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

Abstract

The invention provides for methods of differentiating pancreatic endocrine cells into pancreatic beta cells expressing PDX1, NKX6.1, MAFA, UCN3 and SLC2A. These pancreatic beta cells may be obtained by step-wise differentiation of pluripotent stem cells. The pancreatic beta cells exhibit glucose-dependent mitochondrial respiration and glucose-stimulated insulin secretion similar to islet cells.

Description

(19) *EP004691479A2* (11) EP 4 691 479 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: 11.02.2026 Bulletin 2026 / 07 (21) Application number: 25208870.3 (22) Date of filing: 14.06.2017 (51) International Patent Classification (IPC): A61K 35 / 39 (2015.01) (52) Cooperative Patent Classification (CPC): C12N 5 / 0678; A61K 35 / 39; C12N 5 / 0677; C12N 2500 / 25; C12N 2500 / 32; C12N 2500 / 34; C12N 2500 / 38; C12N 2501 / 105; C12N 2501 / 115; C12N 2501 / 117; C12N 2501 / 119; C12N 2501 / 15; C12N 2501 / 335; C12N 2501 / 345; C12N 2501 / 375; (Cont.) (84) Designated Contracting States: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR Designated Extension States: BA ME Designated Validation States: MA MD (30) Priority: 21.06.2016 US 201662352968 P (62) Document number(s) of the earlier application(s) in accordance with Art. 76 EPC: 22213072.6 / 4 194 548 17815945.5 / 3 472 303 (71) Applicant: Janssen Biotech, Inc. Horsham, PA 19044 (US) (72) Inventors: • RIECK, Sebastian San Diego 92130 (US) • REZANIA, Alireza Wellesley 02481 (US) (74) Representative: Vossius & Partner Patentanwälte Rechtsanwälte mbB Siebertstraße 3 81675 München (DE) Remarks: •This application was filed on 15‑10‑2025 as a divisional application to the application mentioned under INID code 62. •Claims filed after the date of filing of the application / after the date of receipt of the divisional application (Rule 68(4) EPC). (54) GENERATION OF HUMAN PLURIPOTENT STEM CELL DERIVED FUNCTIONAL BETA CELLS SHOWING A GLUCOSE‑DEPENDENT MITOCHONDRIAL RESPIRATION AND TWO‑PHASE INSULIN SECRETION RESPONSE (57) The invention provides for methods of differen- tiating pancreatic endocrine cells into pancreatic beta cells expressing PDX1, NKX6.1, MAFA, UCN3 and SLC2A. These pancreatic beta cells may be obtained by step-wise differentiation of pluripotent stem cells. The pancreatic beta cells exhibit glucose-dependent mito- chondrial respiration and glucose-stimulated insulin se- cretion similar to islet cells. EP 4 69 1 47 9 A 2 Processed by Luminess, 75001 PARIS (FR) (Cont. next page) (52) Cooperative Patent Classification (CPC): (Cont.) C12N 2501 / 415; C12N 2501 / 727; C12N 2506 / 02; C12N 2533 / 90 2 EP 4 691 479 A2 Description CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application 62 / 352,968 (filed on June 21, 2016), which is incorporated by reference in its entirety. FIELD OF THE INVENTION

[0002] The present invention relates to methods for producing in vitro functional pancreatic beta cells, and populations resulting from, the differentiation of pluripotent stem cells. In particular, the invention relates to beta cells or a population of beta cells that exhibit mitochondrial respiration / activity response and a two-phase insulin secretion response. BACKGROUND OF THE INVENTION

[0003] Advances in cell-replacement therapy for Type I diabetes mellitus and a shortage of transplantable islets of Langerhans have focused interest on developing sources of insulin-producing cells, or beta (β) cells, appropriate for engraftment. One approach is the generation of functional beta cells from pluripotent stem cells, such as, embryonic stem cells or induced pluripotent cells.

[0004] In vertebrateembryonic development, apluripotent cell gives rise toagroupof cells comprising threegerm layers (ectoderm, mesoderm, and endoderm) in a process known as gastrulation. Tissues such as, thyroid, thymus, pancreas, gut, and liver will develop from the endoderm via an intermediate stage.

[0005] D’Amour et al. described the production of enriched cultures of human embryonic stem cell-derived definitive endoderm in the presence of a high concentration of activin and low serum (Nature Biotechnology 2005, 23:1534‑1541; U.S. Patent No. 7,704,738). Transplantation of these cells under the kidney capsule of mice resulted in differentiation into more mature cells with characteristics of endodermal tissue (U.S. Patent No. 7,704,738). Human embryonic stem cell- deriveddefinitiveendodermcells canbe further differentiated intoPDX1positive cells after additionofFGF‑10and retinoic acid (U.S. Patent App. Pub. No. 2005 / 0266554). Subsequent transplantation of these pancreatic precursor cells in the fat pad of immune deficient mice resulted in the formation of functional pancreatic endocrine cells following a 3‑4 month maturation phase (U.S. Patent Nos. 7,534,608 and 7,993,920).

[0006] Small molecule inhibitors have been used for induction of pancreatic endocrine precursor cells. For example, small molecule inhibitors of TGF-β receptor and BMP receptors (Development 2011, 138:861‑871; Diabetes 2011, 60:239‑247) have been used to enhance the number of pancreatic endocrine cells. In addition, small molecule activators have also been used to generate definitive endoderm cells or pancreatic endoderm cells (Curr. Opin. Cell Biol. 2009, 21:727‑732, Nature Chem. Biol. 2009, 5:258‑263).

[0007] In general, the process of differentiating progenitor cells to functional beta cells progresses through various stages and strides have been made in improving protocols to generate pancreatic cells from progenitor cells, such as human pluripotent stem cells. Despite these advances in research, each step in the process of differentiating progenitor cells presents a unique challenge. As such, there is still a need for further differentiation protocol development for the purpose of producing functional pancreatic endocrine cells and, in particular, functional beta cells. In particular, it is desirable to provide for in vitrogeneration of glucose responsive insulin-producing cells capable of the rapid and regulated glucose-stimulated insulin secretion ("GSIS") observed with functional beta-cells. Specifically, it is desirable to provide a methodof producing in vitro functional beta-cells exhibitingan increase inmitochondrial respiration / activity followedby the first phase and second phase of insulin secretion.

