Differentiation and functional maturation of pancreatic islet tissue from pluripotent stem cells by ai-designed epigenetic modifiers

Abstract

Methods are provided for generating pancreatic endocrine cells from pluripotent stem cells (PSCs) using epigenetic modifiers. A fusion protein comprising catalytically inactivated Cas9 (dCas9) and an embryonic ectoderm development (EED)-binding domain (EBdCas9) is used with guide RNAs (gRNAs) to target regulatory regions of transcriptional regulators including EOMES, PDX1, and NGN3. Sequential targeting of PDX1 and NGN3 promoters with EBdCas9 removes repressive H3K27me3 marks, increases activating H3K27ac modifications, and accelerates differentiation into insulin-producing β-cells. The methods significantly increase β-cell yields (~4-6 fold), reduce non-pancreatic lineage contaminants (CDX2+, SOX2+, enteroendocrine cells), enhance mitochondrial function, and produce glucose-responsive cells with superior functional maturation in vivo. The epigenetic interventions enforce pancreatic developmental fates while activating NGN3-dependent transcriptional programs necessary for endocrine cell differentiation, insulin secretion, and metabolic maturation. Cells and compositions produced by these methods are also provided.

Description

MBHB Ref. No. 24-1170-WO UW 49850.02WO2 SUPERIOR DIFFERENTIATION AND FUNCTIONAL MATURATION OF PANCREATIC ISLET TISSUE FROM PLURIPOTENT STEM CELLS BY AI- DESIGNED EPIGENETIC MODIFIERSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to and the benefit of U. S. Provisional Application No. 63 / 723,049, filed November 20, 2024, the disclosure of which is incorporated by reference in its entirety herein for all purposesSTATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with Government support under Federal Grant nos. RC2DK 140839-01 and 1RC2DK144787-01, awarded by the National Institutes of Health. The Federal Government has certain rights to this invention.REFERENCE TO ELECTRONIC SEQUENCE LISTING

[0003] The application contains a Sequence Listing which has been submitted electronically in. XML format and is hereby incorporated by reference in its entirety. Said. XML copy, created on November 18, 2025, is named “24-1170-WO_sequence_listing.xml” and is 71,065 bytes in size. The sequence listing contained in this. XML file is part of the specification and is hereby incorporated by reference herein in its entirety.BACKGROUND

[0004] The pancreatic islets of Langerhans are small micro-organs scattered throughout the pancreas composed of -3,000 cells. They are composed of endocrine a-cells, P-cells, 5-cells, y-cells, and s-cells, producing glucagon, insulin, somatostatin, pancreatic polypeptide, and ghrelin, respectively. Over the past two decades, significant progress has been made to produce these endocrine cells in vitro from PSC. Current strategies are based on factor-directed approaches of PSC differentiation, designed to activate in a spatiotemporal manner the expression of a cascade of transcription factors that recapitulate sequentially the specification of PSCs into Definitive Endoderm (DE), next into Pancreatic Progenitors (PP), and then into Endocrine Progenitors (EP), eventually leading to SC differentiation into hormone-positiveMBHB Ref. No. 24-1170-WO UW 49850.02WO2 cells. Yet, despite significant improvements, the process remains lengthy and highly inefficient as progression through the DE, PP, and EP bottlenecks (FIG. 1) end up yielding just a fraction of cells exhibiting features of P-like cells after >5 weeks of culture. Instead, many cells resulting from this differentiation are short-lived polyhormonal cells, held at progenitor-like stages that can unpredictably drift toward alternative lineages and / or functionally immature. These challenges highlight the need to devise more efficient protocols to control the yield and quality of SC-derived islet cell types.

[0005] Mounting evidence indicates that epigenetic changes of the chromatin, such as histone methylation and acetylation, as well as DNA methylation, are critical determinants of stem cell fate choices, lineage specification, and differentiation. Indeed, genome-wide maps of chromatin states in undifferentiated and differentiated stem cells have delineated unique epigenetic dynamics regulating chromatin accessibility to transcription factors and their binding at the promoter and enhancer regions of genes that regulate developmental choices. For instance, histone modifications such as trimethylation of histone H3 at lysine 27 (i.e., H3K27me3) are preferentially enriched at repressed loci, while active promoters usually exhibit H3K4me3 or H3K27ac marks. On the other end, active enhancers are preferentially marked by H3K4mel and H3k27ac histone modifications. In addition, unique patterns of repressive H3K27me3 co-existing with activating H3K4mel marks are often observed at the promoters of developmental regulators such as transcription factors, creating "bivalent" chromatin states which poise developmental genes for activation once the repressive marks are removed. Unlike histone methylation, the DNA methylation status of many genes tend to remain stable in terminally differentiated cells. However, changes in DNA methylation / demethylation may occur in adaptive cellular responses to environmental stresses or with aging. Repressive H3K27me3 modifications are primarily established through the histone methyltransferase activity of EZH2 subunits of Polycomb Repressive Complex 2 (PRC2), whereas regulation of DNA methylation is mediated by methylating (i.e., Dnmtl / Dnmt3a / b) and demethylating (e.g., Tetl / Tet2) enzymes.

[0006] Of direct relevance to the regulation of pancreatic P-cells differentiation from stem cells, several studies in the last decade have uncovered a prominent modulation of repressive H3K27m3 marks during directed differentiation. Specifically, a progressive loss of repressive H3K27m3 marks at gene loci encoding for transcriptional regulators of the DE (e.g., GSC, EOMES, FOXA2, SOX17 and GATA6), PE (e g., PDX, NKX6.1, and PAX4) and PP (e g.,MBHB Ref. No. 24-1170-WO UW 49850.02WO2 Ngn3, NKX2.2 and NEURODI) stages has been observed, indicating a possible role of the PRC2 complex in influencing these key cell lineage choices through restraining chromatin remodeling of those loci. Knock-down of EZH2 at the PP stage in mice has been shown to lead to the premature up-regulation of Ngn3, and ultimately an increased number of endocrine progenitors and P-cells in vivo. Many of the genes undergoing loss of H3K27m3 repressive marks at the DE, PP and EP stages were found to harbor "bivalent" H3k4me3 active marks at the undifferentiated stage which were maintained. Bivalency early in developmental stem cell programs may thus poise cells for gene activation later during differentiation. Nevertheless, it was noted that under factor-directed differentiation of current protocols only 30% of bivalent genes at EP stage loose H3K27me3 repressive marks in the final EP / SC-P-cell cell stage; whereas, the majority of them lose these repressive marks after engraftment, as cells become functionally mature in vivo. Genes remaining inappropriately repressed are associated with hormone processing and secretion as well as transcriptional networks relevant to P-cell function, consistent with an incomplete mature phenotype of PSC-derived P-cells that current protocols are unable to resolve.

[0007] Further studies have provided evidence for a dynamic regulation of DNA methylation at genomic regions distal (50-500 kB) to promoters, indicating a possible role in modulation of enhancers' activities during differentiation of islet cell types from IPSC. Enhancer DNA methylation is maximal at undifferentiated and DE stages, minimal at PP and EP stage, and is returned to intermediated levels at the final endocrine stage (FIG. 2). Based on H3K4mel marking and activity defined by ATAC-seq and RNA-seq experiments, >34,000 putatively active enhancer regions of DE, PP and EP signature genes have been defined providing a valuable resource for epigenetic targeting of select genes of interest. Furthermore, it has been shown that competence to execute lineage fate decision is first marked by gain of H3K4mel and then H3K27ac, and that a hot spot for this remodeling at lineage-restricted enhancers is at the PP to EP branching point. This remodeling appears to affect preferentially alpha-specific enhancers in polyhormonal [3-like cells creating an overall bias toward development of glucagon-expressing cells after transplantation in vivo. Hence a-cell identity may be the default epigenetic endocrine state during islet differentiation from iPSC if the polyhormonal phenotype of terminally differentiated P-cell fails to be resolved. Lastly, it was shown that P-cell endocrine functions depend on DNMT3a-mediated DNA methylation and silencing of "disallowed" genes, specifically of HK1 / 2 and LDHA which are glucose secretion decoupling genes.MBHB Ref. No. 24-1170-WO UW 49850.02WO2

[0008] The inventors hypothesized that DNA methylation of lineage-specific enhancers regulates [3-cell identity and that there may be a time window of opportunity for epigenetic interventions aimed at controlling transcriptional programs driving a- over [3-cell developmental choices (e.g., ARX, PAX4) to re-direct endocrine cell identity toward [3-cell phenotypes at the EP stage (FIG. 2). Accordingly, described herein are methods for making stem cell-derived pancreatic islets of improved homogeneity through enforced activation of epigenetically-induced islet cell developmental programs. It is also described herein that these epigenetic interventions significantly shorten the time required to generate P-cells, result in significantly higher endocrine cell yields, and generate endocrine tissue with superior hormone secretory functions and metabolic performance in vitro and in vivo.SUMMARY OF THE DISCLOSURE

[0009] The present disclosure relates to methods of epigenetically editing human pluripotent stem cells (hPSC) lines with small protein epigenetic binders (EpiBinders) to enhance the efficiency of pancreatic islet cells specification and functional maturation. In an aspect, the disclosure provides a method of generating an endocrine progenitor (EP) stage cell from a pluripotent stem cell (PSC), including (i) inducing expression in the PSC of a transcriptional regulator of a definitive endoderm (DE) stage cell to differentiate the PSC into a DE stage cell, into a primitive gut tube (PGT) stage cell, and then to a posterior foregut (PF) stage cell; (ii) inducing expression in the PF stage cell of a transcriptional regulator of a pancreatic progenitor (PP) stage cell to differentiate the PF stage cell into PP stage cell; and (iii) inducing expression in the PP stage cell of a transcriptional regulator of a EP stage cell to differentiate the PP stage cell into a EP stage cell; in which binding of a guide RNA (gRNA) complementary to a regulatory region of the transcriptional regulator of the DE stage cell, of the PP stage cell, or the EP stage cell and also a fusion protein (EBdCas9) comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain together induce expression of the transcriptional regulator of the DE stage cell, of the PP stage cell, or the EP stage cell.

[0010] In embodiments, the disclosure provides a method for generating an endocrine progenitor (EP) stage cell from a primitive gut tube (PGT) stage cell or a posterior foregut (PF) stage cell, including (i) inducing expression in the PGT stage cell or PF stage cell of a transcriptional regulator of a pancreatic progenitor (PP) stage cell to differentiate the PGTMBHB Ref. No. 24-1170-WO UW 49850.02WO2 stage cell or PF stage cell into PP stage cell; and (ii) inducing expression in the PP stage cell of a transcriptional regulator of a EP stage cell to differentiate the PP stage cell into a EP stage cell; in which binding of (i) a guide RNA (gRNA) complementary to a regulatory region of the transcriptional regulator of the PP stage cell, or the EP stage cell and (ii) a fusion protein (EBdCas9) comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain together induce expression of the transcriptional regulator of the PP stage cell, or the EP stage cell.

[0011] In another aspect, the disclosure provides a method of inducing differentiation of a pluripotent stem cell (PSC) in culture to a definitive endoderm (DE) stage cell, the method including (i) introducing to the PSC a gRNA for a transcriptional regulator of a DE stage cell selected from GSC, EOMES, FOXA2, SOX17, and GATA6; (ii) introducing to the PSC a fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain; and (iii) inducing expression of the transcriptional regulator.

[0012] In some embodiments of the method of inducing differentiation of a pluripotent stem cell (PSC) in culture to a definitive endoderm (DE) stage cell, (a) inducing expression in the PSC of a transcriptional regulator of a DE stage cell of step (i) is performed on day 0-3; (b) inducing expression in the PF stage cell of a transcriptional regulator of a PP stage cell of step (ii) is performed on between about day 5 to day 10; and (c) inducing expression in the PP stage cell of a transcriptional regulator of a EP stage cell of step (iii) is performed between about 2 to 5 days after step (b).

[0013] In another aspect, the disclosure provides a method of inducing differentiation of a primitive gut tube (PGT) stage cell or a posterior foregut (PF) stage cell in culture to a pancreatic progenitor (PP) stage cell, the method including (i) introducing to the PF stage cell a gRNA for a transcriptional regulator of a PP stage cell selected from PDX1, NKX6.1, and PAX4; (ii) introducing to the PF stage cell a fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain; and (iii) inducing expression of the transcriptional regulator. In some embodiments, the transcriptional regulator of a PP stage cell is PDX1, and the gRNA is complementary to a regulatory region of PDX1. In some embodiments of the method of inducing differentiation of a primitive gut tube (PGT) stage cell or a posterior foregut (PF) stage cell in culture to a pancreatic progenitor (PP) stage cell, inducing expression in the PP stage cell of a transcriptional regulator of a EPMBHB Ref. No. 24-1170-WO UW 49850.02WO2 stage cell of step (ii) is performed between about 2 to 5 days after inducing expression in the PF stage cell of a transcriptional regulator of a PP stage cell.

[0014] In another aspect, the disclosure provides a method of inducing differentiation of a pancreatic progenitor (PP) stage cell to an endocrine progenitor (EP) stage cell, including (i) introducing to the PP stage cell a gRNA for a transcriptional regulator of a EP stage cell selected from NGN3, NKX2.2 and NEURODI; (ii) introducing to the PP stage cell a fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain; and (iii) inducing expression of the transcriptional regulator. In some embodiments, the transcriptional regulator of a EP stage cell is NGN3, and the gRNA is complementary to a regulatory region of NGN3.

[0015] In some of the various aspects and embodiments of the disclosure the include EOMES as a transcriptional regulator of a DE stage cell, the gRNA complementary to a regulatory region for EOMES utilized in such aspects and embodiments comprises a sequence at least about 90% identical, or at least about 95% identical to SEQ ID NO:24 and SEQ ID NO:28. In some embodiments, the gRNA complementary to a regulatory region for EOMES is selected from the group consisting of SEQ ID NO:24 and SEQ ID NO:28.

[0016] In some of the various aspects and embodiments of the disclosure the include PDX1 as a transcriptional regulator of a PP stage cell, the gRNA complementary to a regulatory region for PDX1 utilized in such aspects and embodiments comprises a sequence at least about 90% identical, or at least about 95% identical to SEQ ID NO:9, SEQ ID NO: 10, and SEQ ID NO: 11. In some embodiments, the gRNA complementary to a regulatory region for PDX1 is selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11.

[0017] In some of the various aspects and embodiments of the disclosure the include NGN3 as a transcriptional regulator of a EP stage cell, the gRNA complementary to a regulatory region for NGN3 utilized in such aspects and embodiments comprises a sequence at least about 90% identical, or at least about 95% identical to SEQ ID NO: 17. In some embodiments, the gRNA complementary to a regulatory region for NGN3 is SEQ ID NO: 17.

[0018] In the various aspects and embodiments of the disclosure, the the fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain is originally transcribed from a nucleic acid sequence that comprises aMBHB Ref. No. 24-1170-WO UW 49850.02WO2 sequence at least about 90% identical, or at least about 95% identical, or at least about 99% identical to SEQ ID NO:32, or is originally transcribed from a nucleic acid sequence that comprises SEQ ID NO: 32.

[0019] In the various aspects and embodiments of the disclosure, the fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain includes an amino acid sequence that is at least about 90% identical, or at least about 95% identical, or at least about 99% identical to SEQ ID NO:53, and in some aspects and embodiments, is SEQ ID NO:53.

[0020] In another aspect, the disclosure provides a cell produced according to the various methods described herein. In some embodiments, the cell is an a-cell, [3-cell, 5-cell or y-cell. In some embodiments, the cell is a [3-cell, and in some embodiments, the cell produces insulin.

[0021] In another aspect, the disclosure provides a composition comprising cells produced according to any of the methods described herein. In some embodiments, the composition includes a pharmaceutically acceptable excipient.

[0022] In another aspect, the disclosure provides a method of treating diabetes in a subject in need thereof, including (i) obtaining cells produced according to a method described herein, or a composition as described herein; and (ii) administering the cells or composition to the subject in an amount effective to treat diabetes.

[0023] In another aspect, the disclosure provides a method including (i) obtaining cells produced according to a method described herein, or a composition as described herein; and (ii) administering the cells or composition to the subject in an amount effective to treat diabetes; wherein the administered cells or composition increase insulin production in a subject.

[0024] In another aspect, the disclosure provides a method including (i) obtaining cells produced according to a method described herein that results in the differentiation of a PSC into an a-cell or composition thereof; and (ii) administering the cells or composition to a subject in an amount effective to replace dysfunctional glucagon-producing cells in the subject.

[0025] In another aspect, the disclosure provides a method including (i) obtaining cells produced according to a method described herein that results in the differentiation of a PSCMBHB Ref. No. 24-1170-WO UW 49850.02WO2 into a 5-cell or composition thereof; and (ii) administering the cells or composition to a subject in an amount effective to replace dysfunctional somatostatin-producing cells in the subject.

[0026] In another aspect, the disclosure provides that the fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain and the gRNA increase acetylation of histone 3, lysine 27 (H3K27ac) at genomic loci proximal to or distal from the targeted regulatory region. In embodiments, the increased H3K27ac modifications occur at promoter, enhancer, or intronic regions of genes selected from the group consisting of NGN3, NKX2.2, GCK, NEURODI, and genes involved in exocytosis, insulin secretion, mitochondrial function, or metabolism.

[0027] In another aspect, the disclosure provides that the fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain and the gRNA selectively reduce the frequency of uncommitted PDXl-negative / NKX6.1-negative cells in differentiating cell populations. In a related aspect, the fusion protein and gRNA selectively reduce the frequency of CDX2-positive / PDXl -negative gut lineage cells in differentiating cell populations, wherein in embodiments the gRNA is complementary to a regulatory region of PDX1.

[0028] In another aspect, the disclosure provides that the fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain and the gRNA selectively reduce the frequency of SOX2-positive / PDXl -negative gastric or anterior foregut lineage cells in differentiating cell populations, wherein in embodiments the gRNA is complementary to a regulatory region of NGN3. In a related aspect, the fusion protein and gRNA selectively reduce the frequency of enteroendocrine lineage cells in differentiating cell populations, wherein in embodiments the enteroendocrine lineage cells express tyrosine hydroxylase (TH) and wherein sequential targeting of PDX1 followed by NGN3 reduces enteroendocrine cell frequency compared to targeting NGN3 alone.

[0029] In another aspect, the disclosure provides methods that result in EP stage cells exhibiting increased expression of NGN3 target genes involved in exocytosis, insulin secretion, mitochondrial function, or metabolism compared to comparable controls. In embodiments, the NGN3 target genes comprise at least 10, at least 50, at least 100, or at least 150 genes selected from a set of 1,263 predicted or validated NGN3 target genes.MBHB Ref. No. 24-1170-WO UW 49850.02WO2

[0030] In another aspect, the disclosure provides methods that result in EP stage cells or differentiated endocrine cells exhibiting enhanced mitochondrial function compared to comparable controls. In embodiments, the enhanced mitochondrial function comprises at least one of: (a) increased ATP production-coupled respiration; (b) reduced proton leak; or (c) increased coupling efficiency.

[0031] In another aspect, the disclosure provides methods that result in insulin-producing cells that exhibit glucose-stimulated insulin secretion in vitro. In embodiments, the glucose-stimulated insulin secretion is measured in response to D-Glucose, L-Arginine, or KC1. In a related aspect, the methods result in insulin-producing cells that, when transplanted into an immunodeficient animal, exhibit glucose-responsive insulin secretion in vivo within 3 months of transplantation. In embodiments, the insulin-producing cells exhibit (a) lower basal insulin secretion at fasting compared to control cells produced without gRNA-mediated targeting; and (b) increased insulin secretion in response to glucose challenge compared to control cells. In further embodiments, the insulin-producing cells exhibit a stimulation index (ratio of C-peptide secreted after glucose load to C-peptide at fasting) that is significantly higher than control cells produced without gRNA-mediated targeting.

[0032] In another aspect, the disclosure provides that the introducing of the fusion protein and / or the gRNA is performed by transient mRNA transfection. In embodiments, the fusion protein is introduced as mRNA encoding the fusion protein and the mRNA is modified to comprise Nl-Methyl-Pseudouri dine.

[0033] In another aspect, the disclosure provides that the PSC is a human embryonic stem cell (hESC) or a human induced pluripotent stem cell (iPSC). In embodiments, the method is reproducible across multiple independent PSC lines of diverse genetic backgrounds.

[0034] In another aspect, the disclosure provides that the PSC is engineered to comprise a nucleic acid encoding the fusion protein under control of an inducible promoter integrated at a safe harbor locus. In embodiments, the safe harbor locus is the AAVS1 locus and the inducible promoter is a doxycycline-inducible promoter. In further embodiments, the nucleic acid encoding the fusion protein further comprises a nucleic acid encoding a detectable marker, wherein in embodiments the detectable marker is mCherry.MBHB Ref. No. 24-1170-WO UW 49850.02WO2

[0035] In another aspect, the disclosure provides a method of generating insulin-producing P-cells from a pluripotent stem cell (PSC), comprising: (i) differentiating the PSC to a posterior foregut (PF) stage cell; (ii) at about day 8 of differentiation, transiently inducing expression of a fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain and introducing a gRNA complementary to a regulatory region of PDX1; (iii) differentiating the PF stage cell to a pancreatic progenitor (PP) stage cell; (iv) at about day 11-12 of differentiation, transiently inducing expression of the fusion protein and introducing a gRNA complementary to a regulatory region of NGN3; and (v) differentiating the PP stage cell to an endocrine progenitor (EP) stage cell and then to an insulin-producing [3-cell; wherein the sequential targeting of PDX1 and NGN3 promoters results in: (a) increased yield of insulin-producing P-cells compared to control; (b) reduced frequency of CDX2-positive gut lineage cells compared to control; (c) reduced frequency of SOX2-positive gastric lineage cells compared to control; and (d) reduced frequency of enteroendocrine lineage cells compared to control. In embodiments, the gRNA complementary to a regulatory region of PDX1 comprises a combination of gRNAs selected from SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11. In further embodiments, the gRNA complementary to a regulatory region of NGN3 comprises SEQ ID NO: 17. In still further embodiments, the insulin-producing P-cells are obtained by day 19-20 of differentiation and comprise at least 20%, at least 25%, or at least 30% of the cell population.

[0036] In another aspect, the disclosure provides a method of producing pancreatic endocrine cells with reduced contamination by non-pancreatic lineages, comprising: (i) differentiating a pluripotent stem cell (PSC) toward pancreatic lineages; (ii) at a primitive gut tube or posterior foregut stage, introducing to the differentiating cells: (a) a gRNA complementary to a regulatory region of PDX1; and (b) a fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain; wherein the method selectively reduces the frequency of CDX2-positive / PDXl -negative posterior gut lineage cells and / or SOX2-positive / PDXl -negative anterior foregut lineage cells compared to control differentiation without gRNA-mediated targeting. In embodiments, the method further comprises at a pancreatic progenitor stage, introducing to the differentiating cells: (a) a gRNA complementary to a regulatory region of NGN3; and (b) the fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain; wherein the method further selectively reduces the frequency of SOX2-positive anterior foregut lineage cells and enteroendocrine lineage cells compared to control.MBHB Ref. No. 24-1170-WO UW 49850.02WO2

[0037] In another aspect, the disclosure provides a method for enhancing genome-wide chromatin remodeling in differentiating pancreatic endocrine cells, comprising: (i) differentiating a pluripotent stem cell (PSC) toward pancreatic endocrine lineages; (ii) sequentially introducing to the differentiating cells at defined developmental stages: (a) a first gRNA complementary to a regulatory region of PDX1 and a fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain; and (b) a second gRNA complementary to a regulatory region of NGN3 and the fusion protein; wherein the sequential targeting results in genome-wide deposition of H3K27ac marks at genomic loci associated with endocrine cell differentiation, insulin secretion, mitochondrial function, and metabolic maturation. In embodiments, the genome-wide deposition of H3K27ac marks comprises at least 5,000, at least 10,000, or at least 14,000 unique H3K27ac peaks compared to control cells that received the fusion protein without gRNA targeting. In further embodiments, the genomic loci with increased H3K27ac marks are enriched for genes involved in: (a) small GTPase-mediated signaling; (b) Rapl and cAMP signaling; (c) establishment of apical / basal cell polarity; (d) negative regulation of Wnt signaling; or (e) regulation of sodium ion transport.

