may be evaluated by any method known to those skilled in the art, such as immunochemistry or quantitative RT-PCR using anti-Pdx1 antibody. As used herein, the term "Pdx1 positive, NKX6-1 positive pancreatic progenitor cell" or "Pdx1+, NKX6-1+ pancreatic progenitor cell" refers to a cell that is a pancreatic endoderm (PE) cell. Pdx1 positive, NKX6-1 positive pancreatic progenitor cells express markers Pdx1 and NKX6-1.Other markers include, but are not limited to, Cdcp1, or Ptf1a, or HNF6, or NRx2.2.NKX6-1 expression may be evaluated by methods known to those skilled in the art, such as immunochemistry or quantitative RT-PCR using anti-NKX6-1 antibody.

[0022] The terms "stem cell-derived beta cells", "SC-beta cells", and "mature SC-beta cells" refer to cells (e.g., pancreatic beta cells) that exhibit at least one marker indicative of a pancreatic beta cell, express insulin, and exhibit a GSIS response characteristic of endogenous mature beta cells. In some embodiments, "SC-beta cells" include mature pancreatic beta cells. It will be understood that the methods of the present disclosure can use any cell as a starting point to derive SC-beta cells from any insulin-positive endocrine cell or its precursor, and thus the SC-beta cells need not be derived (e.g., directly) from stem cells (e.g., embryonic stem cells, induced pluripotent stem cells, progenitor cells, partially reprogrammed somatic cells (e.g., somatic cells partially reprogrammed to an intermediate state between induced pluripotent stem cells and the somatic cells from which they are derived), pluripotent cells, totipotent cells, retrodifferentiated versions of any of the aforementioned cells, etc. can be used, as the invention is not intended to be so limited). Furthermore, it should be understood that the SC-β cells of the present invention are non-native, i.e., non-naturally occurring, non-endogenous cells, and have at least one characteristic that distinguishes them from native / naturally occurring / endogenous cells. Examples of SC-β cells, and methods for obtaining such SC-β cells, are described in WO2015 / 002724 and WO2014 / 201167, both of which are incorporated herein by reference in their entireties.

[0023] The terms "stem cell-derived α-cells," "SC-α-cells," and "mature SC-α-cells" refer to cells (e.g., pancreatic α-cells) that exhibit at least one marker indicative of pancreatic α-cells, express and secrete glucagon, and exhibit an ultrastructure similar to cadaveric α-cells. In some embodiments, "SC-α-cells" include mature pancreatic α-cells. It will be understood that the methods of the present disclosure can use any cell as a starting point to derive SC-α-cells from any insulin-positive endocrine cell or precursor thereof, and thus the SC-α-cells need not be derived (e.g., directly) from stem cells (e.g., embryonic stem cells, induced pluripotent stem cells, progenitor cells, partially reprogrammed somatic cells (e.g., somatic cells partially reprogrammed to an intermediate state between induced pluripotent stem cells and the somatic cells from which they are derived), pluripotent cells, totipotent cells, retrodifferentiated versions of any of the foregoing cells, and the like can be used, as the invention is not intended to be so limited). Furthermore, it should be understood that the SC-α cells of the present invention are non-native, i.e., non-naturally occurring, non-endogenous cells, and have at least one characteristic that distinguishes them from native / naturally occurring / endogenous cells. Examples of SC-α cells, and methods for obtaining such SC-α cells, are described in WO2019 / 217487, which is incorporated herein by reference in its entirety.

[0024] The terms "stem cell-derived enterochromaffin cells", "SC-EC cells", and "mature SC-EC cells" refer to cells (e.g., enterochromaffin cells) that exhibit at least one marker indicative of enterochromaffin cells, express SLC18A1, and are capable of producing and releasing serotonin (5-HT). In some embodiments, "SC-EC cells" include mature enterochromaffin cells. It will be understood that the methods of the present disclosure can use any cell as a starting point to derive SC-EC cells from any progenitor cell, and thus the SC-EC cells need not be derived (e.g., directly) from stem cells (e.g., embryonic stem cells, induced pluripotent stem cells, progenitor cells, partially reprogrammed somatic cells (e.g., somatic cells partially reprogrammed to an intermediate state between induced pluripotent stem cells and the somatic cells from which they are derived), pluripotent cells, totipotent cells, retrodifferentiated versions of any of the aforementioned cells, and the like can be used, as the invention is not intended to be so limited). Furthermore, it should be understood that the SC-EC cells of the present invention are non-native, i.e., non-naturally occurring, non-endogenous cells, and have at least one characteristic that differs from native / naturally occurring / endogenous cells. Examples of SC-EC cells, and methods for obtaining such SC-EC cells, are described in WO2019 / 217493, which is incorporated herein by reference in its entirety.

[0025] The term "triple negative cells" or "TN cells" refers to cells that may be present in a cluster of differentiated pancreatic cells but do not express INS, GCG and SLC18A1. TN cells may include cells derived from progenitor or stem cells that are not SC-β, SC-α or SC-EC cells.

[0026] As used herein, the term "exocrine cells" refers to cells of exocrine glands, i.e., glands that excrete secretions through ducts. In certain embodiments, exocrine cells refer to exocrine pancreatic cells, which are pancreatic cells that produce enzymes that are secreted into the small intestine. These enzymes aid in the digestion of food as it passes through the digestive tract. Exocrine pancreatic cells, also called the islets of Langerhans, secrete two hormones: insulin and glucagon.

[0027] The term "phenotype" refers to the totality of biological characteristics, or many, that define a cell or organism under a particular set of environmental conditions and factors, regardless of the actual genotype.

[0028] As used herein, the term "pluripotent" refers to a cell that has the ability to differentiate under different conditions into one or more differentiated cell types, preferably into cell types characteristic of all three germ cell layers. Pluripotent cells are primarily characterized by their ability to differentiate into one or more cell types, preferably into all three germ cell layers, using, for example, a nude mouse teratoma formation assay. Pluripotency can also be demonstrated by expression of embryonic stem (ES) cell markers, but the preferred test of pluripotency is the demonstration of the ability to differentiate into cells of each of the three germ layers. It should be noted that simply culturing such cells does not, in and of itself, make them pluripotent. Reprogrammed pluripotent cells (e.g., iPS cells as defined herein) are also characterized by the ability to be passaged for extended periods without loss of proliferation potential, compared to their parent primary cells, which generally only undergo a limited number of divisions in culture.

[0029] As used herein, the terms "iPS cells" and "induced pluripotent stem cells" are used interchangeably and refer to pluripotent stem cells that have been artificially derived (e.g., by induction or complete reversal) from non-pluripotent cells, typically adult somatic cells, for example, by inducing forced expression of one or more genes.

[0030] The terms "progenitor cell" or "precursor" cell are used interchangeably herein and refer to a cell that has a more primitive cellular phenotype (i.e., earlier in a developmental pathway or progression than a fully differentiated cell) compared to the cell it may give rise to by differentiation. In many cases, progenitor cells also have significant, or even very high, proliferative potential. Progenitor cells can give rise to multiple different differentiated cell types, or a single differentiated cell type, depending on the developmental pathway and environment in which the cell develops and differentiates.

[0031] As used herein, the term "stem cell" refers to an undifferentiated cell that is capable of proliferation and of giving rise to more progenitor cells, which in turn have the capacity to give rise to a number of mother cells, which in turn can give rise to differentiated or differentiable daughter cells. The daughter cells themselves can be induced to proliferate and give rise to progeny that subsequently differentiate into one or more mature cells, while at the same time retaining one or more cells of the parent's developmental potential. The term "stem cell" refers to a subset of progenitor cells that, under certain conditions, have the capacity or potential to differentiate into a more specialized or differentiated phenotype, and also retain the capacity to proliferate without substantial differentiation, under certain conditions. In one embodiment, the term stem cell refers to a naturally occurring mother cell whose progeny (progeny cells) often specialize in different directions by differentiation, e.g., by acquiring entirely separate characteristics, as they occur in the progressive diversification of embryonic cells and tissues. Cell differentiation is a complex process, and typically occurs through many cell divisions. Differentiated cells may be derived from pluripotent cells, which themselves are derived from pluripotent cells, and so on. Although each of these pluripotent cells may be considered stem cells, the range of cell types each can give rise to may vary considerably. Some differentiated cells also have the ability to give rise to more developmentally potent cells. This ability may be natural or may be induced artificially by treatment with various factors. In many biological examples, stem cells are "pluripotent" because they can give rise to more than one different type of cell progeny, but this does not require "stemness". Self-renewal is another classical part of the definition of a stem cell, and is mandatory as used in this document. In theory, self-renewal can occur primarily by one of two main mechanisms. Stem cells may divide asymmetrically, with one daughter maintaining the stem cell state and the other daughter expressing some other specific function and phenotype. Alternatively, some of the stem cells in a population can divide symmetrically into two stem cells, thus maintaining some stem cells in the population as a whole, while other cells in the population give rise only to differentiated progeny.Formally, cells that begin as stem cells progress toward a differentiated phenotype, but may then "reverse" and re-express the stem cell phenotype, a term often referred to by those skilled in the art as "dedifferentiation" or "reprogramming," or "redifferentiation." As used herein, the term "pluripotent stem cells" includes embryonic stem cells, induced pluripotent stem cells, placental stem cells, and the like.

[0032] The term "embryonic stem cells" is used to refer to pluripotent stem cells of the inner cell mass of blastocysts (see U.S. Patent Nos. 5,843,780 and 6,200,806). Such cells can also be obtained from the inner cell mass of blastocysts derived from somatic cell nuclear transfer (see, e.g., U.S. Patent Nos. 5,945,577, 5,994,619, and 6,235,970). The distinguishing characteristics of embryonic stem cells define the phenotype of the embryonic stem cells. Thus, a cell has an embryonic stem cell phenotype if it possesses one or more of the distinctive characteristics of embryonic stem cells that distinguish the cell from other cells. Exemplary distinguishing characteristics of embryonic stem cells include, but are not limited to, gene expression profile, proliferation potential, differentiation potential, karyotype, responsiveness to specific culture conditions, and the like.

[0033] The term "adult stem cell" or "ASC" refers to pluripotent stem cells derived from non-embryonic tissues, including fetal, juvenile and adult tissues. Stem cells have been isolated from a variety of adult tissues, including blood, bone marrow, brain, olfactory epithelium, skin, pancreas, skeletal and muscle. Each of these stem cells has been characterized based on gene expression, factor responsiveness, and morphology in culture. Exemplary adult stem cells include neural stem cells, neural crest stem cells, mesenchymal stem cells, hematopoietic stem cells and pancreatic stem cells. As discussed above, stem cells have been found to be present in virtually every tissue. Thus, the present invention recognizes that stem cell populations can be isolated from virtually every animal tissue.

[0034] As used herein, the term "reprogramming" refers to the process of changing or reversing the differentiation state of a somatic cell. Prior to reprogramming, the cell may be partially or terminally differentiated. Reprogramming includes the complete reversion of the differentiation state of a somatic cell to a pluripotent cell. Such a complete reversal of differentiation produces an induced pluripotent (iPS) cell. As used herein, reprogramming also includes the partial reversal of the differentiation state of a cell, e.g., to a pluripotent state, or to a somatic cell that is neither pluripotent nor multipotent, but has lost one or more specific characteristics of the differentiated cell from which it was derived, e.g., characteristics that directly reprogram a differentiated cell to a different somatic cell type. Reprogramming generally involves changing, e.g., reversing, at least a portion of the genetic patterns, such as nucleic acid modifications (e.g., methylation), chromatin condensation, epigenetic changes, genomic imprinting, etc., that occur during cell differentiation as a zygote develops into an adult.

[0035] As used herein, the term "agent" refers to any compound or substance, such as, but not limited to, small molecules, nucleic acids, polypeptides, peptides, drugs, ions, and the like. An "agent" can be any chemical, entity, or moiety, including, but not limited to, synthetic and naturally occurring proteinaceous and non-proteinaceous entities. In some embodiments, the agent is a nucleic acid, a nucleic acid analog, a protein, an antibody, a peptide, an aptamer, an oligomer of nucleic acid, an amino acid, or a carbohydrate, including, but not limited to, a protein, an oligonucleotide, a ribozyme, a DNAzyme, a glycoprotein, an siRNA, a lipoprotein, an aptamer, and modifications and combinations thereof. In certain embodiments, the agent is a small molecule having a chemical moiety. For example, the chemical moiety includes unsubstituted or substituted alkyl, aromatic, or heterocyclic moieties, including macrolides, leptomycin, and related natural products or analogs thereof. The compound may be known to have the desired activity and / or characteristics, or may be selected from a diverse library of compounds.

[0036] As used herein, the term "contacting" (i.e., contacting at least one endocrine cell or precursor thereof with a maturation factor or combination of maturation factors) is intended to include culturing the cells together with the maturation factor in vitro (e.g., adding the maturation factor to the cells in culture). In some embodiments, the term "contacting" is not intended to include in vivo exposure of the cells to a compound as disclosed herein that may occur naturally in a subject (i.e., exposure that may occur as a result of a natural physiological process). As in the embodiments described herein, the step of contacting at least one endocrine cell or precursor thereof with a maturation factor can be performed in any suitable manner. For example, the cells can be treated in adherent or suspension culture. In some embodiments, the cells can be treated under conditions that promote cell clustering. The present disclosure contemplates any condition that promotes cell clustering. Examples of conditions that promote cell clustering include, but are not limited to, suspension culture in low-attachment tissue culture plates, spinner flasks, or aggrewell plates. In some embodiments, the inventors have observed that the clusters are stably maintained in medium containing 10% serum. In some embodiments, the conditions that promote cluster formation include low serum medium.

[0037] It is understood that cells contacted with a maturation factor may be simultaneously or subsequently contacted with another agent, such as a growth factor or other differentiation agent or environment, to stabilize the cells or to further differentiate the cells.

[0038] As used herein, a "cell culture medium" (also referred to herein as "culture medium" or "culture medium") is a medium for culturing cells that contains nutrients that maintain cell viability and support growth. Cell culture media may contain any of the following in appropriate combinations: salt(s), buffer(s), amino acids, glucose or other sugar(s), antibiotics, serum or serum substitutes, and other components such as peptide growth factors. Cell culture media commonly used for particular cell types are known to those of skill in the art.

[0039] The term "cell line" refers to a population of largely or substantially identical cells, typically derived from a single ancestral cell or from a population of distinct and / or substantially identical ancestral cells. A cell line may have been or may be maintained in culture for extended periods (e.g., months, years, indefinitely). A cell line may have undergone a spontaneous or induced process of transformation that confers unlimited culture life to the cells. A cell line includes all cell lines recognized in the art as such. It is recognized that at least some characteristics of individual cells of a cell line may differ from one another, as cells acquire mutations over time and, in some cases, undergo epigenetic changes. In some embodiments, a cell line comprises cells derived from stem cells as described herein.

[0040] The term "exogenous" refers to a substance present in a cell or organism other than its native source. For example, the term "exogenous nucleic acid" or "exogenous protein" refers to a nucleic acid or protein that is introduced by a human-mediated process into a biological system, such as a cell or organism, in which it is not normally present or is present in low abundance. A substance is considered exogenous if it is introduced into a cell or an ancestor of a cell that inherits the substance. In contrast, the term "endogenous" refers to a substance that is native to a biological system.

[0041] The term "expression" refers to the cellular processes involved in producing RNA and proteins and, if appropriate, secreting proteins, including, but not limited to, transcription, translation, folding, modification, and processing, as applicable. "Expression product" includes RNA transcribed from a gene and polypeptides resulting from translation of mRNA transcribed from a gene.

[0042] As used herein, the term "genetically modified" or "engineered" cell refers to a cell into which an exogenous nucleic acid has been introduced by a process mediated by the hand of man (or the progeny of such a cell that inherits at least a portion of the nucleic acid). The nucleic acid may, for example, contain a sequence exogenous to the cell, or may contain a native sequence (i.e., a sequence naturally found in the cell), but may also contain a sequence that does not naturally occur (e.g., a coding region linked to a promoter from a different gene), or a modified version of a native sequence, etc. Introducing the nucleic acid into the cell can be accomplished by any suitable technique. Suitable techniques include calcium phosphate or lipid-mediated transfection, electroporation, and introduction or infection with a viral vector. In some embodiments, the polynucleotide or a portion thereof is integrated into the genome of the cell. The nucleic acid may then be removed or excised from the genome, provided that such removal or excision results in a detectable change in the cell compared to an unmodified but otherwise equivalent cell. It should be recognized that the term genetic modification is intended to include the direct introduction of modified RNA into the cell (e.g., synthetic modified RNA). Such synthetic modified RNAs include modifications to prevent rapid degradation by endonucleases and exonucleases and to avoid or reduce cellular innate immune or interferon responses to RNA. Modifications include, for example, (a) terminal modifications, such as 5'-terminal modifications (phosphorylation, dephosphorylation, splicing, reverse linkage, etc.), 3'-terminal modifications (splicing, DNA nucleotides, reverse linkage, etc.), (b) base modifications, such as modified bases, stabilizing bases, destabilizing bases, or bases that base-pair with an expanded repertoire of partners, or substitutions with conjugate bases, (c) sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions, and (d) internucleoside linkage modifications, including modifications or substitutions of phosphodiester linkages. To the extent that such modifications disrupt translation (i.e., translation is reduced by 50% or more relative to the absence of the modification, for example, in a rabbit reticulocyte in vitro translation assay), the modifications are not suitable for the methods and compositions described herein.