[0008] GSISbeginsby the import of glucose into thebeta-cell viaaglucose transporter (solute carrier family 2member1; SLC2A1;or commonly referred toasglucose transporter 1;GLUT1 forhumanbetacells), and themetabolismofglucose to pyruvate, through a process named glycolysis. The import of pyruvate into the mitochondria, its metabolism through the TCA (tricarboxylic acid, and subsequent activation of the electron transport chain ("ETC" and referred to here as "mitochondrial activity" or "mitochondrial respiration") cycle is tightly coupled with insulin exocytosis to ensure the rapid and correct quantity of insulin release.

[0009] Functional beta cells within an islet have been shown to secrete insulin upon a sudden increase in glucose concentration in twosequential phases (Henquin et al. Diabetologia (2009) 52(5):739‑751). Theamplitude andduration of both phases is regulated by the kinetics of the intracellular Ca2+ signal or additive secretion coupling factors. First phase (1st) insulin secretion represents the exocytosis of a small pool of bound and readily releasable insulin granules. The second phase (2nd) of insulin secretion, lower in amplitude but longer in duration, represents the translocation of granules from a reserve pool of granules, and their docking / priming for release. Biphasic GSIS, a key marker of maturation in beta cells, is not detected until the postnatal phase of human development, and is in contrast to the monophasic GSIS seen in immature beta-cells (Otonkoski et al. Diabetes (1988) 37:286‑291). 3 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55

[0010] In type 2 diabetics, the 1st phase of GSIS is absent; and the 2nd phase of GSIS is also reduced. Type 1 diabetics, whosebeta-cell number is severely reduced via autoimmuneattack, are reported to lacka robust biphasicGSIS (Krogvold et al. Diabetes (2015) 64: 2506‑2512). BRIEF SUMMARY OF THE INVENTION

[0011] As embodied and fully described, the invention provides methods for producing in vitro functional beta-cells (functional pancreatic beta-cells), and populations of cells resulting from, the differentiation of pluripotent stem cells In particular, the invention relates to generating functional pancreatic beta-cells (insulin producing cells) or a population of functional beta-cells that exhibit mitochondrial respiration / activity response and a two-phase insulin secretion response.

[0012] One aspect of the invention is a method of differentiating pluripotent stem cells into functional beta-cells. A particular aspect of the invention is a method of differentiating pancreatic endoderm cells into functional beta-cells expressing PDX1, NKX6.1, MAFA, UCN3 and SLC2A1. In embodiments, the method comprises culturing pancreatic endocrine cells in a culturemedium supplemented with one or more small molecules of UNC0638, UNC0642, UCN0646, TC-E5003, A366, PF03814735, ZM447439, SB747651A, PFI1, LY303511, MS436, AZT, DEZA, pyroxamide, C19994 or MC1568.

[0013] An embodiment of the invention is in vitro population of functional pancreatic beta-cells expressing single hormonal insulin, PDX1, NKX6.1 and MAFA obtained by in vitro differentiation of pancreatic endocrine cells. In some embodiments, the population of functional beta-cells also co-express UCN3 and SLC2A1. In embodiments, MAFA expression is increased as compared to immature beta-cells. In embodiments, the in vitro population of functional beta cells is obtained by culturing pancreatic endocrine cells in a medium supplemented with one or more small molecules of UNC0638, UNC0642, UCN0646, TC-E5003, A366, PF03814735, ZM447439, SB747651A, PFI1, LY303511, MS436, AZT, DEZA, pyroxamide, CI9994 or MC1568.

[0014] A further embodiment of the invention is amethodof differentiatingpluripotent stemcells into functional beta cells expressing PDX1, NKX6.1, MAFA, UCN3 and SLC2A comprising the steps of differentiating pluripotent stem cells into pancreatic endocrine cells, and differentiating the pancreatic endocrine cells into functional beta cells expressing PDX1, NKX6.1, MAFA, UCN3 and SLC2A1. In embodiments, MAFA expression is increased as compared to immature beta- cells. In embodiments, themethod comprises culturing pancreatic endocrine cells in a culturemediumsupplementedwith one or more small molecules of UNC0638, UNC0642, UCN0646, TC-E5003, A366, PF03814735, ZM447439, SB747651A, PFI1, LY303511, MS436, AZT, DEZA, pyroxamide, CI9994 or MC1568.

[0015] In each of the embodiments as described above, the culturemedium is further supplementedwith one ormore of ZM447439, heparin, N-Acetyl Cysteine, and Formulation I. In embodiments above, the culture medium is further supplemented with one or more of T3, T4 and analogue thereof. In embodiments above, the culture medium is supplemented with an ALK5 inhibitor. In some embodiments above, the culture medium lacks an ALK5 inhibitor. In embodiments of the invention above, the culture medium is supplemented with AZT or DEZA. In some embodiments above, the culture medium is supplemented with both AZT and DEZA.

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

[0017] Further in embodiments of the invention described above, the functional beta-cells are obtained by step-wise differentiation of one or more of cells selected from the group: cells expressing markers characteristic of the definitive endoderm; cells expressing markers characteristic of primitive gut tube cells; cells expressing markers characteristic of foregut endoderm cells; cells expressing markers characteristic of pancreatic endoderm cells; pancreatic endocrine precursor cells; and cells expressing markers characteristic of immature beta cells (pancreatic endocrine cells).

[0018] In each of the embodiments described above, themethod comprises culturing the cells at the air-liquid interface. In some embodiments described above, themethod comprises culturing the cells in suspension, for example cell clusters in suspension.