[0038] In another aspect, the disclosure provides a method of activating NGN3 -dependent transcriptional programs in differentiating pancreatic cells, comprising: (i) introducing to pancreatic progenitor cells: (a) a gRNA complementary to a regulatory region of NGN3; and (b) a fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain; wherein the method results in activation of at least 100, at least 150, or at least 200 NGN3 target genes compared to control cells. In embodiments, the activated NGN3 target genes comprise genes involved in exocytosis, insulin secretion, mitochondrial function, metabolism, transcription factors, or development.

[0039] In another aspect, the disclosure provides a method comprising comparing the efficacy of epigenetic-based gene activation versus synthetic transactivator-based gene activation for enhancing pancreatic [3-cell differentiation, comprising: (i) introducing to a first population of differentiating pancreatic cells a gRNA and a fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain (EBdCas9); (ii) introducing to a second population of differentiating pancreatic cells the same gRNA and a fusion protein comprising a catalytically inactivated Cas9 and VP64 transactivator domain (VP64dCas9); (iii) comparing yields of PDXl-positive / NKX6.1 -positive endocrineMBHB Ref. No. 24-1170-WO UW 49850.02WO2 progenitors and insulin-positive P-cells between the first and second populations; wherein the EBdCas9-treated first population exhibits significantly higher yields of endocrine progenitors and P-cells compared to the VP64dCas9-treated second population.

[0040] In another aspect, the disclosure provides that the methods produce pancreatic endocrine cells within 19-25 days of differentiation, representing an acceleration of at least 10 days, at least 15 days, or at least 20 days compared to control differentiation protocols.

[0041] In another aspect, the disclosure provides a cell population produced according to the methods described herein, wherein the cell population comprises by Day 20 of differentiation: (a) at least 20% insulin-positive P-cells; (b) less than 2% CDX2-positive / PDXl -negative gut lineage cells; (c) less than 2% SOX2-positive / PDXl -negative gastric lineage cells; and (d) less than 10% tyrosine hydroxylase-positive enteroendocrine lineage cells. In embodiments, the cell population comprises at least 25%, at least 30%, or at least 40% insulin-positive P-cells.

[0042] In another aspect, the disclosure provides a composition comprising the cell population described above and a pharmaceutically acceptable excipient suitable for transplantation.

[0043] The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.

[0044] These and other aspects of the disclosure are set forth in more detail in the description of the disclosure below.BRIEF DESCRIPTION OF THE DRAWINGS

[0045] FIG. 1 is a diagram illustrating bottlenecks in islet differentiation from stem cells.

[0046] FIG. 2 is an illustration showing genes of interest targeted by Epi-Binders based on reported Methylome in stem cells differentiating toward islet cells.

[0047] FIG. 3A to FIG. 3D show the generation of MELl-DoubleFOXA2 / INSreporter lines and differentiation of DoubleFOXA2INScells into DE and insulin producing P-cells. FIG. 3A shows a fluorescence microscopy image (left panel) and flow cytometry histograms (right panel) ofMBHB Ref. No. 24-1170-WO UW 49850.02WO2 BFP expressed in MELl-DoubleFOXA2 / INScells differentiated to definitive endoderm stage, showing uniform FOXA2-driven expression of the fluorescent reporter in all cells. FIG. 3B shows bright and fluorescence microscopy images (left panels) and flow cytometry histograms (right panel) of MELl-DoubleFOXA2 / INScell clusters at stage 7 of differentiation (day 35), showing expression of the GFP reporter of insulin expression in ~ 30% of the cells. Transplantation in NSG mice leads to glucose-responsive P-cells upon an intraperitoneal GTT (FIG. 3C). FIG. 3D shows the targeting construct used to target BFP downstream of the FOXA2 promoter via gRNA-guided CRISPR-mediated recombination.

[0048] FIG. 4A to FIG. 4D show engineering of a PRC2 EpiBinder inhibitor EBdCas9 into the AAVS1 safe harbor of hESC and iPSC lines. FIG. 4A illustrates a construct for the EBdCas9-mCherry used to target the AAVS1 locus. FIG. 4B-C show DOX-inducible expression of the construct in MELl-DoubleFOXA2INSlines as detected by fluorescence microscopy (FIG. 4B) and flow cytometric analysis (FIG. 4C) of the mCherry reporter. FIG.4D shows a timeline of DOX induction and qPCR analysis of EB-transcription.

[0049] FIG. 5A to FIG. 5J show screening of transcription factors4promoters in iPSC using EBdCas9 and gRNAs. FIG 5A shows a schematic of the dCas9-EB and guide RNA system used to stimulate gene transcription. FIG. 5A to FIG. 5J show gRNA-mediated targeting of EBdCas9 to EOMES, PDX-1 and NGN3 promoters leads to gene activation and, for PDX-1 and NGN3, chromatin remodeling of the targeted genomic sites is demonstrated. FIG. 5A illustrates mode of action of EBdCas9. FIG. 5B, FIG. 5D, and FIG. 5G show genomic coordinates of gRNAs designed for targeting the EOMES (FIG. 5B), PDX1 (FIG. 5D) and NGN3 (FIG. 5G) promoters overlay ed onto integrative genomic viewer of H3K27me3 marks and EZH2 -binding sites of the corresponding genomic regions. FIG. 5E, F, H show RT-qPCR analysis of PDX1 and NGN3 transcription detected 48 hours (PDX1) and 24 hours (NGN3) in PSC after DOX induction of EBdCas9 alone or in combination with transfection of the indicated gRNAs. RT-qPCR of 18S of the same samples are shown as housekeeping controls.FIG. 51, J show ChlP-qPCR analysis showing loss of H3K27me3 marks and EZH2 occupancy in targeted PDX-1 and NGN3 promoters. In contrast non-targeted Hl 9 gene loci remain unaffected.

[0050] FIG. 6A to FIG. 6D show EBdCas9 / gRNA-mediated sequential activation of PDX1 and NGN3 promoters accelerates the development and increases the output of beta cells across multiple PSC clones. FIG. 6A shows a flow chart of EBdCas9 induction and gRNA deliveryMBHB Ref. No. 24-1170-WO UW 49850.02WO2 during directed differentiation of PSC toward pancreatic islet lineages. FIG. 6B shows fluorescence microscopy of islet clusters from one MEL-EBdCas9 hESC clone (clone #2) at day 19 of differentiation showing cell expression of EBdCas9mCherry and INS-GFP fluorescent reporters, as well as representative flow cytometric analysis of GFP+ Insulin+ cells from the clone. FIG. 6C and FIG 6D mimic FIG. 6B, but for clones #4 and #5, respectively.

[0051] FIG. 8A and FIG. 8B show a FACS analysis of undifferentiated IPSCs transfected with Fluoro-gRNA (FIG. 8A) or with an EBdCas9mCherry mRNA (FIG. 8B).

[0052] FIG. 9A illustrates a protocol followed for PDX1 and NGN3 gRNA transfection in conjunction with EBdCas9 induction. FIG.9B shows qPCR results forPDXl andNGN3. FIG.9C shows qPCR results for NEURODI and NKX2.2. FIG. 9D shows quantification of P-cells by FACS. FIG. 9E shows insulin secretory function in response to D-Glucose, L-Arginine, and KC1.

[0053] FIG. 10A to FIG. 10C show effects of EBdCas9-mediated epigenetic editing. FIG.10A shows EBdCas9-mediated epigenetic editing produces SC-islet cells that exhibit a higher ATP production-coupled Respiration. FIG. 10B demonstrates a resulting lower Proton Leak.FIG. 10C shows higher overall Coupling Efficiency in response to Leucine / Glutamine. Bars indicate mean ±SD of OCR reads in 8 replicas / condition normalized to DNA content. *p<0.05.

[0054] FIG. 11 shows an assessment of in vivo function of EBdCas9»gRNAs-edited SCs transplanted at day 24 and day 43 of differentiation. At 3 -months post-transplantation, EpiBinder edited SCs responded to glucose both when transplanted at Day 24 and 43 of differentiation.

[0055] FIG. 12A shows a faster normalization of glycemia in mice transplanted with EBdCas9»gRNAs-edited SC-derived islet cells, compared to control, after IPGTT. FIG. 12B shows measurement of plasma human insulin C-peptide in response to an IPGTT. Note the enhanced insulin secretory response of EBdCas9»gRNAs-edited SC-islet cells, compared to control at 3-6 months post-transplantation.

[0056] FIG. 13A shows immunostaining for Sox 17 and FOXA2 DE cells at day 3 of differentiation after DOX-induced EBdCas9 and transfection with two EOMES-specific gRNAs. FIG. 13B shows quantification of Soxl7+FOXA2+ DE cells.MBHB Ref. No. 24-1170-WO UW 49850.02WO2

[0057] FIG. 14A-H show sequential activation of PDX1 and NGN3 promoters by EBdCas9 leads to extensive remodeling of H3K27Ac chromatin modifications in differentiating PSC.FIG. 14A shows timeline of EBdCas9 interventions during PSC differentiation and cell harvesting for CUT& Tag. FIG. 14B-D show genome browser view showing enhanced H3K27ac signal within the promoter, enhancer and / or intronic regions of NGN3, NKX2.2 and GCK, in PDX1 / NGN3 -guided EBdCas9-treated samples compared to control. Two biologic replicas (Rep) per condition are shown. FIG. 14E shows heatmaps of H3K27Ac signal within the PDX1 and NGN3 gene loci. The averaged signal of the two biological replicas is shown.FIG. 14F shows heatmaps of H3K27Ac signal showing unique peak calling of PDX1 / NGN3-guided EBdCas9-treated samples (upper panels, 14,695 peaks) and EBdCas9-treated controls (lower panels, 28,033 peaks). For each condition, the top 5% significant peaks and averaged signal of two biological replicas are shown. Composite plots of normalized H3K27Ac signal are shown at the top of each heatmap. TSS: Transcriptional Start Site. TES: Transcriptional End Site. FIG. 14G-H show enrichment pathway and cell type analysis performed on the nearest neighboring genes to the top 1% unique H3K27Ac peaks, using the indicated gene data sets from Enrichr.

[0058] FIG. 15A-J show EBdCas9-mediated epigenetic interventions increase the efficiency of SC commitment to Pancreatic Endocrine Progenitors with developmental bias toward beta cell types. FIG. 15A shows schematic of EBdCas9 induction and gRNA transfection. FIG.15B-D show morphometric analysis of SC clusters at day 20 of differentiation showing cell cluster areas (FIG. 15B), number of PDX1+ cells per cluster area (FIG. 15C), and number of PDX1+ / NKX6.1+ Endocrine Progenitors per cluster area (FIG. 15D) under the indicated treatments. FIG. 15E-G show cumulative (FIG. 15E) and fractional representation of Insulin+ (FIG. 15F) and Glucagon+ (FIG. 15G) areas detected in the clusters at day 20 reveals increased yield of Insulin-positive cells significantly induced by sequential targeting of EBdCas9 to PDX1 and NGN3 promoters. Box-and-whiskers-plots show the distribution of measurements in individual clusters. The thick line in each box represents the median. FIG.15H shows flow cytometric analysis of alpha and beta cell subpopulations detected in multiple independent experiments using hESCs and iPSCs at day 20 of differentiation confirms the increased yield of beta cell types under EBdCas9+PDX / NGN3 gRNA treatment. Bars are mean ± SEM (n=4-ll). FIG. 15I-J show flow cytometric analysis of insulin single positive cells (FIG. 151) and insulin / glucagon double positive cells (FIG. 15J) in follow-up experiments (n=6-8). Enrichment of insulin single positive beta cells in EBdCas9+PDX / NGN3 gRNA-MBHB Ref. No. 24-1170-WO UW 49850.02WO2 treated samples relative to EBdCas9 controls is detected at both stage 5 and stage 7 of differentiation (day 35).

[0059] FIG. 16A-G show sequential, EBdCas9-driven epigenetic remodeling of PDX1 and NGN3 promoters fosters the activation of NGN3 -dependent transcriptional programs. FIG.16A-C show Gene Set Enrichment Analysis (GSEA) of transcripts, concordantly regulated in Hl and MEL hESCs differentiated to day 20, identifies differentially expressed Reactome Pathways in cultures of PDX1-, NGN3- and PDX1 / NGN3 -guided EBdCas9-treated samples as compared to EBdCas9 only control. FIG. 16D shows fraction of predicted NGN3 target genes concordantly up-regulated in Hl and MEL-hESCs epigenetically manipulated with EBdCas9+PDX / NGN3 gRNAs and differentiated to day 20, as detected by bulk RNA-seq (223 of 1263 NGN3 target genes). FIG. 16E-F show heatmaps of NGN3 target genes comprised within the Exocytosis / Insulin Secretion and Mitochondrial function / Metabolism categories shown in FIG. 16D. Heatmaps represent fold changes of normalized gene counts averaged in the indicated gRNA-treated vs EBdCas9 only Hl and MEL-hESC samples. FIG. 16G shows heatmap of Enteroendocrine (EC)-specific gene signatures. A gene signature strongly biased toward enteroendocrine lineages is detected in NGN3-guided EBdCas9-treated samples.

[0060] FIG. 17A-H show gene-guided EBdCas9 selects against uncommitted cells, enforcing pancreatic over gut developmental fates. FIG. 17A-B show representative fluorescence microscopy (FIG. 17A) and morphometric analysis (FIG. 17B) of cell clusters at day 20 of differentiation, showing the frequency of uncommitted PDX1- / NKX6.1 -negative cell types (red labelled nuclei) normalized to cluster areas under each treatment. FIG. 17C shows uncommitted cells include PDX-neg / CDX2+ gut epithelium (arrows and circles). FIG. 17D shows schematic representation of gastric (SOX2+), pancreatic (PDX1+ / CDX2+) and gut (PDX-neg / CDX2+) domains developing from primitive gut tube cells in mouse embryogenesis and during directed hESC differentiation toward pancreatic lineages. High levels of PDX1 are critical to repress CDX2 and intestinal fate choices within posterior Hindgut. FIG. 17E-F show morphometric analysis of PDXl-neg / CDX2+ cell types counted per cluster area at day 20 of directed differentiation of MEL (FIG. 17E) and Hl hESC (FIG. 17F). FIG. 17G-H show morphometric analysis of PDXl-neg / SOX2+ cell types counted per cluster area at day 20 of directed differentiation of MEL (FIG. 17G) and Hl hESC (FIG. 17H).

[0061] FIG. 18A-C show Stage 7 islet clusters epigenetically modified by PDX / NGN3 -guided EBdCas9 are glucose responsive in vivo. FIG. 18A shows time course of in vivo experiments.MBHB Ref. No. 24-1170-WO UW 49850.02WO2 FIG. 18B shows levels of human C-peptide measured in IL2R- / - SCID mice in response to intraperitoneal glucose tolerance tests (IPGTT, 2 mg / kg) after 5 hours fasting after transplantation of stage 7 islet clusters. Bars are mean ± SD of n=2 experimental mice per group, tested sequentially at 3- and 6-months post-transplantation. *P<0.05. FIG. 18C shows human C-peptide detected in 5 independent experiments during IPGTT performed 3-6 months post-transplantation, showing significant increased insulin secretory responses over basal secretion in mice transplanted with islet clusters resulting from treatments with PDX / NGN3-guided EBdCas9. **P<0.01.DETAILED DESCRIPTION

[0062] The present disclosure is explained in greater detail below. This description is not intended to be a detailed catalog of all the different ways in which the disclosure may be implemented, or all the features that may be added to the instant disclosure. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. Numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant disclosure. Hence, the following specification is intended to illustrate some particular embodiments of the disclosure, and not to exhaustively specify all permutations, combinations, and variations thereof.

[0063] Unless the context indicates otherwise, it is specifically intended that the various features of the disclosure described herein can be used in any combination. Moreover, the present disclosure also contemplates that in some embodiments of the disclosure, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0064] All publications, patent applications, patents, nucleotide sequences, amino acid sequences and other references mentioned herein are incorporated by reference in their entireties for all purposes.MBHB Ref. No. 24-1170-WO UW 49850.02WO2

[0065] A number of terms are introduced below, which are used to describe the invention of the present disclosure. In instances where a technical or scientific term is not specifically defined herein, they will have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. In case of conflict, the present disclosure, including definitions, will control.

[0066] As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and / or “comprising,” when used in this specification, specify the presence of stated features, elements, and / or components, but do not preclude the presence or addition of one or more other features, elements, components, and / or groups thereof. As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items. As used herein, phrases such as “between X and Y” and “between about X and Y” should be interpreted to include X and Y. As used herein, phrases such as “between about X and Y” mean “between about X and about Y.” As used herein, phrases such as “from about X to Y” mean “from about X to about Y.”

[0067] Furthermore, the term “about,” as used herein when referring to a measurable value such as an amount of a compound or agent of this disclosure, dose, time, temperature, and the like, is meant to encompass variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.

[0068] As used herein, the transitional phrase “consisting essentially of’ is to be interpreted as encompassing the recited materials or steps and those that do not materially affect the basic and novel characteristic(s) of the disclosure. Thus, the term “consisting essentially of’ as used herein should not be interpreted as equivalent to “comprising.”

[0069] As used herein, the terms “pluripotent”, “pluripotential”, and the like refers to a cell that is positive for one or more of the pluripotent markers such as but are not limited to Oct-4, Nanog, and Sox-2 and the cell has the potential to differentiate to at least a subset of the mammalian body's approximately 260 cell types upon appropriate stimulation, such as by temporally appropriate gene expression or exposure to specific growth factors.MBHB Ref. No. 24-1170-WO UW 49850.02WO2

[0070] As used herein, the term “stem cell” refers to a master cell that can reproduce indefinitely to form the specialized cells of tissues and organs. A stem cell is a developmentally pluripotent or multipotent cell. A stem cell can divide to produce two daughter stem cells, or one daughter stem cell and one progenitor (“transit”) cell, which then proliferates into the tissue's mature, fully formed cells. The “stem cell” used herein includes “progenitor cells” unless otherwise noted.

[0071] The term “subject” refers to any living organism susceptible to or suffering from an adverse or abnormal condition (e.g., diabetes) and, as such, in need of treatment for such adverse condition. The term subject includes, but is not limited to, humans, nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. In some embodiments, the subject is a mammal, including humans and non-human mammals. In preferred embodiments, the subject is a human.

[0072] As used herein “diabetes” refers to a disease resulting either from an absolute deficiency of insulin (Type 1 diabetes) due to a defect in the biosynthesis or production of insulin, or a relative deficiency of insulin in the presence of insulin resistance (type 2 Diabetes), i.e., impaired insulin action, in an organism. The diabetic subject (or patient) thus has absolute or relative insulin deficiency, and displays, among other symptoms and signs, elevated blood glucose concentration, presence of glucose in the urine and excessive discharge of urine.

[0073] As used herein, the term “treating” refers to inhibiting or arresting the development of a disease, disorder or condition and / or causing the reduction, remission, or regression of a disease, disorder or condition in a subject suffering from, or diagnosed with, the disease, disorder or condition. Those of skill in the art will be aware of various methodologies and assays which can be used to assess the development of a disease, disorder or condition, and similarly, various methodologies and assays which can be used to assess the reduction, remission or regression of a disease, disorder or condition.

[0074] As used herein, the term “pharmaceutically acceptable” refers to compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction when administered to a subject, preferably a human or a non-human subject. Preferably, asMBHB Ref. No. 24-1170-WO UW 49850.02WO2 used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of a federal or state government or listed in the U. S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0075] As used herein, the terms "effective amount", “therapeutically effective amount”, and the like, refer to the amount of a therapy, which is sufficient to reduce or ameliorate the severity and / or duration of a disorder or one or more symptoms thereof, inhibit or prevent the advancement of a disorder, cause regression of a disorder, inhibit or prevent the recurrence, development, onset or progression of one or more symptoms associated with a disorder, detect a disorder, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy (e.g., prophylactic or therapeutic agent). An effective dose can refer to, e.g., a cell population number in a subject that provides the desired specific pharmacological effect, e.g. to express insulin in a subject with Type I diabetes, or an increased concentration of a biomarker in a subject having been treated with the cells or compositions of the disclosure. A therapeutically effective amount can include a therapeutically significant reduction in a symptom, e.g., at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150% or more in a measured parameter as compared to a control or non-treated subject. Measured or measurable parameters include clinically detectable markers of disease, for example, elevated or depressed levels of a biological marker (e.g., insulin), as well as parameters related to a clinically accepted scale of symptoms or markers for a disease or disorder. It will be understood that dosing and usage of the cells and compositions disclosed herein will be decided by the attending physician within the scope of sound medical judgment. The exact amount required will vary depending on factors such as the type of disease being treated.

[0076] An effective amount can require more than one dose. As used herein, the term "prophylaxis" refers to preventing or reducing the progression of a disorder, either to a statistically significant degree or to a degree detectable by a person of ordinary skill in the art.

[0077] Effective amounts may vary depending upon the biological effect desired in the individual, condition to be treated, and / or the specific characteristics of the composition according to the present invention and the individual. In this respect, any suitable dose of the composition can be administered to the patient (e.g., human), according to the type of disease to be treated. Various general considerations taken into account in determining the "effectiveMBHB Ref. No. 24-1170-WO UW 49850.02WO2 amount" are known to those of skill in the art and are described, e.g., in Gilman et al., eds., Goodman And Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990; and Remington's Pharmaceutical Sciences, 17th Ed., Mack Publishing Co., Easton, Pa., 1990, each of which is herein incorporated by reference.

[0078] As used herein, the terms “administration” or “administering” are used to describe the process by which cells produced according to methods of the present invention are delivered to a subject. The cells can be administered a number of ways including intraepatic, intradermal, mesenteric, and intramuscular, among others which term allows the cells to migrate to the site where needed.

[0079] Administration will often depend upon the disease or condition treated and can preferably be via a parenteral route, for example, intravenously, by administration into the cerebral spinal fluid or by direct administration into the affected tissue in the brain or other body site.

[0080] As used herein, the terms “grafting”, “transplanting”, “graft”, “transplantation”, and the like, are used synonymously to describe the process by which the cells of the invention are delivered to the site where the cells are intended to exhibit a favorable effect, such as treating autoimmune diseases (e.g., Type 1 diabetes), treating diabetes, and the like. The cells of the invention can also be delivered in a remote area of the body by any mode of administration as described above, relying on cellular migration to the appropriate area in the body to effect transplantation.

[0081] The term “undifferentiated” as used herein refers to pluripotent stem cells which have not developed a characteristic of a more specialized cell. As will be recognized by one of skill in the art, the terms “undifferentiated” and “differentiated” are relative with respect to each other. A stem cell which is “differentiated” has a characteristic of a more specialized cell. Differentiated and undifferentiated cells are distinguished from each other by several well-established criteria, including morphological characteristics such as relative size and shape, ratio of nuclear volume to cytoplasmic volume; and expression characteristics such as detectable presence of known markers of differentiation. A marker of differentiation indicating that cells are differentiated or undifferentiated includes a protein, carbohydrate, lipid, nucleic acid, functional characteristic and / or morphological characteristic which is specific to a differentiated cell.MBHB Ref. No. 24-1170-WO UW 49850.02WO2

[0082] As used herein, the term “substantially homogeneous” when applied to cells, refers to a population of cells, wherein at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% of the cells in the population are of the same cell type. Examples of cell types include, but are not limited to, pluripotent stem cells, Definitive Endoderm (DE), Pancreatic Progenitors (PP), Endocrine Progenitors (EP), primitive gut tube (PGT) cells, posterior foregut (PF) cells, P-cells, P-cell-like insulin-producing cells, and the like. In some embodiments, the term “substantially homogeneous” describes a population of cells wherein at least about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% of the cells in the population are undifferentiated. In a further embodiment a substantially homogeneous population of cells is one in which more than 95% of the cells are undifferentiated. In another embodiment, a substantially homogeneous population of cells is one in which more than 99% of the cells are undifferentiated. A population of cells can be assayed for one or more markers of differentiation to determine whether the population of cells is substantially homogeneous.