[0043] As used herein, the term "identity" refers to the degree to which two or more nucleic acid or polypeptide sequences are the same. The percent identity between a sequence of interest and a second sequence over an evaluation range, such as the length of a sequence of interest, can be calculated by aligning the sequences, determining the number of residues (nucleotides or amino acids) in the evaluation range opposite the identical residues, allowing for the introduction of gaps to maximize identity, dividing by the total number of residues in the sequence of interest or the second sequence (whichever is larger) that fall within the range, and multiplying by 100. When calculating the number of identical residues required to achieve a particular percent identity, fractions are rounded to the nearest whole number. Percent identity can be calculated using various computer programs known in the art. For example, computer programs such as BLAST2, BLASTN, BLASTP, Gapped BLAST generate alignments and provide percent identity between sequences of interest. The algorithm of Karlin and Altschul (Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:22264-2268, 1990), modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993, has been incorporated into the NBLAST and XBLAST programs of Altschul et al. (Altschul, et al., J. MoI. Biol. 215:403-410, 1990). To obtain gapped alignments for comparison, Gapped BLAST is utilized as described by Altschul et al. (Altschul, et al. Nucleic Acids Res. 25:3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs can be used. PAM250 or BLOSUM62 matrices may be used. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI).For these programs, see the website with the URL World Wide Web address "ncbi.nlmnih.gov." In a specific embodiment, the percent identity is calculated using BLAST2 with the default parameters as provided by NCBI.

[0044] As used herein, the terms "isolated" or "partially purified" refer to a nucleic acid or polypeptide that has been separated from at least one other component (e.g., nucleic acid or polypeptide) that is present with the nucleic acid or polypeptide in a natural source and / or that would be present with the nucleic acid or polypeptide when expressed by a cell or that would be secreted, in the case of a secreted polypeptide. Chemically synthesized nucleic acids or polypeptides or those synthesized using in vitro transcription / translation are considered "isolated."

[0045] As used herein, the term "isolated cell" refers to a cell that has been removed from the organism in which it was originally found, or the progeny of such a cell. Optionally, the cell has been cultured in vitro, for example in the presence of other cells. Optionally, the cell is subsequently introduced into a second organism, or the cell (or the progeny of the cell) is reintroduced into the organism from which it was isolated.

[0046] As used herein, the term "isolated population" with respect to an isolated population of cells refers to a cell population that is removed and separated from a mixed or heterogeneous cell population. In some embodiments, an isolated population is a substantially pure cell population compared to the heterogeneous population from which the cells are isolated or enriched.

[0047] The term "substantially pure" with respect to a particular cell population refers to a cell population that is at least about 75%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% pure with respect to the cells that make up the total cell population.

[0048] The terms "enrich" or "enriched" are used interchangeably herein and mean that the yield (proportion) of a type of cell is increased by at least 10% over the proportion of that type of cell in the starting culture or preparation.

[0049] As used herein, the terms "renewal" or "self-renewal" or "proliferation" are used interchangeably and refer to the ability of a stem cell to renew itself by dividing into the same non-specific cell type over an extended period of time and / or over a period of months to years. In some cases, proliferation refers to the expansion of a cell by repeatedly dividing into two identical daughter cells.

[0050] As used herein, the term "lineage" refers to cells that share a common ancestor or a common developmental fate. For example, in the context of a cell that is of endoderm origin or of "endodermal lineage," it means that the cell is derived from an endoderm cell and can differentiate along one or more developmental lineage pathways that are restricted to the endodermal lineage, such as giving rise to a committed endoderm cell, which can then differentiate into liver cells, thymus, pancreas, lung, and intestine.

[0051] As used herein, a "marker" is used to describe the characteristics and / or phenotype of a cell. Markers can be used to select cells containing a characteristic of interest. Markers vary depending on the particular cell. A marker is a characteristic, such as a morphological, functional, or biochemical (enzymatic) characteristic of a particular cell type of cell or a molecule expressed by a cell type. Preferably, such a marker is a protein, more preferably having an epitope for an antibody or other binding molecule available in the art. However, a marker may be composed of any molecule found within a cell, including, but not limited to, proteins (peptides and polypeptides), lipids, polysaccharides, nucleic acids, and steroids. Examples of morphological characteristics or traits include, but are not limited to, shape, size, and nucleus to cytoplasm ratio. Examples of functional characteristics or traits include, but are not limited to, the ability to adhere to a particular substrate, the ability to take up or exclude a particular dye, the ability to migrate under particular conditions, and the ability to differentiate along a particular lineage. Markers can be detected by any method available to one of skill in the art. A marker can also be the absence of a morphological feature, or the absence of a protein, lipid, etc. A marker can be a combination of a panel of unique features related to the presence and absence of polypeptides, as well as other morphological features.

[0052] The term "modulate" is used as it is in the art, i.e., to cause or promote a qualitative or quantitative change, alteration, or modification in a process, pathway, or phenomenon of interest. Without being limited thereto, such a change can be an increase, decrease, or change in the relative strength or activity of different components or branches of the process, pathway, or phenomenon. A "modulator" is an agent that causes or promotes a qualitative or quantitative change, alteration, or modification in a process, pathway, or phenomenon of interest.

[0053] As used herein, the term "DNA" is defined as deoxyribonucleic acid.

[0054] The term "polynucleotide" is used interchangeably with "nucleic acid" herein to refer to a polymer of nucleosides. Typically, the polynucleotides of the present invention are nucleosides found naturally in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) linked by phosphodiester bonds. However, the term encompasses molecules containing nucleosides, whether or not found in naturally occurring nucleic acids, or nucleoside analogs, including chemically or biologically modified bases, modified backbones, and the like, which may be preferred for certain applications. When the present application refers to polynucleotides, it is understood that DNA, RNA, and in each case both single-stranded and double-stranded forms (and the complement of each single-stranded molecule) are provided. As used herein, "polynucleotide sequence" can refer to the polynucleotide material itself and / or the sequence information (i.e., the sequence of letters used as abbreviations for bases) that biochemically characterize a particular nucleic acid. Polynucleotide sequences presented herein are presented in the 5' to 3' orientation unless otherwise indicated.

[0055] As used herein, the term "polypeptide" refers to a polymer of amino acids. The terms "protein" and "polypeptide" are used interchangeably herein. A peptide is a relatively short polypeptide, typically about 2-60 amino acids in length. A polypeptide as used herein typically contains amino acids, such as the 20 L-amino acids most commonly found in proteins. However, other amino acids and / or amino acid analogs known in the art can be used. One or more amino acids in a polypeptide can be modified, for example, by the addition of a chemical entity, such as a carbohydrate group, a phosphate group, a fatty acid group, a linker for conjugation, functionalization, etc. Polypeptides to which non-polypeptide moieties are covalently or non-covalently attached are also considered to be "polypeptides". Exemplary modifications include glycosylation and palmitoylation. Polypeptides can be purified from natural sources, produced using recombinant DNA technology, synthesized by chemical means such as conventional solid-phase peptide synthesis, etc. As used herein, the terms "polypeptide sequence" or "amino acid sequence" can refer to the polypeptide material itself and / or to the sequence information that biochemically characterizes the polypeptide (i.e., the sequence of letters or three-letter codes used as an abbreviation for the names of amino acids). The sequences of the polypeptides presented herein are presented in the N-terminal to C-terminal direction unless otherwise indicated.

[0056] The term "variant" when referring to a polypeptide may be, for example, a polypeptide that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the full-length polypeptide. A variant may be a fragment of a full-length polypeptide. A variant may be a naturally occurring splice variant. A variant may be a polypeptide that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to a fragment of a polypeptide, where the fragment is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% in length of the full-length wild-type polypeptide or a domain thereof that has the desired activity. In some embodiments, the domain is at least 100, 200, 300, or 400 amino acids in length, beginning at any amino acid position in the sequence and extending toward the C-terminus. Variants known in the art that eliminate or substantially reduce the activity of a protein are preferably avoided. In some embodiments, the variant lacks the N-terminal and / or C-terminal portions of the full-length polypeptide, e.g., up to 10, 20, or 50 amino acids from either end. In some embodiments, the polypeptide has the sequence of a mature (full-length) polypeptide, meaning a polypeptide from which one or more portions, such as a signal peptide, have been removed during normal intracellular proteolytic processing (e.g., during or after co-translational processing). In some embodiments, where the protein is produced by other than purification from a cell that naturally expresses it, the protein is a chimeric polypeptide, meaning that it contains portions from two or more different species. In some embodiments, where the protein is produced by other than purification from a cell that naturally expresses it, the protein is a derivative, meaning that it contains additional sequences not associated with the protein, so long as those sequences do not substantially reduce the biological activity of the protein.

[0057] As used herein, the term "functional fragment" refers to a polypeptide having a substantially homologous amino acid sequence but smaller in size than the polypeptide from which the fragment is derived, where the functional fragment polypeptide sequence has at least 50%, or 60%, or 70%, or 80%, or 90%, or 100% or more, e.g., 1.5-fold, 2-fold, 3-fold, 4-fold, or more than 4-fold, effective biological activity of the polypeptide from which the fragment is derived. Functional fragment polypeptides can have additional functions including reduced antigenicity, increased DNA binding (such as a transcription factor), or altered RNA binding (such as regulating RNA stability or degradation).

[0058] The term "vector" refers to a carrier DNA molecule into which a DNA sequence can be inserted for introduction into a host cell. Preferred vectors are capable of autonomous replication and / or expression of the nucleic acid to which it is linked. A vector capable of directing the expression of a gene to which it is operably linked is referred to herein as an "expression vector." Thus, an "expression vector" is a specialized vector that contains the necessary regulatory regions for the expression of a gene of interest in a host cell. In some embodiments, the gene of interest is operably linked to another sequence in the vector. The vector may be a viral vector or a non-viral vector. When a viral vector is used, it is preferred that the viral vector is replication-deficient, which can be achieved, for example, by removing all viral nucleic acid that codes for replication. Replication-deficient viral vectors still retain infectious properties and enter cells in a manner similar to replicating adenoviral vectors, but once inside the cell, replication-deficient viral vectors do not replicate or grow. Vectors also include means of delivering DNA molecules to cells, such as liposomes and nanoparticles.

[0059] The term "operably linked" means that the control sequences necessary for expression of a coding sequence are positioned in a DNA molecule in the appropriate position relative to the coding sequence to effect expression of the coding sequence. This same definition may also be applied to the positioning of coding sequences and transcription control elements (e.g., promoters, enhancers, and termination elements) in an expression vector. The term "operably linked" includes having an appropriate initiation signal (e.g., ATG) in front of the polynucleotide sequence to be expressed, and maintaining the correct reading frame to allow expression of the polynucleotide sequence under the control of the expression control sequences, and production of the desired polypeptide encoded by the polynucleotide sequence.

[0060] The term "viral vector" refers to the use of a virus or virus-associated vector as a carrier of a nucleic acid construct into a cell. The construct may be incorporated and packaged into an adenovirus, adeno-associated virus (AAV), or herpes simplex virus (HSV), or other non-replicating defective viral genome, including retroviral and lentiviral vectors, to infect or introduce into a cell. The vector may or may not be integrated into the genome of the cell. The construct may optionally include viral sequences for transfection. Alternatively, the construct may be incorporated into a vector capable of episomal replication, such as EPV and EBV vectors.

[0061] The terms "control sequence" and "promoter" are used interchangeably herein and refer to nucleic acid sequences, such as initiation signals, enhancers, and promoters, that induce or control the transcription of a protein coding sequence to which they are operably linked. In some examples, transcription of a recombinant gene is under the control of a promoter sequence (or other transcription control sequence) that controls expression of the recombinant gene in a cell type in which expression is intended. It is also understood that a recombinant gene can be under the control of a transcription control sequence that is the same or different from the sequence that controls transcription of the naturally occurring protein. In some cases, the promoter sequence is recognized by the synthetic machinery of the cell, or introduced synthetic machinery, necessary to initiate transcription of a particular gene.

[0062] As used herein, the term "transcription factor" refers to a protein that binds to a specific portion of DNA using a DNA-binding domain and is part of a system that controls the transfer (or transcription) of genetic information from DNA to RNA. As used herein, "proliferate" and "proliferation" refer to an increase in the number of cells in a population (growth) by cell division. Cell proliferation is generally understood to result from the coordinated activation of multiple signaling pathways in response to an environment that includes growth factors and other mitogens. Cell proliferation can also be promoted by relief from the action of intracellular or extracellular signals and mechanisms that inhibit or adversely affect cell proliferation.

[0063] The term "selectable marker" refers to a gene, RNA, or protein that, when expressed, confers on a cell a selectable phenotype, such as resistance to cytotoxic or cytostatic agents (e.g., antibiotic resistance), trophogenicity, or expression of a specific protein that can be used as a basis for distinguishing cells that express the protein from those that do not. Proteins whose expression can be easily detected ("detectable markers"), such as fluorescent or luminescent proteins, or enzymes that act on a substrate to produce a colored, fluorescent, or luminescent substance, constitute a subset of selectable markers. The presence of a selectable marker linked to expression control elements native to genes that are normally selectively or exclusively expressed in pluripotent cells allows for the identification and selection of somatic cells that have been reprogrammed to the pluripotent state. Various selectable marker genes can be used, such as the neomycin resistance gene (neo), puromycin resistance gene (puro), guanine phosphoribosyltransferase (gpt), dihydrofolate reductase (DHFR), adenosine deaminase (ada), puromycin-N-acetyltransferase (PAC), hygromycin resistance gene (hyg), multidrug resistance gene (mdr), thymidine kinase (TK), hypoxanthine-guanine phosphoribosyltransferase (HPRT), and hisD gene. Detectable markers include green fluorescent protein (GFP), blue, sapphire, yellow, red, orange, and cyan fluorescent proteins, and mutants of any of these. Luminescent proteins such as luciferase (e.g., firefly or Renilla luciferase) are also useful. As will be appreciated by those skilled in the art, the term "selectable marker" as used herein can refer to a gene or the expression product of a gene, such as an encoded protein.

[0064] In some embodiments, a selectable marker confers a growth and / or survival advantage to cells expressing it compared to cells that do not express it or express it at a significantly lower level. Such a growth and / or survival advantage usually occurs when cells are maintained under specific conditions, i.e., "selective conditions". To ensure effective selection, a population of cells can be maintained for a sufficient period of time under conditions such that cells that do not express the marker do not grow and / or survive and are eliminated from the population or are reduced in number to a small fraction of the population. The process of selecting cells expressing a marker that confers a growth and / or survival advantage by maintaining a population of cells under selective conditions that largely or completely eliminate cells that do not express the marker is referred to herein as "positive selection" and a marker is said to be "useful for positive selection". Negative selection and markers useful for negative selection are also of interest in certain methods described herein. Expression of such a marker confers a growth and / or survival disadvantage on cells expressing the marker relative to cells that do not express the marker or express the marker at significantly lower levels (or, viewed alternatively, cells that do not express the marker have a growth and / or survival advantage relative to cells that express the marker.) Thus, cells expressing the marker can be largely or completely eliminated from a cell population when maintained under selective conditions for a sufficient period of time.

[0065] As used herein, "reporter gene" includes any gene genetically introduced into a cell that adds to the stem cell phenotype. The reporter genes disclosed in the present invention are intended to include fluorescent genes, luminescent genes, enzyme genes, and resistance genes, but also include other genes that can be easily detected by a person skilled in the art. In some embodiments of the present invention, reporter genes are used as markers to identify specific stem cells, cardiovascular stem cells, and their differentiated progeny. Reporter genes are generally operably linked to sequences that control their expression in a manner that depends on one or more conditions monitored by measuring the expression of the reporter gene. In some cases, the expression of the reporter gene is determined in living cells. When a live cell reporter gene assay is used, the expression of the reporter gene can be monitored at multiple time points, for example, 2, 3, 4, 5, 6, 8, or 10 or more time points. In some cases, when a live cell reporter assay is used, reporter gene expression is monitored at a frequency of at least about 10 minutes to about 24 hours, e.g., 20 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, or another frequency from any integer between about 10 minutes and about 24 hours.

[0066] The terms "subject" and "individual" are used interchangeably herein and refer to an animal, e.g., a human, from which cells can be obtained and / or to which cell therapy (including prophylactic therapy) as described herein is provided. For the treatment of an infection, condition, or disease state specific to a particular animal, such as a human subject, the term subject refers to that particular animal. As used interchangeably herein, "non-human animals" and "non-human mammals" include mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The term "subject" also encompasses any vertebrate, including, but not limited to, mammals, reptiles, amphibians, and fish. However, advantageously, the subject is a mammal, such as a human, or other mammal, such as a domesticated mammal, e.g., dog, cat, horse, or a production mammal, e.g., cow, sheep, pig, etc.

[0067] When applied to isolated cells, the terms "treat," "treating," "treatment," and the like include subjecting the cells to any process or condition, or performing any operation or procedure on the cells. When applied to a subject, these terms refer to the medical or surgical treatment, care, or management of an individual, who is usually ill or injured, or at increased risk of developing a disease compared to the average member of the population, and who is in need of such attention, care, or management.

[0068] As used herein, the terms "treat" and "treatment" refer to administering an effective amount of a composition to a subject so that the subject achieves at least one symptom of the disease or an improvement in the disease, e.g., a beneficial or desired clinical outcome. For purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, the reduction of one or more symptoms, the reduction in the extent of the disease, the stabilization of the disease state (i.e., not worsening), the delay or slowing of the progression of the disease, the improvement or mitigation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. Treating may mean extending survival compared to the expected survival in the absence of treatment. Thus, one of skill in the art will recognize that treatment may improve the disease state but may not result in a complete cure of the disease. As used herein, the term "treatment" includes prevention. Alternatively, treatment is "effective" if the progression of the disease is inhibited or halted. "Treatment" may also mean extending survival compared to the expected survival in the absence of treatment.