[0019] In the embodiments above, the differentiated functional beta-cell expresses MAFA at a higher level than an immature beta-cell. In embodiments, the differentiated functional beta-cell expresses UCN3 at a higher level than an immature beta-cell. In some embodiments above, expression of MAFA andUCN3 are increased compared to expression in an immature beta-cell.

[0020] In the embodiments of the invention above, the population of functional beta-cells exhibits glucose-stimulated insulin secretion and glucose-dependent mitochondrial respiration. In embodiments described above, the glucose- stimulated insulin secretion and glucose-dependent mitochondrial respiration is similar to that of human islet cells. In embodiments of the invention described above, the functional beta-cells secrete insulin in multiple phases.

[0021] In the embodiments described above, the glucose-dependentmitochondrial respiration has amaximumoxygen consumption rate response following glucose stimulation in the range of about 20% to about 80% over basal oxygen consumption rate. Inembodiments, theoxygenconsumption rate responseoccurredat least 15minutes followingglucose 4 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 stimulation.

[0022] In the embodiments above, the glucose-stimulated insulin secretion comprises a rapid, bi-phasic insulin secretion in response to glucose stimulation. In embodiments, a first phase of the bi-phasic insulin secretion has at least a four-fold increase to at least an eight-fold increase over baseline secretion, and a second phase of the bi-phasic insulin secretion has at least a two-fold to at least a four-fold increase over baseline secretion. In embodiments, the insulin secretion occurs at least five minutes to at least ten minutes following the glucose stimulation.

[0023] In some embodiments, themethod of differentiating pluripotent stem cells comprises the steps of: differentiating the pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm ("Stage 1 cells"); differentiating the Stage 1 cells into cells expressing markers characteristic of primitive gut tube cells ("Stage 2 cells"); differentiating the Stage 2 cells into cells expressing markers characteristic of foregut endoderm cells ("Stage 3 cells"); differentiating theStage 3 cells into cells expressingmarkers characteristic of pancreatic endoderm cells ("Stage 4 cells"); differentiating the Stage 4 cells into pancreatic endoderm / endocrine precursor cells (cells expressing markers char- acteristic of one or both of pancreatic endoderm cells and pancreatic endocrine precursor cells; "Stage 5"); differentiating the Stage 5 cells into immature beta-cells (pancreatic endocrine cells; "Stage 6"); and differentiating the Stage 6 cells into functional beta-cells (pancreatic endocrine cells; "Stage 7").

[0024] Inembodiments, themethodofdifferentiatingcomprisesdifferentiatingpluripotent stemcells intoStage1cellsby culturing the pluripotent stem cells in a medium supplemented with MCX compound and GDF‑8.

[0025] In embodiments, themethodcomprises differentiating theStage1cells intoStage2 cells by culturing theStage1 cells in a medium supplemented with FGF7 and ascorbic acid.

[0026] In embodiments, themethodcomprises differentiating theStage2cells intoStage3 cells by culturing theStage2 cells in a medium supplemented with FGF7, retinoic acid, SANT‑1, a PKC activator, a BMP inhibitor and ascorbic acid.

[0027] In embodiments, themethodcomprises differentiating theStage3cells intoStage4 cells by culturing theStage3 cells in a medium supplemented with FGF7, retinoic acid, SANT‑1, a PKC activator, a BMP inhibitor and ascorbic acid.

[0028] In embodiments, themethodcomprises differentiating theStage4cells intoStage5 cells by culturing theStage4 cells in a medium supplemented with SANT‑1, a PKC activator, a BMP inhibitor, ascorbic acid. In embodiments, the medium is further supplemented with one or more of T3, T4 or analogue thereof. In some embodiments, the medium is further supplemented with an ALK5 inhibitor.

[0029] In embodiments, themethodcomprises culturing theStage5cells into pancreatic endocrine cells by culturing the Stage 5 cells in amedium supplemented with a BMP inhibitor, ascorbic acid, one ormore of T3, T4 or analogue thereof. In embodiments, the medium is supplemented with T3. In some embodiments, the T3 is in the range of 1 nM to 1 µM. In certain embodiments, the T3 is in the range of 1nM to 100nM. In someembodiments, themedium is further supplemented with an ALK 5 inhibitor. In other embodiments, the culture medium is not supplemented with an ALK5 inhibitor. In some embodiments, the medium is further supplemented with a gamma secretase inhibitor. In certain embodiments, the medium is supplementedwith one ormore of ZM447439, heparin, N-Acetyl Cysteine, andFormulation I. In embodiments, the culturemedium is supplemented with AZTor DEZA. In some embodiments, the culture medium is supplemented with both AZT and DEZA. In the embodiments, the method comprises culturing the cells at the air-liquid interface. In some embodiments, the method comprises culturing the cells in suspension, for example cell clusters in suspension.

[0030] In each of the embodiments of the method of differentiation described above, the pancreatic endoderm cells ("Stage 4 cells") may be cryopreserved. In some embodiments, the method of differentiating pancreatic endoderm cells into pancreatic endocrine precursor cells comprises culturing cryopreserved cells.

[0031] In each of the embodiments described above, the pluripotent stem cells may be human pluripotent stem cells such as human embryonic stem cells. In one embodiment, the pluripotent stem cells may be human H1 or H9 cells.

[0032] Another embodiment of the invention is amethod of differentiating pluripotent stemcells into functional beta cells expressing PDX1, NKX6.1, MAFA, UCN3 and SLC2A comprising: (a) differentiating pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm obtained by culturing pluripotent stem cells in a medium supplemented with activin A and WNT3A; (b) differentiating the cells expressing markers characteristic of the definitive endoderm into immature beta-cells; and (c) differentiating the immature beta-cells into functional beta-cells expressing PDX1, NKX6.1, MAFA, UCN3 and SLC2A1 by culturing immature beta-cells in amedium supplementedwith one ormore of UNC0638, UNC0642, UCN0646, TC-E5003, A366, PF03814735, ZM447439, SB747651A, PFI1, LY303511, MS436, AZT, DEZA, pyroxamide, CI9994 or MC1568. The pluripotent stem cells used in this method may be human pluripotent stem cells, such as human embryonic stem cells. In one embodiment, the pluripotent stem stem cells are human CyT49 cells.