[0083] The production and / or maintenance of a substantially homogeneous population of embryonic-like stem cells and / or a differentiated cell type may be measured by assessing the proportion of cells for particular markers of undifferentiated cells and / or differentiated cells. For example, relative ratios of transcription products for markers of undifferentiated cells such as Oct4, or HNFla for functional P-cells, can be assessed by quantitative RT-PCR. Also, production and localization of markers of undifferentiated cells can be assessed by immunocytochemistry.

[0084] Markers of undifferentiated stem cells and differentiated cells are assayed by any of various methods such as antibody-based detection techniques using an antibody specific for a particular marker. Antibody-based techniques include immunofluorescence and immunoblotting. Further assays include assays for detection of mRNAs encoding a particular marker. Such assays include polymerase chain reaction, Northern blots, and in situ hybridization. Details of these and other such assays are described herein and in standard references including J. Sambrook and D. W. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd ed., 2001; F. M. Ausubel, Ed., Short Protocols in Molecular Biology, Current Protocols; 5th ed., 2002; and E. Harlow and D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988.MBHB Ref. No. 24-1170-WO UW 49850.02WO2

[0085] As used herein, the term “culture medium” or “medium” refers generally to any substance or preparation used for the cultivation of living cells. A “cell culture” refers to a growth of cells in vitro; although the cells proliferate, they do not organize into tissue per se.

[0086] The term "nucleic acid" as used herein typically refers to an oligomer or polymer (preferably a linear polymer) of any length composed essentially of nucleotides. A nucleotide unit commonly includes a heterocyclic base, a sugar group, and at least one, e.g. one, two, or three, phosphate groups, including modified or substituted phosphate groups. Heterocyclic bases may include inter alia purine and pyrimidine bases such as adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) which are widespread in naturally-occurring nucleic acids, other naturally-occurring bases (e.g., xanthine, inosine, hypoxanthine) as well as chemically or biochemically modified (e.g., methylated), non-natural or derivatised bases. Sugar groups may include inter alia pentose (pentofuranose) groups such as preferably ribose and / or 2-deoxyribose common in naturally-occurring nucleic acids, or arabinose, 2-deoxyarabinose, threose or hexose sugar groups, as well as modified or substituted sugar groups. Nucleic acids as intended herein may include naturally occurring nucleotides, modified nucleotides or mixtures thereof. A modified nucleotide may include a modified heterocyclic base, a modified sugar moiety, a modified phosphate group or a combination thereof. Modifications of phosphate groups or sugars may be introduced to improve stability, resistance to enzymatic degradation, or some other useful property. The term "nucleic acid" further preferably encompasses DNA, RNA, and DNA-RNA hybrid molecules, specifically including hnRNA, pre-mRNA, mRNA, gRNA, cDNA, genomic DNA, amplification products, oligonucleotides, and synthetic (e.g., chemically synthesized) DNA, RNA or DNA-RNA hybrids. A nucleic acid can be naturally occurring, e.g., present in or isolated from nature; or can be non-naturally occurring, e.g., recombinant, i.e., produced by recombinant DNA technology, and / or partly or entirely, chemically or biochemically synthesized. A "nucleic acid" can be double-stranded, partly double stranded, or single-stranded. Where singlestranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear.

[0087] The term "peptide", "polypeptide", "protein", and the like, as used herein, refers to a molecule that is formed using naturally occurring L-amino acids or analogs thereof, like D-amino acids, or N-alkylated amino acids, or the like. Preferred amino acids are selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, He, Leu, Lys, Met, Phe, Pro,MBHB Ref. No. 24-1170-WO UW 49850.02WO2 Ser, Thr, Trp, Tyr, and Vai. Additionally, modifications such as fluorescence dyes or biotin are also contemplated.

[0088] " Functional derivatives" of proteins are also contemplated, in which a functional derivative refers to a "chemical derivative," "fragment," "polymorph" or "variant" of the polypeptide or nucleic acid of the invention. A functional derivative retains at least a portion of the function of the protein, which permits its utility in accordance with the invention. It is well known in the art that, due to the degeneracy of the genetic code, numerous different nucleic acid sequence can code for the same amino acid sequence. It is also well known in the art that conservative changes in amino acid can be made to arrive at a protein or polypeptide that retains the functionality of the original. In both cases, all permutations are intended to be covered by this disclosure.

[0089] Another functional derivative intended to be within the scope of the present invention is a "variant" polypeptide, which either lacks one or more amino acids or contains additional or substituted amino acids relative to the native polypeptide. Such variants having added, substituted and / or additional amino acids retain the functional portion of the original polypeptide. A functional derivative of a protein with deleted, inserted and / or substituted amino acid residues may be prepared using standard techniques well-known to those of ordinary skill in the art (e.g., site-directed mutagenesis). Alternatively, proteins with amino acid deletions, insertions and / or substitutions may be conveniently prepared by direct chemical synthesis, using methods well-known in the art.

[0090] The terms "identity", "identical", "similar", "similarity", "homology", "homologous", and the like, refer to the "likeness" or "sameness" of two or more sequences, e.g., between two nucleic acid sequences or two peptide sequences, often expressed as a percentage. Sequence identity is the amount of characters which match exactly between two different sequences, where gaps are not counted and the measurement is relational to the shorter of the two sequences. This has the effect that sequence identity is not transitive, i.e. if sequence A=B and B=C then A does not necessarily equal C (in terms of the identity distance measure): Consider exemplary sequences A: AAGGCTT, B: AAGGC, and C: AAGGCAT. Here identity(A, B)=100% (5 identical nucleotides / min(length(A),length(B))). Identity(B, C)=100%, but identity(A, C)=85% ((6 identical nucleotides / 7)). So 100% identity does not necessarily indicate two sequences are the same. Sequence similarity accounts for sequence identity and conservative substitutions with positive scores in substitution matrices.MBHB Ref. No. 24-1170-WO UW 49850.02WO2 E.g., a leucine to isoleucine substitution would receive partial "similarity" credit while not for a sequence identity calculation. Methods for aligning sequences for comparison are well-known in the art, and a detailed consideration of sequence alignment methods and homology calculations can be found in, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-10.

[0091] Included within the scope of the invention are functional equivalents of the herein-described isolated nucleic acid molecules. The degeneracy of the genetic code permits substitution of certain codons by other codons that specify the same amino acid and hence would give rise to the same protein. The nucleic acid sequence can vary substantially since, with the exception of methionine and tryptophan, the known amino acids can be coded for by more than one codon. The encoded amino acid sequence thereof would, however, be preserved.

[0092] In addition, the nucleic acid sequence may comprise a nucleotide sequence which results from the addition, deletion or substitution of at least one nucleotide to the 5’-end and / or the 3 ’-end, provided that its addition, deletion or substitution does not alter the amino acid sequence described herein, which is encoded by the nucleotide sequence. For example, the nucleic acid molecule of the present invention may have restriction endonuclease recognition sites added to its 5’-end and / or 3’-end.

[0093] Further, it is possible to delete codons or to substitute one or more codons with codons other than degenerate codons to produce a structurally modified polypeptide, but one which has substantially the same utility or activity as the polypeptide produced by the unmodified nucleic acid molecule. As recognized in the art, the two polypeptides are functionally equivalent, as are the two nucleic acid molecules that give rise to their production, even though the differences between the nucleic acid molecules are not related to the degeneracy of the genetic code.

[0094] As will be described in greater detail herein, the present disclosure generally relates to epigenetically editing human pluripotent stem cells with small protein epigenetic binders (EpiBinders) to enhance the efficiency of pancreatic islet cells specification and functional maturation. Such disclosure generally involves methods for directing differentiation of PSCs toward endocrine cells from less mature cell types (e.g., definitive endoderm cells) to more mature cell types such as pancreatic progenitors and immature endocrine progenitors (e.g., immature a-cells, P-cells, 5-cells, and y-cells), and, in embodiments, to mature, functional endocrine cells, e.g., P-cells.MBHB Ref No. 24-1170-WO UW 49850.02WO2

[0095] More specifically, the disclosure provides evidence that the sequential, targeted epigenetic activation of both PDX1 and NGN3 using the EBdCas9 / gRNA system, not just bulk or untargeted epigenetic modulation, is critical for both achieving higher [3-cell yields and depleting off-target lineages. The disclosure further provides a demonstration that PRC2 inhibition via EBdCas9, rather than simply activating transcription, uniquely overcomes barriers not addressed by previous approaches (e.g., VP64 transactivation), resulting in durable epigenetic and transcriptional reprogramming. Such results are shown to be applicable in both transgenic / engineered and "wild-type" PSC lines (the latter by, e.g., mRNA / gRNA transfection), extending the described platform's compatibility.

[0096] In some embodiments, the disclosure provides methods of epigenetically editing human pluripotent stem cells (hPSC) lines with small protein epigenetic binders (EpiBinders) to enhance the efficiency of pancreatic islet cells specification and functional maturation. In one aspect, the disclosure provides a method of generating an endocrine progenitor (EP) stage cell from a pluripotent stem cell (PSC), including (i) inducing expression in the PSC of a transcriptional regulator of a definitive endoderm (DE) stage cell to differentiate the PSC into a DE stage cell, into a primitive gut tube (PGT) stage cell, and then to a posterior foregut (PF) stage cell; (ii) inducing expression in the PF stage cell of a transcriptional regulator of a pancreatic progenitor (PP) stage cell to differentiate the PF stage cell into PP stage cell; and (iii) inducing expression in the PP stage cell of a transcriptional regulator of a EP stage cell to differentiate the PP stage cell into a EP stage cell; wherein binding of a guide RNA (gRNA) complementary to a regulatory region of the transcriptional regulator of the DE stage cell, of the PP stage cell, or the EP stage cell and also a fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain together induce expression of the transcriptional regulator of the DE stage cell, of the PP stage cell, or the EP stage cell.

[0097] Regarding stage-specific transcriptional regulators, in some embodiments, the transcriptional regulator of a DE stage cell comprises GSC, EOMES, FOXA2, SOX17, or GATA6, and in some embodiments, the transcriptional regulator of a PP stage cell comprises PDX1, NKX6.1, or PAX4. In some embodiments, the transcriptional regulator of a EP stage cell comprises NGN3, NKX2.2 or NEURODI.

[0098] In some embodiments, the transcriptional regulator of a DE stage cell is EOMES, the transcriptional regulator of a PP stage cell is PDX1, and the transcriptional regulator of a EPMBHB Ref. No. 24-1170-WO UW 49850.02WO2 stage cell is NGN3. In some embodiments, the transcriptional regulator of a DE stage cell is EOMES, the transcriptional regulator of a PP stage cell is NKX6.1, and the transcriptional regulator of a EP stage cell is NGN3. In some embodiments, the transcriptional regulator of a DE stage cell is EOMES, the transcriptional regulator of a PP stage cell is PAX4, and the transcriptional regulator of a EP stage cell is NGN3. In some embodiments, the transcriptional regulator of a DE stage cell is EOMES, the transcriptional regulator of a PP stage cell is PDX1, and the transcriptional regulator of a EP stage cell is NKX2.2. In some embodiments, the transcriptional regulator of a DE stage cell is EOMES, the transcriptional regulator of a PP stage cell is PDX1, and the transcriptional regulator of a EP stage cell is NEURODI. In some embodiments, the transcriptional regulator of a DE stage cell is EOMES, the transcriptional regulator of a PP stage cell is NKX6.1, and the transcriptional regulator of a EP stage cell is NKX2.2. In some embodiments, the transcriptional regulator of a DE stage cell is EOMES, the transcriptional regulator of a PP stage cell is NKX6.1, and the transcriptional regulator of a EP stage cell is NEURODI. In some embodiments, the transcriptional regulator of a DE stage cell is EOMES, the transcriptional regulator of a PP stage cell is PAX4, and the transcriptional regulator of a EP stage cell is NKX2.2. In some embodiments, the transcriptional regulator of a DE stage cell is EOMES, the transcriptional regulator of a PP stage cell is PAX4, and the transcriptional regulator of a EP stage cell is NEURODI.

[0099] In some embodiments, the transcriptional regulator of a DE stage cell is GSC, the transcriptional regulator of a PP stage cell is PDX1, and the transcriptional regulator of a EP stage cell is NGN3. In some embodiments, the transcriptional regulator of a DE stage cell is GSC, the transcriptional regulator of a PP stage cell is NKX6.1, and the transcriptional regulator of a EP stage cell is NGN3. In some embodiments, the transcriptional regulator of a DE stage cell is GSC, the transcriptional regulator of a PP stage cell is PAX4, and the transcriptional regulator of a EP stage cell is NGN3. In some embodiments, the transcriptional regulator of a DE stage cell is GSC, the transcriptional regulator of a PP stage cell is PDX1, and the transcriptional regulator of a EP stage cell is NKX2.2. In some embodiments, the transcriptional regulator of a DE stage cell is GSC, the transcriptional regulator of a PP stage cell is PDX1, and the transcriptional regulator of a EP stage cell is NEURODI. In some embodiments, the transcriptional regulator of a DE stage cell is GSC, the transcriptional regulator of a PP stage cell is NKX6.1, and the transcriptional regulator of a EP stage cell is NKX2.2. In some embodiments, the transcriptional regulator of a DE stage cell is GSC, the transcriptional regulator of a PP stage cell is NKX6.1, and the transcriptional regulator of a EPMBHB Ref. No. 24-1170-WO UW 49850.02WO2 stage cell is NEURODI. In some embodiments, the transcriptional regulator of a DE stage cell is GSC, the transcriptional regulator of a PP stage cell is PAX4, and the transcriptional regulator of a EP stage cell is NKX2.2. In some embodiments, the transcriptional regulator of a DE stage cell is GSC, the transcriptional regulator of a PP stage cell is PAX4, and the transcriptional regulator of a EP stage cell is NEURODI.

[0100] In some embodiments, the transcriptional regulator of a DE stage cell is FOXA2, the transcriptional regulator of a PP stage cell is PDX1, and the transcriptional regulator of a EP stage cell is NGN3. In some embodiments, the transcriptional regulator of a DE stage cell is FOXA2, the transcriptional regulator of a PP stage cell is NKX6.1, and the transcriptional regulator of a EP stage cell is NGN3. In some embodiments, the transcriptional regulator of a DE stage cell is FOXA2, the transcriptional regulator of a PP stage cell is PAX4, and the transcriptional regulator of a EP stage cell is NGN3. In some embodiments, the transcriptional regulator of a DE stage cell is FOXA2, the transcriptional regulator of a PP stage cell is PDX1, and the transcriptional regulator of a EP stage cell is NKX2.2. In some embodiments, the transcriptional regulator of a DE stage cell is FOXA2, the transcriptional regulator of a PP stage cell is PDX1, and the transcriptional regulator of a EP stage cell is NEURODI. In some embodiments, the transcriptional regulator of a DE stage cell is FOXA2, the transcriptional regulator of a PP stage cell is NKX6.1, and the transcriptional regulator of a EP stage cell is NKX2.2. In some embodiments, the transcriptional regulator of a DE stage cell is FOXA2, the transcriptional regulator of a PP stage cell is NKX6.1, and the transcriptional regulator of a EP stage cell is NEURODI. In some embodiments, the transcriptional regulator of a DE stage cell is FOXA2, the transcriptional regulator of a PP stage cell is PAX4, and the transcriptional regulator of a EP stage cell is NKX2.2. In some embodiments, the transcriptional regulator of a DE stage cell is FOXA2, the transcriptional regulator of a PP stage cell is PAX4, and the transcriptional regulator of a EP stage cell is NEURODI.

[0101] In some embodiments, the transcriptional regulator of a DE stage cell is SOX17, the transcriptional regulator of a PP stage cell is PDX1, and the transcriptional regulator of a EP stage cell is NGN3. In some embodiments, the transcriptional regulator of a DE stage cell is SOX17, the transcriptional regulator of a PP stage cell is NKX6.1, and the transcriptional regulator of a EP stage cell is NGN3. In some embodiments, the transcriptional regulator of a DE stage cell is SOX17, the transcriptional regulator of a PP stage cell is PAX4, and the transcriptional regulator of a EP stage cell is NGN3. In some embodiments, the transcriptionalMBHB Ref. No. 24-1170-WO UW 49850.02WO2 regulator of a DE stage cell is SOX17, the transcriptional regulator of a PP stage cell is PDX1, and the transcriptional regulator of a EP stage cell is NKX2.2. In some embodiments, the transcriptional regulator of a DE stage cell is SOX17, the transcriptional regulator of a PP stage cell is PDX1, and the transcriptional regulator of a EP stage cell is NEURODI. In some embodiments, the transcriptional regulator of a DE stage cell is SOX17, the transcriptional regulator of a PP stage cell is NKX6.1, and the transcriptional regulator of a EP stage cell is NKX2.2. In some embodiments, the transcriptional regulator of a DE stage cell is SOX17, the transcriptional regulator of a PP stage cell is NKX6.1, and the transcriptional regulator of a EP stage cell is NEURODI. In some embodiments, the transcriptional regulator of a DE stage cell is SOX17, the transcriptional regulator of a PP stage cell is PAX4, and the transcriptional regulator of a EP stage cell is NKX2.2. In some embodiments, the transcriptional regulator of a DE stage cell is SOX17, the transcriptional regulator of a PP stage cell is PAX4, and the transcriptional regulator of a EP stage cell is NEURODI.

[0102] In some embodiments, the transcriptional regulator of a DE stage cell is GATA6, the transcriptional regulator of a PP stage cell is PDX1, and the transcriptional regulator of a EP stage cell is NGN3. In some embodiments, the transcriptional regulator of a DE stage cell is GATA6, the transcriptional regulator of a PP stage cell is NKX6.1, and the transcriptional regulator of a EP stage cell is NGN3. In some embodiments, the transcriptional regulator of a DE stage cell is GATA6, the transcriptional regulator of a PP stage cell is PAX4, and the transcriptional regulator of a EP stage cell is NGN3. In some embodiments, the transcriptional regulator of a DE stage cell is GATA6, the transcriptional regulator of a PP stage cell is PDX1, and the transcriptional regulator of a EP stage cell is NKX2.2. In some embodiments, the transcriptional regulator of a DE stage cell is GATA6, the transcriptional regulator of a PP stage cell is PDX1, and the transcriptional regulator of a EP stage cell is NEURODI. In some embodiments, the transcriptional regulator of a DE stage cell is GATA6, the transcriptional regulator of a PP stage cell is NKX6.1, and the transcriptional regulator of a EP stage cell is NKX2.2. In some embodiments, the transcriptional regulator of a DE stage cell is GATA6, the transcriptional regulator of a PP stage cell is NKX6.1, and the transcriptional regulator of a EP stage cell is NEURODI. In some embodiments, the transcriptional regulator of a DE stage cell is GATA6, the transcriptional regulator of a PP stage cell is PAX4, and the transcriptional regulator of a EP stage cell is NKX2.2. In some embodiments, the transcriptional regulator of a DE stage cell is GATA6, the transcriptional regulator of a PP stage cell is PAX4, and the transcriptional regulator of a EP stage cell is NEURODI.MBHB Ref. No. 24-1170-WO UW 49850.02WO2

[0103] In some embodiments of the methods of generating an EP stage cell from a pluripotent stem cell (PSC), (a) the inducing expression in the PSC of a transcriptional regulator of a DE stage cell of step (i) is performed on day 0; (b) the inducing expression in the PF stage cell of a transcriptional regulator of a PP stage cell of step (ii) is performed on between about day 7 to about day 10; and (c) the inducing expression in the PP stage cell of a transcriptional regulator of a EP stage cell of step (iii) is performed between about 2 to about 5 days after step (b). In some embodiments, the inducing expression in the PSC of a transcriptional regulator of a DE stage cell of step (i) is performed on day 0-3. In some embodiments, the inducing expression in the PF stage cell of a transcriptional regulator of a PP stage cell of step (ii) is performed on between about day 5 to about day 10.

[0104] In some embodiments of the methods of generating an EP stage cell from a pluripotent stem cell (PSC), the transcriptional regulator of a DE stage cell is EOMES, and the gRNA complementary to a regulatory region for EOMES is a sequence at least about 90% identical, or at least about 95% identical to SEQ ID NO:24 and SEQ ID NO:28. In some embodiments of the methods of generating an EP stage cell from a PSC, the transcriptional regulator of a DE stage cell is EOMES, and the gRNA complementary to a regulatory region for EOMES is selected from the group consisting of SEQ ID NO:24 and SEQ ID NO:28.

[0105] In some embodiments of the methods of generating an EP stage cell from a PSC, the transcriptional regulator of a PP stage cell is PDX1, and the gRNA complementary to a regulatory region for PDX1 is a sequence at least about 90% identical, or at least about 95% identical to SEQ ID NO:9, SEQ ID NO: 10, and SEQ ID NO:11. In some embodiments of the methods of generating an EP stage cell from a PSC, the transcriptional regulator of a PP stage cell is PDX1, and the gRNA complementary to a regulatory region for PDX1 is selected from the group consisting of SEQ ID NO:9, SEQ ID NO: 10, and SEQ ID NO: 11.

[0106] In some embodiments of the methods of generating an EP stage cell from a PSC, the transcriptional regulator of a EP stage cell is NGN3, and the gRNA complementary to a regulatory region for NGN3 is a sequence at least about 90% identical, or at least about 95% identical to SEQ ID NO: 17, and in some embodiments, the transcriptional regulator of a EP stage cell is NGN3, and the gRNA complementary to a regulatory region for NGN3 is SEQ ID NO:17.MBHB Ref. No. 24-1170-WO UW 49850.02WO2

[0107] In some embodiments of the methods of generating an EP stage cell from a PSC, the fusion protein (EBdCas9) comprising a catalytically inactivated Cas9 (dCas9) and an embryonic ectoderm development (EED)-binding domain is originally transcribed from a nucleic acid sequence that comprises a sequence at least about 90% identical, or at least about 95% identical, or at least about 99% identical to SEQ ID NO:32, while in some embodiments, the fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain is originally transcribed from a nucleic acid sequence that comprises SEQ ID NO: 32.

[0108] In some embodiments of the methods of generating an EP stage cell from a PSC, the fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain comprises an amino acid sequence that is at least about 90% identical, or at least about 95% identical, or at least about 99% identical to SEQ ID NO:53, while in some embodiments, the fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain comprises SEQ ID NO:53.

[0109] In another aspect, the disclosure provides a method of inducing differentiation of a pluripotent stem cell (PSC) in culture to a definitive endoderm (DE) stage cell, the method including (i) introducing to the PSC a gRNA for a transcriptional regulator of a DE stage cell selected from GSC, EOMES, FOXA2, SOX17, and GATA6; (ii) introducing to the PSC a fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain; and (iii) inducing expression of the transcriptional regulator. In some embodiments of the method of inducing differentiation of a PSC in culture to a DE stage cell, the transcriptional regulator of a DE stage cell for which a gRNA is introduced to the PSC is SOX17. In some embodiments, the transcriptional regulator of a DE stage cell for which a gRNA is introduced to the PSC is EOMES. In some embodiments, the transcriptional regulator of a DE stage cell for which a gRNA is introduced to the PSC is FOXA2. In some embodiments, the transcriptional regulator of a DE stage cell for which a gRNA is introduced to the PSC is GATA6. In some embodiments, the transcriptional regulator of a DE stage cell for which a gRNA is introduced to the PSC is GSC.

[0110] In another aspect, the disclosure provides a method of inducing differentiation of a posterior foregut (PF) stage cell in culture to a pancreatic progenitor (PP) stage cell, the method including (i) introducing to the PF stage cell a gRNA for a transcriptional regulator of a PP stage cell selected from PDX1, PAX4, and NKX6.1; (ii) introducing to the PF stage cell aMBHB Ref. No. 24-1170-WO UW 49850.02WO2 fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain; and (iii) inducing expression of the transcriptional regulator. In some embodiments, the transcriptional regulator of a PP stage cell is PDX1, and in some embodiments, the transcriptional regulator of a PP stage cell is NKX6.1, and in some embodiments, the transcriptional regulator of a PP stage cell is PAX4.[OHl] In another aspect, the disclosure provides a method of inducing differentiation of a pancreatic progenitor (PP) stage cell to a endocrine progenitor (EP) stage cell, including (i) introducing to the PP stage cell a gRNA for a transcriptional regulator of a EP stage cell selected from NGN3, NKX2.2 and NEURODI; (ii) introducing to the PP stage cell a fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain; and (iii) inducing expression of the transcriptional regulator. In some embodiments, the transcriptional regulator of a EP stage cell is NGN3. In some embodiments, the transcriptional regulator of a EP stage cell is NKX2.2, and in some embodiments, the transcriptional regulator of a EP stage cell is NEURODI.