[0069] As used herein, the terms "administer", "introduce" and "implant" are used interchangeably in the context of introducing the cells of the present invention into a subject in a manner or route such that the introduced cells are at least partially localized at a desired site. The cells can be directly implanted into the pancreas or digestive tract, or can be administered by a suitable route such that at least some of the implanted cells or components of the cells remain viable and are delivered to the desired site in the subject. The viability of the cells after administration to the subject can be as short as a few hours, e.g., 24 hours, to as long as several days or years. In some cases, the cells can be administered subcutaneously, e.g., in a capsule (e.g., microcapsule) to maintain the implanted cells at the implanted site and avoid migration of the implanted cells.

[0070] As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. As used herein, the phrases "systemic administration", "administered systemically", "peripheral administration", "administered peripherally" refer to administration of stem cell-derived cells and / or their progeny and / or compounds and / or other substances other than directly into the central nervous system, such that it enters the animal's body and thus undergoes metabolic and other similar processes, e.g., subcutaneous administration.

[0071] The term "tissue" refers to a group or layer of specialized cells that together perform a particular specialized function. The term "tissue-specific" refers to the source of cells that are derived from a particular tissue.

[0072] As used herein, the terms "reduce", "reduced", "reduction", "reduction" or "inhibit" are all commonly used to mean a statistically significant amount of reduction. However, for the avoidance of doubt, "reduced", "reduction" or "reduce" or "inhibit" means a reduction of at least 10% compared to the reference level, for example, a reduction of at least about 20%, or a reduction of at least about 30%, or a reduction of at least about 40%, or a reduction of at least about 50%, or a reduction of at least about 60%, or a reduction of at least about 70%, or a reduction of at least about 80%, or a reduction of at least about 90%, or a reduction of up to 100% (i.e., absent levels compared to the reference sample), or any reduction between 10-100% compared to the reference level.

[0073] The terms "increased", "increase", "enhance" or "activate" are all used herein to generally mean an increase of a statistically significant amount. For the avoidance of any doubt, the terms "increase", "increase", "enhance" or "activate" mean an increase of at least 10% compared to a reference level, for example, an increase of at least about 20%, or an increase of at least about 30%, or an increase of at least about 40%, or an increase of at least about 50%, or an increase of at least about 60%, or an increase of at least about 70%, or an increase of at least about 80%, or an increase of at least about 90%, or an increase of up to 100%, or any increase between 10-100% compared to a reference level, or an increase of at least about 2-fold compared to a reference level, or an increase of at least about 3-fold, or an increase of at least about 4-fold, or an increase of at least about 5-fold, or an increase of at least about 10-fold, or any increase between 2-fold and 10-fold or more compared to a reference level.

[0074] The term "statistically significant" or "significantly" refers to statistical significance, which generally means that the concentration of the marker is two standard deviations (2SD) or lower than the normal value. The term refers to statistical evidence that there is a difference. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true. It is often determined using a p-value.

[0075] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods, and their respective component(s) that are essential to the invention, but are open to the inclusion of unspecified elements, whether essential or not.

[0076] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0077] The term "comprising" refers to compositions, methods, and components thereof described herein, which do not include any element not recited in that description of an embodiment.

[0078] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more methods, and / or steps of the type described herein and / or that will become apparent to those skilled in the art upon reading this disclosure, etc.

[0079] stem cells Stem cells are cells that retain the ability to renew themselves by mitotic cell division and can differentiate into a variety of specialized cell types. There are two types of mammalian stem cells: embryonic stem (ES) cells, found in blastocysts, and adult stem cells, found in adult tissues. In the developing embryo, stem cells can differentiate into all specialized embryonic tissues. In adult organisms, stem and progenitor cells act as the body's repair system, not only replenishing specialized cells but also maintaining the normal turnover of regenerative organs such as blood, skin, and intestinal tissues. Pluripotent stem cells can differentiate into cells derived from any of the three germ cell layers.

[0080] Below, certain embodiments are described with respect to the use of stem cells, however, germ cells can be used in place of or together with stem cells to provide at least one differentiated cell using protocols similar to the exemplary protocols described herein. Suitable germ cells can be prepared, for example, from primordial germ cells present in human fetal material harvested approximately 8-11 weeks after the last menstrual period. Exemplary methods for preparing germ cells are described, for example, in Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998 and U.S. Patent No. 6,090,622.

[0081] ES cells, such as human embryonic stem cells (hESCs) or mouse embryonic stem cells (mESCs), have a virtually infinite replicative capacity and can differentiate into most cell types, and thus, in principle, provide an infinite starting material for generating differentiated cells for clinical therapy (stemcells.nih.gov / info / scireport / 2006report.htm, 2006).

[0082] hESC cells are described, for example, in Cowan et al. (N Engl. J. Med. 350:1353, 2004) and Thomson et al. (Science 282:1145, 1998). Embryonic stem cells from other primates, such as rhesus monkey stem cells (Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844, 1995), marmoset stem cells (Thomson et al., Biol. Reprod. 55:254, 1996), and human embryonic germ (hEG) cells (Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998), can also be used in the methods disclosed herein. mESCs are described, for example, in Tremml et al. (Curr Protoc Stem Cell Biol. Chapter 1:Unit 1C.4, 2008). Stem cells may be, for example, unipotent, totipotent, pluripotent, or multipotent. In some examples, any cell from a primate that can produce progeny that are derivatives of at least one germ cell layer, or all three germ cell layers, can be used in the methods disclosed herein.

[0083] In certain embodiments, ES cells can be isolated as described, for example, in Cowan et al. (N Engl. J. Med. 350:1353, 2004) and U.S. Patent No. 5,843,780, and Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844, 1995. For example, hESC cells can be prepared from human blastocyst cells using techniques described by Thomson et al. (U.S. Patent No. 6,200,806; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133 ff., 1998) and Reubinoff et al., Nature Biotech. 18:399, 2000. Equivalent cell types to hESCs include their pluripotent derivatives, such as primitive ectoderm-like (EPL) cells, as reviewed, for example, in WO01 / 51610 (Bresagen). hESCs can also be obtained from human preimplantation embryos. Alternatively, in vitro fertilized (IVF) embryos can be used, or human one-cell embryos can be expanded to the blastocyst stage (Bongso et al., Hum Reprod 4:706, 1989). Embryos are cultured to the blastocyst stage in G1.2 and G2.2 media (Gardner et al., Fertil. Steril. 69:84, 1998). The zona pellucida is removed from the developed blastocysts by brief exposure to pronase (Sigma). The inner cell mass may be isolated by immunosurgery, in which blastocysts are exposed to a 1:50 dilution of rabbit anti-human spleen cell antiserum for 30 min, washed three times for 5 min with DMEM, and exposed to a 1:5 dilution of guinea pig complement (Gibco) for 3 min (Solter et al., Proc. Natl. Acad. Sci. USA 72:5099, 1975). After two additional washes with DMEM, lysed trophectoderm cells are removed from the intact inner cell mass (ICM) by gentle pipetting, and the ICM is plated onto an mEF feeder layer.After 9-15 days, the inner cell mass-derived outgrowths can be dissociated into clumps by exposure to phosphate-buffered saline (PBS) with 1 mM EDTA without calcium and magnesium, by exposure to dispase or trypsin, or by mechanical dissociation with a micropipette, and then replated on mEFs in fresh medium. Growing colonies with undifferentiated morphology can be individually selected with a micropipette, mechanically dissociated into clumps, and replated. ES-like morphology is characterized as small colonies with a clearly high nuclear to cytoplasmic ratio and prominent nucleoli. The resulting hESCs can be split periodically every 1-2 weeks, for example, by brief trypsinization, exposure to Dulbecco's PBS (containing 2 mM EDTA), exposure to collagenase type IV (approximately 200 U / mL; Gibco), or by selection of individual colonies with a micropipette. In some examples, clump sizes of approximately 50-100 cells are optimal. mESC cells can be prepared, for example, using the techniques described by Conner et al. (Curr. Prot. in Mol. Biol. Unit 23.4, 2003).

[0084] Embryonic stem cells can be isolated from blastocysts of members of the primate species (U.S. Patent No. 5,843,780; Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844, 1995). Human embryonic stem cells (hES) can be prepared from human blastocyst cells using techniques described by Thomson et al. (U.S. Patent No. 6,200,806; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133 ff., 1998) and Reubinoff et al., Nature Biotech. 18:399, 2000. Equivalent cell types to hES cells include their pluripotent derivatives, such as primitive ectoderm-like (EPL) cells, as reviewed in WO01 / 51610 (Bresagen).

[0085] Alternatively, in some embodiments, hESCs can be obtained from human preimplantation embryos. Alternatively, in vitro fertilized (IVF) embryos can be used, or human one-cell embryos can be expanded to the blastocyst stage (Bongso et al., Hum Reprod 4:706, 1989). Embryos are cultured to the blastocyst stage in G1.2 and G2.2 media (Gardner et al., Fertil. Steril. 69:84, 1998). The zona pellucida is removed from the developed blastocysts by brief exposure to pronase (Sigma). The inner cell mass is isolated by immunosurgery by exposing blastocysts to a 1:50 dilution of rabbit anti-human spleen cell antiserum for 30 min, followed by three 5-min washes in DMEM, and a 3-min exposure to a 1:5 dilution of guinea pig complement (Gibco) (Solter et al., Proc. Natl. Acad. Sci. USA 72:5099, 1975). After two additional washes in DMEM, lysed trophectoderm cells are removed from the intact inner cell mass (ICM) by gentle pipetting, and the ICM is plated onto mEF feeder layers.

[0086] After 9–15 days, the inner cell mass-derived outgrowths can be dissociated into clumps by exposure to phosphate-buffered saline (PBS) containing 1 mM EDTA without calcium and magnesium, by exposure to dispase or trypsin, or by mechanical dissociation with a micropipette, and then replated on mEFs in fresh medium. Growing colonies with undifferentiated morphology can be individually selected with a micropipette, mechanically dissociated into clumps, and replated. ES-like morphology is characterized as small colonies with a clearly high nuclear to cytoplasmic ratio and prominent nucleoli. The resulting ES cells are routinely split every 1–2 weeks by brief trypsinization, exposure to Dulbecco's PBS (containing 2 mM EDTA), exposure to collagenase type IV (approximately 200 U / mL; Gibco), or by selection of individual colonies with a micropipette. A clump size of approximately 50–100 cells is optimal.

[0087] In some embodiments, human embryonic germ (hEG) cells are pluripotent stem cells that can be used to differentiate into primitive endoderm cells in the manner disclosed herein. hEG cells can be prepared from primordial germ cells present in human fetal material harvested approximately 8-11 weeks after the last menstrual period. Suitable preparation methods are described in Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998, and U.S. Patent No. 6,090,622, which are incorporated herein by reference in their entireties.

[0088] Briefly, genital ridges are treated to form disaggregated cells. EG growth medium is DMEM, 4500 mg / L D-glucose, 2200 mg / L mM NaHCO3, 15% ES-qualified fetal bovine serum (BRL), 2 mM glutamine (BRL), 1 mM sodium pyruvate (BRL), 1000-2000 U / mL human recombinant leukemia inhibitory factor (LIF, Genzyme), 1-2 ng / mL human recombinant bFGF (Genzyme), and 10 µM forskolin (10% in DMSO). 96-well tissue culture plates are prepared with a subconfluent layer of feeder cells (e.g., STO cells, ATCC number CRL1503) cultured for 3 days in modified EG growth medium without LIF, bFGF, or forskolin inactivated by 5000 rad gamma irradiation, and approximately 0.2 mL of primary germ cell (PGC) suspension is added to each well. The first passage is performed after 7-10 days in EG growth medium, transferring each well into one well of a 24-well culture dish previously prepared with irradiated STO mouse fibroblasts. Culture the cells with daily changes of medium until the morphology of the cells is consistent with EG cells, typically after 7-30 days or 1-4 passages.

[0089] In some examples, the stem cells may be undifferentiated cells (e.g., cells that are not committed to a particular lineage) prior to exposure to at least one maturation factor according to the methods as disclosed herein, while in other examples, it may be desirable to differentiate the stem cells into one or more intermediate cell types prior to exposure to at least one maturation factor(s) as described herein. For example, the stem cells may exhibit morphological, biological, or physical characteristics of undifferentiated cells that can be used to distinguish them from differentiated cells of embryonic or adult origin. In some examples, undifferentiated cells may be seen in colonies of cells with high nuclear / cytoplasmic ratios and prominent nucleoli when viewed in two dimensions under a microscope. The stem cells may be used by themselves (e.g., in the substantial absence of undifferentiated cells) or in the presence of differentiated cells. In some examples, the stem cells may be cultured in the presence of appropriate nutrients and, optionally, other cells, to allow the stem cells to proliferate and, optionally, differentiate. For example, embryonic fibroblasts or fibroblast-like cells may be present in the culture to support the growth of the stem cells. Fibroblasts may be present at one stage of stem cell proliferation, but not necessarily at all stages, for example, fibroblasts may be added to the stem cell culture medium during an initial culture stage, but not during one or more subsequent culture stages.

[0090] The stem cells used in all aspects of the invention can be any cell derived from any type of tissue (e.g., embryonic tissue, such as fetal or pre-fetal tissue, or adult tissue), characterized in that under appropriate conditions they can produce progeny of different cell types, such as all derivatives of at least one of the three germ layers (endoderm, mesoderm, and ectoderm). These cell types can be provided in the form of established cell lines, or can be obtained directly from primary embryonic tissue and used immediately for differentiation. Cells listed in the NIH Human Embryonic Stem Cell Registry include hESBGN-01, hESBGN-02, hESBGN-03, hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3, HES-4, HES-5, HES-6 (ES Cell International); Miz-hES1 (MizMedi Hospital-Seoul National University); HSF-1, HSF-6 (University of California at San Francisco); and H1, H7, H9, H13, H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute)). In some embodiments, the source of human stem cells or pluripotent stem cells used for chemically induced differentiation into stem cell-derived cells did not involve the destruction of human embryos.

[0091] In another embodiment, stem cells can be isolated from tissues including solid tissues. In some embodiments, the tissue is skin, fat tissue (e.g., adipose tissue), muscle tissue, heart or cardiac tissue. In other embodiments, the tissue is, for example, but not limited to, umbilical cord blood, placenta, bone marrow, or cartilage.

[0092] Stem cells of interest also include various types of embryonic cells, such as human embryonic stem (hES) cells, as described by Thomson et al. (1998) Science 282:1145; embryonic stem cells from other primates, such as rhesus monkey stem cells (Thomson et al. (1995) Proc. Natl. Acad. Sci. USA 92:7844); marmoset stem cells (Thomson et al. (1996) Biol. Reprod. 55:254); and human embryonic germ (hEG) cells (Shambloft et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). Also of interest are lineage-committed stem cells such as mesodermal stem cells and other early cardiac progenitors (see, e.g., Reyes et al. (2001) Blood 98:2615-2625; Eisenberg & Bader (1996) Circ Res. 78(2):205-16). Stem cells can be obtained from any mammalian species, e.g., human, horse, cow, pig, dog, cat, rodent, e.g., mouse, rat, hamster, primate, etc. In some embodiments, human embryos were not destroyed for the source of pluripotent cells used in the methods and compositions as disclosed herein.

[0093] ES cells are considered undifferentiated if they are not committed to a particular lineage of differentiation. Such cells display morphological characteristics that distinguish them from differentiated cells of embryonic or adult origin. Undifferentiated ES cells are easily recognized by those skilled in the art and are usually seen in colonies of cells with high nuclear / cytoplasmic ratios and prominent nucleoli when viewed two-dimensionally under a microscope. Undifferentiated ES cells express genes that can be used as markers to detect the presence of undifferentiated cells, and whose polypeptide products can be used as markers for negative selection. See, e.g., U.S. Application No. 2003 / 0224411A1; Bhattacharya (2004) Blood 103(8):2956-64; and Thomson (1998), supra., each of which is incorporated herein by reference. Human ES cell lines express cell surface markers that characterize undifferentiated non-human primate ES cells and human EC cells, including stage-specific embryonic antigen (SSEA)-3, SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase. The globo-series glycolipid GL7, which carries the SSEA-4 epitope, is formed by the addition of sialic acid to the globo-series glycolipid GbS, which carries the SSEA-3 epitope. Thus, GL7 reacts with antibodies against both SSEA-3 and SSEA-4. Undifferentiated human ES cell lines did not stain with SSEA-1, whereas differentiated cells stained strongly with SSEA-I. Methods for expanding undifferentiated hES cells are described in WO99 / 20741, WO01 / 51616, and WO03 / 020920.

[0094] A mixture of cells from a suitable source of endothelial, muscle, and / or neural stem cells can be harvested from a mammalian donor by methods known in the art. A suitable source is the hematopoietic microenvironment. For example, circulating peripheral blood, preferably mobilized (i.e., recruited) circulating peripheral blood, may be removed from a subject. Alternatively, bone marrow may be harvested from a mammal, such as a human patient undergoing an autologous transplant. In some embodiments, stem cells can be obtained from adipose tissue of a subject, for example, using Cytori's CELUTION™ SYSTEM, as disclosed in U.S. Pat. Nos. 7,390,484 and 7,429,488, which are incorporated herein by reference in their entireties.