[0033] These functional beta cellsmayhaveglucose-dependentmitochondrial respiration or glucose-stimulated insulin secretion similar to human islet cells. The functional beta cells may also secrete insulin in multiple phases.

[0034] In certain embodiments of this method, the culture medium lacks an ALK5 inhibitor. In some embodiments, the culture medium is further supplemented with heparin, N‑ Acetyl Cysteine, Formulation I and one or more of T3, T4 or an analogue thereof. In other embodients, the culture medium is supplemented with T3. In alternate embodiments, the medium is supplemented with ZM447439, heparin, N‑ Acetyl Cysteine and one or more of T3, T4 or an analogue thereof 5 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 and contains no ALK5 inhibitor. The medium may be supplemented with T3, AZT or DEZA.

[0035] The method may include culturing the immature beta cells at the air-liquid interface, in suspension clusters, in roller bottlesoronmicrocarriers. Inoneembodiment, themethod includesculturing the immaturebetacells in roller bottles. In another embodiment, the method includes culturing the immature beta cells in roller bottles on microcarriers.

[0036] The differentiating of the pluripotent stem cells into immature beta-cells in the method may comprise: (a) differentiating the pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm ("Stage 1 cells") by culturing the pluripotent stem cells in a medium supplemented with activin A andWNT3A; (b) differentiating the Stage 1 cells into cells expressingmarkers characteristic of primitive gut tube cells ("Stage 2 cells"); (c) differentiating the Stage 2 cells into cells expressingmarkers characteristic of foregut endoderm cells ("Stage 3 cells"); (d) differentiating the Stage 3 cells into cells expressingmarkers characteristic of pancreatic endoderm cells ("Stage 4 cells"); (e) differentiating the Stage 4 cells into pancreatic endocrine precursor cells ("Stage 5"); and (f) differentiating the Stage 5 cells into the immature beta-cells. In certain embodiments, steps e. and f. include culturing at the air-liquid interface, in suspension clusters, in roller bottles or on micocarriers. In other embodiments, steps e. and f. comprise culturing the cells in roller bottles. In yet other embodiments, the steps e. and f. include culturing the immature beta cells in roller bottles on microcarriers.

[0037] In certain embodiments, the method includes differentiating the Stage 1 cells into Stage 2 cells by culturing the Stage 1 cells in a medium supplemented with FGF7 and ascorbic acid. In other embodiments, the method also includes differentiating the Stage 2 cells into Stage 3 cells by culturing the Stage 2 cells in a medium supplemented with FGF7, retinoic acid, SANT‑1, aPKCactivator andascorbic acid. In certain embodiments, themedium lacksaBMP inhibitor. In yet another embodiment, themethodcomprisesdifferentiating theStage3cells intoStage4cells byculturing theStage3cells in a medium supplemented with FGF7, retinoic acid, SANT‑1, a PKC activator and ascorbic acid. Again, in certain embodiments, the medium lacks a BMP inhibitor.

[0038] In another embodiment, themethod comprises differentiating the Stage 4 cells into Stage 5 cells by culturing the Stage 4 cells in a medium supplemented with SANT‑1, a PKC activator, a BMP inhibitor, and ascorbic acid. The medium may be further supplemented one or more of T3, T4 or analogue thereof.

[0039] In an alternate embodiment, the method comprises culturing the Stage 5 cells into immature beta-cells by culturing theStage5cells inamediumsupplementedwithaBMP inhibitor, ascorbicacid, oneormoreofT3,T4oranalogue thereof. The medium may be further supplemented with an ALK 5 inhibitor or a gamma secretase inhibitor. The method mayalso includedifferentiating the immaturebeta-cells into functional beta cells expressingPDX1,NKX6.1,MAFA,UCN3 and SLC2A by culturing immature beta-cells in a medium lacking an ALK5 inhibitor and supplemented with ZM447439, AZT, N-acetyl cysteine, DEZA, Formulation I and one or more of T3, T4 or analogue thereof.

[0040] In each of the embodiments of the method of differentiation described above, the culture may be a suspension culture or a cell aggregate suspension culture. In certain embodiments, the culturingmaybe carried out in roller bottles, on microcarriers or on microcarriers in roller bottles. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIGS. 1A‑ 1H show results of screening for small molecules that upregulateMAFAor UCN3 expression at Stage 7. FIGS. 1A to 1D demonstrate that the Stage 7 expression of MAFAwas significantly upregulated as compared to S6D7, or untreated or DMSO treated cultures at Stage 7 after treatment with UNC0638 (selective G9a and GLP histone lysine methyltransferase inhibitor), UNC0646 (potent and selective (G9a / GLP inhibitor), UNC0642 (potent and selective G9a and GLP histone lysine methyltransferase inhibitor), TC-E5003 (selective PILMT1 arginine methyltransferase inhibitor), A366 (potent and selective G9a / GLP histone lysine methyltransferase inhibitor), PF03814735 (Aurora kinase A and B inhibitor), ZM447439 (Inhibits Aurora kinase B), SB747651A dihydrochloride (Potent MSK1 inhibitor; also inhibits other AGC group kinases), PFI1 (BET bromodomain inhibitor), LY303511 (BRD2, BRD3 and BRD4 inhibitor), MS436 (Potent and selective BRD4 bromodomain inhibitor), and MC1568 (selectively inhibits HDAC class II (IIa)). FIG. 1E to 1H demonstrate that theStage7expressionofUCN3 increasedascompared toS6D7, or untreatedorDMSOtreatedcultures atStage7after theadditionof 5-Azacytidine ("AZT") (DNAmethyltransferase inhibitor), Pyroxamide (Histonedeacetylase inhibitor), and CI994 (Histone deacetylase inhibitor). All conditions S7D7; ALI clusters.