[0112] In the various aspects and embodiments described herein, the fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain and the gRNA reduce methylation of histone 3, lysine 27 (H3K27me3) to induce expression of a transcriptional regulator. In some embodiments, the fusion protein comprising a catalytically inactivated Cas9 and an EED-binding domain and the gRNA reduce histone methyltransferase activity of EZH2 subunits of Polycomb Repressive Complex 2 (PRC2) to induce expression of a transcriptional regulator.

[0113] In another aspect, the disclosure provides a cell produced according to the various methods described herein. In some embodiments, the cell is an a-cell, [3-cell, 5-cell or y-cell. In some embodiments, the cell is a [3-cell, and in some embodiments, the [3-cell produces insulin. In some embodiments, the cell is a a-cell, and in some embodiments, the a-cell produces glucagon. In some embodiments, the cell is a 5-cell, and in some embodiments, the 5-cell produces somatostatin, and in some embodiments, the cell is a y-cell, and in some embodiments, the y-cell produces pancreatic polypeptide.

[0114] In another aspect, the disclosure provides a method of treating diabetes in a subject in need thereof, including (i) obtaining cells produced according to a method described herein, orMBHB Ref. No. 24-1170-WO UW 49850.02WO2 a composition as described herein; and (ii) administering the cells or composition to the subject in an amount effective to treat diabetes.

[0115] In another aspect, the disclosure provides a method including (i) obtaining cells produced according to a method described herein, or a composition as described herein; and (ii) administering the cells or composition to the subject in an amount effective to treat diabetes; wherein the administered cells or composition increase insulin production in a subject.

[0116] In another aspect, the disclosure provides a method including (i) obtaining cells produced according to a method described herein that results in the differentiation of a PSC into an a-cell or composition thereof; and (ii) administering the cells or composition to a subject in an amount effective to replace dysfunctional glucagon-producing cells in the subject.

[0117] In another aspect, the disclosure provides a method including (i) obtaining cells produced according to a method described herein that results in the differentiation of a PSC into a 5-cell or composition thereof; and (ii) administering the cells or composition to a subject in an amount effective to replace dysfunctional somatostatin-producing cells in the subject.

[0118] In various aspects and embodiments of the method of generating an EP stage cell from a PSC described herein, the EP stage cell is an insulin-producing [3-cell. In the methods described herein, differentiation into EP stage cells is accelerated over conventional differentiation protocols known in the art. In some embodiments, the at least about 20 percent, about 25 percent, about 30 percent, about 35 percent, about 40 percent, about 45 percent, or about 50 percent of EP stage cells are insulin-producing [3-cell by day 19 after inducing expression in the PSC of a transcriptional regulator of a DE stage cell. In some embodiments, at least about 25 percent of EP stage cells are insulin-producing [3-cell by day 19 after inducing expression in the PSC of a transcriptional regulator of a DE stage cell.

[0119] In various methods of generating an EP stage cell from a PSC described herein, the EP stage cells are selectively enriched for P-cells and / or a-cells over 5-cells and y-cells. In some embodiments, [3-cell abundance is increased at least about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold more than comparable control differentiations. In some embodiments, [3-cell abundance is increased at least about 4-fold, about 5-fold, about 6-fold, about 7-fold, or about 8-fold over comparable controls, and inMBHB Ref. No. 24-1170-WO UW 49850.02WO2 some embodiments, P-cell abundance is increased at least about 3-fold, about 4-fold, about 5-fold, or about 6-fold over comparable controls.

[0120] Formulations and Administration

[0121] In another aspect, the disclosure provides a composition comprising cells produced according to any of the methods described herein. In some embodiments, the composition includes a pharmaceutically acceptable excipient. Cells produced according to the methods of the disclosure can be administered to a subject by a several methods available to the art, including but not limited to intraepatic, intradermal, mesenteric, and intramuscular routes.

[0122] A method to potentially increase cell survival is to incorporate cells of the disclosure into a biopolymer or synthetic polymer. Depending on the patient's condition, the site of injection might prove inhospitable for cell seeding and growth because of scarring or other impediments. Examples of biopolymer include, but are not limited to cells mixed with fibronectin, fibrin, fibrinogen, thrombin, collagen, and proteoglycans. This could be constructed with or without additional factors such as cytokines, differentiation factors, angiogenesis factors and / or anti-apoptosis factors. Additionally, these could be in suspension. Another alternative is a three-dimension gel with cells entrapped within the interstices of the cell biopolymer admixture. Again one or more additional factors such as cytokines, differentiation factors, angiogenesis factors and / or anti-apoptosis factors may be included within the gel. Such gels may be deployed by injection via various routes described herein, via catheters or other surgical procedures.

[0123] The quantity of cells to be administered will vary for the subject being treated. In some embodiments, between about 103to about 109, or about 104to about 108, or about 105to about 107PBSCs can be administered to a human subject. The precise determination of what would be considered an effective dose may be based on factors individual to each patient, including their size, age, disease or injury, size of damage caused by the disease or injury and amount of time since the damage occurred.

[0124] When administering a therapeutic composition of the present invention, it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion). The doses may be single doses or multiple doses over a period of several days. The pharmaceutical formulations suitable for injection include sterile aqueous solutions and dispersions. The carrierMBHB Ref. No. 24-1170-WO UW 49850.02WO2 can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof.

[0125] Additionally, various additives which enhance the stability, sterility, and isotonicity of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. In many cases, it will be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the present invention, however, any vehicle, diluent, or additive used would have to be compatible with the cells.

[0126] Sterile injectable solutions can be prepared by incorporating the cells utilized in practicing the present invention in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired.

[0127] In one embodiment, cells of the methods described herein can be administered initially, and thereafter maintained by further administration of cells. For instance, cells of the disclosure can be administered by one method of injection, and thereafter further administered by a different or the same method.

[0128] Examples of compositions comprising cells of the disclosure include liquid preparations for administration, including suspensions; and, preparations for direct or intravenous administration (e.g., injectable administration), such as sterile suspensions or emulsions. Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as Remington, The Science And Practice of Pharmacy (9. sup. th Ed. 1995), incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation. In the manufacture of a pharmaceutical formulation according to the invention, the cells are typically admixed with an acceptable carrier. The carrier must, ofMBHB Ref. No. 24-1170-WO UW 49850.02WO2 course, be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the patient. The carrier may be a solid or a liquid, or both (e.g., hydrogels), and may be formulated with the cells as a unit-dose formulation. In one embodiment the cells are provided as a suspension in the carrier to reduce clumping of the cells.

[0129] Compositions of the invention are conveniently provided as liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions or viscous compositions, which may be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues.

[0130] The choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e.g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form, such as a time release form or liquid-filled form).

[0131] Solutions, suspensions and gels normally contain a significant amount of water (preferably purified, sterilized water) in addition to the cells. Minor amounts of other ingredients such as pH adjusters (e.g., a base such as NaOH), emulsifiers or dispersing agents, buffering agents, preservatives, wetting agents and jelling agents (e.g., methylcellulose), may also be present. The compositions can be isotonic, i.e., they can have the same osmotic pressure as blood and lacrimal fluid.

[0132] The desired isotonicity of the compositions of this invention may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is preferred particularly for buffers containing sodium ions.

[0133] Viscosity of the compositions, if desired, can be maintained at the selected level using a pharmaceutically acceptable thickening agent. Methylcellulose is preferred because it is readily and economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, PVA, ethyl cellulose,MBHB Ref. No. 24-1170-WO UW 49850.02WO2 hydroxypropyl cellulose, carbomer, and the like. The preferred concentration of the thickener will depend upon the agent selected and the desired viscosity. Viscous compositions are normally prepared from solutions by the addition of such thickening agents.

[0134] A pharmaceutically acceptable preservative or cell stabilizer can be employed to increase the life of the compositions. Preferably, they will not affect the viability or efficacy of the cells as described in the present invention.

[0135] Compositions can be administered in dosages and by techniques available to those skilled in the medical and veterinary arts taking into consideration such factors as the age, sex, weight, and condition of the particular subject, and the composition form used for administration (e.g., solid vs. liquid).

[0136] Collectively, to date, the results described herein have mapped out targetable genomic sequences in the promoter regions of stage-specific transcription factors (e.g., EOMES, PDX1, and NGN3), effectively driving Al-designed epigenetic regulators such as EBdCas9 to unlock DE, PP, and EP islet developmental programs in SCs, as governed by each one of these transcription factors. Timing and methods of EpiBinder induction have been identified herein, which can significantly step-up and increase the efficacy of factor-based directed differentiation of SCs into islet cells, as well as accelerate SC maturation into functional islet tissue for transplantation in diabetes.

[0137] Having described the present disclosure, the same will be explained in greater detail in the following examples, which are included herein for illustration purposes only, and which are not intended to be limiting to the disclosure.EXAMPLES

[0138] The Examples that follow are illustrative of specific embodiments of the disclosure, and various uses thereof. They are set forth for explanatory purposes only and should not be construed as limiting the scope of the disclosure in any way.EXAMPLE 1: Derivation and Characterization of new hESC lines to monitor pancreatic islets' developmental stages

[0139] To monitor the impact of epigenetic manipulations during the differentiation of SCs along the islet cell lineage, new reporter lines were generated that facilitate the identificationMBHB Ref. No. 24-1170-WO UW 49850.02WO2 of Definitive endoderm (DE), Endocrine Progenitors (EP) and islet P-cells in live cultures. To produce these lines, the original hESC MELl / InsGFPcell line was used in which a polycistronic construct FOXA2; T2A-nucBFP2 was engineered to target BFP at the endogenous F0XA2 locus, to generate double F0XA2BFPInsulinGFPlines. Henceforth, for simplicity, this new line is referred to as DoubleFOXA2INS. or MELl-DoubleFOXA2INS. As shown in FIG.3A-D, this new cell line can be efficiently used to generate up to 96.5% of F0XA2+ DE cells (FIG. 3A), and more than 28% of Insulin-producing GFP+ P-cells by day 30 of differentiation (FIG. 3B).Transplantation of these cells in NSG mice (NOD. Cg-PrkdcscldH2rgtmlwjl / SzJ mice) leads to their functional maturation into glucose-responsive islet P-cells following an intraperitoneal GTT P-cells (FIG. 3C). Histologic assessment of the grafts reveals the presence of all major islet cell types, producing insulin, glucagon and somatostatin.

[0140] The targeting construct consisted of a bicistronically (T2A) expressed mTagBFP2 (FOXA2), immediately followed by a loxP-flanked Puromycin resistance cassette. It was synthesized with Ikb homology arms from the target locus, such that Cas9-mediated integration occurs in-frame with the target gene downstream of the full open reading frame and immediately 5' of the endogenous stop codon. Guide RNAs were designed using the CRISPOR design tool. A guide RNA targeting the FOXA2 locus was synthesized by Synthego as single guide RNAs (sgRNAs) with the sequence GAAGCCGTCGTCTTCTTAAG (SEQ ID NO:1). PSCs were transfected using the P3 Primary Cell kit on a Nucleofector 4D unit (Lonza V4XP-3012), with 1 pg of Cas9 mRNA (Tri-Link), 2 pg of synthesized sgRNA, and 1 pg of plasmid. Cells were cultured under Puromycin selection for 14 days. The Puromycin cassette was then excised using Cre mRNA (Tri-Link) transfected into the hPSCs using RNAiMax (Thermofisher) following the manufacturer instructions. PCR of the insertion site, followed by Sanger sequencing, was used to confirm the fluorescent reporter was integrated in-frame. For sequencing we used a FOXA2-specific forward primer 5'- GAGCTGAAGGGGACGCCG-3' (SEQ ID NO:2) and a mTagBFP2-specific reverse primer 5'-ACTCCGCCATCTTCATACGT (SEQ ID NO:3). The transfected hPSC pool was clonally expanded following single-cell deposition into 96-well plates using a Sony MA900 cell sorter. Clonally expanded lines were screened for puromycin sensitivity to confirm excision of the selection cassette. Cell clones were further quality controlled for appropriate up-regulated expression of BFP and GFP expression at the DE and EP stages of PSC differentiation, respectively, as measured by fluorescence microscopy and flow cytometry (FIG. 3B-D).MBHB Ref. No. 24-1170-WO UW 49850.02WO2 EXAMPLE 2: Engineering a PRC2 EpiBinder inhibitor EBdCas9 into the AAVS1 safe harbor of DoubleFOXA2INSSCs and other PSC lines

[0141] Previous work by the inventors has led to generation of a robust, PRC2-specific EpiBinder. This Al-designed PRC2 inhibitor binds to EED with high affinity inhibiting the binding of a catalytic component of PRC2, EZH2. The designed EED binder (referred herein as EB) when fused to catalytically dead Cas9 (dCas9) for gRNA-mediated gene targeting, is sufficient to activate the targeted gene by reducing the PRC2 dependent repressive histone post-translational modification, H3K27me3. To assess the effects of this EpiBinder during SC differentiation, the DoubleFOXA2INSSCs were engineered with a polycistronic Doxycycline (DOX)-inducible EBdCas9mCherry construct (FIG. 4A) into the safe-harbor AAVS1 site.

[0142] The targeting construct uses EB fused to dCas9-NLS-2A-mCherry under control of the AAVS1-TREG inducible promoter. In the construct, EB is fused with a 30 amino acid residue 6x5 (SGGGG)n linker. Nucleofection of MELl-DoubleFOXA2INShESC with targeting plasmid, gRNA (GGGGCCACTAGGGACAGGAT (SEQ ID NO:4)) and CAS9 mRNA followed standard protocols. For EBdCas9 targeting into Hl hESC and CV-2A iPSC, TALENs were used to enforce recombinant homology at the AAVS1 site on chromosome 19.

[0143] Multiples clones of this new reporter line, termed DoubleFOXA2INS; EBdCas9, were then tested for their responsiveness to DOX. FIG. 4B shows representative images of the mCherry reporter in two independent clones of DOX-induced, undifferentiated DoubleFOXA2INS; EBdCas9 cells. These results validate the functionality of the construct.

[0144] Clones of iPSC; EBdCas9, Hl; EBdCas9 and CV-2A; EBdCas9 were hand-picked under sterile conditions, expanded and banked. Validation of the correct insertion of the transgene included verification of mCherry expression following Dox treatment by flow cytometry (FIG.4C), and genotyping by PCR using forward and reverse primers binding upstream and downstream of the 5' and 3' homology arms of the transgene. The nucleotide sequence of the primers was: Fl: TCGACTTCCCCTCTTCCGATG (SEQ ID NO:5); Rl: CTCAGGTTCTGGGAGAGGGTAG (SEQ ID NO:61); R2: GAGCCTAGGGCCGGGATTCTC (SEQ ID NO:6). The F1 / R2 primer set was used to detect the insertion of the construct into AAVS1 locus (PCR amplicon 1.2kb). The Fl / Rl primer set was used to detect unmodified AAVS 1 (PCR amplicon 1,4kb). The co-amplification of the 1.2-and 1.4-kb fragments was used to identify mono- and biallelic insertion at the AAVS1 locusMBHB Ref. No. 24-1170-WO UW 49850.02WO2 (FIG. 4E-F). Chromosome banding and karyotyping was performed by Diagnostic Cytogenetics, Inc. (Seattle, WA, USA) and confirmed numerically and structurally normal chromosomes in all tested lines.

[0145] qPCR analysis of the EpiBinder transcript expressed in these lines confirmed its transient expression upon DOX withdrawal (FIG. 4D). Altogether, these lines provided a unique set of stem cell models to test the wide applicability of epigenetic interventions to control islet cell production in diverse genetic backgrounds.EXAMPLE 3: Identification of gRNAs specific for EOMES, PDX1 and NGN3

[0146] Next, to identify sequences suitable for targeting by EBdCas9 and gRNAs, promoter regions of key transcription factors relevant to DE, EP and PP developmental stages were screened. EOMES was selected for DE, and PDX1 and NGN3 for the PP and EP stages, respectively. FIG. 5A shows the model of mechanism of action of EBdCas9 and gRNAs. ENCODE UCSC genome browser was used to inspect H3K27me3 and EZH2 tracks of Hl hESC at promoter regions of the selected transcription factors and designed candidate targeting gRNAs proximal to regulatory TATA boxes within each of those promoters (FIG. 5B, FIG.5D, FIG. 5G)

[0147] Within the 1.5 kb upstream of the transcriptional start site (TSS) of PDX1 and NGN3, candidate targeting gRNAs were designed proximal to predicted regulatory TATA boxes and / or specific to chromatin regions enriched for bivalent H3K27me3 / H3K4me3 histone marks (FIG. 5D, FIG. 5G). For each gene, sets of 8-9 guides were synthesized (Synthego). The nucleotide sequence and genomic distance from the TSS of the screened gRNAs is provided in the sequence listing (see Tables 1 and 2 for details, below).

[0148] Undifferentiated EBdCas9 iPSCs were induced with DOX and transfected with individual gRNAs using lipofectamine. Controls (DOX only) were treated with lipofectamine only. After 72 hours, RT-qPCR analysis of EOMES transcription (normalized to 18S and calculated as relative fold change compared with no gRNA (-g, induced with DOX) identified several guides capable of inducing this transcription factor (FIG.5C; +C: DE positive control).

[0149] A similar approach was used to tile the promoter regions of PDX1 and NGN3, except, for these genes, culture conditions of undifferentiated SC were switched to serum-containing medium (DMEM-10% FCS for PDX1 promoter screening or TeSR for NGN3 promoterMBHB Ref. No. 24-1170-WO UW 49850.02WO2 screening) to allow the cells exit pluripotency. Semiquantitative PCR analysis (PDX1) and RT-qPCR (NGN3) of RNA extracted from these cultures identified a cocktail of gRNAs (#3, 4, and 5, i.e., combination 1 (Cl) (FIG. 5E, F) and gRNA#3 (FIG. 5H), as most effective for the induction of PDX1 and NGN3, respectively (C+ in FIG. 5F and FIG. 5H: Positive Control cDNA from human islets; -C: H2O).

[0150] Full length EBdCas9-mCherry mRNA was synthesized by Trilink Biotech using sequences modified with full substitution of Nl-Methyl-Pseudo-U, Capped (Cap 1) and purified using CleanCap M6, Polyadenylated (120A), DNase Treatment, Oligo dT Purification, and elution in 1 mM Sodium Citrate, pH 6.4 solution. mRNA sequences are provided in Table 1, below.EXAMPLE 4: Validation of chromatin remodeling at PDX1 and NGN3 loci by EBdCas9

[0151] To validate the efficacy of EBdCas9 to relieve PDX1 and NGN3 promoters from the repressive action of PRC2, experiments of ChlP-qPCR in DoubleFOXA2INS; EBdCas9 ESCs were conducted to detect the displacement of EZH2 and depletion of H3K27me3 at select chromatin regions of those genes. The cells were first plated and cultured overnight in TeSRplus medium, and next switched to medium containing fetal bovine serum (FBS) to promote their spontaneous differentiation, and Doxycycline to induce EBdCas9 expression. The next day, cells were transfected with either PDX1 -specific or NGN3 -specific gRNAs. Samples at 24, 48, and 72 hours of culture post gRNA-transfection were then processed for ChlP-qPCR.

[0152] Chromatin immunoprecipitation using antibodies specific for EZH2 and H3K27me3 followed by qPCR of DNA regions flanking the genomic regions targeted by each gRNA revealed significant reduction of EZH2 and depletion of H3K27me3 at the promoter regions of PDX1 (FIG. 7A and FIG. 51) by day 2-3 post-transfection and at the promoter regions of NGN3 after just day 1-3 post-gRNA transfection (FIG. 7B and FIG. 5 J). Similar results were obtained in chromatin precipitation experiments using antibodies specific for H3K27me3, indicating progressive loss of these repressive marks driven by EBdCas9 recruited to those sites. Importantly, the same analysis conducted for the Hl 9 (Imprinting Control Regions) revealed no significant effects (FIG. 7C and FIG. 5I-J), indicating the specificity of the intervention by using the PDX1 and NGN3 gRNAs.MBHB Ref. No. 24-1170-WO UW 49850.02WO2

[0153] For ChlP-qPCR, chromatin samples were added to UV-treated polypropylene 96-well microplates in blocking buffer and incubated with matrix-ChIP antibodies. The antibodies used for Matrix ChIP were: H3K27me3 (Active Motif 39155), EZH2 (Cell Signaling D2C9), H3K27ac (Active Motif 39133), and control Mouse or Rabbit IgGs (Vector Lab). Matrix ChIP experiments were performed in triplicate followed by qPCR in 4-8 replicates. Primers used for ChlP-qPCR were: PDX-1 Fw: CGTTCAGGAGTGTGCAGCAA (SEQ ID NO:55); PDX1- Rev: CTAAGAGGCTAGGCCCAGGT (SEQ ID NO:56); NGN3 Fw: CGCACAGGAAGATAGTGGCA (SEQ ID NO:57); NGN3 Rev: GAGCAGGGCGTCCTTTAGAA (SEQ ID NO:58); H19-ICR Fw: GAGCCGCACCAGATCTTCAG (SEQ ID NO:59); H19 ICR Rev: TTGGTGGAACACACTGTGATC (SEQ ID NO:60).

[0154] These results demonstrate that the EBdCas9-mediated approach to epigenetically regulate the expression of pancreatic islet-specific genes in iPSCs described herein is highly specific and efficient.EXAMPLE 5: EOMES-guided EBdCas9 increases the efficiency of DE differentiation of IPSCs, the first bottleneck to the production of islet cell types from SC

[0155] Having identified gRNAs that drive EBdCas9 to unlock EOMES transcription, it was assessed whether induction of this transcription factor could increase the number of FOXA2+Soxl7+ bona fide DE cells under iPSC-directed differentiation (see FIG. 6A, Stage 1). For transfection with EOMES-specific gRNAs, an iPSC line (WTC1) expressing a DOX-inducible EBdCas9 and endowed with a low propensity to differentiate into DE was specifically chosen. These experiments revealed that EBdCas9 driven to the EOMES promoter by select gRNAs (e.g., #1 and #5) caused a significant increase in the fraction of FOXA2+Soxl7+ DE cells, as compared to EBdCas9 control cells (DOX, no gRNAs) (FIG.13A and FIG. 13B)

[0156] For induction of EOMES at the DE stage, undifferentiated EBdCas9-engineered lines were lifted with TripleE and replated at a density of 1.5-2X106cells / well on Cultrex-coated 6 well plates in 2 ml of TeSRPlus medium containing 10 pM ROCKi and DOX (2 pg / ml) for induction of EBdCas9. Two hundred and fifty microliters of Optimem medium containing 2 pg EOMES-specific gRNA (gRNA #1 or 5) and 10 pl of lipofectamine RNAiMax transfection reagent were then dropped on the cell suspension and plates centrifuged at 1,400 rpm for 7MBHB Ref. No. 24-1170-WO UW 49850.02WO2 minutes. The next day, medium was changed to Stage 1 medium containing DOX (2 pg / ml) and cultures were continued up to Stage 3 (d7) with the appropriate culture media. At day 7, cells were harvested for histologic and flow cytometric assessment of DE markers (E-cadherin, SOX17, F0XA2, CXCR4).

[0157] These results demonstrate that the EBdCas9-mediated approach to epigenetically regulate the expression of DE-specific genes in iPSCs described herein is effective and can be exploited to overcome the first bottleneck of SC differentiation into DE lineages (FIG. 1).EXAMPLE 6: EBdCas9 / gRNA-mediated activation of PDX1 and NgN3 promoters in DoubleFOXA2INS; EBdCas9 SCs increases -cells output

[0158] To test the ability of EBdCas9 to drive the activation of PDX1 in posterior foregut (PF) cells, and NGN3 in pancreatic progenitors (PP), three distinct clones of the DoubleFOXA2INS; EBdCas9 cell line were used in experiments of differentiation. At the end of “Stage 3” (Day 8 of differentiation), PF cells were treated with DOX to induce EBdCas9 and transfected with gRNAs (FIG. 6A) that were identified as capable of inducing PDX1 transcription. Subsequently, at the end of “Stage 4” (Day 12), the induction of EBdCas9 by DOX was repeated in PP cells in conjunction with the transfection with NGN3-specific gRNAs (FIG. 6A), identified as inducers of NGN3 transcription. Following these interventions, by Day 19 of differentiation, these cultures revealed a strikingly high number of Ins / GFP+ cells when compared to control cultures that received DOX alone, without gRNAs transfection (FIG. 6B-D, bright light and fluorescence images). Validation of these results by quantitative flow cytometry revealed that all three ESC clones tested consistently exhibited a significant (~3 to ~7-fold, FIG. 6B-D, flow cytometric graphs at bottom of page) increase in the number of P-cells (FIG. 6C).