[0095] In some embodiments, human umbilical cord blood cells (HUCBCs) are useful in the methods disclosed herein. Human UBC cells are recognized as a rich source of hematopoietic and mesenchymal progenitor cells (Broxmeyer et al., 1992 Proc. Natl. Acad. Sci. USA 89:4109-4113). Previously, umbilical cord blood and placental blood were generally considered waste products that were discarded at the birth of the infant. Umbilical cord blood cells have been used as a source of transplantable stem and progenitor cells, as well as a source of bone marrow repopulating cells, to treat malignant diseases (i.e., acute lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndromes, and neuroblastoma), as well as non-malignant diseases such as Fanconi anemia and aplastic anemia (Kohli-Kumar et al., 1993 Br. J. Haematol. 85:419-422; Wagner et al., 1992 Blood 79;1874-1881; Lu et al., 1996 Crit. Rev. Oncol. Hematol 22:61-78; Lu et al., 1995 Cell Transplantation 4:493-503). A distinct advantage of HUCBC is that these cells have immature immune properties very similar to fetal cells, thus significantly reducing the risk of rejection by the host (Taylor & Bryson, 1985 J. Immunol. 134:1493-1497).Human umbilical cord blood contains mesenchymal, hematopoietic, and endothelial progenitor cells that can be expanded in tissue culture (Broxmeyer et al., 1992 Proc. Natl. Acad. Sci. USA 89:4109-4113; Kohli-Kumar et al., 1993 Br. J. Haematol. 85:419-422; Wagner et al., 1992 Blood 79:1874-1881; Lu et al., 1996 Crit. Rev. Oncol. Hematol 22:61-78; Lu et al., 1995 Cell Transplantation 4:493-503; Taylor & Bryson, 1985 J. Immunol. 134:1493-1497; Broxmeyer, 1995 Transfusion 4:493-503). 35:694-702;Chen et al.,2001 Stroke 32:2682-2688;Nieda et al.,1997 Br.J.Haematology 98:775-777;Erices et al.,2000 Br.J.Haematology 109:235-242). The total hematopoietic progenitor cell content in umbilical cord blood is equal to or greater than that in bone marrow; moreover, highly proliferative hematopoietic cells are eight times more abundant in HUCBC than in bone marrow and express hematopoietic markers such as CD14, CD34, and CD45 (Sanchez-Ramos et al., 2001 Exp. Neuro. 171:109-115; Bicknese et al., 2002 Cell Transplantation 11:261-264; Lu et al., 1993 J. Exp Med. 178:2089-2096).

[0096] In another embodiment, the pluripotent cells are cells in a hematopoietic microenvironment, such as circulating peripheral blood, preferably the peripheral blood mononuclear fraction of a mammal, umbilical cord blood, bone marrow, fetal liver, or yolk sac. Stem cells, particularly neural stem cells, may be derived from the central nervous system, including the meninges.

[0097] In another embodiment, pluripotent cells present in embryoid bodies are formed by harvesting ES cells with brief protease digestion and growing small clumps of undifferentiated human ESCs in suspension culture. Differentiation is induced by removal of conditioned medium. The resulting embryoid bodies are plated on a semi-solid substrate. Formation of differentiated cells is observed after about 7 days to about 4 weeks. To select viable differentiated cells from in vitro cultures of stem cells, embryoid bodies or similar structures are partially dissociated to obtain cell aggregates. Aggregates containing cells of interest are selected for phenotypic characteristics using methods that substantially maintain contact between cells in the aggregates.

[0098] In another embodiment, the stem cells can be reprogrammed stem cells, such as stem cells derived from somatic cells or differentiated cells. In such embodiments, the dedifferentiated stem cells can be, for example, but not limited to, neoplastic cells, tumor cells, and cancer cells, or alternatively derived reprogrammed cells, such as induced pluripotent stem cells, or iPS cells.

[0099] Cloning and cell culture Exemplary methods of molecular genetics and genetic engineering that can be used in the techniques described herein can be found, for example, in Molecular Cloning: A Laboratory Manual (Sambrook et al., Cold Spring Harbor), Gene Transfer Vectors for Mammalian Cells (Miller & Calos eds.), and current editions of Current Protocols in Molecular Biology (FMA Usubel et al. eds., Wiley & Sons). Cell biology, protein chemistry, and antibody techniques can be found, for example, in Current Protocols in Protein Science (JE Colligan et al. eds., Wiley & Sons), Current Protocols in Cell Biology (JS Bonifacino et al., Wiley & Sons), and Current Protocols in Immunology (JE Colligan et al. eds., Wiley & Sons). Exemplary reagents, cloning vectors, and kits for genetic manipulation are commercially available from, for example, BioRad, Stratagene, Invitrogen, ClonTech, and Sigma-Aldrich Co.

[0100] Suitable cell culture techniques can be found, for example, in the current edition of Culture of Animal Cells: A Manual of Basic Technique (RI Freshney ed., Wiley & Sons), General Techniques of Cell Culture (MA Harrison & IF Rae, Cambridge Univ. Press), and Embryonic Stem Cells: Methods and Protocols (K. Turksen ed., Humana Press). Suitable tissue culture supplies and reagents are commercially available, for example, from Gibco / BRL, Nalgene-Nunc International, Sigma Chemical Co., and ICN Biomedicals.

[0101] Pluripotent stem cells can be continuously expanded in culture by those skilled in the art using culture conditions that promote proliferation without promoting differentiation. An exemplary serum-containing ES medium is made with 80% DMEM (e.g., Knock-Out DMEM, Gibco), 20% either defined fetal bovine serum (FBS, Hyclone) or serum replacement (WO98 / 30679), 1% non-essential amino acids, 1 mM L-glutamine, and 0.1 mM β-mercaptoethanol. Human bFGF is added to 4 ng / mL immediately before use (WO99 / 20741, Geron Corp.). Traditionally, ES cells are cultured on a layer of feeder cells, typically fibroblasts derived from embryonic or fetal tissue.

[0102] Geron scientists have discovered that pluripotent stem cells can be maintained in an undifferentiated state even without feeder cells. The environment for feeder-free culture includes a suitable culture substrate, especially an extracellular matrix such as Matrigel® or laminin. Typically, the enzymatic digestion is stopped before the cells are completely dispersed (e.g., about 5 min with collagenase IV). Then, clumps of about 10 to 2,000 cells are plated directly on the substrate without further dispersion.

[0103] Feeder-free culture is supported by a nutrient medium containing factors that support the growth of the cells without causing differentiation. Such factors can be introduced into the medium by culturing the medium with cells that secrete such factors, such as irradiated (approximately 4,000 rad) primary mouse embryonic fibroblasts, telomerized mouse fibroblasts, or fibroblast-like cells derived from pPS cells. The medium is cultured with approximately 5-6 × 10 cells in a serum-free medium such as KO DMEM supplemented with 20% serum replacement and 4 ng / mL bFGF. 4 cm -2 The medium can be conditioned by plating the feeders at a density of 100-200 nm. The medium conditioned for 1-2 days is further supplemented with bFGF and used to support pluripotent SC culture for 1-2 days. The characteristics of feeder-free culture methods are further discussed in International Patent Publication No. WO 01 / 51616 and Xu et al., Nat. Biotechnol. 19:971, 2001.

[0104] Under the microscope, ES cells appear with a high nuclear / cytoplasmic ratio, prominent nucleoli, and small colony formation with poorly discernible cell junctions. Primate ES cells express markers detectable with antibodies designated stage-specific embryonic antigens (SSEA) 3 and 4, as well as Tra-1-60 and Tra-1-81 (Thomson et al., Science 282:1145, 1998). Mouse ES cells can be used as a positive control for SSEA-1 and as a negative control for SSEA-4, Tra-1-60, and Tra-1-81. SSEA-4 is consistently present in human embryonic carcinoma (hEC) cells. Differentiation of pluripotent stem cells in vitro results in loss of expression of SSEA-4, Tra-1-60, and Tra-1-81, and increased expression of SSEA-1, which is also seen in undifferentiated hEG cells.

[0105] Methods for generating stem cell-derived cells Aspects of the present disclosure relate to the generation of stem cell-derived cells (e.g., SC-β cells, SC-EC cells, SC-α cells, etc.). Generally, at least one stem cell-derived cell or its precursor, e.g., pancreatic precursor, produced according to the methods disclosed herein can include a mixture or combination of different cells, e.g., a mixture of cells such as Pdx1+ pancreatic progenitor cells, pancreatic progenitor cells co-expressing Pdx1 and NKX6-1, Ngn3-positive endocrine precursor cells, endocrine cells (e.g., β-like cells, α-like cells, EC-like cells), non-endocrine cells, and / or other pluripotent or stem cells.

[0106] In some embodiments, somatic cells, e.g., fibroblasts, can be isolated from a subject, e.g., as a tissue biopsy, e.g., a skin biopsy, and reprogrammed into induced pluripotent stem cells for further differentiation to produce at least one stem cell-derived cell or a precursor thereof for use in the compositions and methods described herein. In some embodiments, the somatic cells, e.g., fibroblasts, are maintained in culture by methods known to those of skill in the art, and in some embodiments, expanded before being converted into a stem cell-derived cell by the methods disclosed herein.

[0107] In some embodiments, the progenitor cells are genetically modified before being converted into stem cell-derived cells by the methods as disclosed herein. In some embodiments, the progenitor cells are genetically modified to inhibit or knock out essential factors or regulators, thereby inhibiting the development of specific cell types (e.g., SC-EC cells and TN cells). In some embodiments, the stem cells are genetically modified to inhibit or knock out essential factors or regulators, thereby increasing the development of specific cell types (e.g., SC-β cells or SC-α cells). In one embodiment, the stem cells are genetically modified to inhibit or knock out essential factors or regulators, thereby increasing the development of SC-β cells. In one embodiment, the stem cells are genetically modified to inhibit or knock out essential factors or regulators, thereby increasing the development of SC-α cells. In one embodiment, the stem cells are genetically modified to inhibit or knock out essential factors or regulators, thereby increasing the development of SC-EC cells. In one embodiment, the stem cells are genetically modified to inhibit or knock out essential factors or regulators, thereby decreasing the development of SC-EC cells. In some embodiments, the targeting of the essential or regulatory factor is carried out using any gene editing tool known to those skilled in the art (e.g., TALENS, CRISPR, etc.). In some embodiments, the gene editing tool is delivered to stem cells using retrovirus (e.g., lentivirus).

[0108] In some embodiments, one or more genes can be identified as controlling cell fate during differentiation protocol. In some embodiments, one or more genes can be targeted using gene editing (e.g., CRISPR) to regulate the expression of one or more genes and thereby control the fate of the differentiation process. In some aspects, a first gene can be knocked out or inhibited in progenitor cells before the progenitor cells are converted into stem cell-derived cells. In some aspects, a first gene and a second gene are knocked out or inhibited in progenitor cells before the progenitor cells are converted into stem cell-derived cells.

[0109] In some aspects, the differentiation of a progenitor cell population is directed towards an SC-β cell fate by knocking down or knocking out expression of one or more genes listed in Table 1. Knocking down or knocking out expression of one or more genes listed in Table 1 in progenitor cells can direct cell differentiation of progenitor cells towards SC-β cells and away from triple negative cells. In some embodiments, the one or more genes are selected from the group including FBXL14, BCORL1, SHOC2, CCDC6, B3GALT6, HOXA1, DDX3X, CARM1, EXT2, EXT1, DYRK1A, SCAF1, SCAF8, CAND1, NDST1, EYA3, GLCE, DYRK1B, PRDM16, ALG3, CXXC4, SMURF1, PHF21A, SOX4, and TET2. In some embodiments, the one or more genes are selected from the group including FBXL14, BCORL1, SHOC2, CCDC6, B3GALT6, HOXA1, DDX3X, CARM1, EXT2, and EXT1. In some aspects, the differentiation of a progenitor cell population is directed toward an SC-β cell fate by knocking down or knocking out expression of one or more genes listed in Table 2. Knocking down or knocking out expression of one or more genes listed in Table 2 in progenitor cells can direct cell differentiation of progenitor cells toward SC-β cells and away from SC-EC cells. In some embodiments, the one or more genes are selected from the group including SOX4, BCORL1, FBXL14, CCDC6, SOX1, CARM1, TNRC18, CAND1, TET2, HOXA1, ASCL1, ARID2, SIRT6, FBXO22, FLVCR1, FOXA1, COPS9, ELAVL1, SSBP3, PROSER1, PROX1, SMURF1, SCAF1, HELLS, and DACH1. In certain embodiments, the one or more genes are selected from the group including SOX4, BCORL1, FBXL14, CCDC6, SOX1, CARM1, TNRC18, CAND1, TET2, and HOXA1. [Table 1-1]

Table 1-2

Table 2-1

Table 2-2

[0110] In some aspects, knocking down or knocking out expression of one or more genes listed in Table 3 directs differentiation of a progenitor cell population towards an SC-α cell fate. Knocking down or knocking out expression of one or more genes listed in Table 3 in progenitor cells can direct cell differentiation of progenitor cells towards SC-α cells and away from triple negative cells. In some embodiments, the one or more genes are selected from the group including PDX1, CCDC6, HES1, PHF21A, PAX4, DYRK1B, DYRK1A, BCORL1, TET2, DDX3X, PROSER1, PBX1, HELLS, CAND1, EYA3, MYT1, AFF4, FBXL14, HOXA1, ZC3H15, SCAF8, PRDM16, HEXIM1, TTC14, ZRANB1, and B3GALT6. In some embodiments, the one or more genes are selected from the group including PDX1, CCDC6, HES1, PHF21A, PAX4, DYRK1B, DYRK1A, BCORL1, TET2, and DDX3X. In some aspects, knocking down or knocking out expression of one or more genes listed in Table 4 directs differentiation of a progenitor cell population toward an SC-α cell fate. Knocking down or knocking out expression of one or more genes listed in Table 4 in progenitor cells can direct cell differentiation of progenitor cells toward SC-α cells and away from SC-EC cells. In some embodiments, the one or more genes are selected from the group including PAX4, HES1, CCDC6, SOX4, ZBTB10, PHF21A, PBX1, ARID2, TET2, BCORL1, TTC14, CAND1, PROSER1, SOX1, FBXO22, HELLS, DYRK1B, ZRANB1, DYRK1A, ASCL1, ZC3H15, SETBP1, FAM58A, MYT1, and RALGAPB. In certain embodiments, the one or more genes are selected from the group including PAX4, HES1, CCDC6, SOX4, ZBTB10, PHF21A, PBX1, ARID2, TET2, and BCORL1. [Table 3-1] [Table 3-2] [Table 4-1] [Table 4-2]

[0111] In some aspects, an increased population of SC-β cells is generated by inhibiting the development of SC-EC cells. In some aspects, an increased population of SC-β cells is generated by inhibiting the development of TN cells. In some aspects, an increased population of SC-α cells is generated by inhibiting the development of SC-EC cells. In some aspects, an increased population of SC-α cells is generated by inhibiting the development of TN cells. By inhibiting the production of SC-EC cells and / or the production of TN cells during differentiation, the resulting population of differentiated cells exhibits an increased yield of SC-β cells and / or SC-α cells. In some embodiments, overexpression of one or more transcription factors disrupts SC-EC formation. In some aspects, overexpression of one or more transcription factors alters or modifies the ratio of endocrine cells. In some embodiments, overexpression of one or more transcription factors results in an increased population of SC-α cells and / or SC-β cells. In one embodiment, overexpression of ISL1 results in an increase in SC-β cells and / or SC-α cells and reduces the formation of SC-EC cells.

[0112] In some embodiments, at least one stem cell-derived cell or a precursor thereof is maintained in culture by methods known to those of skill in the art, and in some embodiments expanded, prior to being converted to a stem cell-derived cell by the methods disclosed herein.

[0113] Furthermore, the at least one stem cell-derived cell or its precursor, such as a pancreatic precursor, can be from any mammalian species, including, but not limited to, murine, bovine, simian, porcine, equine, ovine, or human cells. Although the description of the methods herein refers to at least one stem cell-derived cell or its precursor from a mammal for clarity and simplicity, it should be understood that all methods described herein can be readily applied to at least one stem cell-derived cell or its precursor from other cell types. In some embodiments, the at least one stem cell-derived cell or its precursor is from a human individual.

[0114] The at least one stem cell-derived cell or precursor thereof can be produced according to any suitable culture protocol for differentiating a stem cell or pluripotent cell to a desired differentiation stage, hi some embodiments, the at least one stem cell-derived cell or precursor thereof is produced by culturing at least one pluripotent cell for a period of time and under conditions suitable for the at least one pluripotent cell to differentiate into at least one stem cell-derived cell or precursor thereof.

[0115] In some embodiments, at least one stem cell-derived cell or its precursor is a substantially pure population of stem cell-derived cells or its precursors. In some embodiments, the population of stem cell-derived cells or its precursors comprises a mixture of pluripotent or differentiated cells (e.g., a mixture of SC-β cells, SC-α cells, SC-EC cells, and / or other differentiated cell types, also referred to herein as triple negative or TN cells). In some embodiments, the population of SC-β cells or its precursors is substantially free of or devoid of embryonic stem cells or pluripotent cells or iPS cells. In some embodiments, the population of SC-α cells or its precursors is substantially free of or devoid of embryonic stem cells or pluripotent cells or iPS cells. In some embodiments, the population of SC-EC cells or its precursors is substantially free of or devoid of embryonic stem cells or pluripotent cells or iPS cells.

[0116] In some embodiments, stem cell-derived cells (e.g., pancreatic stem cell-derived cells) may be produced using methods known to those of skill in the art. In certain embodiments, stem cell-derived cells may be produced using methods disclosed in WO2015 / 002724, WO2014 / 201167, WO2019 / 217493, and / or WO2019 / 217487, all of which are incorporated herein by reference.

[0117] Transcriptional profiling at each step of the differentiation protocol In some aspects of the present disclosure, single cell sequencing (e.g., high-throughput single cell RNA sequencing) is used to provide detailed characterization of the entire transcriptome of all cell populations produced using an in vitro differentiation protocol (e.g., an in vitro beta cell or alpha cell differentiation protocol). In some embodiments, specific genes are identified as enriched in a single population or combination of cells. In some aspects, single cell sequencing is performed at all stages of the in vitro differentiation protocol. In some embodiments, sequencing is performed at the end of stage 6 of the differentiation protocol (e.g., a beta cell or alpha cell differentiation protocol).