[0042] FIGS. 2A and 2B demonstrate the robustness of selected small molecules as either MAFA or UCN3 up- regulators, as their effect wasmaintained across different Stage 7 conditioning protocols. 3-Deazaneplanocin A ("DEZA") was found tobeaneffectiveup-regulator ofMAFA,butnotUCN3(FIG.2A).AZTwasconfirmedasanUCN3,butnotMAFA, up-regulator during Stage 7 (FIG. 2B). All conditions S7D7; ALI clusters.

[0043] FIGS. 3A‑3Mdemonstrate an increase in geneexpression, after sevendays of improvedStage7conditioning, of a set of maturation markers in ALI cell clusters to levels observed in human islets. Improvements utilized during Stage 7 differentiation include: (i) removal ofALK5 inhibitor II; (ii) loweringof T3 concentration; (iii) addition ofDEZA; (iv) addition of AZT; (v) addition of ZM; (vi) lowering glucose concentration to 5.56 mM; and (vii) the addition of a defined cocktail of vitamins, non-essential aminoacids, lipids, sodiumpyruvate, and traceelements (Formulation I -TableXII). FIG.3Ashows 6 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 that INHBB was enriched above the expression seen in human islets in conditions utilizing ALK5 inhibitor II, and the removal of ALK5 inhibitor in the absence or presence of AZT / DEZA decreased INHBB to levels seen in human islets. FIG. 3B shows that INHA expression was increased to human islet levels in ALI clusters by the removal of ALK5 inhibitor II. While MAFA and SLC2A1 expression, shown in FIGS. 3C and 3D respectively, are decreased by the removal of ALK5 inhibitor II, the addition of AZT / DEZA rescues MAFA and SLC2A1 expression to human islet levels. UCN3 (FIG. 3E), G6PC2 (Glucose‑6-phosphatase catalytic subunit 2; FIG. 3F), and PDK1 (Pyruvate dehydrogenase kinase 1; FIG. 3G) expression was increased to levels at or above human islets by the addition of AZT / DEZA and removal of ALK5 inhibitor II inALI clusters. The expression of INS (Insulin; FIG. 3H),GJD2 (Gap junctionprotein, delta 2; alsoCX36 / connexin 36; FIG. 3I), SIX2 (SIX homeobox 2; FIG. 3J), and PDX1 (FIG. 3K) increased in a gradual step-wise manner, first by removal of ALK5 inhibitor II and secondby theaddition of AZT / DEZA.Theexpression ofNKX6.1 (FIG. 3L), andGLPIR (Glucagon like peptide 1 receptor; FIG. 3M) did not significantly change across conditions, but their expression in ALI clusters were consistently at or above levels observed in human islets. All conditions S7D7; ALI clusters.

[0044] FIGS. 4A‑4E demonstrate the generation of C-PEPTIDE cells in ALI cell clusters that co-express, in terms of protein presence, maturation markers: PDX1 (FIG. 4A), NKX6.1 (FIG. 4B), MAFA (FIG. 4C), SLC2A 1 (FIG. 4D), and UCN3 (FIG. 4E) at S7D7. Representative human islet staining is shown in the left column. No ALK5, low T3, ZM, H, NAC, AZT / DEZA, FI, BLAR001 condition is shown in the middle column. No ALK5, low T3, ZM, H, NAC, AZT / DEZA, FI, BLAR004 condition is shown in the right column. All conditions S7D7; ALI clusters.

[0045] FIGS. 5A‑5F demonstrate the generation of C-PEPTIDE cells at S7D7 that exhibit human-islet-similar glucose- dependent mitochondrial respiration kinetics on ALI, specifically by Stage 7 specific ’No ALK5, low T3, ZM, H, NAC, AZT, DEZA,FI,BLAR001’ conditioning.FIG.5Ashows that human islets rapidly responded tohighD-glucose, demonstratedby anOCR123.3%± 12.92 over baseline after 15min post-injection ("ip"), andmaintained a highOCRover time (131.5%± 11.32; 72 min ip). Conversely, S6D7 ALI clusters, which were enriched for immature C-PEPTIDE positive cells, lacked a rapid OCR response to high D-glucose (104.4%± 3.37; 15 min ip) and exhibit a relatively weak OCR response over time (113.3%±4.51; 72min ip). FIG. 5Ddemonstrates thatwithin theS7D7ALI cluster grouponly the ’NoALK5, lowT3, ZM,H, NAC, AZT / DEZA, FI, BLAR001’ condition exhibited human-islet-similar glucose-dependent mitochondrial respiration kinetics (112.4% ± 3.25 - 15 min ip; 129.5% ± 3.78 - 72 min ip). S7D7 ALI cluster conditions that exhibit a glucose- dependent mitochondrial kinetics indistinguishable from immature S6D7 ALI clusters: ’ALK5, T3, ZM, H, NAC, FI, BLAR001’ (100.3% = 4.04 - 15 min ip; 107.8% ± 6.51 - 72 min ip) (FIG. 5B); ’No ALK5, low T3, ZM, H, NAC, FI, BLAR001’ (96.9%± 3.06 - 15min ip; 109.0%± 4.58 - 72min ip) (FIG. 5C); ’No ALK5, low T3, ZM, H, NAC, FI, BLAR004’ (103.4% ± 4.76 - 15 min ip; 113.6% ± 6.72 - 72 min ip) (FIG. 5E); and ’No ALK5, low T3, ZM, H, NAC, AZT / DEZA, FI, BLAR004’ (102.1 ± 4.04 - 15 min ip; 112.7% ± 3.38 - 72 min ip) (FIG. 5F).