[0159] For induction of PDX1, cells at day 8 of differentiation were treated with DOX (2 pg / ml) to induce EBdCas9. The next day, cells from each 6 well were lifted with TripleE and replated in the appropriate culture medium in the presence of 10 pM ROCKi and DOX (2 pg / ml) on a fresh Cultrex-coated 6 well in 2.5 ml of medium; 125 microliters of Optimem medium containing a combination of PDX-1 -specific gRNAs (gRNA #3, 4, 5; 1 pg / each) and 5 pl of lipofectamine RNAiMax transfection reagent were then dropped on the cell suspension and plates centrifuged at 1,400 rpm for 7 minutes. For induction of NGN3, cells at day 11-12 of differentiation were lifted with TripleE and replated in Aggrewells in 2.5 ml of medium inMBHB Ref. No. 24-1170-WO UW 49850.02WO2 the presence of 10 pM ROCKi. One hundred and fifty microliters of Optimem medium containing NGN3 -specific gRNA (gRNA#; 1 pg) and 5 pl of lipofectamine RNAiMax transfection reagent were then dropped on the cell suspension and plates centrifuged at 1,400 rpm for 7 minutes. To minimize toxicity, culture media were replaced 8 hours after transfection.

[0160] Collectively, these results demonstrate that EBdCas9-mediated inhibition of the PRC2 complex at select PDX1 and NGN3 promoter regions can unleash a stepwise specification of FG cells toward PDX1+ PP cells, and subsequently promote a robust specification toward a P-cell phenotype.EXAMPLE 7: Testing the efficacy of “EBdCas9 mRNA and gRNAs” transfection in the differentiation of non-engineered SC lines: transient targeting EBdCas9 to PDX1 and NGN3 promoters leads to (3-cells with enhanced insulin secretory functions

[0161] To broaden the applicability of the epigenetic editing intervention to non-genetically manipulated pluripotent SC lines that do not express a DOX-inducible EBdCas9, the feasibility of co-transfecting EBdCas9»mRNA and gRNAs in SCs was tested. As a first step, transfection conditions were optimized and the efficiency of gRNA and mRNA transfection was determined in SC using Fluoro-labelled gRNAs and an mCherry-EBdCas9 mRNA construct in iPSCs. Flow cytometric analysis revealed that the Fluoro-labelled gRNAs can be successfully targeted to >85% of the cells (FIG. 8A), while by this approach the mCherry-EBdCas9 mRNA is detectable in about 43% of the cells (FIG. 8B).

[0162] The DoubleFOXA2INScells were transfected with EBdCas9»mRNA and PDXbgRNAs at Day 8 of the differentiation protocol (PF, end of “Stage 3”; FIG.9A), and subsequently with an EBdCas9»mRNA and a NGN3»gRNAs transfection at Day 12 (PP, end of “Stage 4”; FIG.9 A). It was found that at “Stage 5”, transfected cells significantly upregulated PDX1 and NGN3 transcripts (Fig.9B). Importantly, by Stage 7, NEURODI and NKX2.2 transcripts, two known downstream targets of PDX1 and NGN3 were also found significantly upregulated in cells transfected with EBdCas9»mRNA and NGN3»gRNAs (Fig. 9C). These results point to a feedforward developmental program triggered by EBdCas9 which follow the expected hierarchy of transcriptional regulatory loops.MBHB Ref. No. 24-1170-WO UW 49850.02WO2

[0163] By day 19, flow cytometric analysis revealed a striking increase in the fraction of INS+GFP+ cells, which continued to increase, as compared to control samples transfected with EBdCas9mRNA alone (i.e., 25% vs 5.7% at day 19; 48% vs 29% at day 31; Fig. 9D).

[0164] These results recapitulate the effect observed in the engineered lines and validate the transient mRNA delivery approach for non-engineered PSC lines.

[0165] To test whether the increased yield of P-cells observed with EBdCas9 interventions correlated with improved endocrine cell function, in vitro glucose-stimulated insulin secretion (GSIS) assays were performed. Islet clusters at Stage 7 (day 35) of differentiation were challenged with high glucose (20 mM D-Glucose), L-Arginine (10 mM), or potassium chloride (30 mM KC1) to assess their insulin secretory capacity. Results showed that EBdCas9-mRNA+gRNAs-edited SC-derived islet cells exhibited robust insulin secretion in response to all three secretagogues, with significantly enhanced responses compared to control samples (FIG. 9E) These data demonstrate that the epigenetic editing approach not only increases P-cell yield but also enhances their functional maturation and secretory competence.EXAMPLE 8: EBdCas9-mediated epigenetic editing produces SC-islet cells with enhanced mitochondrial function

[0166] Metabolic coupling of glucose sensing to insulin secretion is a hallmark of functionally mature P-cells and requires efficient mitochondrial oxidative phosphorylation. To assess whether EBdCas9-mediated epigenetic interventions impact mitochondrial function, oxygen consumption rates (OCR) were measured using Seahorse metabolic flux analysis on Stage 7 (Day 35) islet clusters.

[0167] Analysis revealed that EBdCas9-mediated epigenetic editing produced SC-islet cells that exhibited significantly higher ATP production-coupled respiration (FIG. 10A), a lower proton leak (FIG. 10B), and higher overall coupling efficiency (FIG. 10C) in response to leucine / glutamine stimulation. These results indicate that epigenetically edited SC-islet cells display improved mitochondrial metabolic performance, which is consistent with enhanced functional maturity of P-cells.EXAMPLE 9: EpiBinder-treated SC-derived islet cells exhibit accelerated insulin-secretory functions in vivoMBHB Ref. No. 24-1170-WO UW 49850.02WO2

[0168] To evaluate the ability of SC-derived islet cells to engraft and respond to glucose challenges in vivo, EBdCas9mRNA»gRNAs-edited and control cells generated from day 24 (early) and day 43 (terminal) of the differentiation protocol were transplanted in vivo into immunodeficient NSG mice. At three months after transplantation, mice were fasted and subjected to measurements of circulating human-C peptide in response to an intraperitoneal Glucose Tolerance Tests (IPGTT) to test the grafts’ endocrine function.

[0169] These experiments revealed a higher basal insulin secretion and no response to glucose challenge in animals carrying control day 24 and day 43 cell preparations (Fig. 11), whereas animals carrying the epigenetically edited cell preparations showed a 2-fold induction of human C-peptide for both day 24 and day 43 cell transplants. Interestingly, competence to respond to glucose was already evident in the grafts of epigenetically edited islet cell preparations from day 24 of differentiation, although levels of circulating human C-peptide were lower compared to those detected in animals harboring day 43 grafts (FIG. 11).

[0170] Upon repeated IPGTT at monthly intervals, a consistently faster normalization of the glycemia in response to glucose challenges was also measured in mice harboring the epigenetically edited islets as compared to controls (Fig. 12A). Overall, across 5 experiments, insulin secretion in response to glucose challenge over basal secretion was higher in mice carrying epigenetically edited islet grafts (Fig. 12B).

[0171] These results indicate that, as compared to islet tissue resulting from conventional differentiation protocols, the methods of epigenetic editing described in this invention leads not only to a higher efficiency of SCs differentiation into islet tissue, but also to [3-cell preparations with a superior glucose-responsive insulin secretory function. These results have significant translational implications as they demonstrate a more efficient approach for derivation of functional islet tissue for transplantation in diabetes, an approach that will (1) accelerate the time required to produce islet tissue in vitro, (2) quantitatively enhance islet tissue produced, and (3) promote a faster functional maturation, both in vitro and in vivo, following their transplantation.EXAMPLE 10: Engineering and validation of EBdCas9 across multiple PSC lines of diverse genetic backgroundsMBHB Ref. No. 24-1170-WO UW 49850.02WO2

[0172] To demonstrate the broad applicability of the EBdCas9 epigenetic editing approach across genetically diverse stem cell lines, EBdCas9 constructs were engineered into multiple human PSC lines, including the MELl-DoubleFOXA2INShESC line, Hl hESC, and CV-2A iPSC lines (FIG. 5A and FIG. 4A-D).

[0173] The targeting construct used EB fused to dCas9-NLS-2A-mCherry under control of the AAVS1-TREG inducible promoter (FIG. 4A). Nucleofection of MELl-DoubleFOXA2INShESC with targeting plasmid, gRNA (GGGGCCACTAGGGACAGGAT (SEQ ID NO:4)), and CAS9 mRNA followed standard protocols. For EBdCas9 targeting into Hl hESC and CV-2A iPSC, TALENs were used to enforce recombinant homology at the AAVS1 site on chromosome 19.

[0174] Multiple clones of each engineered line were hand-picked, expanded, and banked. Validation of correct insertion included verification of mCherry expression following DOX treatment by flow cytometry (FIG. 4C) and genotyping by PCR. DOX-inducible expression of the construct was confirmed in MELl-DoubleFOXA2INSlines as detected by fluorescence microscopy (FIG. 4B) and flow cytometric analysis of the mCherry reporter (FIG. 4C). qPCR analysis of the EpiBinder transcript expressed in these lines confirmed its transient expression upon DOX withdrawal (FIG. 4D). Chromosome banding and karyotyping confirmed numerically and structurally normal chromosomes in all tested lines.

[0175] Altogether, these engineered lines provided a unique set of stem cell models to test the wide applicability of epigenetic interventions to control islet cell production across diverse genetic backgrounds.EXAMPLE 11: gRNA-mediated targeting of EBdCas9 leads to chromatin remodeling and gene activation at PDX1 and NGN3 loci

[0176] To comprehensively validate the mechanism of EBdCas9 action, genomic coordinates of gRNAs were designed for targeting the PDX1 and NGN3 promoters and overlaid onto integrative genomic viewer tracks showing H3K27me3 marks and EZH2 -binding sites of the corresponding genomic regions (FIG. 5D, G).

[0177] Within the 1.5 kb upstream of the transcriptional start site (TSS) of PDX1 and NGN3, candidate targeting gRNAs were designed proximal to predicted regulatory TATAMBHB Ref. No. 24-1170-WO UW 49850.02WO2 boxes and / or specific to chromatin regions enriched for bivalent H3K27me3 / H3K4me3 histone marks. For each gene, sets of 8-9 guides were synthesized (Synthego).

[0178] RT-qPCR analysis of PDX1 and NGN3 transcription detected 48 hours (PDX1) and 24 hours (NGN3) in PSCs after DOX induction of EBdCas9 alone or in combination with transfection of the indicated gRNAs showed robust gene activation (FIG.5E, F, H). RT-qPCR of 18S of the same samples served as housekeeping controls.

[0179] ChlP-qPCR analysis demonstrated loss of H3K27me3 marks and EZH2 occupancy in targeted PDX1 and NGN3 promoters (FIG. 5I-J). In contrast, non-targeted H19 gene loci remained unaffected, demonstrating the specificity of the intervention (FIG. 5I-J).

[0180] These comprehensive mechanistic studies identify specific regions of PDX1 and NGN3 promoters that are sensitive to the derepressive-remodeling epigenetic action of EBdCas9.EXAMPLE 12: EBdCas9 / gRNA-mediated sequential activation accelerates beta cell development across multiple PSC clones

[0181] To demonstrate reproducibility of the EBdCas9 epigenetic editing approach, the sequential activation of PDX1 and NGN3 promoters was tested across multiple independent PSC clones of diverse genetic backgrounds.

[0182] A flow chart of EBdCas9 induction and gRNA delivery during directed differentiation of PSCs toward pancreatic islet lineages is shown in FIG. 6A. Specifically, at day 8 of differentiation (posterior foregut stage), cells were induced with DOX to express EBdCas9 and transfected with PDXl-specific gRNAs (gRNA #3, 4, 5). Subsequently, at day 11-12 (pancreatic progenitor stage), EBdCas9 induction was repeated and cells were transfected with NGN3-specific gRNA (#3).

[0183] Fluorescence microscopy of islet clusters from three independent MEL-EBdCas9 hESC clones at day 19 of differentiation showed robust co-expression of EBdCas9- mCherry and INS-GFP fluorescent reporters (FIG. 6B-D, fluorescence images). Representative flow cytometric analysis of GFP+ insulin+ cells from these samples confirmed consistently increased [3-cell yields across all three clones tested (FIG. 6B-D, flow cytometric graphs at bottom of page for each clone).MBHB Ref. No. 24-1170-WO UW 49850.02WO2

[0184] These results demonstrate that the EBdCas9-mediated epigenetic editing approach is highly reproducible across independent clones and can consistently enhance [3-cell differentiation efficiency..EXAMPLE 13: Sequential activation of PDX1 and NGN3 by EBdCas9 leads to extensive H3K27Ac chromatin remodeling

[0185] To investigate genome-wide chromatin changes induced by EBdCas9 interventions, CUT& Tag assays were performed to profile H3K27ac histone modifications — a mark of active chromatin — in differentiating PSCs.

[0186] The timeline of EBdCas9 interventions during PSC differentiation and cell harvesting for CUT& Tag is shown in FIG. 14A. Duplicate samples of stem cell clusters, induced to express EBdCas9 alone or PDX1 / NGN3 -guided EBdCas9, were harvested 48 hours after NGN3 gRNA transfection (day 14) and processed for CUT& Tag using H3K27ac-specific antibodies.

[0187] Genome browser views showed enhanced H3K27ac signal within the promoter, enhancer, and / or intronic regions of NGN3, NKX2.2, and GCK in PDX1 / NGN3 -guided EBdCas9-treated samples compared to control (FIG. 14B-D). Two biological replicas per condition are shown. Heatmaps of H3K27ac signal within the PDX1 and NGN3 gene loci confirmed robust activation marks at these targeted loci (FIG. 14E).

[0188] Peak calling analysis identified 14,695 unique H3K27ac peaks in PDX1 / NGN3-guided EBdCas9-treated samples and 28,033 unique peaks in EBdCas9-treated controls (FIG.14F). For each condition, the top 5% significant peaks and averaged signal of two biological replicas are shown. Composite plots of normalized H3K27ac signal are shown at the top of each heatmap.

[0189] Enrichment pathway and cell type analysis was performed on the nearest neighboring genes to the top 1% unique H3K27ac peaks using gene data sets from Enrichr (FIG. 14G-H) Gene Ontology and KEGG pathway analysis of H3K27ac histone modifications unique to PDX1 / NGN3 -guided EBdCas9 samples indicated enrichment for genes involved in endocrine progenitor differentiation, including small GTPase-mediated signaling, Rapl and cAMP signaling, establishment of apical / basal cell polarity, negative regulation of Wnt signaling, and regulation of sodium ion transport. The topmost significantMBHB Ref. No. 24-1170-WO UW 49850.02WO2 cell phenotype detected by the Descartes cell type dataset aligned with pancreatic endocrine cells.

[0190] In contrast, H3K27ac histone modifications unique to EBdCas9 control samples were most significantly enriched for cell growth-related genes (e.g., cellular response to growth stimulus, TGFP-receptor signaling, Hippo signaling pathways) and axogenesis, as well as pancreatic ductal and splenic cellular phenotypes.

[0191] These genome-wide profiling results demonstrate that EBdCas9-mediated epigenetic interventions induce extensive chromatin remodeling that favors pancreatic endocrine cell developmental programs while suppressing alternative lineage fates..EXAMPLE 14: EBdCas9 increases Endocrine Progenitor commitment with developmental bias toward beta cells

[0192] To quantitatively assess the impact of EBdCas9 interventions on pancreatic progenitor and endocrine cell populations, comprehensive morphometric and flow cytometric analyses were performed on differentiating PSC clusters.

[0193] A schematic of EBdCas9 induction and gRNA transfection is shown in FIG. 15A.Morphometric analysis of SC clusters at day 20 of differentiation revealed that PDX1 / NGN3-guided EBdCas9 treatment resulted in significantly smaller cluster areas (FIG. 15B), but with markedly increased numbers of PDX1+ cells per cluster area (FIG. 15C) and PDX1+ / NKX6.1+ endocrine progenitors per cluster area (FIG. 15D).

[0194] Cumulative and fractional representation of insulin+ and glucagon+ areas detected in the clusters at day 20 revealed significantly increased yields of insulin-positive cells induced by sequential targeting of EBdCas9 to PDX1 and NGN3 promoters (FIG. 15E-G). Box-and-whiskers plots show the distribution of measurements in individual clusters, with the thick line in each box representing the median.

[0195] Flow cytometric analysis of alpha and beta cell subpopulations detected in multiple independent experiments using hESCs and iPSCs at day 20 of differentiation confirmed the increased yield of beta cell types under EBdCas9+PDXl / NGN3 gRNA treatment (FIG. 15H).Bars represent mean ± SEM (n=4-l 1).MBHB Ref. No. 24-1170-WO UW 49850.02WO2

[0196] Follow-up flow cytometric analysis of insulin single-positive cells (FIG. 151) and insulin / glucagon double-positive cells (FIG. 15J) in additional experiments (n=6-8) showed enrichment of insulin single-positive beta cells in EBdCas9+PDXl / NGN3 gRNA-treated samples relative to EBdCas9 controls detected at both stage 5 and stage 7 of differentiation (day 35).

[0197] These comprehensive phenotypic analyses demonstrate that EBdCas9-mediated epigenetic interventions significantly increase the efficiency of SC commitment to pancreatic endocrine progenitors with a strong developmental bias toward beta cell types.EXAMPLE 15: EBdCas9-driven epigenetic remodeling fosters activation of NGN3-dependent transcriptional programs

[0198] To investigate the transcriptional programs activated downstream of EBdCas9 interventions, bulk RNA-seq was performed on differentiated Hl and MEL hESCs at day 20, induced to express EBdCas9 alone, EBdCas9 guided sequentially by PDX1 / NGN3 gRNAs, or EBdCas9 guided by either PDX1 orNGN3 gRNAs individually.

[0199] Gene Set Enrichment Analysis (GSEA) of transcripts concordantly regulated in Hl and MEL hESCs identified differentially expressed Reactome pathways in cultures of PDX1-, NGN3-, and PDX1 / NGN3 -guided EBdCas9-treated samples compared to EBdCas9-only control (FIG. 16A-C). NGN3-guided EBdCas9 samples were positively enriched in molecular functions associated with early endocrine development and function (e.g., beta cell development, voltage-gated potassium channels) and neuronal -related functions (e.g., neurotransmitter receptors and transmission across chemical synapses). PDX1 and PDX1 / NGN3 -guided samples were positively enriched for metabolic functions (e.g., glucose metabolism, triglyceride catabolism, FOXO-mediated transcription, amino acid regulation of mTORCl) and negatively enriched for genes involved in neurotransmission and cell-matrix interactions (e.g., GABA receptor activation, integrin signaling, extracellular matrix organization).

[0200] Analysis of predicted NGN3 target genes showed that 223 of 1,263 NGN3 target genes were concordantly upregulated in Hl and MEL-hESCs epigenetically manipulated with EBdCas9+PDXl / NGN3 gRNAs (FIG. 16D). The top three most represented classes of theseMBHB Ref. No. 24-1170-WO UW 49850.02WO2 genes were related to exocytosis / insulin secretion, mitochondrial function / metabolism, and transcription factors / development.

[0201] Heatmaps of NGN3 target genes within the exocytosis / insulin secretion and mitochondrial function / metabolism categories showed robust upregulation in PDX1 / NGN3-guided samples (FIG. 16E-F). Heatmaps represent fold changes of normalized gene counts averaged in the indicated gRNA-treated vs. EBdCas9-only Hl and MEL-hESC samples.

[0202] Interestingly, analysis of enteroendocrine (EC)-specific gene signatures revealed a gene signature strongly biased toward enteroendocrine lineages detected in NGN3 -guided EBdCas9-treated samples, but not in PDX1 / NGN3 sequentially guided samples (FIG. 16G).This finding suggests that sequential PDX1 followed by NGN3 targeting selects against enteroendocrine fates while promoting pancreatic beta cell specification.

[0203] These transcriptomic analyses demonstrate that EBdCas9-driven epigenetic remodeling activates comprehensive NGN3 -dependent transcriptional programs that promote beta cell development and function.EXAMPLE 16: Gene-guided EBdCas9 enforces pancreatic over gut developmental fates

[0204] To investigate whether EBdCas9 interventions affect lineage commitment decisions, the presence of uncommitted and alternative lineage cell types was assessed in differentiating PSC clusters.

[0205] Representative fluorescence microscopy and morphometric analysis of cell clusters at day 20 of differentiation showed significantly reduced frequency of uncommitted PDX1- / NKX6.1 -negative cell types (red-labeled nuclei) normalized to cluster areas under gene-guided EBdCas9 treatments (FIG. 17A-B).

[0206] Immunostaining revealed that uncommitted cells included PDXl-neg / CDX2+ gut epithelium (arrows and circles, FIG. 17C). A schematic representation of gastric (SOX2+), pancreatic (PDX1+ / CDX2+), and gut (PDXl-neg / CDX2+) domains developing from primitive gut tube cells in mouse embryogenesis and during directed hESC differentiation toward pancreatic lineages is shown in FIG. 17D. High levels of PDX1 are critical to repress CDX2 and intestinal fate choices within posterior hindgut.MBHB Ref. No. 24-1170-WO UW 49850.02WO2

[0207] Morphometric analysis of PDXl-neg / CDX2+ cell types counted per cluster area at day 20 of directed differentiation of MEL (FIG. 17E) and Hl hESC (FIG. 17F) confirmed that PDX1 -guided EBdCas9 as well as EBdCas9 guided sequentially to PDX1 and NGN3 promoters effectively selected against CDX2+ / PDXl-neg gut lineage cells.

[0208] Similarly, morphometric analysis of PDXl-neg / SOX2+ cell types counted per cluster area at day 20 of directed differentiation of MEL (FIG. 17G) and Hl hESC (FIG. 17H) showed that these gastric / anterior foregut contaminants were significantly reduced in samples treated with EBdCas9 guided by NGN3 gRNA alone and were lowest in samples that received sequential guidance with PDX1 followed by NGN3 gRNAs.

[0209] These findings demonstrate the ability of EBdCas9 interventions to enforce engagement of pancreatic over gut lineage choices during PSC-directed differentiation toward islet cell lineages, implicating PRC2-dependent repression of PDX1 and NGN3 promoters in differentially influencing posterior vs. anterior gut developmental choices.EXAMPLE 17: NEW - Comparison of EBdCas9 with VP64-mediated gene activation

[0210] To investigate whether gene activation alone, independent of epigenetic remodeling, is sufficient to enhance islet cell differentiation, the effects of VP64dCas9 — a synthetic transactivator that recruits transcription factors without modifying chromatin states — were compared to EBdCas9.

[0211] VP64 was engineered to replace EB fused to dCas9 in the mCherry reporter plasmid, and this construct was used to synthesize VP64dCas9mCherry mRNA for cell delivery.

[0212] Initially, to validate its function in inducing PDX1 and NGN3 transcription, this mRNA was used in transfections of undifferentiated iPSCs to screen for gRNAs targeting PDX1 and NGN3 promoter regions proximal to the transcriptional start sites of each gene. The same PDXl-specific gRNA pool (i.e., gRNA #3, 4, 5) previously used for EBdCas9 and a new NGN3 -specific gRNA were identified as effective at inducing VP64-mediated upregulation of PDX1 and NGN3 mRNA, respectively, as compared to VP64dCas9 mRNA-treated controls.

[0213] Next, the VP64dCas9- mCherry mRNA alone or in combination with the selected PDX1- and NGN3-specific gRNAs were transfected into differentiating PSCs following theMBHB Ref. No. 24-1170-WO UW 49850.02WO2 same timeline used for EBdCas9. Western blotting analysis of cell extracts confirmed robust and sustained expression of dCas9 delivered by these transfections up to 7 days post-NGN3 gRNA transfection.