[0118] In some aspects of the present disclosure, upon completion of the differentiation protocol (e.g., after stage 6), clusters are formed. In some embodiments, the clusters comprise one or more cell types. In some aspects, the clusters are screened to identify the various cells contained within the clusters. In some embodiments, the clusters are screened using single-cell sequencing (e.g., high-throughput single-cell RNA sequencing) to identify cells associated with the clusters. In some aspects, the clusters comprise one or more of SC-β cells, SC-α cells, SC-δ cells, SC-EC cells, and TN cells.

[0119] Pancreatic stem cell-derived cells In some aspects of the disclosure, stem cell-derived cells (e.g., pancreatic stem cell-derived cells) are provided. In some embodiments, the stem cell-derived cells are SC-β cells, SC-α cells, and / or SC-δ cells. The stem cell-derived cells disclosed herein share many distinguishing characteristics of native pancreatic cells, but differ in certain aspects. In some embodiments, the stem cell-derived cells are non-native, i.e., non-endogenous cells that do not occur in nature. As used herein, "non-native" means that the stem cell-derived cells are significantly different in some aspects from cells that occur in nature, i.e., native cells. However, it should be recognized that, although such significant differences may cause the stem cell-derived cells to exhibit certain differences, the stem cell-derived cells may nevertheless behave similarly to native cells, with certain functions altered (e.g., improved) compared to native cells.

[0120] The present invention is not intended to be limited by the starting cell from which the stem cell-derived cells are derived, and thus the stem cell-derived cells may be differentiated in vitro from any starting cell. Exemplary starting cells include, but are not limited to, NKX6-1+ pancreatic progenitor cells, Pdx1+ pancreatic progenitor cells, and endocrine cells or precursors thereof, such as pluripotent stem cells, embryonic stem cells, and induced pluripotent stem cells. In some embodiments, the stem cell-derived cells are differentiated in vitro from reprogrammed cells, partially reprogrammed cells (i.e., somatic cells, e.g., fibroblasts that are partially reprogrammed to exist in an intermediate state between an induced pluripotent cell and the somatic cell from which it is derived), transdifferentiated cells. In some embodiments, the stem cell-derived cells disclosed herein may be differentiated in vitro from endocrine cells or precursors thereof. In some embodiments, the stem cell-derived cells are differentiated in vitro from precursors selected from the group including NKX6-1+ pancreatic progenitor cells, Pdx1+ pancreatic progenitor cells, and pluripotent stem cells. In some embodiments, the pluripotent stem cells are selected from the group including embryonic stem cells and induced pluripotent stem cells. In some embodiments, the stem cell-derived cells or the pluripotent stem cells from which the stem cell-derived cells are derived are human. In some embodiments, the stem cell-derived cells are human.

[0121] In some embodiments, the stem cell-derived cells are not genetically modified. In some embodiments, the stem cell-derived cells acquire characteristics in common with native cells in the absence of genetic modification of the cells. In some embodiments, the stem cell-derived cells are genetically modified.

[0122] In some aspects, the disclosure provides cell lines comprising the stem cell-derived cells described herein. In some aspects, the disclosure provides SC-islets comprising the stem cell-derived cells described herein (e.g., SC-β cells, SC-α cells, and / or SC-δ cells).

[0123] In some embodiments, the cells described herein, e.g., a population of stem cell-derived cells, can be transplanted, e.g., the population of stem cell-derived cells can be administered to a subject. In some embodiments, the subject to which the population of stem cell-derived cells is administered is the same subject from which the pluripotent stem cells used for differentiation into stem cell-derived cells were obtained (e.g., for autologous cell therapy). In some embodiments, the subject is a different subject. In some embodiments, the subject suffers from an intestinal disease, such as enteritis, or is a normal subject. For example, the cells for transplantation (e.g., a composition comprising a population of stem cell-derived cells) can be in a form suitable for transplantation, e.g., organ transplantation.

[0124] The method may further comprise administering the cells to a subject in need thereof, e.g., a mammalian subject, e.g., a human subject. The source of the cells may be a mammal, preferably a human. The source or recipient of the cells may also be a non-human subject, e.g., an animal model. The term "mammal" includes organisms including mice, rats, cows, sheep, pigs, rabbits, goats, horses, monkeys, dogs, cats, and preferably humans. Similarly, transplantable cells may be obtained from any of these organisms, including non-human transgenic organisms. In one embodiment, the transplantable cells are genetically engineered, e.g., the cells are genetically engineered to contain an exogenous gene or to inactivate or modify an endogenous gene.

[0125] A composition comprising a population of stem cell-derived cells (e.g., pancreatic stem cell-derived cells, such as SC-β cells and / or SC-α cells) can be administered to a subject using an implantable device. Implantable devices and related technologies are known in the art and are useful as delivery systems where continuous or time-release delivery of the compounds or compositions detailed herein is desired. In addition, implantable device delivery systems are useful for targeting a specific point (e.g., localized site, organ) of delivery of the compound or composition (Negrin et al., Biomaterials, 22(6):563 (2001)). Time-release technologies, including alternative delivery methods, can also be used in the present invention. For example, time-release formulations based on polymeric technologies, sustained release technologies, and encapsulation technologies (e.g., polymers, liposomes) can also be used for delivery of the compounds and compositions detailed herein.

[0126] For administration to a subject, a cell population, e.g., a population of stem cell-derived cells, produced by a method as disclosed herein can be administered to a subject, e.g., in a pharmaceutically acceptable composition, which comprises a therapeutically effective amount of such a stem cell-derived cell population, formulated with one or more pharmaceutically acceptable carriers (additives) and / or diluents.

[0127] As described in detail below, the pharmaceutical compositions of the present invention can be specially formulated for administration in solid or liquid form, including those suitable for: (1) oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), troches, dragees, capsules, pills, tablets (e.g., intended for buccal, sublingual and systemic absorption), boli, powders, granules, pastes for application to the tongue; (2) parenteral administration, e.g., as a sterile solution, or suspension, or as a sustained release formulation, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection; (3) topical application, e.g., as a cream, ointment, or controlled release patch or spray applied to the skin; (4) vaginal or rectal administration, e.g., as a pessary, cream, or foam; (5) sublingual; (6) intraocular; (7) transdermal; (8) transmucosal; or (9) intranasal. Additionally, the compounds can be implanted into the patient or injected using a drug delivery system. See, e.g., Urquhart, et al., Ann. Rev. Pharmacol. Toxicol. 24:199-236 (1984); Lewis, ed. "Controlled Release of Pesticides and Pharmaceuticals" (Plenum Press, New York, 1981); U.S. Pat. No. 3,773,919; and U.S. Pat. No. 353,270,960.

[0128] As used herein, the term "pharmacologically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0129] As used herein, the term "pharmaceutical acceptable carrier" refers to a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricants, magnesium talc, calcium or zinc stearate, or stearic acid), or solvent encapsulating material, that is involved in the transport or transfer of a compound of interest from one organ or part of the body to another. Each carrier should be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Examples of substances which may function as pharma- ceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) lubricants, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil. (10) oils, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; (24) C2-C 12Alcohol, e.g., ethanol; and (25) other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, coloring agents, releasing agents, coating agents, sweetening agents, flavoring agents, perfumes, preservatives and antioxidants may also be present in the formulation. The terms "excipient", "carrier", "pharmaceutical acceptable carrier" and the like are used interchangeably herein.

[0130] The phrase "therapeutically effective amount" as used herein with respect to a cell population refers to an amount of a cell population, e.g., stem cell-derived cells, or relevant cells in a composition comprising the stem cell-derived cells of the present invention, effective to produce some desired therapeutic effect on at least a subpopulation of cells in an animal, with a reasonable benefit / risk ratio applicable to any medical treatment. For example, the administration of a population of stem cell-derived cells to a subject is sufficient to produce a statistically significant and measurable change in at least one symptom of type 1, type 1.5 or type 2 diabetes, e.g., glycosylated hemoglobin level, fasting blood glucose level, hypoinsulinemia, etc. Determination of a therapeutically effective amount is well within the capabilities of one of ordinary skill in the art. In general, a therapeutically effective amount may vary depending on the subject's medical history, age, condition, sex, as well as the severity and type of the condition in the subject, and the administration of other pharmacologic active agents.

[0131] As used herein, the term "administering" refers to placing a composition in a subject by a method or route that results in at least partial localization of the composition at a desired site so that a desired effect occurs. The compounds or compositions described herein may be administered by any suitable route known in the art, including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual) administration.

[0132] Exemplary modes of administration include, but are not limited to, injection, infusion, instillation, inhalation, or oral ingestion. "Injection" includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion. In a preferred embodiment, the composition is administered by intravenous infusion or injection.

[0133] "Treating," "preventing," or "ameliorating" a disease or disorder means delaying or preventing the onset of such disease or disorder, reversing, alleviating, ameliorating, inhibiting, slowing or halting the progression or severity of the conditions associated with such disease or disorder. In one embodiment, symptoms of the disease or disorder are alleviated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%.

[0134] Treatment of diabetes is determined by standard medical methods. The goal of diabetes treatment is to lower sugar levels as close to normal as safely possible. Commonly set goals are 80-120 milligrams per deciliter (mg / dl) before meals and 100-140 mg / dl at bedtime. A particular physician may set different goals for a patient depending on other factors such as how often the patient has hypoglycemic reactions. Useful medical tests include testing the patient's blood and urine to measure blood glucose levels, testing for glycosylated hemoglobin levels (HbA1c; an indicator of average blood glucose levels over the past 2-3 months, normal range is 4-6%), testing for cholesterol and fat levels, and testing for urinary protein levels. Such tests are standard tests known to those skilled in the art (see, for example, American Diabetes Association, 1998). A successful treatment program can also be determined by the patient's low incidence of diabetes-related complications, such as eye disease, kidney disease, or nerve disease.

[0135] Delaying the onset of diabetes in a subject refers to delaying the onset of at least one symptom of diabetes, e.g., hyperglycemia, hypoinsulinemia, diabetic retinopathy, diabetic nephropathy, blindness, memory loss, renal failure, cardiovascular disease (including coronary artery disease, peripheral artery disease, cerebrovascular disease, atherosclerosis, and hypertension), neuropathy, autonomic dysfunction, hyperglycemic hyperosmolar coma, or a combination thereof, for at least 1 week, at least 2 weeks, at least 1 month, at least 2 months, at least 6 months, at least 1 year, at least 2 years, at least 5 years, at least 10 years, at least 20 years, at least 30 years, at least 40 years or more, and can include the entire lifespan of the subject.

[0136] In certain embodiments, the subject is a mammal, such as a primate, such as a human. The terms "patient" and "subject" are used interchangeably herein. Preferably, the subject is a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. A non-human mammal may be advantageously used as a subject representing an animal model of type 1 diabetes, type 2 diabetes, or a pre-diabetic condition. Furthermore, the methods described herein may be used to treat livestock animals and / or pets. The subject may be male or female. The subject may be one who has previously been diagnosed or identified as suffering from or having diabetes (e.g., type 1 or type 2), one or more complications associated with diabetes, or a pre-diabetic condition, and optionally, need not have already been treated for diabetes, one or more complications associated with diabetes, or a pre-diabetic condition. The subject may also be one who does not suffer from diabetes or a pre-diabetic condition. The subject may also be one who has been diagnosed with or identified as suffering from diabetes, one or more complications associated with diabetes, or a pre-diabetic condition, but who has received one or more treatments for diabetes, one or more complications associated with diabetes, or a pre-diabetic condition, and as a result shows improvement in known diabetes risk factors. Alternatively, the subject may not have been previously diagnosed with diabetes, one or more complications associated with diabetes, or a pre-diabetic condition. For example, the subject may be a subject who shows one or more risk factors for diabetes, one or more complications associated with diabetes, or a pre-diabetic condition, or a subject who does not show risk factors for diabetes, or a subject who is asymptomatic for diabetes, one or more complications associated with diabetes, or a pre-diabetic condition. The subject may also be one who has or is at risk of developing diabetes or a pre-diabetic condition. The subject may also be one who has been diagnosed with or identified as having one or more complications associated with diabetes or a pre-diabetic condition, as defined herein, or the subject may not have been previously diagnosed with or identified as having one or more complications associated with diabetes or a pre-diabetic condition.

[0137] As used herein, "subject in need of pancreatic stem cell-derived cells" refers to a subject who has been diagnosed or identified as suffering from, having, or at risk of developing diabetes (e.g., type 1, type 1.5, or type 2), one or more complications associated with diabetes, or a pre-diabetic condition.

[0138] The subject who needs the population of cells derived from pancreatic stem cells can be identified by any method used for diagnosing diabetes.For example, type 1 diabetes can be diagnosed by glycosylated hemoglobin (A1C) test, random blood glucose test, and / or fasting blood glucose test.The parameters for diagnosing diabetes are known in the art and can be obtained by those skilled in the art without much effort.

[0139] In some embodiments, the method of the present invention further comprises selecting the subject identified as needing additional pancreatic stem cell-derived cells.The subject needing the population of pancreatic stem cell-derived cells can be selected based on the symptoms presented, such as type 1, type 1.5 or type 2 diabetes symptoms. Exemplary symptoms of diabetes include, but are not limited to, excessive thirst (polydipsia), frequent urination (polyuria), extreme hunger (polyphagia), extreme fatigue, weight loss, hyperglycemia, low insulin levels, hyperglycemia (e.g., blood glucose levels above 250 mg, above 300 mg), presence of ketones in the urine, fatigue, dry and / or itchy skin, blurred vision, slow healing of cuts or sores, more infections than usual, numbness and tingling in the feet, diabetic retinopathy, diabetic nephropathy, blindness, memory loss, kidney failure, cardiovascular disease (including coronary artery disease, peripheral artery disease, cerebrovascular disease, atherosclerosis, high blood pressure), neuropathy, autonomic dysfunction, hyperglycemic hyperosmolar coma, and combinations thereof.

[0140] In some embodiments, compositions comprising a population of stem cell-derived cells for administration to a subject may further comprise a pharma- ceutically active agent, such as an agent known in the art as a treatment for diabetes and / or having antihyperglycemic activity, for example, dipeptidyl peptidase 4 (DPP-4) inhibitors (e.g., alogliptin, linagliptin, saxagliptin, sitagliptin, vildagliptin, and berberine), biguanides (e.g., metformin, buformin, and phenformin), peroxisome proliferator-activated receptor (PPAR) modulators such as thiazolidinediones (TZDs) (e.g., pioglitazone, rivoglitazone, rosiglitazone, and troglitazone), dual PPAR agonists (e.g., aleglitazar, muraglitazar, and tesaglitazar), sulfonylureas (e.g., acetohexafluorophosphate, sorbitol, sorbitol iodine, and sorbitol 200 mg), and the like. amide, carbutamide, chlorpropamide, gliclazide, tolbutamide, tolazamide, glibenclamide (glyburide), glipizide, gliquidone, glyclopyramide, and glimepiride), meglitinides ("glinides") (e.g., nateglinide, repaglinide, and mitiglinide), glucagon-like peptide-1 (GLP-1) and its analogs (e.g., exendin-4, exenatide, liraglutide, albiglutide), insulin and insulin analogs (e.g., insulin lispro, insulin aspart, insulin glargine, insulin detemir, Exubera, and NPH insulin), α-glucosidase inhibitors (e.g., acarbose, miglitol, and voglibose), amylin analogs (e.g., pramlintide), sodium-dependent glucose cotransporter T2 (SGLT T2) inhibitors (e.g., dapriflozin, remogliflozin, and sergliflozin), and others (e.g., benfluorex, and trestat).

[0141] The composition comprising stem cell-derived cells can be administered to a subject at the same time as the administration of a pharma- ceutically active agent or a composition comprising the same, or at different times. When administered at different times, the composition comprising a population of stem cell-derived cells and / or the pharma- ceutically active agent for administration to a subject can be administered within 5 minutes, 10 minutes, 20 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, 24 hours of the other administration. When the composition comprising a population of stem cell-derived cells and the composition comprising a pharma- ceutically active agent are administered as different pharmaceutical compositions, the routes of administration can be different. In some embodiments, the subject is administered a composition comprising stem cell-derived cells. In other embodiments, the subject is administered a composition comprising a pharma- ceutically active agent. In another embodiment, the subject is administered a composition comprising a population of stem cell-derived cells mixed with a pharma- ceutical active agent. In another embodiment, the subject is administered a composition comprising a population of stem cell-derived cells and a composition comprising a pharma- ceutical active agent, the administrations being substantially simultaneous or subsequent to each other.

[0142] Toxicity and therapeutic efficacy of administration of a composition comprising a population of stem cell-derived cells can be determined by standard pharmaceutical procedures using cell cultures or experimental animals, for example to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). Compositions comprising a population of stem cell-derived cells that exhibit large therapeutic indices are preferred.

[0143] The amount of the composition comprising a population of stem cell-derived cells can be tested using a number of established animal models.

[0144] Nonobese diabetic (NOD) mice have a genetic defect that causes insulitis at a few weeks of age (Yoshida et al., Rev. Immunogenet. 2:140, 2000). 60-90% of female mice develop overt diabetes by 20-30 weeks of age. The immune-related pathology appears to be similar to that of human type 1 diabetes. Other models of type 1 diabetes include transgenic and knockout mutant mice (Wong et al., Immunol. Rev. 169:93, 1999). Recently, Lenzen et al. reported a rat model of spontaneous type 1 diabetes (Diabetologia 44:1189, 2001). Hyperglycemia can also be induced in mice (glucose >500 mg / dL) by a single intraperitoneal injection of streptozotocin (Soria et al., Diabetes 49:157, 2000) or by continuous administration of low doses of streptozotocin (Ito et al., Environ. Toxicol. Pharmacol. 9:71, 2001). To examine the efficacy of the transplanted islet cells, mice are monitored for the return of glucose to normal levels (<200 mg / dL).