[0046] FIGS. 6A‑6L demonstrate Aggrewell™ cell clusters differentiated in suspension culture to immature pancreatic beta cells. FIG. 6A depicts the procedure through which a (i) fresh S4D3 monolayer or S4D3 cryopreserved cells was assembled (ii) via the Aggrewell™ method into (iii) cell clusters. FIG. 6B shows that high protein presence of pancreatic endodermTFPDX1 (top, left), andNKX6.1 (top,middle), but low protein presence of endocrine TFNEUROD1 (top, right); alternate non-pancreatic endoderm lineage allocating TFSOX2 (bottom,middle), andCDX2 (bottom, right) was detected inS4D5Aggrewell™clusters. In FIG. 6C,FACSanalysis shows that 99.3±0.1%of the cellswerePDX1+ (left),84.4±0.1% NKX6.1+ (middle), but 2.2±0.4% NKX6.1+ NEUROD1+ (right) or 1.35±0.55% NKX6.1+ CHGA+ (middle) (S4DS Aggrewell™ clusters). FIG. 6D shows that the robust pancreatic endoderm characteristics were maintained in S4D4 Aggrewell™ clusters derived fromS4D3cryopreserved cells, as thehigh geneexpression of PDX1 (top, left),NKX6.1 (top, right), and low expression of NEUROD1 (bottom, leff), and CHGA (bottom, right) wasmaintained as compared to a S4D3 monolayer. FIG. 6E depicts the procedure through which a (i) fresh S4D3 monolayer or S4D3 cryopreserved cells was assembled (ii) via the Aggrewell™ method into cell clusters, and (iii) differentiated in suspension culture to (iv) immature beta-cells byS6D6.FACSanalysis ofS6D6Aggrewell™clusters showsa robust immaturebeta-cell proteinprofile, as78.5 ±1.87% of cells were NKX6.1+ CHGA+ (left, FIG. 6F), 73.6±4.34% NKX6.1+ NEUROD1+ (right, FIG. 6F), and 38.4 ±5.96% NKX6.1+ INSULIN+ (left, FIG. 6G). The majority of the INSULIN-positive population (50.2±6.92% of total cells were INSULIN+)wasNKX6.1+ (left,FIG. 6G)andnotGLUCAGON-positive (7.43±1.49% INSULIN+GLUCAGON+; (right, FIG. 6G). FIG. 6H shows by IF that at S6D6 the majority of C-PEPTIDE-positive cells in Aggrewell™ clusters were both PDX1+ (left) andNKX6.1+ (middle).FIG. 6I shows that the protein presence of thematuration gatekeeper TFMAFA (right) was already easily detected at S6D6, and was expressed at a higher level than previous ALI clusters at S6D36-S6D7 (NatureBiotechnology,2014 (32) 11, 1121‑1133) (seealsoFIG.3C forALIS7D7comparison). Theexpressionof INSULIN (FIG. 6J), PDX1 (FIG. 6K), and NKX6.1 (FIG. 6L) was higher than previous ALI clusters at S6D6-S6D7.

[0047] FIGS. 7A‑7M demonstrate generation of S7D7 Aggrewell™ clusters by suspension culture tomature pancreatic beta cells. FIG. 7A depicts the procedure through which a (i) fresh S4D3 monolayer or S4D3 cryopreserved cells were assembled into Aggrewell™ clusters, conditioned by suspension culture through (ii & iii) Stages 5, 6 and 7 to generate (iv) S7D7Aggrewell™ clusters. S7D7Aggrewell™ clusters cultured by ’NOALK5LOWT3ZMHNACDEZAAZTFIBLAR001’ during Stage 7maintained a baseline beta-cell protein profile (FIG. 7B‑7C). 89% of cells were NKX6.1+ CHGA+ (left, FIG. 7B), 77.7% NKX6.1+ NEUROD1+ (right, FIG. 7B), and 40.8% NKX6.1+ INSULIN+ (left, FIG. 7C). The majority of the 7 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 INSULIN-positive population (46.4% of total cells were INSULIN+) was NKX6.1+ (left, FIG. 7C) and not GLUCAGON- positive (3.9% INSULIN+ GLUCAGON+; (right, FIG. 7C). The gene expression of maturation markers MAFA (FIG. 7D), UCN3 (FIG. 7E), SLC2A1 (FIG. 7F),G6PC2 (FIG. 7G), INSULIN (FIG. 7H), andNKX6.1 (FIG. 7I), were at or above human islet levels in Stage 7 ’NOALK5 LOWT3 ZMHNACDEZA AZT FI BLAR001 or BLAR004’ conditioned S7D7 Aggrewell™ clusters. The expression levels S7D7 of maturation marker such as MAFA (FIG. 3C), G2PC2 (FIG. 3F), INSULIN (FIG. 3H), and NKX6.1 (FIG. 3L) are much greater in Aggrewell™ (’NO ALK5 LOW T3 ZM H NAC DEZA AZT FI BLAR001 or BLAR004’) than in ALI clusters. FIGS. 7J - 7Mdemonstrate that the addition of DEZAandAZT to ’NOALK5 LOWT3ZMH NAC FI BLAR004’ Stage 7 conditioning, generated a significant number of non-GLUCAGON (FIG. 7J; bottom, right) C- PEPTIDE cells that co-expressed at S7D7, in terms of protein presence, PDX1 (FIG. 7J; bottom, left), NKX6.1 (FIG. 7J; bottom, middle), MAFA (FIG. 7K; bottom, left), UCN3 (FIG. 7K; bottom, middle), and SLC2A1 (FIG. 7K; bottom, right). FIGS. 7L‑7Mdemonstrate thegeneration, by ’NOALK5LOWT3ZMHNACFIBLAR004’ -specificStage7conditioning, of C-PEPTIDE cells that co-expressed, in terms of protein presence, by S7D7maturation markers: PDX1 (FIG. 7L; bottom, left), NKX6.1 (FIG. 7L; bottom, middle), MAFA (FIG. 7M; bottom, left), SLC2A1 (FIG. 7M; bottom, right), but not GLUCAGON (FIG. 7L; bottom, right) or UCN3 (FIG. 7M; bottom, middle).