[0214] However, immunofluorescence and morphometric analysis of PDX1 and NKX6.1+ cells at day 20 of differentiation showed that none of the gene-guided VP64dCas9 interventions increased the yield of PDX1+ / NKX6.1+ endocrine progenitors nor significantly decreased the frequency of PDXl-neg / NKX6.1-neg cells. In addition, morphometric analysis of insulin and glucagon+ cells at stage 7 in samples treated sequentially with PDX1 and NGN3 -guided VP64dCas9 revealed no significantly enhanced yield of cells positive for either hormone.

[0215] Hence, VP64-mediated activation and EB-driven epigenetic remodeling of PDX1 and NGN3 promoters are not functionally equivalent in driving downstream developmental decisions. These comparative findings support the idea that the PRC2 remodeling action of EBdCas9 is critical for the observed effects on pancreatic lineage commitment, not merely gene activation.EXAMPLE 18: Stage 7 islet clusters epigenetically modified by PDXl / NGN3-guided EBdCas9 are glucose responsive in vivo

[0216] To compare the ability of PDX1 / NGN3 -guided EBdCas9 and EBdCas9-only control samples to respond to glucose in vivo, cell clusters at Stage 7 of differentiation were transplanted under the kidney capsule of immunodeficient IL2R- / - SCID mice (FIG. 18A).

[0217] Sequential measurements of circulating human C-peptide in response to intraperitoneal GTTs (IPGTT) at 3 and 6 months after transplantation revealed a consistently lower basal insulin secretion at fasting and increased insulin secretion in response to glucose already at 3 months post-transplantation in mice carrying the epigenetically edited cells (FIG.18B). Bars represent mean ± SD of n=2 experimental mice per group, tested sequentially at 3-and 6-months post-transplantation (P<0.05).

[0218] In contrast, mice carrying EBdCas9-only treated grafts exhibited higher basal insulin secretion at fasting at all time points and variable responses to glucose loads at 6 months post-transplantation.MBHB Ref. No. 24-1170-WO UW 49850.02WO2

[0219] In experiments using 3 distinct PSC lines, by 6 months after transplantation, human C-peptide detected in 5 independent experiments during IPGTT performed 3-6 months posttransplantation showed significant increased insulin secretory responses over basal secretion in mice transplanted with islet clusters resulting from treatments with PDX1 / NGN3 -guided EBdCas9 (FIG. 18C, P<0.01).

[0220] Hence, the reportedly immature endocrine features of PSC transplants — namely high basal insulin secretion and a blunted response to glucose — are resolved more rapidly and across genetically diverse backgrounds in epigenetically manipulated PSC samples. These results demonstrate that PDXl / NGN3-guided EBdCas9 interventions produce SC-islet cells with accelerated functional maturation and superior glucose-responsive insulin secretory function in vivo.METHODS

[0221] Derivation of human PSC reporter lines of pancreatic islet differentiation

[0222] CRISPR / Cas9 technology and homology-directed recombination (HDR) were used to engineer a polycistronic construct FOXA2; T2A- nucBFP2 targeting BFP at the endogenous FOXA2 locus of the original hESC MELl / InsGFP. The targeting construct consisted of a bicistronically (T2A) expressed mTagBFP2 (FOXA2), immediately followed by a loxP-flanked Puromycin resistance cassette. The targeting construct was synthesized with Ikb homology arms from the target locus, such that Cas9-mediated integration occurs in-frame with the target gene downstream of the full open reading frame and immediately 5’ of the endogenous stop codon. The targeting plasmid was amplified in bacteria using standard procedures, with final preparation using endotoxin- free column purification (Qiagen Midi Plus Plasmid kit).

[0223] Guide RNAs were designed using the CRISPOR design tool, optimizing for 1) proximity to the HDR integration site, 2) minimal off-target sites, and 3) high predicted Cas9 activity. Guide RNAs were synthesized by Synthego as single guide RNAs (sgRNAs) with modified ends. The gRNA sequence used for targeting to the FOXA2 locus was: GAAGCCGTCGTCTTCTTAAG (SEQ ID NO:1).

[0224] For PSC transfection, four hours prior to nucleofection, a full media change was performed with mTeSRPlus supplemented with Y-27632 [10uM], PSCs were then dissociatedMBHB Ref. No. 24-1170-WO UW 49850.02WO2 into single cells using TrypLE Express, and nucleofected using a Nucleofector 4D with the P3 Primary Cell kit following the manufacturer instructions (Lonza V4XP-3012) using lug of Cas9 mRNA (Tri-Link), 2ug of synthesized sgRNA, and lug of plasmid. The nucleofected hPSCs were immediately neutralized with mTeSR Plus supplemented with Y-27632 [lOuM], and plated in a single well of a 6-well plate. The following day, the mTeSR Plus media was replaced with fresh media containing Puromycin [500ng / ml] and without Y-27632. Successfully targeted cells were cultured under Puromycin selection for approximately 7-14 days (until hPSCs recover and are ready to passage again). At this point, the Puromycin cassette was excised using Cre mRNA (Tri-Link) transfected into the hPSCs using RNAiMax (Thermofisher) following the manufacturer instructions.

[0225] PCR of the insertion site, followed by Sanger sequencing, was used to confirm the fluorescent reporter was integrated in-frame (without error). For sequencing a FOXA2-specific forward primer (5'- GAGCTGAAGGGGACGCCG-3') (SEQ ID NO:2) and a mTagBFP2 -specific reverse primer (51ACTCCGCCATCTTCATACGT) (SEQ ID NO:3) were used.

[0226] The transfected hPSC pool was then clonally expanded following single-cell deposition into 96-well plates using a Sony MA900 cell sorter. Clonally expanded lines were screened for puromycin sensitivity to confirm excision of the selection cassette. Cell clones were further quality controlled for appropriate up-regulated expression of BFP and GFP expression at the DE and EP stages of PSC differentiation, respectively, as measured by fluorescence microscopy and flow cytometry.

[0227] Engineering of EBdCas9 in the AAVS1 site of hPSC lines

[0228] The DoubleFOXA2INSPSCs generated above were engineered with a polycistronic Doxycycline (DOX)-inducible EBdCas9mCherryconstruct (Fig. 4A) targeted to the AAVS1 safe-harbor site. The targeting EbCas9 plasmid was published previously. The sequence of gRNA used for targeting to the AAVS1 locus was: GGGGCCACTAGGGACAGGAT (SEQ ID NO:4). Nucleofection of PSC with targeting plasmid, gRNA and CAS9 mRNA followed the same strategy outlined above. After neomycin selection select clones were picked, expanded and banked. For EbdCas9 targeting into Hl hESC and CV-A2 iPSC a plasmid in which EB was fused to dCas9-NLS-2A-mCherry (Mandegar et al., 2016) with a 30 aa residue 6x5 (SGGGG) (SEQ ID NO: 54) linker under control of the AAVS1-TREG inducible promoter was used. The EBdCas9 construct was transfected to HlhESC and CV-2A iPSC usingMBHB Ref. No. 24-1170-WO UW 49850.02WO2 TALENS to enforce recombinant homology at the AAVS1 site on chromosome 19. After antibiotic selection, single clones of the new reporter lines, termed DoubleFOXA2INS; EBdCas9, Hl; EBdCas9 and CV-2A; EBdCas9 were hand-picked under sterile conditions, expanded and banked. Validation of the correct insertion of EBdCas9 constructs included verification of mCherry expression following Dox treatment by flow cytometry, and genotyping by PCR using forward and reverse primers binding upstream and downstream of the 5' and 3' homology arms of the transgene, thereby confirming that cells were targeted and that the rtTA components were functional. The nucleotide sequence of the forward primer was: TCGACTTCCCCTCTTCCGATG(SEQ ID NO:5); the one of the reverse primer was: GAGCCTAGGGCCGGGATTCTC (SEQ ID NO: 6).

[0229] Guide RNA design, EBdCas9 mRNA synthesis and transfection

[0230] To identify sequences suitable for targeting of EBdCas9 to the promoter regions of EOMES, PDX1 and NgN3, ENCODE UCSC genome browser was used to inspect H3K27me3 and EZH2 tracks of Hl hESC within the ~1.7 Kb promoter region proximal to the transcriptional start site of each transcription factors' gene. For each gene, sets of 8-9 guides were synthesized (Synthego) and screened. This screen identified two EOMES-, three PDX1, and one NGN3 -specific gRNAs effective in inducing transcription factor up-regulation, as measured by qPCR. The nucleotide sequence and position of the screened gRNAs is shown below. The ones selected as inducers of gene expression are in bold print and underlined.Table 1:SEQ ID Base# from TSS- gRNA# SequenceNO: gRNA namegRNAs for EBdCas9 targetingPDX1-1 7 60 PDX1 CGCGGAGCCTATGGTGCGGC PDX1-2 8 114PDX1 CTTAGTGCGGCCAGCCAGGC PDX1-3 9 219 PDX1 AT T T TGGGGAGCACCGCCAG PDX1-4 io 472 PDX1 TGC T TO TGACC TAGAGAGC T PDX1-5 11 739 PDX1 CTCGCTGTATTGGGAAGCTA PDX1-6 12 827 PDX1 C G G C C C G T AGAGAG T C G T C A PDX1-7 13 977 PDX1 AGCACAGAT GT TAT CAT GGAMBHB Ref. No. 24-1170-WO UW 49850.02WO2 PDX1-8 14 1186PDX1 i-LC G GT T G CJ C G T T '1' C G AGGAGAG NGN3-1 15 101 NGN3 C G C AG G G CACAG C T G GAT T C NGN3-2 16 310NGN3 TTCCTTAGCATCTGTCCTGG NGN3-3 17 461 NGN3 C TGAC TAGCAGCC TGGAGAA NGN3-4 18 555 NGN3 AT G T C C C T T C CAT T C G C AG A NGN3-5 19 649 NGN3 A C C C C C AT AG AT C C C AC TGC NGN3-6 20 799 NGN3 T T C T G G C GAG GAAC C G T G AC NGN3-7 21 1589 NGN3 GGTTTAAGTCTCCTTAGGCG NGN3-8 22 1712 NGN3 TAGT GATAGC C T C T TACC GC NGN3-9 23 381 NGN3 T GGC T T GT C T GAAAT CGCAC EOMES-1 24 EOMES 1153 ACGCCGCCAGTAATCGCTCC EOMES-2 25 EOMES 2150 CCGCGTTATTCGCGCCAGAG EOMES-3 26 EOMES 1093 GGCACGTTTCGTTGCGGATA EOMES-4 27 EOMES 1799 TTGAGCTCGCGTACCCCTCA EOMES-5 28 EOMES 287 CTGAGATGACTAATGCGCCA EOMES-6 29 EOMES 458 TGAAGTTACAGCTTCCGACG EOMES-7 30 EOMES 816 AAGGGACGGAATCCGGGATC EOMES-8 31 EOMES 518 AAAGTGGTTCTGTGCGTTGA gRNAs for VP64 targetingA 7 60 PDX1 CGCGGAGCCTATGGTGCGGC B 8 114 PD X1 C T T AG T G C G G C CAG C CAG G C C 9 219 PDX1 ATTTTGGGGAGCACCGCCAG D 62 72 PDX1 GTTCAGCCGGGGGCCGTGAT E 63 7 PDX1 AACCCACAGCCAGCGCGGACA 64 43 NGN3 C G C C AC C G G C C AAT GAG C G C B 65 157NGN3 C G C T C T G T T T G C T C T C T C GA C 15 101 NGN3 CGCAGGGCACAGCTGGATTC D 66 16 NGN3 GAG C C G G G GAG G C AC G C T C CMBHB Ref. No. 24-1170-WO UW 49850.02WO2

[0231] Full length EBdCas9-mCherry mRNA was synthesized by Trilink Biotech using the sequence below (SEQ ID NO:32).ATGATTAACGAGATCAAGAAGAACGCTCAAGAGCGGATGGACGAGACTGTCGAACAGCTGAAGAACGA GCTGAGCAAGGTGAGGACCGGGGGAGGGGGAACAGAAGAGCGACGCTTGGAGCTGGCCAAGCAGGTCG TGTTTGCAGCCAATCGAGCTTTGATCCGGGTCCGCACTATTGCCCTCGAAGCGGCATGGCGCCTTAGA ATGCTGGGTAGTGATAAAGAGGTGAACAAAAGAGATATATCACAGGCTCTGGAAGAAATCGAGAAGTT GACCAAAGTGGCGGCGAAAAAGATCAAGGAGGTACTGGAGGCTAAGATAAAGGAGCTCAGAGAGGTGA TGGCCGTTAACTCCGGCGGAGGGGGTTCCCGCGGAGGGGGTTCCGGCGGAGGGGGTTCCGGCGGAGGG GGTTCCGGCGGAGGGGGTTCCGGCGGAGGGGGTATGGACAAGAAGTACAGCATCGGCCTGGCCATCGG CACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGC TGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCGCCCTGCTGTTCGACAGCGGAGAA ACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTG CTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAG AGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAG GTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAA GGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCG AGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAAC CAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACT GAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCG GCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGAT GCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGA CCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGA GAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCAC CAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTT CGACCAGAGCAAGAACGGCTACGCCGGCTACATCGATGGCGGAGCCAGCCAGGAAGAGTTCTACAAGT TCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGAC CTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCA CGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGA TCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATG ACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCCAGCGC CCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGC ACAGCCTGCTGTACGAGTACTTCACCGTGTACAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGA ATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAAGCCATCGTGGACCTGCTGTTCAAGACCAA CCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGG AAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATC AAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACMBHB Ref. No. 24-1170-WO UW 49850.02WO2 ACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAG TGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGC ATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAA CTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCG GCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATC CTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGT GATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGC GGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAG CTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACT GGACATCAACCGGCTGTCCGACTACGATGTGGACGCTATCGTGCCTCAGAGCTTTCTGAAGGACGACT CCATCGATAACAAAGTGCTGACTCGGAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAA GAGGTCGTGAAGAAGATGAAGAACTACTGGCGCCAGCTGCTGAATGCCAAGCTGATTACCCAGAGGAA GTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGA GACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACT AAGTACGACGAGAACGACAAACTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTC CGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACG CCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTG TACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGC TACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACG GCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACAGGCGAGATCGTGTGGGATAAGGGC CGGGACTTTGCCACCGTGCGGAAAGTGCTGTCTATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGT GCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGACAAGCTGATCGCCAGAA AGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTG GTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCAT CATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGA AAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATG CTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCT GTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTG T GG AAC AGC AC AAAC AC T ACC T GGACG AG AT CAT C GAGC AG AT C AGC GAGT T C T C C AAG AG AGT G AT C CTGGCCGACGCTAATCTGGACAAGGTGCTGAGCGCCTACAACAAGCACAGAGACAAGCCTATCAGAGA GCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACT TTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCAC CAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACGCCTATCCCTA TGACGTGCCCGATTATGCCAGCCTGGGCAGCGGCTCCCCCAAGAAAAAACGCAAGGTGGAAGATCCTA AGAAAAAGCGGAAAGTGGACGGCATTGGTAGTGGGAGCAACGGCAGCAGCGGATCCGCTACTAACTTC AGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTATGGTGAGCAAGGGCGAGGAGGA TAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGMBHB Ref. No. 24-1170-WO UW 49850.02WO2 AGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTG ACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGC CTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGG AGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGC GAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAA GACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCA AGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAG AAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGA CTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGT ACAAGTAA

[0232] EBdCas9 mRNA sequence - Protein sequence (SEQ ID NO:53):MINEIKKNAQERMDETVEQLKNELSKVRTGGGGTEERRLELAKQVVFAANRALIRVRTIALEAAWRLR MLGSDKEVNKRDISQALEEIEKLTKVAAKKIKEVLEAKIKELREVMAVNSGGGGSRGGGSGGGGSGGG GSGGGGSGGGGMDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGE TAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDE VAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYN QLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAED AKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHH QDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWM TRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEG MRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLING IRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGI LQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQ LQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSE EVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNT KYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFV YGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKG RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLV VAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRM LASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVI LADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIH QSITGLYETRIDLSQLGGDAYPYDVPDYASLGSGSPKKKRKVEDPKKKRKVDGIGSGSNGSSGSATNF SLLKQAGDVEENPGPMVSKGEEDNMAI IKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVMBHB Ref. No. 24-1170-WO UW 49850.02WO2 TKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDG EFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAK KPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK

[0233] VP64dCas9 mRNA sequence follows (SEQ ID NO: 67)GACGCATTGGACGATTTTGATCTGGATATGCTGGGAAGTGACGCCCTCGATGATTTTGACCTTGACAT GCTTGGTTCGGATGCCCTTGATGACTTTGACCTCGACATGCTCGGCAGTGACGCCCTTGATGATTTCG ACCTGGACATGCTGTCCGGCGGAGGGGGTTCCCGCGGAGGGGGTTCCGGCGGAGGGGGTTCCGGCGGA GGGGGTTCCGGCGGAGGGGGTTCCGGCGGAGGGGGTATGGACAAGAAGTACAGCATCGGCCTGGCCAT CGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGG TGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCGCCCTGCTGTTCGACAGCGGA GAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGAT CTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGG AAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGAC GAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGA CAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGA TCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTAC AACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAG ACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGT TCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAG GATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGG CGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCC TGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCAC CACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTT CTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATCGATGGCGGAGCCAGCCAGGAAGAGTTCTACA AGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAG GACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCT GCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGA AGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGG ATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCCAG CGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCA AGCACAGCCTGCTGTACGAGTACTTCACCGTGTACAACGAGCTGACCAAAGTGAAATACGTGACCGAG GGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAAGCCATCGTGGACCTGCTGTTCAAGAC CAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCG TGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATT ATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTMBHB Ref. No. 24-1170-WO UW 49850.02WO2 GACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACA AAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAAC GGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAG AAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGT CCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGC ATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACAT CGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGA AGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACC CAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGA ACTGGACATCAACCGGCTGTCCGACTACGATGTGGACGCTATCGTGCCTCAGAGCTTTCTGAAGGACG ACTCCATCGATAACAAAGTGCTGACTCGGAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCC GAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGCCAGCTGCTGAATGCCAAGCTGATTACCCAGAG GAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCA AGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAAC ACTAAGTACGACGAGAACGACAAACTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGT GTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACG ACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTC GTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAA GGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCA ACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACAGGCGAGATCGTGTGGGATAAG GGCCGGGACTTTGCCACCGTGCGGAAAGTGCTGTCTATGCCCCAAGTGAATATCGTGAAAAAGACCGA GGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGACAAGCTGATCGCCA GAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTG GTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCAC CATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAG TGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGA ATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTT CCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGT TTGTGGAACAGCACAAACACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTG ATCCTGGCCGACGCTAATCTGGACAAGGTGCTGAGCGCCTACAACAAGCACAGAGACAAGCCTATCAG AGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGT ACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATC CACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACGCCTATCC CTATGACGTGCCCGATTATGCCAGCCTGGGCAGCGGCTCCCCCAAGAAAAAACGCAAGGTGGAAGATC CTAAGAAAAAGCGGAAAGTGGACGGCATTGGTAGTGGGAGCAACGGCAGCAGCGGATCCGCTACTAAC TTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTATGGTGAGCAAGGGCGAGGA GGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCMBHB Ref. No. 24-1170-WO UW 49850.02WO2 ACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAG GTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAA GGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGT GGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGAC GGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAA GAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGA TCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCC AAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGA GGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGC TGTACAAGTAA

[0234] The EBdCas9 mRNA was modified with full substitution of N1 -Methyl -Pseudo-U Capped (Cap 1) and purified using CleanCap® M6 Polyadenylated (120A) DNase Treatment, Oligo dT Purification, and elution in 1 mM Sodium Citrate, pH 6.4 solution. For cell transfection, 5 pg of the purified EBdCas9-mCherry mRNA was incubated in 250 pl Optimem (Fisher Scientific) together with lOul of Lipofectamine MessengerMax transfection reagent (Invitrogen, LMRNA003) for 7 minutes and transfected onto 2 ml of cell suspension per well of a 6 well plate. For transfection of gRNAs, 2 pg of each guide RNA were incubated in 250 ul Optimem together with lOul of lipofectamine RNAiMax transfection reagent (Invitrogen, 13778075) for 7 minutes and transfected onto 2 ml of cell suspension per well of a 6 well plate. For transfections making use of both EBdCas9 mRNA and gRNAs, the two sets of transfections were prepared individually with the respective transfection reagents, and both were dropped onto each cell suspension.

[0235] PSC lines maintenance and differentiation into islet cell types

[0236] DoubleFOXA2INSPSCs and Hl, and engineered hESC DoubleFOXA2INS; EBdCas9, Hl; EBdCas9, and WTC1 l; EBdCas9 iPSC previously generated as described above were routinely maintained by culturing them on Cultrex-coated dishes (diluted 1:30 in medium) in TeSERPlus medium (Clonetics) in a 5% CO2, 20% oxygen incubator. Medium was changed every 24 hours. When cells reached -80% confluency, cells were passed with TripleE, blocked in medium, pelleted and replated onto fresh Cultrex-coated plates in the presence of lOuM ROCKi for the first 8-16 hours and used in differentiation experiments up to 15-20 passages.

[0237] To differentiate these lines into islet cell types, a seven-stage differentiation protocol modified from our previous studies, was followed. The culture media and supplements specificMBHB Ref. No. 24-1170-WO UW 49850.02WO2 to each stage and their timed application are listed in Table 2. From Stage 1 to 6, all media and supplements were replaced every 24 hours. At stage 7, culture medium was replaced every 48 hours.

[0238] Cultures were started by lifting PSC with Triple E, blocking in culture medium and replating 1.5-2xl06PSC / well on Cultrex- coated 6 well plates in 3 ml of TeSERPlus medium in the presence of lOuM ROCKi (day 0). The next day, stage 1 culture medium was applied. At day 11, cell monolayers were lifted by TripleE treatment, blocked, counted and replated at a cell density of 1.5xl06in 6-wells Aggrewells (Stem Cells Technology), in 4 ml of the appropriate stage medium.

[0239] Prior to cell replating, Aggrewells were treated with anti-adherence cell rinsing solution (Stem Cell Technology cat#07010), centrifuged at 2,400 RPMI to eliminate air bubble, and washed x 3 with PBS. After cell seeding, plates were centrifuged at 1,400 RPMI for 7 minutes, and then carefully moved to a 5% CO2, 20% oxygen incubator.