[0145] Large animals provide a good model for tracking the sequelae of chronic hyperglycemia. Dogs can be made insulin-dependent by removing the pancreas (J. Endocrinol.158:49,2001) or feeding galactose (Kador et al.,Arch.Opthalmol.113:352,1995). The Keeshond is a genetic model for type 1 diabetes (Am.J.Pathol.105:194,1981). Early studies using the dog model (Banting et al.,Can.Med.Assoc.J.22:141,1922) led to the discovery in February 1925 of a group of two or three Canadians who made a long voyage to Stockholm.

[0146] In some embodiments, data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dosage of such compounds is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. Dosages can vary within this range depending on the dosage form employed and the route of administration utilized.

[0147] The therapeutically effective dose of a composition comprising a stem cell-derived cell population can be estimated initially from cell culture assays, or the effect of a particular dose can be monitored by a suitable bioassay.

[0148] With regard to duration and frequency of treatment, a skilled clinician will typically monitor the subject to determine when a therapeutic benefit is being achieved and decide whether to increase or decrease the dosage, increase or decrease the frequency of administration, discontinue treatment, resume treatment, or make other changes to the treatment regimen. Administration schedules can vary from once a week to daily, depending on various clinical factors, such as the subject's sensitivity to the stem cell-derived cells. The desired dosage can be administered all at once, or in smaller doses, e.g., 2-4 doses, administered over a period of time, e.g., at suitable intervals throughout the day, or on any other suitable schedule. Such smaller doses can be administered as unit dosage forms. In some embodiments, administration is chronic, e.g., administered once or multiple times daily over a period of several weeks or months. Exemplary administration schedules are daily, twice daily, three times daily, or four or more times daily for a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months or more.

[0149] In another aspect of the invention, a method provides for the use of an isolated population of stem cell-derived cells as disclosed herein. In one embodiment of the invention, an isolated population of stem cell-derived cells as disclosed herein can be used for the manufacture of a pharmaceutical composition for use in transplantation into a subject in need of treatment, such as, for example, a subject having diabetes or at risk of developing diabetes, including, but not limited to, subjects having congenital and acquired diabetes. In one embodiment, the isolated population of stem cell-derived cells can be genetically modified. In another aspect, the subject has or is at risk for diabetes and / or metabolic disorders. In some embodiments, the isolated population of stem cell-derived cells disclosed herein can be autologous and / or allogeneic. In some embodiments, the subject is a mammal, and in other embodiments, the mammal is a human.

[0150] The use of an isolated population of stem cell-derived cells as disclosed herein provides an advantage over existing methods since the population of stem cell-derived cells can be differentiated from stem cell-derived endocrine precursor cells or their precursors, such as iPS cells obtained or harvested from a subject administered the isolated population of stem cell-derived cells. This is highly advantageous in that it provides a renewable source of stem cell-derived cells that can be differentiated into endocrine precursor cells from stem cells by methods generally known to those of skill in the art, and further differentiated into pancreatic alpha-like cells, pancreatic beta-like cells, or cells having characteristics of pancreatic alpha-cells or pancreatic beta-cells, in particular a substantially pure population of mature pancreatic alpha-like cells or pancreatic beta-like cells, without the risks and limitations of cells derived from other lineages, by the methods described herein, for transplantation into a subject.

[0151] One embodiment of the invention relates to a method of treating diabetes or a metabolic disorder in a subject comprising administering to the subject having diabetes and / or a metabolic disorder an effective amount of a composition comprising a population of stem cell-derived cells (e.g., pancreatic stem cell-derived cells) as disclosed herein. In a further embodiment, the invention provides a method of treating diabetes comprising administering to a subject having or at increased risk of developing diabetes a composition comprising a population of stem cell-derived cells as disclosed herein.

[0152] In one embodiment of the above method, the subject is a human and the population of stem cell-derived cells as disclosed herein are human cells. In some embodiments, the present invention contemplates that the population of stem cell-derived cells as disclosed herein are administered directly to the pancreas of the subject or administered systemically. In some embodiments, the population of stem cell-derived cells as disclosed herein can be administered to any suitable location within the subject, for example, within the capsule of a blood vessel or liver.

[0153] The present invention is also directed to methods of treating subjects with diabetes or metabolic disorders resulting from genetic defects, physical injury, external stimuli or environmental conditioning, poor health, obesity, and other diabetes risk factors commonly known to those of skill in the art. The effectiveness of treatment of a subject administered a composition comprising a population of stem cell-derived cells (e.g., pancreatic stem cell-derived cells) can be monitored by clinically accepted standards and tests, including, for example: (i) Glycosylated hemoglobin (A1C) test, which indicates the subject's average blood glucose level over the past 2-3 months by measuring the percentage of blood glucose attached to hemoglobin, the oxygen-carrying protein in red blood cells. The higher the blood glucose level, the more sugar is attached to the hemoglobin. An A1C value of 6.5% or higher on two tests indicates diabetes. A test value between 6-6.5% suggests that the subject has prediabetes. (ii) Random blood glucose tests. A blood sample is drawn from the subject at random times, and a random blood glucose value of 200 milligrams per deciliter (mg / dL), or 11.1 millimoles per liter (mmol / L), or greater, indicates that the subject has diabetes. (iii) Fasting Blood Glucose Test. The subject's blood is drawn after fasting overnight. A fasting blood glucose value between 70 and 99 mg / dL (3.9 and 5.5 mmol / L) is normal. A fasting blood glucose value of 126 mg / dL (7 mmol / L) or greater on two tests indicates diabetes. A blood glucose value between 100 and 125 mg / dL (5.6 and 6.9 mmol / L) indicates that the subject has prediabetes. (iv) Oral Glucose Tolerance Test. Blood is drawn after the subject has fasted for at least 8 hours or overnight, then ingests a glucose solution, and blood glucose is measured two hours later. A blood glucose value less than 140 mg / dL (7.8 mmol / L) is normal. A person is considered to have prediabetes if their blood sugar level is between 140 and 199 mg / dL (7.8 and 11 mmol / L). This is sometimes called impaired glucose tolerance (IGT). A blood sugar level of 200 mg / dL or higher may indicate diabetes.

[0154] In some embodiments, the effects of administration of a stem cell-derived cell population (e.g., pancreatic stem cell-derived cells) as disclosed herein to a subject in need thereof are associated with improved exercise tolerance or other quality of life measures, and reduced mortality. The effects of cell therapy with stem cell-derived cell populations are evident within days to weeks following treatment. However, beneficial effects may be observed as early as hours following treatment and may persist for several years. In some embodiments, the effects of cell therapy with stem cell-derived cell populations are evident within two weeks following procedure.

[0155] In some embodiments, stem cell-derived cell populations (e.g., pancreatic stem cell-derived cells) as disclosed herein may be used for tissue reconstruction or regeneration in human patients or other subjects in need of such treatment. In some embodiments, compositions of stem cell-derived cell populations may be administered in a manner that allows them to attach or migrate to the intended tissue site and reconstruct or regenerate the functionally deficient site. Specialized devices are available that are suitable for administering cells capable of reconstructing pancreatic cell populations (e.g., alpha cells and / or beta cells) to the pancreas or another desired site. Thus, stem cell-derived cells may be administered by injection into the pancreas of the recipient subject or by intramuscular injection. EXAMPLES

[0156] Example 1: Charting cell identity during human in vitro β-cell differentiation Human stem cells can be differentiated in vitro to generate pancreatic β-cells, an insulin-secreting cell type whose loss is responsible for type 1 diabetes. As a step towards mastering this process, we provide a report on the transcriptional profiling of over 100,000 cells harvested during in vitro β-cell differentiation and describe the cells that emerge. Populations representing β-cells, α-like polyhormonal cells, non-endocrine cells resembling pancreatic exocrine cells, and an unreported population resembling enterochromaffin cells were isolated. Endocrine cells were shown to maintain their identity in culture without exogenous growth factors, and gene expression changes associated with β-cell maturation in vivo were recapitulated in vitro. To deplete non-endocrine cells and to identify CD49a / ITGA1 as a surface marker for β-cell populations, a scalable reaggregation technique was implemented, allowing magnetic sorting to 80% purity. Finally, we utilize a high-resolution sequencing time course to characterize gene expression dynamics during human pancreatic endocrine induction, from which we develop a lineage model of in vitro β-cell differentiation. This study provides a deeper perspective on the current state of human stem cell differentiation and will guide future efforts in pancreatic islet cell differentiation and their application in regenerative medicine.

[0157] In the SC-β protocol, human pluripotent stem cells grown in 3D clusters are differentiated in six stages with specific inducers to produce "SC-islets" containing stem cell-derived β cells. Progression and efficiency are measured using immunofluorescence microscopy and flow cytometry (Figure 10A). The first three stages of differentiation generate a nearly homogenous population (~90%) of progenitor cells expressing the master transcription factor PDX1. Different populations are then identified by staining for C-peptide (a fragment of proinsulin), the pan-endocrine marker CHGA, and the β cell transcription factor NKX6.1 (Figure 10A, Figure 15A).

[0158] Here, we apply single-cell RNA sequencing and computational analysis to gain a deeper understanding of in vitro β-cell differentiation (Figure 10B). We identify emerged cell types at each stage of differentiation through their global gene expression profiles, creating a precise cell-by-cell description of in vitro β-cell differentiation. These are critical steps in advancing stem cell differentiation toward diabetes treatment.

[0159] There are four major cell types in the SC islet We sequenced 40,444 cells sampled at the end of stages 3 to 6 from differentiations performed with the two modified protocols and used their whole transcriptomes to define cell populations. 1 A subset of v1 stage 3 and 4 factors was used, resulting in different population ratios at stage 4 (Figures S15D-15E, Figure S26). Throughout this study, the fact that SC-β differentiation is performed in 3D suspension culture was exploited to repeatedly sample the same differentiation over time.

[0160] The main populations identified (Figures 10C-10G, Figure 23) were progenitor cells (stages 3 and 4), three types of endocrine cells (stages 4, 5, and 6), and one type of non-endocrine cell (stages 5 and 6). In both protocols, stage 3 cells gave rise to repopulating pancreatic progenitor cells (PDX1 + At the end of stage 4, the first α-like cells as well as the NKX6.1 +Progenitor cells are also observed. Finally, at stages 5 and 6, three types of CHGA+ endocrine cells are observed: (i) SC-β cells expressing INS, NKX6.1, ISL1 and other β-cell markers, (ii) α-like cells expressing GCG, ARX, IRX2 but also INS, and (iii) an endocrine cell type expressing CHGA, TPH1, LMX1A, SLC18A1 that most resembles enterochromaffin cells (SC-EC, Figure 15B). At stages 5 and 6, SOX9+ non-endocrine cells (Figure 15C) form the final population with significant heterogeneity. Thus, two translationally relevant cell populations corresponding to adult pancreatic islet cell types (SC-β cells and SC-α cells) were identified along with two other populations (SC-EC and non-endocrine cells).

[0161] Besides these major populations, both protocols contained small populations of SST+ / HHEX+ / ISL1+ cells that appeared as early as the end of stage 4. A single population labeled with high levels of FOXJ1+ was present in only one protocol (Figure 27). Although protocol variants showed the expected large differences in cell type ratios (Figures 10D-10G, Figures 15F-15I), all cell types shared across protocols showed similar gene expression signatures (Figure 15J). We conclude that population ratios can be significantly influenced by protocol modifications without altering cell type identity.

[0162] Finally, stage 6 cells produced from differentiation of embryonic stem cells (ESCs, HUES8 line) were compared with induced pluripotent stem cells (iPSCs, 1016 / 31 line), and a high correlation was observed between the corresponding cell types (Figures 15K-15M). Taken together, these results demonstrate that the in vitro beta cell differentiation protocol induces lineage progression that is robust to perturbations of differentiation factors and stem cell lines.

[0163] SC-β cells stably maintain their identity Key features of SC-β cells are their glucose responsiveness and transcriptional similarity to endogenous human β cells. These features were characterized over several weeks of stage 6 using serum-free medium without exogenous signaling factors (Protocol v8). Single-cell RNA sequencing and in vitro glucose-stimulated insulin secretion (GSIS) studies were performed on samples from three differentiations at weekly intervals over several weeks of stage 6 (Figure 11A).

[0164] SC-islets acquire glucose-responsive insulin secretion during the first week of stage 6 and retain this ability for approximately 4 more weeks (Figures 11B-11C, Figure 16). The stimulation indices observed were in the same range as human islet controls, but the magnitude of secretion was greater in the islets. These results indicate that glucose responsiveness is a stable trait and does not require exogenous factors or serum.

[0165] In parallel, we assessed whether stage 6 cell populations maintained their identity over extended periods of culture. As in the previous dataset, SC-β, SC-α, SC-EC cells, and non-endocrine cells were identified (Figures 11D-11E, Figures 17A-17B). Small and rare populations (Figure 27) are only present at week 0 and then disappear (PHOX2A+) or are only detected later in stage 6 (GAP43+, ONECUT3+). SST+ / HHEX+ cells, similar to delta cells, also constitute a small population. There was no absolute (r 2 A relatively high correlation was observed for both endocrine cells and progenitor cells (>0.8) (Figure 11F). Importantly, there was no evidence of dedifferentiation to a progenitor state or reversal of differentiation to alternative fates for endocrine cells during stage 6. It was therefore concluded that the global transcriptional profile indicative of identity is maintained during long-term culture at stage 6.

[0166] Consistent with their glucose responsiveness, SC-β cells have β-cell identity 15 , Metabolic Sensing and Signaling 16 , insulin synthesis, packaging and secretion17 We observe that the NKX6.1+ progenitor cells express key genes related to cell cycle regulation. Broadly speaking, these genes are expressed in both cadaveric islet β cells and SC-β cells, but not in late NKX6.1+ progenitor cells (Figures 17C-17F). Cell replication appears to be minimal, as evidenced by negligible expression of cell cycle-related genes (TOP2A) and high expression of the cell cycle inhibitor CDKN1C.

[0167] Finally, we attempted to describe the refinement of SC-β gene expression over time. Pseudotime analysis was applied to order cells according to their transcriptional state and reverted gene expression using pseudotime to identify dynamic genes (Figures S1G-S1H). Genes increasing along pseudotime included IAPP, and HOPX. 13 , N.E.F.M. 18 , SIX2 13,18 Several markers of maturation or age (UCN3, 4-HT1C, 5-HT1C, 6-HT2C, 7-HT3C, 8-HT4C, 9-HT4C, 10-HT4C, 11-HT4C, 12-HT4C, 13-HT4C, 14-HT4C, 15-HT4C, 16-HT4C, 17-HT4C, 18-HT4 19 , M.A.F.A. 18 and SIX3 18 ) was not expressed. As a downregulated gene, its repression is required for proper metabolic sensing. 20 LDHA, and IGF2, a secreted peptide downstream of the INS gene, suggesting better transcriptional regulation of the insulin genomic locus. In summary, relatively subtle changes are observed in the SC-β transcriptome during stage 6, some of which correspond to known markers of maturation.

[0168] Early SC-α cells express insulin Multihormonal cells expressing both insulin and glucagon have been reported in several in vitro pancreatic differentiation protocols. Besides glucagon, these cells express many markers of islet α cells, but atypically express insulin. On this basis, and because insulin expression is corrected during stage 6 (Figure 18A), these cells are referred to as SC-α cells. To investigate the in vivo similarity of SC-α and SC-β cells, we identified genes that are differentially expressed between adult cadaveric α and β cells. 5 (Figure 18B). Genes highly expressed in α cells were also highly expressed in SC-α cells, whereas β cell-enriched genes were highly expressed in SC-β cells (Figures 18C-18D). This result is consistent with previous findings that in vitro-derived polyhormonal cells convert to monohormonal glucagon-expressing cells. 21 Insulin and glucagon co-expressing cells have been observed in two contexts: in the developing human fetal pancreas, where INS+ / GCG+ / ARX+ cells have been described as alpha precursors. 22 , and type 2 diabetes, where INS+ / GCG+ cells are described as dedifferentiated β cells. 23 Given the evidence for a transient state for monohormonal SC-α cells, it is likely that the in vitro polyhormonal cells correspond to nascent INS+ / GCG+ / ARX+ cells.

[0169] Stem cell-derived enterochromaffin cells This study identified an endocrine cell population that expresses TPH1, NKX6.1, and low levels of insulin, but lacks the beta cell markers G6PC2, NPTX2, ISL1, and PDX1. These cells are hypothesized to be stem cell-derived enterochromaffin cells (SC-ECs). Enterochromaffin cells synthesize and secrete serotonin (5-HT) in the intestine, functioning as chemosensors. 24 The transcriptome was analyzed in mouse intestinal epithelium. 25 and organoids 26The SC-EC cells were characterized by single-cell sequencing of serotonin. Compared to SC-β cells (Figure 12A), SC-EC cells express genes required for serotonin synthesis (TPH1, DDC, SLC18A1, Figure 19A), as well as markers such as LMX1A, ADRα2A, FEV, TAC1, and CXCL14. Expression of these genes is enriched in SC-EC cells compared to both other in vitro and in vivo pancreatic populations (Figure 12B). Immunostaining (Figures 12C-12D) confirmed that SC-EC cells co-express TPH1, LMX1A, and SLC18A1 and contain serotonin (5-HT). Like SC-β cells, these cells survive transplantation into the kidney capsule of mice (Figure 12E). SC-islets release serotonin when depolarized with KCl, but not when stimulated with high glucose (Figure 19B). This is consistent with the expected behavior of EC cells. 27 SC-EC cells are observed in all data sets in this study. In addition, bulk expression data from iPSC differentiation using different protocols 28 Expression of the SC-EC gene was also observed in (FIGS. 19C-19E), suggesting that EC cells are also present in other β cell protocols and pluripotent cell lines.