[0048] FIGS. 8A‑8I demonstrate generation of pluripotent stem-cell derivedmature pancreatic beta cells by suspension culture with human islet-similar glucose-dependent mitochondrial respiration and GSIS kinetics. FIGS. 8A‑8B both show that human islet cells rapidly responded to high D-glucose, demonstrated by anOCR 123.3%± 12.92 over baseline after 15 min ip and maintained a high OCR over time (131.5%± 11.32; 72 min ip). S6D7 ALI clusters, which were enriched for immature C-PEPTIDE positive cells, lacked a rapid OCR response to high D-glucose (104.4% ± 3.37; 15 min ip) and exhibit a relatively weak OCR response over time (113.3% ± 4.51; 72 min ip). Human islet similar glucose-dependent mitochondrial respiration kineticswereobserved in the ’NoALK5, lowT3,ZM,H,NAC,FI, BLAR004’ conditionatS7D13 in the context of Aggrewell™ clusters in suspension (110.7%± 2.46 - 15 min ip; 125.9%± 2.27 - 72 min ip) (FIG. 8A). ’No ALK5, low T3, ZM, H, NAC, AZT, DEZA, FI, BLAR004’ Aggrewell™ clusters in suspension consumed oxygen in response tohighglucosestimulationabovehuman islet levels (162.0%±11.51 -15min ip; 177.1%±0.99 -72min ip) (FIG.8B).FIG. 8C shows that matured beta cells within human islets exhibited the ability for multiple rounds of rapid bi-phasic insulin secretion in response to glucose stimuli. Human islets demonstrated the ability for multiple rounds of "on-off’ switching of insulin secretion. All conditions tested exhibited a strong insulin secretion response to KC1 (FIGS. 8C‑8I). The addition of Exendin‑4did not increase the amplitude of theGSIS response in human islets shown, butwas included for comparison to ALI or Aggrewell™ GSIS profiles (FIG. 8D). S7D21 - ALI clusters conditioned during Stage 7 in ’No ALK5, low T3, ZM, H, NAC FI,’ whether in BLAR001 (FIG. 8E) or BLAR004 (FIG. 8F), did show a first (~4‑10 fold first phase of first GSIS response), but not a second, and a relatively slow, bi-phasicGSIS response. ALI clusters did not have the ability to shut off the second phase of insulin secretion upon re-perifusion of 3mM D-glucose after stimulus. S7D14 Agarewell™ clusters conditioned during Stage 7 in ’NoALK5, low T3, ZM, H, NAC, FI BLAR004,’ exhibited a strong first bi-phasicGSIS (~5 fold first phase of first GSIS response) followed by an ability to completely shut-downGSIS, and a weak secondmono-phasic response (FIG. 8G). With the addition of DEZA and AZT to ’No ALK5, low T3, ZM, H, NAC, FI BLAR004’ conditioning, S7D14 Aggrewell™ clusters exhibited multiple rounds of human-islet-similar bi-phasic GSIS (~5‑7 fold first phase of first GSIS response), and the ability to completely shut-down GSIS in between high glucose pulses (FIGS. 8H‑8I).

[0049] FIGS. 9A‑9Qdemonstrate the generation of pancreatic endodermderived fromCyT49hESC inmultiple types of suspension culture. FIG. 9A depicts the step-wise differentiation protocol to generate CyT49 hESC-derived pancreatic endoderm. Important transcription factors ("TF") are indicated for each stage. FIG. 9B shows the number of S4D3 cells generated relative to hESC input in millions of cells per ml. FIG. 9C shows the S4D3 yields for 0.1 PBSMini and 0.5 PBS Mini. For example, for the 0.5 PBS suspension culture format generated 4.08±0.854 cells per one hESC cell, markedly higher than PEC‑01 d12 (2.23±0.090). FIG. 9C demonstrates an approximately 6-fold increase in S4D3 total cell yield (in millions of cells) when scaled up from 100ml (0.1 PBS) to 500ml (0.5 PBS) media volume. FIG. 9D and 9E depict phase- contrast images and diameter (in microns) of S4D3 aggregates from 2-liter roller bottle (FIG. 9D right), 0.1 PBS (FIG. 9E middle), and 0.5 PBS (FIG. 9E right) suspension formats; 2-liter roller bottle generated PEC‑01 d12 (FIG. 9D left), and PEC‑01 d15 (FIG. 9E left) are shown as examples of prior art. S4D3 aggregates generated by 0.1 PBS and 0.5 PBS suspensioncultureareuniformlysmaller than roller bottlegeneratedaggregates.FIG.9F‑9Lshow thegeneexpression for S4D3 aggregates. The gene expression of NKX6.1 (FIG. 9F), PTF1A (FIG. 9G), PDX1 (FIG. 9H), SOX2 (FIG. 9I), CDX2 (FIG. 9J), NEUROD1 (FIG. 9K), andCHGA (FIG. 9L), relative toCyT49 hESC is shown for S4D3aggregates generated in 2-liter roller bottle, 0.1 PBS and 0.5 PBS, and 2-liter roller bottle generated PEC‑01 d15. FIG. 9F‑9L demonstrates the robust inductionof thepancreatic endodermgeneprogram,while limiting theexpressionof alternative endoderm lineages and early pancreatic endocrine differentiation, relative to PEC‑01 d15 (prior art). FIG. 9N‑9P shows the strong protein co- localization of pancreatic endoderm markers (e.g. PDX1, NKX6.1), but not protein markers of alternative endoderm lineages (e.g. SOX2,CDX2) andmarkers for endocrine differentiation (e.g. CHGA) for S4D3aggregates generated by the new protocol in 2-liter roller bottle (FIG. 9N), 0.1 PBS (FIG. 9O), and 0.5 PBS (FIG. 9P), relative to 2-liter roller bottle generated PEC‑01 d12 aggregates (FIG. 9M), and PEC‑01 d15 aggregates (FIG. 9Q).