[0240] At day 15 of differentiation, the cell aggregates were transferred to cell suspension culture plates in 5 ml of the appropriate stage medium and placed on a rotating platform at 95 RPMI until the end of differentiation (Stage 7). From Stage 1 to 6, all media and supplements were replaced every 24 hours. At stage 7, culture medium was replaced every 48 hours.Table 2Stage Day Medium Supplements dOStgl dl Basal 1 medium Activin A (100(MCDB131, ImM Glutamine, lOmM Glucose, ng / ml) CHIR-99021 1.5 g / 1 NaHCO3, BSA 0.5%) (3 pM)d2Basal 1 medium (same as above) Activin A (100ng / ml) CHIR-99021 (0.3 pM)d3Basal 1 medium (same as above) Activin A (100 ng / ml) Stg2 d4 Basal 1 medium (same as above) Vitamin C (0.25mM) FGF-7 (50 ng / ml)d5 Basal 1 medium (same as above) (same as above)MBHB Ref. No. 24-1170-WO UW 49850.02WO2d6 Basal 1 medium (same as above) (same as above)Stg3 d7 Basal 2 medium Vitamin C (MCDB131, ImM Glutamine, lOmM Glucose, (0.25mM) FGF-7 2.5 g / 1 NaHCO3, BSA 2%, ITS-X 0.5x) (50ng / ml)SANT 1(0.25 pM) Retinoic Acid ( 1 pM) LDN-193189 (100 nM)TPB (200 mM) d8 Basal 2 medium (same as above) Same as above Stg4 d9 Basal 2 medium (same as above) lO uM ROCKi Vitamin C (0.25mM) FGF-7 (2.0 ng / ml)hEGF (100 ng / ml) Activin A (10 ng / ml) Y -27632 (10 pM) Nicotinamide (10 mM) LDN-193189 (200 nM) SANT 1(0.25 pM) TPB (100 nM) Retinoic Acid (0.1 pM) dlO-12 Basal 2 medium (same as above) Same as above Stg5 dl3 Basal 3 Medium Betacellulin (20 ng / ml)(MCDB131, ImM Glutamine, 20mM Glucose, Y -27632 (10 pM) 1.5 g / 1 NaHCO3, BSA 2%, ITS-X 0.5x, 10 pM Retinoic Acid (0.05 pM) ZnSO4, 10 pg / ml heparin, Ix Pen / Strep) SANT1 (0.25 pM) LDN-193189 (100 nM) GC1 (1 pM) GSiXX (lOO nM) ALK5inhII (10 pM) dl4-dl6 (same as above) (same as above) Stg6 d 17-23 Basal 3 Medium Alk5inhII (10 uM) LDN-193189 (100 nM) GC1(1 pM) GSiXX (lOO nM)MBHB Ref. No. 24-1170-WO UW 49850.02WO2 Stg7 d24-45 Basal Stage 7 Medium N-acetylcysteine (CMRL 1066, GlutaMAX lx, lx Pen / Strep, ITS- (ImM) T3 (10 nM) X 0.5x, Sodium Pyruvate 0.5 mM, ZnSO4 10 ZM-447439 (0.5uM) pM, Heparin 10 pg / ml, Lipid Concentrate lx11905-031, Invitrogen), Trace Elements A lx(25-021-CI, Cellgro), Trace Elements B lx (99-175-CI, Cellgro), Endotoxin-free BSA 2%)

[0241] To enforce the transcriptional programs of interest using EBdCas9-engineered lines, the differentiation protocol was modified at select stages (FIG. 6A).

[0242] Briefly, for induction of EOMES at the DE stage, undifferentiated EBdCas9- engineered lines were lifted with TripleE and replated at a density of 1.5-2X106cells / well on Cultrex-coated 6 well plates in 2 ml of TeSERPlus medium containing lOpM ROCKi and DOX (2 pg / ml) for induction of EBdCas9. Two hundred and fifty microliters of Optimem medium containing 2 pg EOMES-specific gRNA (gRNA #1 or 5) and lOul of lipofectamine RNAiMax transfection reagent were then dropped on the cell suspension and plates centrifuged at 1,400 rpm for 7 minutes. The next day, medium was changed to Stage 1 medium containing DOX (2pg / ml) and cultures were continued up to Stage 3 (d7) with the appropriate culture media. At day 7, cells were harvested for histologic and flow cytometric assessment of DE markers (E- cadherin, SOX17, FOXA2, CXCR4).

[0243] To enforce PDX1 and NGN3 transcriptional programs using EBdCas9-engineered lines, the differentiation protocol was modified at select stages. Specifically, for induction of PDX1, cells at day 8 of differentiation were treated with DOX (2 pg / ml) to induce EBdCas9. The next day, cells from each 6-well were lifted with TripleE and replated in the appropriate culture medium in the presence of lOpM ROCKi and DOX (2 pg / ml) on a fresh Cultrex-coated 6-well in 2.5 ml of medium; 125 microliters of Optimem medium containing a combination of PDXl-specific gRNAs (gRNA #3,4,5, 1 pg / each) and 5 pl of lipofectamine RNAiMax transfection reagent were then dropped on the cell suspension and plates centrifuged at 1,400 rpm for 7 minutes.

[0244] For induction of NGN3, cells at day 11-12 of differentiation were lifted with TripleE and replated in Aggrewells in 2.5 ml of medium in the presence of lOpM ROCKi. One hundred and fifty microliters of Optimem medium containing NGN3-specific gRNA (gRNA#3, Ipg) and 5 pl of lipofectamine RNAiMax transfection reagent were then dropped on the cellMBHB Ref No. 24-1170-WO UW 49850.02WO2 suspension and plates centrifuged at 1,400 rpm for 7 minutes. To minimize toxicity, culture media were replaced 8 hours after transfection.

[0245] Induction of the transcriptional programs in non-engineered lines (e.g., Hl and CV-2A iPSCs) followed similar protocols of cell lifting and replating except DOX was omitted. In this case, EBdCas9-mRNA (3 pg) and gRNAs (1 pg) were combined in 125 pl Optimem containing 5 pl of Lipofectamine MessengerMax transfection reagent (Invitrogen, LMRNA003) and the mixture dropped onto the cells in 2.5 ml culture medium. Plates were then centrifuged at 1,400 rpm for 7 minutes and returned to the incubator for 8 hours, after which culture medium was changed.

[0246] RNA extraction and RT-qPCR analysis

[0247] RNA was extracted using RNAeasy (Qiagen) according to the manufacturer's instructions. RNA samples were treated with Rnase-free DNase (Ambion), phenol-chloroform extracted, ethanol precipitated, resuspended in Rnase-free water and quantified using a Nanodrop ND-1000. Reverse transcription was performed using iScript (BioRad). 10 ng of cDNA was used to perform qRT-PCR using Sensimix SYBR Hi-ROX Kit (Meridian Biosciences) and an Applied Biosystems 7300 real-time PCR system.

[0248] The following gene-specific primers were used: EOMES Fw: CTGCCCACTACAATGTGTTCG (SEQ ID NO:33); EOMES Rev: GCGCCTTTGTTATTGGTGAGTTT (SEQ ID NO:34); PDX1 Fw: CAACAAGTACATCTCACGGC (SEQ ID NO:35); PDX1 Rev: CCTCCTCCTTTTTCCACTTCA (SEQ ID NO: 36); NGN3 Fw: CTAAGAGCGAGTTGGCACTGA (SEQ ID NO: 37); NGN3 Rev: GAGGTTGTGCATTCGATTGCG (SEQ ID NO: 38); NEURODI Fw: ATGACCAAATCGTACAGCGAG (SEQ ID NO:39); NEURODI Rev: GTTCATGGCTTCGAGGTCGT (SEQ ID NO:40); NKX2.2 Fw: GTCAGGGACGGCAAACCAT (SEQ ID NO:41); NKX2.2 Rev: GCGCTGTAGGCAGAAAAGG (SEQ ID NO:42); INS Fw: GCAGCCTTTGTGAACCAACAC (SEQ ID NO:43); INS Rev: CCCCGCACACTAGGTAGAGA (SEQ ID NO:44); GCG Fw: CTGAAGGGACCTTTACCAGTGA (SEQ ID NO:45); GCG Rev: CCTGGCGGCAAGATTATCAAG (SEQ ID NO:46); SST Fw:MBHB Ref. No. 24-1170-WO UW 49850.02WO2 ACCCAACCAGACGGAGAATGA (SEQ ID NO:47); SST Rev: GCCGGGTTTGAGTTAGCAGA (SEQ ID NO:48); PPY Fw: CCTGCGTGGCTCTGTTACTAC (SEQ ID NO:49); PPY Rev: CCTGGTCAGCATGTTGATGTATC (SEQ ID NO:50); 18S Fw: GTAACCCGTTGAACCCCATT (SEQ ID N0:51); 18S Rev: CCATCCAATCGGTAGTAGCG (SEQ ID NO:52); qRT-PCR conditions were: stage 1, 50 °C for 2 min; stage 2, 95 °C for lOmin; Stage 3: 95 °C for 15sec, 60 °C for 30 sec (40 Cycles). For each primer combination, amplification efficiency was consistently >95%. Threshold cycle numbers (Ct) were determined using the SDS 2.3 software (Applied Biosystems) and analyzed using the AACt method.

[0249] CUT& Tag assays

[0250] Duplicate samples of stem cell clusters, induced to express EBdCas9 alone or PDX1 / NGN3 -guided EBdCas9, were harvested 48 hours after NGN3 gRNA transfection (day 14) and dissociated into single cells by TrypLE. Five hundred thousand cells per condition were then processed for CUT& Tag using the CUT& Tag-IT kit (Active Motif), as per manufacturer's instructions.

[0251] A Rabbit polyclonal specific for H3K27ac (Active Motif 39133) was used to isolate 300-500 bp chromatin fragments. DNA libraries were PCR amplified using a unique combination of i5 / i7 indexing primers for each sample, cleaned using SPRI beads and pooled into an equimolar library for sequencing.

[0252] Libraries were sequenced with an Illumina NextSeq 2000 (San Diego, CA) using XLEAP chemistry. More than 7 million paired end reads per sample were demultiplexed and converted to fastq format using BCL Convert v2.5.0 software through Illumina's BaseSpace Sequencing Hub.

[0253] FASTQ files were trimmed with TrimGalore and mapped with Bowtie2 to hg38. Mapped files were converted to bed file using BEDTools prior to peak calling with SEACR, selecting the top 95% of regions. Common peaks between replicates were defined as the union regions between replicates. Unique peaks were defined as non-overlapping peaks in one sample compared to another.MBHB Ref. No. 24-1170-WO UW 49850.02WO2

[0254] HOMER was used to annotate peaks to nearest predicted gene target. BAM files were converted to bigwigs using DeepTools for visualization on UCSC Genome Browser. Heatmaps of enrichment were generated using DeepTools.

[0255] Bulk RNA-seq and analysis

[0256] Bulk RNA-seq was performed on differentiated MEL and Hl PSC (day 20), induced to express EBdCas9, EBdCas9 guided sequentially by PDX1 / NGN3 gRNAs or EBdCas9 guided by either PDX1 or NGN3 gRNAs. RNA was collected using an RNAeasy Minikit (Qiagen) and library preparation and sequencing was performed by Novogene (Sacramento, CA, USA).

[0257] Samples were sequenced at an averaged 30 million raw reads using Illumina NovaSeq X Plus platform. Reads were aligned to the human reference genome (hg38) using HISAT2. Gene expression analysis was performed using DESeq2 vl.46.0.

[0258] Gene set enrichment analysis was performed using clusterProfiler v4.14.6 leveraging Reactome gene sets from MSigDB (msigdbr v25.1.1). Nominal enrichment P-value <0.05 was used to designate significance.

[0259] Immunofluorescence and flow cytometry

[0260] SC-derived islet clusters were dissociated into single-cell suspensions by TripleE treatment and fixed in BD Cytofix / Cytoperm solution for 20 minutes on ice. Cells were then washed in lx BD Perm / Wash Buffer, pelleted, resuspended in BD Cytoperm Permeabilization Plus Solution and incubated for 10 minutes on ice.

[0261] After washings in lx BD Perm / Wash Buffer, samples were blocked with rabbit and mouse IgGs for 15 minutes on ice, and then stained with PE-conjugated rabbit monoclonal anti-insulin (clone EPR17359, abeam ab213192) and Alexa647-conjugated mouse anti-Glucagon antibody (Clone 181402, R& D IC1249R) diluted in lx BD Perm / Wash Buffer for 1 hour at room temperature. After washings, cells were resuspended in HBSS / 0.1%BSA and analyzed at a FACScalibur (Becton Dickinson).

[0262] Tissue immunostaining and microscopy

[0263] SC-derived islet clusters were fixed in 4% PFA overnight at 4°C and embedded in paraffin for histology. Seven-micron sections were cut and processed for immunofluorescence.MBHB Ref. No. 24-1170-WO UW 49850.02WO2 Primary antibodies included guinea pig anti-insulin (A0564, Dako), mouse anti-glucagon (Sigma, clone K79bB10), Rabbit anti-Glucagon (Abeam, ab92517), rat anti-Somatostatin (R& D MAB2358), goat-anti-PPY (R& D AF6297), goat anti-PDXl (Abeam, ab47383), mouse anti-E-cadherin Ab (BD 610182, clone 36 / Ecadherin), rabbit anti-Chromogranin (Abeam, ab45179), mouse anti-NKX6.1 (DSHB, clone F55A10), Rabbit anti-CDX2 (Abeam, ab76541), Rat anti-SOX2 (eBioscience, clone Btjce).

[0264] Binding of primary antibodies was revealed with Fab2-species-specific Alexa 647-, Rhodamine- and Alexa 488-conjugated donkey secondary antibodies. After staining, slides and coverslips were counterstained with DAPI, mounted and visualized either at a NIKON Eclipse-190 or at a confocal NIKON AIR microscope equipped with a Spot II CCD camera.

[0265] Morphometric analysis was performed on 10-12 sections per sample collected at approximately 50-pm intervals throughout each block, using the Spot Advanced and ImageProPlus software.

[0266] Tissue transplantation, in vivo Glucose Tolerance Tests and Insulin secretion

[0267] Day 35-45 SC-derived islet clusters were transplanted under the kidney capsule of immunodeficient NSG mice via a minimal 5mm skin incision on the left flank of the animal and exposure of the kidney. Tissue was then injected under the kidney capsule using a blunt plastic microcapillary. The muscle wall was sutured with absorbable sutures and the skin closed with non-absorbable sutures.

[0268] At 3 and 6-months post-transplantation, glucose tolerance tests (GTT) were performed on 5-hour fasted animals by intra-peritoneal injection of a glucose solution (1.5 mg / g of body weight) and glycemia measured at 0, 15, 30, 60, 90 and 120 post glucose load. Blood glucose levels were monitored by tail prick using a FreeStyle glucose monitoring system (Abbott Diabetes Care Inc., Alameda, CA).

[0269] Plasma levels of human C-peptide were measured at 0 and 30' post glucose load using an ultra-sensitive insulin human-specific ELISA kit (Alpco, Salem, NH).

[0270] Chromatin immunoprecipitation and Chip-qPCR

[0271] DoubleFOXA2INSPSCs (300 X106 / well of a 6 well plate) were plated in TeSER in the presence of DOX (2 pg / ml). The next day, medium was replaced with DMEM-10% FCS plusMBHB Ref. No. 24-1170-WO UW 49850.02WO2 DOX (2 ml / well) and cell transfected with PDX1- or NGN3-specific gRNAs using 2 pg of each gRNA and 10 pl of lipofectamine RNAiMax transfection reagent in 250 ul of Optimem.

[0272] 24-72 hours after transfection, cells were harvested using TripleE, blocked in medium and pelleted by centrifugation at 1400 rpm. Cell pellets were washed with PBS and cross-linked by adding 500 ul 1% formaldehyde in PBS for 20min at RT. After centrifugation, cell pellets were resuspended in 500 ul of PBS / glycine (125mM) and incubated for 5 min at RT for quenching. Supernatant was removed and the cells were washed with 500 ul of PBS. PBS was removed and samples were stored at -80°C.

[0273] For shearing, cells were resuspended in 100ul chromatin sheering buffer (Active Motif), transferred into wells of a 96 well plates and placed in PIXUL for shearing. PIXUL parameters were as follows: Cycles = 50; PRF = 1kHz; Burst = 20 for 6 min x 4.

[0274] Sheered chromatin samples were added to a UV- treated polypropylene 96-well microplate in blocking buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, NP- 40 (0.5% vol / vol), Triton X-100 (1.0% vol / vol), 5% BSA, sheared salmon sperm DNA (10pg / pL final)) and incubated in ultrasonic bath for 60 min at 4 °C in the presence of matrix -Chip antibodies. A duplicate set of wells were prepared by coating the plastic with protein A and blocked in the same blocking buffer. The blocking buffer was aspirated from the protein A-coated plate, and the chromatin + antibody mix from the first set of wells was transferred to the protein A-coated wells and incubated in the ultrasonic bath for 60 min at 4°C. The wells were then washed 3 times with immunoprecipitation buffer followed by 3 washes with TE buffer. Finally, elution buffer containing 25 mM Tris base, 1 mM EDTA (pHlO) with proteinase K 200 pg / mL was added to the wells, centrifugated for 30 s at 1400 rpms and incubated for 45 min at 55 °C and then 10 min at 95 °C. After mixing, the 96-well plates were centrifuged for 3 min at ~ 500g at 4 °C and used for PCR. The antibodies used for Matrix ChIP were: H3K27me3 (Active motif 39155), EZH2 (Cell Signaling D2C9), H3K27ac (Active motif 39133) control Mouse IgG (Vector Lab, Cat#: 1-2000). Matrix ChIP experiments were performed in triplicate followed by qPCR in 4-8 replicates. Primers used for Chip-qPCR were: PDX1 Fw: CGTTCAGGAGTGTGCAGCAA (SEQ ID NO:55); PDXl-Rev: CTAAGAGGCTAGGCCCAGGT (SEQ ID NO:56); NGN3 Fw: CGCACAGGAAGATAGTGGCA (SEQ ID NO:57); NGN3 Rev: GAGCAGGGCGTCCTTTAGAA (SEQ ID NO: 58); H19-ICR Fw:MBHB Ref. No. 24-1170-WO UW 49850.02WO2 GAGCCGCACCAGATCTTCAG (SEQ ID NO:59; H19 ICR Rev: TTGGTGGAACACACTGTGATC (SEQ ID NO:60).

[0275] Immunofluorescence and flow cytometric analysis

[0276] SC-derived islet clusters were dissociated into single- cell suspensions by TripleE treatment and fixed in BD Cytofix / Cytoperm™ Solution for 20 minutes in ice. Cells were then washed in lx BD Perm / Wash™ Buffer, pelleted, resuspended in BD Cytoperm™ Permeabilization Plus Solution and incubated for 10 minutes in ice. After washings in lx BD Perm / Wash™ Buffer, samples were blocked with rabbit and mouse IgGs for 15 minutes in ice, and then stained with PE-conjugated rabbit monoclonal anti -Insulin (clone EPR17359, abeam #ab 213192) and Alexa-647-conjugated mouse anti-Glucagon antibody (Clone # 181402, R& D #IC1249R) diluted in lx BD Perm / Wash™ Buffer for 1 hour at room temperature. After washings, cells were resuspended in HBSS / 0.1%BSA and analyzed at a FACScalibur (Beckton Dickinson).

[0277] Tissue Immunostaining and microscopy

[0278] SC-derived islet clusters and tissue transplants were fixed in 4% PFA overnight at 4 °C and either embedded in OCT or paraffin for histology. Five to seven-micron sections were cut and processed for immunofluorescence. Briefly, sections were first subjected to antigen retrieval by boiling in citrate buffer (10 mM citrate 0.05% Tween20, pH 6.0) for 30 minutes followed by cooling to room temperature for 1 hour. Sections were then permeabilized in 0.05% Triton-X 100 and blocked in 50 mM glycine for 10 minutes at room temperature, followed by incubation in PBS / 1% BSA / 2% donkey serum for 1 hour at room temperature. Tissue sections were then incubated overnight at 4°C with primary antibodies in blocking buffer. Primary antibodies included: guinea pig anti-insulin (A0564, Dako), mouse antiglucagon (Sigma, clone K79bB10), rat anti-Somatostatin, R& D MAB2358), goat-anti-PPY (R& D AF6297), goat anti-PDXl (Abeam, ab47383), mouse anti-E-cadherin Ab (BD 610182, clone 36 / Ecadherin), rabbit anti-Chromogranin, mouse anti- NKX6.1 (BCB, AB2021), rabbit anti-MAFA (Bethyl IHC-00352), rabbit-anti-MAFB (Bethyl, IHC-00351), mouse- anti-NKX2.2 (DSHB, #74.5A5), mouse anti-NGN3 (DSHB, F25A1B3). Binding of primary antibodies was revealed with Fab2-species-specific Alexa 647-, Rhodamine- and Alexa 488-conjugated donkey secondary antibodies. After staining, slides and coverslips were counterstained with DAPI, mounted and visualized either at a NIKON Eclipse-i90 or at aMBHB Ref. No. 24-1170-WO UW 49850.02WO2 confocal NIKON AIR microscope equipped with a Spot II CCD camera. Morphometric analysis was performed on at least 12 tissue sections per sample collected at approximately 50 pm intervals throughout each block, using the Spot Advanced and ImageProPlus software.

[0279] Glucose-Stimulated-Insulin-Secretion Assays

[0280] For in vitro glucose stimulation, 20 islet-like clusters from d24 and d35 of SC differentiation, were first cultured for 2 hours in Krebs-Rings Buffer (KRB) (10 mM HEPES, 1.19 mM MgSO4, 1.19 mM NaCl, 4.74 mM KC1, 1.19 mM KH2PO4, 2.54 mM CaCl2-2H2O, 25 mM NaHCO₃, pH 7.4) 2.8 mM glucose, and subsequently incubated for 30 minutes in KRB 2.8 mM glucose followed sequentially by either 30 minutes incubation in KRB-16 mM glucose, 30 minutes incubation in KRB-16 mM glucose plus lOmM Arginine, and finally 30 minute incubation in the presence of 16mM glucose and 30mM KC1. Supernatants resulting from each incubation were collected and stored at -80 °C. For insulin content, the 20 islet-like clusters were lysed by acid / ethanol extraction and frozen at -80°C until analysis. Insulin in the culture supernatants and cell extracts was measured using an insulin ELISA kit (Mercodia, Uppsala, Sweden). Results were normalized to protein content measured by a BCA protein assay (PierceTM, Thermo Scientific).

[0281] Seahorse Cellular Flux assays

[0282] Stage 7 SC-derived islet clusters were dissociated into single cells with TripleE, plated at a density of 20,000 / well on Cultrex-coated Seahorse XF96 cell culture microplates and cultured at 37 °C in Stage 7 medium overnight. The next day, the medium was changed to KRB 3mM Glucose and cells cultured at 37 °C for 90 minutes. Before the assay, medium was changed again to KRB 3mM Glucose after which OCR was measured in response to sequential stimulation of the cells with 3mM Glucose, 16.7 mM Glucose, 10 mM glutamine & 5 mM leucine, Oligomycin (2 pM), FCCP (2 pM) and rotenone (1 pM). For inter-sample comparisons, OCR was normalized to DNA content of each well measured by Hoechst staining using a plate reader equipped with fluorescence filters for 355 nm excitation and 460 nm emission.

[0283] Tissue transplantation, in vivo Glucose Tolerance Tests and Insulin secretion

[0284] Day 24 or Day 35-45 SC-derived islet clusters were transplanted under the kidney capsule of immunodeficient NGS mice via a minimal (~ 5mm) skin incision on the left flankMBHB Ref. No. 24-1170-WO UW 49850.02WO2 of the animal and exposure of the kidney. Tissues was then injected under the kidney capsule using a blunt plastic microcapillary. The muscle wall was sutured with absorbable sutures and the skin closed with non-absorbable sutures.

[0285] At 2-, 4- and 6-months post-transplantation, glucose tolerance tests (GTT) were performed on 5 hours fasted animals by intra-peritoneal injection of a glucose solution (1.5 mg / g of body weight) and glycemia measured at O', 15’, 30’, 60’ 90’ and 120’ post glucose load. Blood glucose levels were monitored by tail prick using a FreeStyle glucose monitoring system (Abbott Diabetes Care Inc., Alameda, CA). Plasma levels of human C-peptide was measured at O' and 30' post glucose load using an ultra-sensitive insulin human-specific ELISA kit (Alpco, Salem, NH).

[0286] The present disclosure demonstrates that the EBdCas9 epigenetic modifier of the disclosure can be used to overcome critical bottlenecks in the differentiation of pluripotent stem cells into pancreatic islet cells. The present disclosure demonstrates (at least) that: (1) EBdCas9-mediated epigenetic interventions are highly reproducible across multiple independent PSC lines of diverse genetic backgrounds, including hESCs (MELI, Hl) and iPSCs (CV-2A); (2) genome-wide chromatin profiling reveals extensive H3K27ac remodeling at developmentally relevant loci, with enrichment of pathways critical for endocrine differentiation, metabolic maturation, and suppression of alternative lineage fates; (3) transcriptomic analysis confirms activation of NGN3 -dependent gene networks, including genes involved in exocytosis / insulin secretion, mitochondrial function / metabolism, and developmental transcription factors; (4) sequential targeting of PDX1 and NGN3 promoters enforces pancreatic over gut lineage choices, significantly reducing CDX2+ intestinal and SOX2+ gastric contaminants while suppressing enteroendocrine cell types; (5) VP64-mediated gene activation is not functionally equivalent to EBdCas9-mediated epigenetic remodeling, highlighting the critical importance of PRC2 inhibition for achieving enhanced differentiation outcomes; and (6) in vivo functional maturation is accelerated, with epigenetically edited SC-islets demonstrating tighter control of basal insulin secretion, enhanced glucose responsiveness, and significantly higher stimulation indexes compared to controls.