[0170] Serotonin is reported to be produced in human β cells. 29 However, TPH1 expression has not been observed in β-cell populations either in vivo or in vitro. 5-9 EC cells have not been found in single-cell profiling of the pancreas. 5-11 Other studies have shown that β-cells produce serotonin in an age- or context-dependent manner, but this has not been explored in existing single-cell data sets. 29-31 However, recently published data have identified signals that induce the serotonin / EC program in β cells from perturbed mice. 32, suggesting a slight "distance" between β and EC fates. Specifically, 25 weeks after β-cell-specific knockout of the polycomb repressive complex 2 (PRC2) component EED, upregulation of the enterochromic marker genes Tph1, Lmx1a, Slc18a1, and Trpa1 was observed (Figure 19F). This analysis indicates that the serotonin / EC program is induced in the β-cell dedifferentiation model, suggesting a link between β and EC fates.

[0171] Non-endocrine cell fate Some cells do not commit to an endocrine fate during stages 4 and 5 (Figure 20). These non-endocrine cells resemble earlier stage pancreatic progenitor cell types in that they express key transcription factors and lack endocrine markers. Whereas endocrine cells both in vivo and in vitro are largely postmitotic, these non-endocrine cells retain expression of cell cycle-related genes (TOP2A, Figure 23). These cells do not commit to an endocrine state or remain as progenitors, but instead appear to differentiate toward an exocrine pancreatic fate. Continuation of culture to stage 6 results in differentiation into populations expressing markers of pancreatic apical, mesenchymal, and ductal cells (Figure 20).

[0172] Purification of endocrine cells and SC-β cells Single-cell dissociation followed by controlled reaggregation is used to culture the neonatal pancreas 33 and in vitro β-cell preparations 34 It has been used to purify endocrine cells from islets. Enzymatic dissociation followed by reaggregation was found to be applicable beyond stage 5. Unlike previous methods, this technique is scalable as it does not require micropatterned surfaces, hanging droplets, or soluble extracellular matrix factors to increase efficiency. Using single cell sequencing, flow cytometry, and GSIS (Figures 21A-21H), this reaggregation procedure was shown to deplete non-endocrine cells while maintaining cellular identity and improving β-cell function. Interestingly, staining of SC-islets after reaggregation shows a pronounced compartmentalization of the endocrine cell population into areas of homogenous cells.

[0173] In addition to enriching endocrine secretion, we sought a method to specifically enrich SC-β cells. As a result of the analysis, ITGA1 (CD49a) was identified as a novel SC-β cell surface marker (Figure 13A). Interestingly, ITGA1 expression in adult islets is not specific to β cells. 5 Using anti-CD49a staining and magnetic microbeads, SC-β cells were labeled and efficiently sorted. This method produces clusters containing up to 80% SC-β cells with less than 5% SC-EC cells (Figures 13B-13C). Furthermore, comparable purification is observed from differentiation of one ESC and two iPSC lines (data not shown). These highly purified SC-islets respond to glucose in vitro (Figure 13D, Figures 21I-21K) and show increased stimulation indices compared to unsorted reaggregated SC-islets in both static and dynamic GSIS, although the magnitude of secretion is both smaller compared to cadaveric islets. Thus, single-cell sequencing data reveal a novel approach to enrich in vitro generated β cells.

[0174] Origin and lineage of SC-β cells Single-cell sequencing can reconstruct complex developmental trajectories from both single snapshots or sequential sampling. SC-β and SC-EC cells are absent at the end of stage 4 and appear midway through stage 5. Shared expression of key genes (e.g., PAX4, NKX6-1) prompted us to determine whether these cells form separately during endocrine induction or whether one is the precursor of the other. To this end, we sequenced approximately 45,000 cells at 1-day intervals during the progression of stage 5 for two independent differentiations.

[0175] From a general perspective, the individual cells in this dataset form a continuum connecting the day 0 and day 7 populations of stage 5. NEUROG3, a transiently expressed master regulator of in vivo endocrine induction, is expressed by cells bridging endocrine and non-endocrine cells in this continuum as different cell types gradually emerge (Figures S14A-14D, S14H, S22A-22B). Some day 0 cells are already endocrine, corresponding to either SC-α cells (ARX+) or δ-like cells showing co-expression of SST and HHEX. Other day 0 cells (marked by FEV+ / ISL- but not NEUROG3-) resemble NEUROG3+ cells of later time points and may indicate partial endocrine induction. The trajectory leading from progenitor cells to SC-β cells includes two branching events, which are explored (arrows in Figure S14C).

[0176] The onset of endocrine induction is the first major branching of cells at stage 5. At day 0, progenitor cells are SOX2+, FRZB+, PDX1 low From NKX6.1+, PTF1A+, PDX1 high At day 1, expression of NEUROG3+ was correlated with NKX6.1+, PTF1A+, PDX1+, and NKX6.1+, PTF1A+, and PDX1+, forming a single heterogeneous population characterized by a gradient to NKX6.1+, PTF1A+, and PDX1+. Pseudotemporal sequencing of these progenitor cells identifies 335 genes that correlate with the gradient. At day 1, expression of NEUROG3+ was correlated with NKX6.1+, PTF1A+, and PDX1+, forming a single heterogeneous population characterized by a gradient to NKX6.1+, PTF1A+, and PDX1+, forming a single heterogeneous population (Figures 22C-22E). high Since they are observed at the edge of the gradient, we speculate that these genes represent the progenitors most ready for endocrine induction. Expression of NEUROG3 is accompanied by changes in many other transcription factors and cell signaling genes (Figure 22F). We also observe an upregulation of CDX2 (Figures 22B, 22D) among the subset of NKX6-1+ cells that have not yet or have not undergone endocrine induction from day 1. From this analysis, we conclude that NKX6.1+, PTF1A+, and PDX1+ are predictive of endocrine induction potential. high This reveals the axis of variation of stage 4 progenitor cells indicated by

[0177] Stage 5 endocrine induction yields primarily SC-β and SC-EC cells, with the earliest cells of these types appearing at day 3. Global clustering and manifold embedding suggest that SC-β and SC-EC fates diverge at later stages. To validate this bifurcation observation, diffusion pseudotimes were calculated for all SC-β, SC-EC and NEUROG3+ cells (Figures S14E-S14G). Fits to each gene are models incorporating both pseudotime and branch assignment as covariates, and these models are compared to those fitted without branch labels. While some genes (such as NEUROG3 and NKX6.1) are dynamically expressed but show little or no branch dependency (Figure S14F), 313 branch-associated genes are identified (q-value < 0.001 and fold change > 4), including many transcription factors as well as important SC-β and SC-EC fate genes. This analysis suggests that SC-β and SC-EC cells emerge from a common NEUROG3+-derived intermediate, rather than one serving as a progenitor for the other, thus representing a second fate branch in the trajectory of SC-β formation. This analysis suggests a model for the lineage of cell types produced by SC-β differentiation (Figure S14I).

[0178] Consideration Beta cells are prime candidates for regenerative medicine. Nevertheless, directed differentiation protocols for beta cells produce other cells alongside beta cells. Here, we use single-cell RNA sequencing experiments to comprehensively characterize the cells formed during SC-beta differentiation.

[0179] The stepwise and synchronous differentiation of millions of cells offers an unprecedented opportunity to study human developmental processes. SC-β cells were shown to respond to glucose in vitro and maintain their identity even under long-term culture without signaling regulators. Dynamic genes include several markers of β-cell maturation. Furthermore, the identity of the polyhormonal cells was previously debated. These cells were concluded to be SC-α-like (SC-α) cells that only transiently misexpress insulin. In the context of transplantation, these cells may improve β-cell function through local interactions within the SC-islets, or through autocrine signaling. Progenitor cells that fail endocrine induction were shown to progress to exocrine pancreatic cell types. These progenitor cells appear to be undesirable, as they may replicate or occupy valuable space within the transplant device. To remove them, we describe a scalable reaggregation method to enrich for endocrine cells. Furthermore, we identify CD49a as a surface marker for SC-β cells and generate highly pure SC-β clusters by magnetic sorting.

[0180] What was unexpected in this analysis was the presence of SC-EC cells in vitro, which are closely related to but fundamentally distinct from SC-β cells and have been shown to arise from a late branch of differentiation. Given this close similarity, and their expression profile with respect to key genes (NKX6.1+ / CHGA+ / GCG-), these cells may be misclassified as either precursors or true β cells when analyzed using methods that use a preselected set of genes. 14 In vivo, enterochromaffin cells have not been observed in studies of mouse and human pancreatic islets. 5-9 Nonetheless, extremely rare reports of primary pancreatic serotonin-secreting carcinoid tumors support the existence of pancreatic enterochromaffin cells. 35 Importantly, purification of CD49a was shown to deplete SC-EC cells.

[0181] This study will inform the development of future β-cell differentiation protocols. For example, hypotheses regarding the control of cell fate by modulation of signaling pathways can be guided by receptor expression patterns or inferred signaling activities. Although SC-β cells are highly similar to cadaveric β cells, differences remain, such as the absence of expression of UCN3, MAFA, and SIX3. These genes, which are likely to be expressed after transplantation in vivo, represent the next milestone in the pursuit of more mature SC-β cells in vitro. In parallel, further milestones in characterizing SC-β differentiation will come from single-cell measurements of proteins, epigenetics, and lineage.

[0182] Overall, a comprehensive and in-depth analysis of stem cell products for human therapy is provided. Such high-resolution single-cell profiling represents a necessary step towards safe and successful treatment.

[0183] method cell culture Maintenance and differentiation of human pluripotent stem cells (hPSCs) was performed as previously described. 1 Pluripotent stem cell lines were obtained from stocks maintained by Melton lab or Semma Therapeutics. Cell lines were identified by DNA fingerprinting (Cell Line Genetics) and all cell lines tested negative for routine mycoplasma contamination. Pluripotent stem cell lines were maintained in cluster suspension culture format in 500 mL spinner flasks (Corning, VWR) rotating at 70 rpm in an incubator at 37 °C, 5% CO2, 100% humidity using mTeSR1 (Stem Cell Technologies, 85850). Cells were passaged every 72 hours: hPSC clusters were dissociated into single cells by mild mechanical disruption using Accutase (Innovative Cell Technologies; AT104-500), counted and then seeded at 0.5 M cells / mL in mTeSR1 + 10 μM Y27632 (DNSK International, DNSK-KI-15-02).

[0184] Differentiation in flasks was initiated 72 hours after passage by removing the mTeSR1 medium and replacing it with medium and growth factor or small molecule supplements appropriate for the protocol (see Figure 26). Small molecules and signaling factors were prepared and stored as single-use aliquots. During feeding, the differentiating clusters were allowed to gravity settle for 5-10 minutes, the medium was aspirated, and 300 mL of pre-warmed medium was added. All experiments involving human cells were approved by the Harvard University IRB and ESCRO committees.

[0185] Flow cytometry Differentiated clusters (1–2 mL) harvested from suspension cultures were dissociated at 37 °C using TrypLE Express (Gibco; 12604013), mechanically disrupted to form single cells, fixed with 4% PFA for 30 min at room temperature, and stored at 4 °C in PBS. For staining, fixed single cells were cultured in inhibition buffer for 1 h, then incubated with primary antibody in inhibition buffer (1 h at room temperature or overnight at 4 °C), washed three times with inhibition buffer, incubated with secondary antibody in inhibition buffer (1 h at room temperature), washed three times, and then resuspended in PBS + 0.5% BSA (Proliant; 68700). Inhibition buffer: PBS + 0.1% saponin (Sigma; 47036) + 5% donkey serum (Jackson Labs; 100181-234). Stained cells were analyzed using an LSR-II, Accuri C6 (BD Biosciences) or Attune NxT (Invitrogen) flow cytometer. An example of the gating strategy is shown in Figure 22. Results shown in this study are representative of over 100 independent v8 differentiations.

[0186] Immunofluorescence microscopy Differentiated clusters were fixed in 4% PFA for 1 h at room temperature, washed, frozen in OCT, and sectioned. Prior to staining, paraffin-embedded samples were treated with Histo-Clear to remove paraffin. All slides were rehydrated in an ethanol gradient and incubated in boiled antigen retrieval reagent (10 mM sodium citrate, pH 6.0) for 30 min. For staining, slides were incubated with primary antibodies in CAS block (ThermoFisher; 008120) overnight at 4°C, washed three times, then incubated with secondary antibodies for 2 h at room temperature, washed, mounted in Vectashield with DAPI (Vector Laboratories; H-1200) or ProLong Diamond Antifade Mountant with DAPI, covered with a coverslip, and sealed with clear nail polish. Representative fields were imaged using a Zeiss.Z2 equipped Apotome or Zeiss CellDiscoverer 7 microscope. Images displayed are representative of similar outcomes in differentiation from at least three biologically distinct, consistent or similar stages.

[0187] antibody Primary antibodies (supplier; catalog number, working dilution): rat anti-C-peptide (DHSB; GN-ID4; 1:100), mouse anti-NKX6.1 (DHSB; F55A12; 1:50), rabbit anti-CHGA (Abcam; ab15160; 1:500), rabbit anti-SLC18A1 (Sigma; HPA063797; 1:300), rabbit anti-LMX1A (Sigma; HPA030088; 1:300), sheep anti-TPH1 (EMD Millipore; AB1541; 1:100), goat anti-5-HT (Immunostar; 20079; 1:1000), rabbit anti-SOX9 (Cell Marque; AC-0284RUO; 1:500), mouse anti-glucagon (Santa Cruz Biotech.; SC-514592; 1:300).

[0188] Secondary antibodies (supplier; catalog number, all used at 1:300 dilution): anti-rat 594 (Life Tech.; A21209), anti-mouse 594 (Life Tech.; A21203), anti-mouse 647 (Life Tech.; A31571), anti-rabbit 488 (Life Tech. A21206), anti-rabbit 594 (Life Tech.; A21209), anti-rabbit 647 (Life Tech.; A31573), anti-goat 647 (Life Tech.; A21447), anti-sheep 488 (Life Tech.; A11015), anti-rat 488 (Jackson labs.; 712-546-153), anti-rat 405 (Abcam; ab175670).

[0189] transplant research Transplantation of differentiated clusters was performed as previously described. 1 Briefly, approximately 500 IEQ of human islets or approximately 5 × 10 6 Stage 6 native (day 10, non-reaggregated) SC-islet clusters were transplanted under the renal capsule of 8-12 week old male SCID beige mice (Jackson labs). At the indicated times post-transplant, kidneys containing grafts were dissected and fixed in 4% PFA overnight at 4°C. Fixed kidneys were embedded in paraffin and sectioned for immunofluorescence staining. Immunofluorescence staining was performed as described above. All animal experiments were approved by the Harvard University IACUC.

[0190] Glucose stimulated insulin and serotonin secretion Human pancreatic islets (approximately 400 IEQ, Prodo Laboratories) or SC-islet clusters (differentiation days 28–60, approximately 4 × 10 6The clusters (equivalent to 1000 cells) were divided into 4 portions for technical triplicate measurements and insulin / serotonin content samples. Krebs buffer (KRB) was prepared: 128 mM NaCl, 5 mM KCl, 2.7 mM CaCl2, 1.2 mM MgSO4, 1 mM Na2HPO4, 1.2 mM KH2PO4, 5 mM NaHCO3, 10 mM HEPES (Life Technologies; 15630080), 0.1% BSA in deionized water. The clusters were washed twice with low glucose (2.8 mM) KRB, then placed in a 24-well plate insert (Millicell Cell Culture Insert; PIXP01250) and fasted in low glucose KRB for 1 hour in a 37°C incubator to remove residual insulin. The clusters were washed once with low glucose KRB, incubated in low glucose KRB for 1 hour, and the supernatant was collected. The clusters were then transferred to high glucose (20 mM) KRB for 1 h and the supernatant was collected. This sequence was repeated once more, and the clusters were washed once between the high and second low glucose cultures to remove residual glucose. Finally, the clusters were cultured in KRB containing 2.8 mM glucose and 30 mM KCl for 1 h (depolarization challenge), and the supernatant was collected. The clusters were then dispersed into single cells using TrypLE Express, the cell numbers were automatically counted using Vi-Cell (Beckman Coulter), and the insulin levels were normalized by the cell number. The supernatant samples containing secreted insulin were processed using Human Ultrasensitive Insulin ELISA (ALPCO, 80-INSHUU-E01.1) and Serotonin ELISA (ALPCO; 17-SERHU-E01-FST).

[0191] Dynamic washout assay of glucose-stimulated insulin secretion Dynamic GSIS is the method already described. 19Assays were performed using a fully automated Perifusion System (BioRep). Prodolabs non-diabetic human islets (25 IEQ islets, 100-250um diameter, manually picked per sample, n=3) and native or purified SC-β clusters (25 clusters, 100-250um diameter, manually picked per sample, n=3) were assayed using a fully automated Perifusion System (BioRep). The chambers were sequentially flushed with 2.8mM or 20mM glucose, or 2.8mM glucose with 30mM KCL in KRB buffer at a flow rate of 100ul / min. The chambers were first flushed with low glucose (2.8mM) for 1 hour for fasting, then challenged with high glucose (20mM) for 30 minutes after 15 minutes of low glucose incubation. Samples were then flushed with low glucose for 15 minutes, followed by low glucose and 30mM KCl for 15 minutes. Insulin concentrations in the supernatants were measured using an Ultrasensitive Insulin ELISA kit (Alpco; 80-INSHUU). Insulin secretion levels were normalized to total cell number (uIU / mL / 1000 cells).

[0192] Reaggregation method to remove non-endocrine cells The re-aggregation step is similar to that described in the previous related art. 34,36-39It was optimized for scalability to ensure it can be deployed at the scale of billions of cells (unlike ). SC-islets were dissociated into single cells at the end of stage 5 differentiation. 300 mL of SC-islet culture was washed with PBS and cultured in 25 mL of TrypLE Express for 20 min at 37°C. Cells were then quenched with DMEM + 10% FBS, spun down, and resuspended in 10 mL of stage 6 medium. Remaining undissociated cell clusters were mechanically dissociated using a P1000 pipette. The single cell suspension was further diluted to 50 mL with stage 6 medium and then passed through a 40 μm mesh filter (pluriSelect) to remove any remaining undissociated clusters. Dissociated single cells were counted and seeded at a density of 1M cells / mL in stage 6 medium in spinner flasks and cultured in a 37°C incubator with agitation at 70 rpm. Endocrine cells self-aggregate into clusters within 24 hours, while progenitor cells remain in the supernatant. After 48 hours of culture, the cells were fed by centrifuging down all the cells and resuspending them in fresh Stage 6 medium. Subsequent medium changes were performed every 48 hours using a 20 μm mesh filter (pluriSelect). Reaggregated clusters enriched for endocrine cells were collected on a 20 μm mesh filter and reseeded in a spinner flask containing the original volume of Stage 6 medium. The supernatant containing single cells that passed through the 20 μm mesh filter was discarded.

[0193] Magnetic enrichment with CD49a / ITGA1 Stage 6 clusters (harvested at 2 weeks of stage 6) were dissociated starting from 75 mL of stage 6 culture as in the reaggregation section above. Dissociated single cells were resuspended in sorting buffer (PBS+1% BSA+2 mM EDTA) and filtered through a 35 μm mesh filter. Cells were counted and resuspended in a 15 mL conical tube at a density of 10M cells per 300 μL. Cells were stained with a 1:100 dilution of anti-human CD49a PE-conjugated (BD#559596) antibody for 20 min at room temperature, protected from light and agitated every 3 min. Stained cells were washed twice with 15 mL of sorting buffer by centrifugation (5 min, 300 g) and resuspended to their initial density of 10M cells per 300 μL. For labeling with microbeads, 40 μL of anti-PE UltraPure MACS microbeads (Miltenyi 130-105-639) were added per 10M cells and the cell solution was incubated for 15 min at 4 °C with agitation every 5 min. Stained cells were washed twice as above and resuspended to a target density of 25M-30M cells per 500 μL. A volume of 500 μL (containing ≤30M cells) was then magnetically separated on an LS column (Miltenyi 130-042-401) in a QuadroMACS separator (Miltenyi 130-090-976) using the recommended protocol. Briefly, 500 μL of cells were added to a pre-washed column and washed three times with 3 mL of sorting buffer before being removed from the separator and washed in a final volume of 5 mL. The final cell fractions from the different columns were combined. Successful PE enrichment was confirmed by live cell flow cytometry using an Attune NxT (Invitrogen) flow cytometer, with typical experiments showing >70% enrichment. An example of a purification result is shown in Figure 1. Although this method was not used in the paper, a second pass through the LS column enriches CD49a+ cells up to 90% (downstream SC-β fraction >90%), but reduces the number of recovered cells. Enriched cells were diluted to 0.5M cells / mL in Stage 6 medium and plated in ultra-low attachment 6-well plates (Corning #3471) at 2mL of medium per well and placed on a rocker at 27 rpm to reaggregate. Clusters were then fed every 48 h according to the usual protocol.Reaggregation controls were performed in rockers for scale reasons, but note that this results in less efficient endocrine enrichment than spinner flasks. Typical yields were approximately 10-15 M purified cells when starting with approximately 150 M total cell numbers. Cells were functionally assessed 7-9 days after purification.

[0194] Preparation of differentiated cells for sequencing Differentiated clusters were prepared for single-cell RNA sequencing as follows: 1–2 mL of suspension culture was harvested from spinner flasks, dissociated with TrypLE Express (5–15 min at 37 °C), quenched in cold PBS + 1% BSA, and gently dispersed with a P1000 pipette. Cells were then centrifuged (300 rpm, 3 min), resuspended in cold PBS + 1% BSA, and filtered through a 70 μm mesh filter. Centrifugation, resuspension, and filtration were repeated a total of three times. Cells were then counted and resuspended in inDrops working dilution (100,000 cells / mL) in 1X PBS with 13% Optiprep (Sigma; D1556).

[0195] inDrops single-cell RNA sequencing Single-cell RNA sequencing was performed using the inDrops platform as previously described. 4,40Most samples were performed using "inDrops v2" barcoded hydrogel beads (1 Cell Bio, Harvard Single Cell Core), and one experiment was performed using "inDrops v3" beads (Harvard Single Cell Core). Following the inDrops protocol, each biological sample was encapsulated and then split into several aliquots containing 1000-3000 cells. At least two library aliquots were prepared separately from each sample, indexed using the recommended indexing sequences, combined, and sequenced on a NextSeq500 (Illumina). In the first set of experiments (stages 3-6 time course), several thousand cells were sequenced per time point to estimate the expected cell type diversity. In the following stage 5 and 6 time course, separate flasks were used as technical replicates, and several thousand cells were measured from each individual time point, increasing the ability to identify subtle changes in rare populations or major cell types.

[0196] inDrops Raw Data Processing Sequencing read data were processed according to the previously published inDrops pipeline (github.com / indrops / indrops / ). To run the pipeline, a reference index was constructed from the Ensembl GRCh38 human genome assembly and the GRCh38.88 transcriptome annotation. Briefly, the pipeline trims the read data using Trimmomatic, maps the read data to the human transcriptome using Bowtie1.1.1, and quantifies the expression counts of transcripts using unique molecular identifiers called UMIFMs. For each library, the UMIFM count matrix was screened as follows: remove genes with counts less than 3; remove mitochondrially encoded and poorly annotated genes; remove cells with UMIFM counts less than 750 (stage 5 and 6 time courses) or 1000 (all other datasets). Variability in the sum of counts of individual cells was removed by normalizing the sum of each cell's counts to 10,000. These normalized counts were used as input for the following and converted to TPM values ​​for data presentation:

[0197] Dimensionality Reduction and Clustering Dimensionality reduction and clustering for each dataset were performed using the method described by Zeisel et al. 2018. 41 This was done loosely following a modified version of the method presented in. Using non-normalized count values, we identified outliers with high coefficients of variation as a function of mean expression, as previously described. 41 , highly variable genes were identified. Then, within each dataset, the (depth-normalized) count values ​​were z-normalized per gene to obtain z-normalized values. The z-normalized values ​​of variable genes (per dataset) were used as input for principal component analysis (PCA). Genes correlated (Pearson correlation > 0.15) with the cell cycle marker TOP2A were identified and excluded when calculating principal components for the stage 5 dataset. Clustering was performed using the Leiden community detection, which was recently published as an improvement over Louvain community detection. 42For crowding detection, a kNN graph was created by retaining only the mutual edges of 250 (stage 5 and 6 time courses) or 100 (other datasets) nearest neighbors of cells in the space of the first 50 PCs. If necessary, crowding detection was repeated on a subset of cells to improve cell annotation. Retaining only the mutual edges improved the ability to identify SST+ / HHEX+ cells, which represent the most difficult clusters to correctly distinguish in the data. For each dataset, this dimensionality reduction procedure followed by clustering was performed twice per dataset. The first run was used to identify clusters with low average library size, clusters without expression markers (defined using the score of Zeisel et al.), or clusters with clear dual expression patterns. For the stage 5 and stage 6 time courses, this first run of sorting was performed once at each time point and once again with the complete dataset (full dataset is used from now on). Sorted cells were ignored in the second round of clustering. After this second round of clustering, individual clusters were assigned an identity (and merged with other clusters, where appropriate) by correlating their expression profiles to a set of marker genes predefined for each population. After the clusters were interpreted, we trained a scikit-learn random forest classifier on the clusters and used bootstrap out-prediction to assign a final label to cells. This classifier was also used to recover cells removed in the first round of sorting, retaining cells whose predicted label had a majority of 66% across random trees, and recovering approximately 5% of cells in the entire dataset. These retained cells were incorporated into downstream analyses but were ignored when determining principal components. tSNE projections were calculated with a Python wrapper around the C Barnes-Hut t-SNE implementation (github.com / lvdmaaten / bhtsne) using the first 25 principal components. To calculate the average gene expression level within a label, we summed the UMIFM counts for all cells assigned to that label and calculated the tpm normalization on these summed counts.The percentage of cells expressing a given gene within a cluster was also calculated using 1% of the maximum expression of that gene (in any cell in the same dataset) as the threshold for qualifying it as expressed. Correlation between cell groups was calculated by first selecting 2000 highly variable genes across the entire dataset, calculating the average expression within each cell group (as above), z-normalizing each gene across the different classes, and then calculating the Pearson r correlation coefficient between samples for these 2000 genes.

[0198] Diffusion pseudotime analysis Diffusion Pseudo Time Analysis (DPT) 43 The Scanpy package 44 , was performed using 100 nearest neighbors in 10 unscaled principal components to find 10 diffusion components. The DPT was then calculated from manually specified root cells and cells were ordered in their rank along the branches of the DPT (if any). For stage 5 branching analysis, cells assigned to the SC-β or SC-EC cluster were assigned to that branch and progenitor cells were randomly assigned to the branches. The pseudotime along each branch is scaled from 0 to 1 corresponding to the ranking of the cells, but the ranking of the progenitors is adjusted so that both branches diverge from a common progenitor with a value of 0.5. To identify genes whose expression is a function of pseudotime, BEAM 45A version of the model was implemented. For unbranched pseudotime trajectories, two negative binomial generalized linear models were fitted using the VGAM R package. The first was a full model incorporating a natural spline function for pseudotime. The other was a reduced model that did not include the spline term for pseudotime. For branched trajectories, the second full model incorporated the branch terms for each cell as regressors. The fold change between branches, or pseudotime trajectories, was then calculated using the regressed values. Each regression was run on all cells analyzed in that particular analysis, resulting in sample sizes for the regressions: 10,034 cells (number of SC-β cells) for the analysis of Figures 11G-11I, 5,131 cells (number of progenitor cells at stage 5, day 0) and 5,109 cells (number of progenitor cells at stage 5, day 1) for the analysis of Figures 22C-22E, and 18,099 cells (number of progenitor cells, endocrine derivatives, SC-ECs or SC-β cells) for the analysis of Figures 14E-14G. As was done in the BEAM publication, the likelihood of the data under the full and reduced models was compared using a likelihood ratio test (with 3 degrees of freedom) and reported as an FDR (α = 0.001) corrected q value. While this provides a useful relative measure of significance, this analysis does not take into account the fact that the pseudotime values ​​for cells were derived from some of the genes originally tested. 46 Note that significance levels are likely inflated due to the large number of fold changes. When reporting fold changes obtained from pseudotime analysis, a lower limit on predicted expression (tpm=10) is enforced to prevent artificially high fold changes. The fold change between the beginning and end of the trajectory is then calculated by comparing the average predicted expression of the first and last 5% of the trajectory.

[0199] Analysis of human islet inDrops data Baron et al. 5 The raw sequence reads of were reprocessed as above and aligned to the same reference as the in vitro sequence data. UMIFM counts were converted to tpm for expression analysis as above. Finally, clustering was performed as above to identify cells in the same classes as in the original paper.

[0200] Reanalysis of beta cell EED2 knockout data The processed RNA-seq data were downloaded from GEO (accession number GSE110648). Read counts were used as input and the Voom 47 and Limma 48 A linear model was created using the . The original data included three different genotypes (WT, heterozygous, and homozygous EED2-floxed alleles) analyzed at two time points (8 and 25 weeks after knockout induction). All conditions had three samples each, for a total of 15 samples, except for two heterozygous and two homozygous samples at week 25. Using designed contrasts parameterization, we first defined replication groups across all six conditions in the dataset, and then identified differentially expressed genes between conditions at 25 weeks after EED2 KO for WT, heterozygous, and homozygous EED2-floxed alleles. The Benjamini-Hochberg FDR procedure with α = 0.05 was used to correct for multiple hypothesis testing.

[0201] Reanalysis of sorted NKX6.1(GFP)+ / - populations Gupta et al. 28 Full statistical analyses were downloaded from the supplementary materials of each publication. Reported mean expression levels, fold changes, and significance values ​​were used directly to generate the relevant figures.

[0202] Gene set enrichment analysis Gene set enrichment analysis (GSEA) was performed using GSEA 3.0 with fold changes between NKX6.1+ progenitors, SC-β cells, and islet β cells, or fold changes tracking SC-β pseudotime expression as inputs, as a "pre-ranking" analysis. The analysis was performed including Hallmark (h.all.v6.2) and CanonicalPathway categories (c2.cp.v6.2) from MSigDB, as well as the custom gene sets defined in Figure 8, in one single analysis to ensure appropriate correction for multiple hypothesis testing. The set size was small at 5 genes, but was otherwise run with default settings.

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Claims

1. 1. A method of inducing differentiation of a cell population, comprising inhibiting expression of a cell fate regulator in progenitor cells, wherein said regulator is selected from Table 1 or Table 2, thereby inducing differentiation of the cell population into SC-β cells; wherein said method is an in vitro method.

2. 2. The method of claim 1, wherein the modulator is selected from the group including FBXL14, BCORL1, SHOC2, CCDC6, B3GALT6, HOXA1, DDX3X, CARM1, EXT2, EXT1, DYRK1A, SCAF1, SCAF8, CAND1, NDST1, EYA3, GLCE, DYRK1B, PRDM16, ALG3, CXXC4, SMURF1, PHF21A, SOX4, and TET2.

3. 2. The method of claim 1, wherein the regulatory factor is selected from the group comprising FBXL14, BCORL1, SHOC2, CCDC6, B3GALT6, HOXA1, DDX3X, CARM1, EXT2, and EXT1.

4. 2. The method of claim 1, wherein the modulator is selected from the group including SOX4, BCORL1, FBXL14, CCDC6, SOX1, CARM1, TNRC18, CAND1, TET2, HOXA1, ASCL1, ARID2, SIRT6, FBXO22, FLVCR1, FOXA1, COPS9, ELAVL1, SSBP3, PROSER1, PROX1, SMURF1, SCAF1, HELLS, and DACH1.

5. 2. The method of claim 1, wherein the regulatory factor is selected from the group including SOX4, BCORL1, FBXL14, CCDC6, SOX1, CARM1, TNRC18, CAND1, TET2, and HOXA1.

6. 10. The method of claim 1, wherein the expression of the cell fate control factor is inhibited by knocking down the control factor using gene editing technology.

7. 7. The method of claim 6, wherein the expression of the cell fate regulator is inhibited by knocking down the regulator using CRISPR.

8. 10. The method of claim 1, wherein the expression of the cell fate control factor is inhibited by knocking out the control factor using gene editing technology.

9. 9. The method of claim 8, wherein the expression of the cell fate regulator is inhibited by knocking out the regulator using CRISPR.

10. 9. The method of claim 6 or claim 8, wherein the CRISPR is introduced into the progenitor cells via a retrovirus.

11. The method of claim 10, wherein the retrovirus is a lentivirus.

12. 10. An enriched population of SC-β cells produced by the method of claim 1.

13. 13. The enriched population of claim 12, wherein the cell population comprises 70% SC-β cells.

14. 13. The enriched population of claim 12, wherein the cell population comprises 80% SC-β cells.

15. 13. An SC-islet comprising an enriched population of SC-β cells according to claim 12.

16. 1. A method of inducing differentiation of a cell population, comprising inhibiting expression of a cell fate regulator in a progenitor cell, wherein said regulator is selected from Table 3 or Table 4, thereby inducing differentiation of the cell population into SC-α cells; wherein said method is an in vitro method.

17. 17. The method of claim 16, wherein the modulator is selected from the group including PDX1, CCDC6, HES1, PHF21A, PAX4, DYRK1B, DYRK1A, BCORL1, TET2, DDX3X, PROSER1, PBX1, HELLS, CAND1, EYA3, MYT1, AFF4, FBXL14, HOXA1, ZC3H15, SCAF8, PRDM16, HEXIM1, TTC14, ZRANB1, and B3GALT6.

18. 17. The method of claim 16, wherein the regulatory factor is selected from the group including PDX1, CCDC6, HES1, PHF21A, PAX4, DYRK1B, DYRK1A, BCORL1, TET2, and DDX3X.

19. 17. The method of claim 16, wherein the modulator is selected from the group including PAX4, HES1, CCDC6, SOX4, ZBTB10, PHF21A, PBX1, ARID2, TET2, BCORL1, TTC14, CAND1, PROSER1, SOX1, FBXO22, HELLS, DYRK1B, ZRANB1, DYRK1A, ASCL1, ZC3H15, SETBP1, FAM58A, MYT1, and RALGAPB.

20. 17. The method of claim 16, wherein the regulatory factor is selected from the group comprising PAX4, HES1, CCDC6, SOX4, ZBTB10, PHF21A, PBX1, ARID2, TET2, and BCORL1.

21. 17. The method of claim 16, wherein the expression of the cell fate control factor is inhibited by knocking down the control factor using gene editing techniques.

22. 22. The method of claim 21, wherein the expression of the cell fate regulator is inhibited by knocking down the regulator using CRISPR.

23. 17. The method of claim 16, wherein the expression of the cell fate control factor is inhibited by knocking out the control factor using gene editing techniques.

24. 24. The method of claim 23, wherein the expression of the cell fate regulator is inhibited by knocking out the regulator using CRISPR.

25. 25. The method of claim 22 or claim 24, wherein the CRISPR is introduced into the progenitor cells via a retrovirus.

26. 26. The method of claim 25, wherein the retrovirus is a lentivirus.

27. 17. An enriched population of SC-β cells produced by the method of claim 16.

28. 28. The enriched population of claim 27, wherein the cell population exhibits an enrichment yield of 70% SC-β cells.

29. 28. The enriched population of claim 27, wherein the cell population exhibits an enrichment yield of 80% SC-β cells.

30. 28. An SC-islet comprising an enriched population of SC-β cells according to claim 27.

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