[0287] Collectively, these findings reveal the existence of PRC2-dependent epigenetic barriers at multiple stages of pancreatic endocrine differentiation and establish gene-targeted epigenetic modifiers as powerful tools to accelerate differentiation, decrease heterogeneity, and refine the cell composition of islet tissue derived from directed differentiation of stem cells.MBHB Ref. No. 24-1170-WO UW 49850.02WO2 The methods described herein have significant translational implications for the production of functional islet tissue for cell replacement therapies in diabetes.

[0288] The foregoing examples are illustrative of the present disclosure and are not to be construed as limiting thereof. Although the disclosure has been described in detail with reference to preferred embodiments, variations and modifications exist within the scope and spirit of the disclosure as described and defined in the following claims.

Claims

MBHB Ref. No. 24-1170-WO UW 49850.02WO2 What is claimed is:In the Claims:

1. A method of generating an endocrine progenitor (EP) stage cell from a pluripotent stem cell (PSC), comprising:i) inducing expression in the PSC of a transcriptional regulator of a definitive endoderm (DE) stage cell to differentiate the PSC into a DE stage cell, to a primitive gut tube (PGT) stage cell, and then to a posterior foregut (PF) stage cell; ii) inducing expression in the PF stage cell of a transcriptional regulator of a pancreatic progenitor (PP) stage cell to differentiate the PF stage cell into PP stage cell;iii) inducing expression in the PP stage cell of a transcriptional regulator of a EP stage cell to differentiate the PP stage cell into a EP stage cell;wherein binding of (i) a guide RNA (gRNA) complementary to a regulatory region of the transcriptional regulator of the DE stage cell, of the PP stage cell, or the EP stage cell and (ii) a fusion protein (EBdCas9) comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain together induce expression of the transcriptional regulator of the DE stage cell, of the PP stage cell, or the EP stage cell.

2. A method of generating an endocrine progenitor (EP) stage cell from a primitive gut tube (PGT) stage cell or a posterior foregut (PF) stage cell, comprising:i) inducing expression in the PGT stage cell or PF stage cell of a transcriptional regulator of a pancreatic progenitor (PP) stage cell to differentiate the PGT stage cell or PF stage cell into PP stage cell;ii) inducing expression in the PP stage cell of a transcriptional regulator of a EP stage cell to differentiate the PP stage cell into a EP stage cell;wherein binding of (i) a guide RNA (gRNA) complementary to a regulatory region of the transcriptional regulator of the PP stage cell, or the EP stage cell and (ii) a fusion protein (EBdCas9) comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain together induce- 77 -MBHB Ref. No. 24-1170-WO UW 49850.02WO2 expression of the transcriptional regulator of the PP stage cell, or the EP stage cell.

3. The method of claim 1, wherein the transcriptional regulator of a DE stage cell comprises GSC, EOMES, SOX17, GATA6, orFOXA2.

4. The method of claim 1 or claim 2, wherein the transcriptional regulator of a PP stage cell comprises PDX1, NKX6.1, or PAX4.

5. The method of claim 4, wherein the transcriptional regulator of a PP stage cell comprises PDX1.

6. The method of any one of claims 1-5, wherein the transcriptional regulator of a EP stage cell comprises NGN3, NKX2.2 or NEURODI.

7. The method of claim 6, wherein the transcriptional regulator of a EP stage cell comprises NGN3.

8. The method of claim 1, wherein the transcriptional regulator of a DE stage cell is EOMES, the transcriptional regulator of a PP stage cell is PDX, and the transcriptional regulator of a EP stage cell is NGN3.

9. The method of any one of claims 1, 3-7, or 8, whereina) the inducing expression in the PSC of a transcriptional regulator of a DE stage cell of step i) is performed on day 0-3;b) the inducing expression in the PF stage cell of a transcriptional regulator of a PP stage cell of step ii) is performed on between about day 5 to day 10;c) the inducing expression in the PP stage cell of a transcriptional regulator of a EP stage cell of step iii) is performed between about 2 to 5 days after step b).

10. The method of any one of claims 2, 4, 5, or 7, whereinMBHB Ref. No. 24-1170-WO UW 49850.02WO2 inducing expression in the PP stage cell of a transcriptional regulator of a EP stage cell of step ii) is performed between about 2 to 5 days after inducing expression in the PF stage cell of a transcriptional regulator of a PP stage cell.

11. The method of any one of claims 1, 3-8, or 9, wherein the transcriptional regulator of a DE stage cell is EOMES, and the gRNA complementary to a regulatory region for EOMES is a sequence at least about 90% identical, or at least about 95% identical to SEQ ID NO:24 and SEQ IDNO:28.

12. The method of any one of claims 1, 3-9, or 10, wherein the transcriptional regulator of a DE stage cell is EOMES, and the gRNA complementary to a regulatory region for EOMES is selected from the group consisting of SEQ ID NO:24 and SEQ ID NO:28.

13. The method of any one of claims 1-12, wherein the transcriptional regulator of a PP stage cell is PDX1, and the gRNA complementary to a regulatory region for PDX1 is a sequence at least about 90% identical, or at least about 95% identical to SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11.

14. The method of any one of claims 1-13, wherein the transcriptional regulator of a PP stage cell is PDX1, and the gRNA complementary to a regulatory region for PDX1 is selected from the group consisting of SEQ ID NO:9, SEQ ID NO: 10, and SEQ ID NO: 11.

15. The method of any one of claims 1-14, wherein the transcriptional regulator of a EP stage cell is NGN3, and the gRNA complementary to a regulatory region for NGN3 is a sequence at least about 90% identical, or at least about 95% identical to SEQ ID NO: 17.

16. The method of any one of claims 1-15, wherein the transcriptional regulator of a EP stage cell is NGN3, and the gRNA complementary to a regulatory region for NGN3 is SEQ ID NO: 17.

17. The method of any one of claims 1-16, wherein the fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain is originally transcribed from a nucleic acid sequence that comprises a sequence atMBHB Ref. No. 24-1170-WO UW 49850.02WO2 least about 90% identical, or at least about 95% identical, or at least about 99% identical to SEQ ID NO:32.

18. The method of any one of claims 1-16, wherein the fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain is originally transcribed from a nucleic acid sequence that comprises SEQ ID NO:32.

19. The method of any one of claims 1-16, wherein the fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain comprises an amino acid sequence that is at least about 90% identical, or at least about 95% identical, or at least about 99% identical to SEQ ID NO:53.

20. The method of any one of claims 1-16, wherein the fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain comprises SEQ ID NO:53.

21. A method of inducing differentiation of a pluripotent stem cell (PSC) in culture to a definitive endoderm (DE) stage cell, comprising:i) introducing to the PSC a gRNA for a transcriptional regulator of a DE stage cell selected from GSC, EOMES, SOX17, GATA6, or FOXA2;ii) introducing to the PSC a fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain;iii) inducing expression of the transcriptional regulator.

22. The method of claim 2, wherein the transcriptional regulator of a DE stage cell is EOMES, and the gRNA complementary to a regulatory region for EOMES is a sequence at least about 90% identical, or at least about 95% identical to SEQ ID NO:24 and SEQ ID NO:28.

23. The method of claim 2 or claim 22, wherein the transcriptional regulator of a DE stage cell is EOMES, and the gRNA complementary to a regulatory region for EOMES is selected from the group consisting of SEQ ID NO:24 and SEQ ID NO:28.MBHB Ref. No. 24-1170-WO UW 49850.02WO2 24. A method of inducing differentiation of a primitive gut tube (PGT) stage cell or a posterior foregut (PF) stage cell in culture to a pancreatic progenitor (PP) stage cell, comprising:i) introducing to the PGT or PF stage cell a gRNA complementary to a regulatory region of a transcriptional regulator of a PP stage cell selected from PDX1, NKX6.1, and PAX4;ii) introducing to the PGT or PF stage cell a fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain;iii) inducing expression of the transcriptional regulator.

25. The method of claim 24, wherein the gRNA for a transcriptional regulator of a PP stage cell comprises a gRNA complementary to a regulatory region of PDX1.

26. The method of claim 24, wherein the gRNA for a transcriptional regulator of a PP stage cell is a gRNA complementary to a regulatory region of PDX1.

27. The method of any one of claims 24-26, wherein the transcriptional regulator of a PP stage cell is PDX1, and the gRNA complementary to a regulatory region for PDX1 is a sequence at least about 90% identical, or at least about 95% identical to SEQ ID NO: 9, SEQ ID NO:10, and SEQ ID NO:1128. The method of any one of claims 24-26, wherein the transcriptional regulator of a PP stage cell is PDX1, and the gRNA complementary to a regulatory region for PDX1 is selected from the group consisting of SEQ ID NO: 9, SEQ ID NO:10, and SEQ ID NO:11.

29. A method of inducing differentiation of a pancreatic progenitor (PP) stage cell to a endocrine progenitor (EP) stage cell, comprising:i) introducing to the PP stage cell a gRNA complementary to a regulatory region of a transcriptional regulator of a EP stage cell selected from NGN3, NKX2.2 and NEURODI;MBHB Ref. No. 24-1170-WO UW 49850.02WO2 ii) introducing to the PP stage cell a fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain;iii) inducing expression of the transcriptional regulator.

30. The method of claim 22, wherein the gRNA for a transcriptional regulator of a EP stage cell comprises a gRNA complementary to a regulatory region of NGN3.

31. The method of claim 22, wherein the gRNA for a transcriptional regulator of a EP stage cell is a gRNA complementary to a regulatory region of NGN3.

32. The method of any one of claims 29-31, wherein the transcriptional regulator of a EP stage cell is NGN3, and the gRNA complementary to a regulatory region for NGN3 is a sequence at least about 90% identical, or at least about 95% identical to SEQ ID NO: 17.

33. The method of any one of claims 29-31, wherein the transcriptional regulator of a EP stage cell is NGN3, and the gRNA complementary to a regulatory region for NGN3 is SEQ ID NO: 17.

34. The method of any one of claims 21-33, wherein the fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain is originally transcribed from a nucleic acid sequence that comprises a sequence at least about 90% identical, or at least about 95% identical, or at least about 99% identical to SEQ ID NO:32.

34. The method of any one of claims 21-33, wherein the fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain is originally transcribed from a nucleic acid sequence that comprises SEQ ID NO:32.

35. The method of any one of claims 21-33, wherein the fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain comprises an amino acid sequence that is at least about 90% identical, or at least about 95% identical, or at least about 99% identical to SEQ ID NO:53.MBHB Ref. No. 24-1170-WO UW 49850.02WO236. The method of any one of claims 21-33, wherein the fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain comprises SEQ ID NO:53.

37. The method of any one of claims 1-36, wherein the fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain and the gRNA reduce methylation of histone 3, lysine 27 (H3K27me3) to induce expression of a transcriptional regulator.

38. The method of any one of claims 1-36, wherein the fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain and the gRNA reduce histone methyltransferase activity of EZH2 subunits of Polycomb Repressive Complex 2 (PRC2) to induce expression of a transcriptional regulator.

39. A cell produced according to the method of any one of claims 1-38.

40. The cell of claim 39, wherein the cell is an a-cell, P-cell, 5-cell or y-cell.

41. The cell of claim 39 or claim 40, wherein the cell is a P-cell.

42. The cell of any one of claims 39-41, wherein the cell produces insulin.

43. The cell of claim 39 or claim 40, wherein the cell is an a-cell.

44. The cell of claim 39 or claim 43, wherein the cell produces glucagon.

45. The cell of claim 39 or claim 40, wherein the cell is an 5-cell.

46. The cell of claim 39 or claim 45, wherein the cell produces somatostatin.

47. A composition comprising cells of any one of claims 39-46.MBHB Ref. No. 24-1170-WO UW 49850.02WO2 48. A composition comprising cells produced according to the method of any one of claims 1-38.

49. The composition of claim 47 or claim 48, further comprising a pharmaceutically acceptable excipient.

50. A method of treating diabetes in a subject in need thereof, comprising:i) obtaining cells of any one of claims 39-42, or a composition of claim 47 or claim 48;ii) administering the cells or composition to the subject in an amount effective to treat diabetes.

51. A method, comprising:i) obtaining cells of any one of claims 39-42, or a composition of claim 47 or claim 48;ii) administering the cells or composition to a subject in an amount effective to treat diabetes;wherein the administered cells increase insulin production in the subject.

52. A method, comprising:i) obtaining cells of any one of claims 39, 40, 43, or 44, or composition of claim 47 or claim 48;ii) administering the cells or composition to a subject in an amount effective to replace dysfunctional glucagon-producing cells in the subject.

53. A method, comprising:i) obtaining cells of any one of claims 39, 40, 45, or 46, or composition of claim 47 or claim 48;ii) administering the cells or composition to a subject in an amount effective to replace dysfunctional somatostatin-producing cells in the subject.

54. The method of any one of claims 1-38, wherein the EP stage cell is an insulin-producing 3-cell.MBHB Ref. No. 24-1170-WO UW 49850.02WO255. The method of any one of claims 1-38, wherein at least about 20 percent, about 25 percent, or about 30% of EP stage cells are insulin-producing P-cell by day 19 after inducing expression in the PSC of a transcriptional regulator of a DE stage cell.

56. The method of claim 55, wherein at least about 25 percent of EP stage cells are insulinproducing P-cell by day 19 after inducing expression in the PSC of a transcriptional regulator of a DE stage cell.

57. The method of any one of claims 1-38, wherein the EP stage cells are selectively enriched for P-cells, a-cells, or P-cells and a-cells over 5-cells and y-cells.

58. The method of claim 57, wherein P-cell abundance is increased at least about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold over comparable controls.

59. The method of claim 58, wherein P-cell abundance is increased at least about 4-fold, about 5-fold, or about 6-fold over comparable controls.

60. The method of any one of claims 1-38, wherein the fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain and the gRNA increase acetylation of histone 3, lysine 27 (H3K27ac) at genomic loci proximal to or distal from the targeted regulatory region.

61. The method of claim 60, wherein the increased H3K27ac modifications occur at promoter, enhancer, or intronic regions of genes selected from the group consisting of NGN3, NKX2.2, GCK, NEURODI, and genes involved in exocytosis, insulin secretion, mitochondrial function, or metabolism.

62. The method of any one of claims 1-38, wherein the fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain and the gRNA selectively reduce the frequency of uncommitted PDX1-negative / NKX6.1 -negative cells in differentiating cell populations.MBHB Ref. No. 24-1170-WO UW 49850.02WO263. The method of any one of claims 1-38, wherein the fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain and the gRNA selectively reduce the frequency of CDX2-positive / PDXl -negative gut lineage cells in differentiating cell populations.

64. The method of claim 63, wherein the gRNA is complementary to a regulatory region of PDX1.

65. The method of any one of claims 1-38, wherein the fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain and the gRNA selectively reduce the frequency of SOX2-positive / PDXl -negative gastric or anterior foregut lineage cells in differentiating cell populations.

66. The method of claim 65, wherein the gRNA is complementary to a regulatory region of NGN3.

67. The method of any one of claims 1-38, wherein the fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain and the gRNA selectively reduce the frequency of enteroendocrine lineage cells in differentiating cell populations.

68. The method of claim 67, wherein the enteroendocrine lineage cells express tyrosine hydroxylase (TH) and wherein sequential targeting of PDX1 followed by NGN3 reduces enteroendocrine cell frequency compared to targeting NGN3 alone.

69. The method of any one of claims 1-38, wherein the method results in EP stage cells exhibiting increased expression of NGN3 target genes involved in exocytosis, insulin secretion, mitochondrial function, or metabolism compared to controls.

70. The method of claim 69, wherein the NGN3 target genes comprise at least 10, at least 50, at least 100, or at least 150 genes selected from a set of 1,263 predicted or validated NGN3 target genes.MBHB Ref. No. 24-1170-WO UW 49850.02WO271. The method of any one of claims 1-38, wherein the method results in EP stage cells or differentiated endocrine cells exhibiting enhanced mitochondrial function compared to comparable controls.

72. The method of claim 71, wherein the enhanced mitochondrial function comprises at least one of:(a) increased ATP production-coupled respiration;(b) reduced proton leak; or(c) increased coupling efficiency.

73. The method of any one of claims 1-38, wherein the method results in insulin-producing cells that exhibit glucose-stimulated insulin secretion in vitro.

74. The method of claim 73, wherein the glucose-stimulated insulin secretion is measured in response to D-Glucose, L- Arginine, or KC1.

75. The method of any one of claims 1-38, wherein the method results in insulin-producing cells that, when transplanted into an immunodeficient animal, exhibit glucose-responsive insulin secretion in vivo within 3 months of transplantation.

76. The method of claim 75, wherein the insulin-producing cells exhibit:(a) lower basal insulin secretion at fasting compared to control cells produced without gRNA-mediated targeting; and(b) increased insulin secretion in response to glucose challenge compared to control cells.

77. The method of claim 75 or claim 76, wherein the insulin-producing cells exhibit a stimulation index (ratio of C-peptide secreted after glucose load to C-peptide at fasting) that is significantly higher than control cells produced without gRNA-mediated targeting, e.g., at least 2-fold higher.MBHB Ref. No. 24-1170-WO UW 49850.02WO2 78. The method of any one of claims 1-38, wherein the introducing of the fusion protein and / or the gRNA is performed by transient mRNA transfection.

79. The method of claim 78, wherein the fusion protein is introduced as mRNA encoding the fusion protein and the mRNA is modified to comprise Nl-Methyl-Pseudouri dine.

80. The method of any one of claims 1-38, wherein the PSC is a human embryonic stem cell (hESC) or a human induced pluripotent stem cell (iPSC).

81. The method of claim 80, wherein the method is reproducible across multiple independent PSC lines of diverse genetic backgrounds.

82. The method of any one of claims 1-38, wherein the PSC is engineered to comprise a nucleic acid encoding the fusion protein under control of an inducible promoter integrated at a safe harbor locus.

83. The method of claim 82, wherein the safe harbor locus is the AAVS1 locus and the inducible promoter is a doxycycline-inducible promoter.

84. The method of claim 82 or claim 83, wherein the nucleic acid encoding the fusion protein further comprises a nucleic acid encoding a detectable marker.

85. The method of claim 84, wherein the detectable marker is mCherry.

86. A method of generating insulin-producing P-cells from a pluripotent stem cell (PSC), comprising:(i) differentiating the PSC to a posterior foregut (PF) stage cell;(ii) at about day 8 of differentiation, transiently inducing expression of a fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain and introducing a gRNA complementary to a regulatory region of PDX1;(iii) differentiating the PF stage cell to a pancreatic progenitor (PP) stage cell;MBHB Ref. No. 24-1170-WO UW 49850.02WO2 (iv) at about day 11-12 of differentiation, transiently inducing expression of the fusion protein and introducing a gRNA complementary to a regulatory region of NGN3; and(v) differentiating the PP stage cell to an endocrine progenitor (EP) stage cell and then to an insulin-producing P-cell;wherein the sequential targeting of PDX1 and NGN3 promoters results in:(a) increased yield of insulin-producing P-cells compared to control;(b) reduced frequency of CD X2 -positive gut lineage cells compared to control; (c) reduced frequency of SOX2-positive gastric lineage cells compared to control;and(d) reduced frequency of enteroendocrine lineage cells compared to control.

87. The method of claim 86, wherein the gRNA complementary to a regulatory region of PDX1 comprises a combination of gRNAs selected from SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11.

88. The method of claim 86 or claim 87, wherein the gRNA complementary to a regulatory region of NGN3 comprises SEQ ID NO: 17.

89. The method of any one of claims 86-88, wherein the insulin-producing P-cells are obtained by day 19-20 of differentiation and comprise at least 20%, at least 25%, or at least 30% of the cell population.

90. A method of producing pancreatic endocrine cells with reduced contamination by non-pancreatic lineages, comprising:(i) differentiating a pluripotent stem cell (PSC) toward pancreatic lineages;(ii) at a primitive gut tube or posterior foregut stage, introducing to the differentiating cells:(a) a gRNA complementary to a regulatory region of PDX1; and (b) a fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain;wherein the method selectively reduces the frequency of CDX2-positive / PDXl- negative posterior gut lineage cells and / or SOX2-positive / PDXl -negative anteriorMBHB Ref. No. 24-1170-WO UW 49850.02WO2 foregut lineage cells compared to control differentiation without gRNA-mediated targeting.

91. The method of claim 90, further comprising at a pancreatic progenitor stage, introducing to the differentiating cells:(a) a gRNA complementary to a regulatory region of NGN3; and(b) the fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain;wherein the method further selectively reduces the frequency of SOX2 -positive anterior foregut lineage cells and enteroendocrine lineage cells compared to control.

92. A method for enhancing chromatin remodeling in differentiating pancreatic endocrine cells, comprising:(i) differentiating a pluripotent stem cell (PSC) toward pancreatic endocrine lineages; (ii) sequentially introducing to the differentiating cells at defined developmental stages:(a) a first gRNA complementary to a regulatory region of PDX1 and a fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain; and(b) a second gRNA complementary to a regulatory region of NGN3 and the fusion protein;wherein the sequential targeting results in deposition of H3K27ac marks at genomic loci associated with endocrine cell differentiation, insulin secretion, mitochondrial function, and metabolic maturation.

93. The method of claim 92, wherein the deposition of H3K27ac marks comprises at least 5,000, at least 10,000, or at least 14,000 unique H3K27ac peaks compared to control cells that received the fusion protein without gRNA targeting.

94. The method of claim 92 or claim 93, wherein the genomic loci with increased H3K27ac marks are enriched for genes involved in:(a) small GTPase-mediated signaling;MBHB Ref. No. 24-1170-WO UW 49850.02WO2 (b) Rapl and cAMP signaling;(c) establishment of apical / basal cell polarity;(d) negative regulation of Wnt signaling; or(e) regulation of sodium ion transport.

95. A method of activating NGN3 -dependent transcriptional programs in differentiating pancreatic cells, comprising:(i) introducing to pancreatic progenitor cells:(a) a gRNA complementary to a regulatory region of NGN3; and (b) a fusion protein comprising a catalytically inactivated Cas9 and an embryonic ectoderm development (EED)-binding domain;wherein the method results in activation of at least 100, at least 150, or at least 200 NGN3 target genes compared to control cells.

96. The method of claim 95, wherein the activated NGN3 target genes comprise genes involved in exocytosis, insulin secretion, mitochondrial function, metabolism, transcription factors, or development.

97. The method of any one of claims 1-38 or 60-96, wherein the method produces pancreatic endocrine cells within 19-25 days of differentiation, representing an acceleration of at least 10 days, at least 15 days, or at least 20 days compared to control differentiation protocols.

98. A cell population produced according to the method of any one of claims 1-38 or 60-96, wherein the cell population comprises:(a) at least 20% insulin-positive P-cells;(b) less than 2% CDX2-positive / PDXl -negative gut lineage cells;(c) less than 2% SOX2-positive / PDXl -negative gastric lineage cells; and(d) less than 10% tyrosine hydroxylase-positive enteroendocrine lineage cells.

99. The cell population of claim 98, wherein the cell population achieves one or more of:(a) at least 20% insulin-positive P-cells;(b) less than 2% CDX2-positive / PDXl -negative gut lineage cells;(c) less than 2% SOX2-positive / PDXl -negative gastric lineage cells; orMBHB Ref. No. 24-1170-WO UW 49850.02WO2 (d) less than 10% tyrosine hydroxylase-positive enteroendocrine lineage cells; by Day 20 of differentiation.

100. The cell population of claim 98, wherein the cell population achieves all of:(a) at least 20% insulin-positive P-cells;(b) less than 2% CDX2-positive / PDXl -negative gut lineage cells;© less than 2% SOX2-positive / PDXl -negative gastric lineage cells; and(d) less than 10% tyrosine hydroxylase-positive enteroendocrine lineage cells; by Day 20 of differentiation.

101. The cell population of any one of claims 98-100, wherein the cell population comprises at least 25%, at least 30%, or at least 40% insulin-positive P-cells.

102. A composition comprising the cell population of any one of claims 98-101 and a pharmaceutically acceptable excipient suitable for transplantation.

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