[0050] Figures 10A‑10AB demonstrate the generation of insulin producing cells derived from CyT49 hESC in multiple 8 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 types of suspension culture. FIG. 10A depicts the disclosed step-wise differentiation protocol to generate CyT49 hESC- derived non-functional insulin-producing cells. Important transcription factors ("TF") are indicated for each stage, beginning with S4D3 CyT49 hESC-derived cells input into Stage 5 conditioning. FIG. 10B shows the number of S6D7 cells generated relative to hESC input in millions of cells per ml. For example, for the 0.5 PBS suspension culture format utilizing the new protocol generated 2.4±0.169 S6D7 cells per one hESC cell. FIG. 10C shows the S6D7 yield of 0.1 PBS and 0.5 PBS cultures. FIG. 10C demonstrates an approximately 7.5-fold increase in S6D7 total cell yield (in millions of cells)whenscaledup from100ml (0.1PBS) to 500ml (0.5PBS)media volume. FIG. 10Ddepicts phase-contrast imagesof S5D3 aggregates from 2-liter roller bottle (FIG. 10D left), 0.1 PBS (FIG. 10D middle), and 0.5 PBS (FIG. 10D right) suspension formats. FIG. 10E depicts phase-contrast images of S6D7 aggregates from 2-liter roller bottle (FIG. 10E left), 0.1 PBS (FIG. 10E middle), and 0.5 PBS (FIG. 10E right) suspension formats with aggregate diameter indicated below eachpicture. FIG. 10Fdepicts phase-contrast imagesofS7D13aggregates from2-liter roller bottle (FIG. 10F left), and0.1 PBS (FIG. 10F right) suspension formats. Suspension culture by 0.1 PBS and 0.5 PBS maintains tight aggregate architecture through Stage 7 Conversely, Stage 6‑7 aggregates loosen to form sheets of cells in 2-liter roller bottle suspension culture. FIG. 10G‑10N show the gene expressions at S4D3, S5D3, S6D7 and S7D7. FIG. 10G‑10N demonstrate that all suspension culture methods using the new protocol induced a robust pancreatic mono-hormonal insulin-producing cell gene signature; 2-liter roller bottle, 0.1PBS and 0.5 PBS suspension methods relatively equally induced the expression of PDX1 (FIG. 10G), NKX6.1 (FIG. 10H), CHGA (FIG. 10I), NEUROD1 (FIG. 1 0J), NGN3 (FIG. 10K), INSULIN (FIG. 10L), MAFA (FIG. 10M), andGLUCAGON (FIG. 10N). FIG. 10O‑10T shows the step-wise formation of protein co-localization of insulin-producing cell markers such as CHGA, NKX6.1, INSULIN, but not GLUCAGON, at S5D3 in 0.5 PBS (FIG. 10O‑10P), at S6D7 in 2-liter roller bottle (FIG. 10Q), at S6D7 in 0.1 PBS (FIG. 10R), at S7D13 in 2- liter roller bottle (FIG. 10S), and at S7D14 in 0.1 PBS (FIG. 10T). FIG. 10U shows the prevelance of NKX6.1 cells co- expressingCHGA, INSULINorGLUCAGONaswell as theprevelanceof INSULINandGLUCAONco-expressing cells for PEC‑01-derived S6D7 cells. FIG. 10U shows high prevalence of poly-hormonal insulin-producing cells, and thus low numbers of mono-hormonal insulin-positive cells, for PEC‑01 derived S6D7 aggregates in 2-liter roller bottle. FIG. 10V shows the co-localization of GLUCAGON and NKX6.1 for: PEC‑01-derived S6D7; 0.1PBS S7D14 and roller bottle (RB) S7D13. FIG. 10V demonstrates that all INSULIN GLUCAGON+ cells are negative for NKX6.1 presence in all conditions tested (PEC‑01-derived S6D7; 0.1PBS S7D14 and RB S7D13 utilizing new protocol). FIG. 10W‑10AB shows the protein co-localization of insulin-producing cell markers such as SYNAPTOPHYSIN (pan-endocrinemarker), NKX6.1, INSULIN, and MAFA at S6D7 in 0.1 PBS (FIG. 10W‑10X), at S7D16 in 0.1 PBS (FIG. 10Y‑10Z), and at S7D23 in 2-liter roller bottle (FIG. 10AA‑10AB). By Stage 7, a robust protein co-localization between SYNAPTOPHYSIN, NKX6.1, INSULIN, and MAFA, indicative of non-functional mono-hormonal insulin-producing cells, was observed. DETAILED DESCRIPTION OF THE INVENTION

[0051] The following detailed description of the invention will be better understood when read in conjunction with the appended figures. Figures are provided for illustrating certain embodiments of the invention. However, the invention is not limited to the precise arrangements, examples, and instrumentalities shown. For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into subsections that describe or illustrate certain features, embodiments or applications of the present invention. A. Definitions

[0052] Stem cells are undifferentiated cells defined by their ability, at the single cell level, to both self-renew and differentiate. Stem cells may produce progeny cells, including self-renewing progenitors, non-renewing progenitors, and terminally differentiated cells. Stemcells are also characterized by their ability to differentiate in vitro into functional cells of various cell lineages frommultiple germ layers (endoderm,mesoderm, and ectoderm). Stem cells also give rise to tissues of multiple germ layers following transplantation and contribute substantially to most, if not all, tissues following injection into blastocysts. Stemcells are classified by their developmental potential. Pluripotent stem cells are able to give rise to all embryonic cell types.

[0053] Differentiation is the process by which an unspecialized ("uncommitted") or less specialized cell acquires the features of a specialized cell, for example a nerve cell or amuscle cell. A differentiated cell is one that has taken on amore specialized ("committed") position within the lineage of a cell. The term "committed", when applied to the process of differentiation, refers to a cell that has proceeded in the differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type. "De-differentiation" refers to the process bywhich a cell reverts to a less specialized (or committed) positionwithin the lineage of a cell. As used herein, the lineageofa cell defines theheredity of thecell, i.e.,whichcells it came fromand towhat cells it cangive rise.The lineage of a cell places the cell within a hereditary schemeof development and differentiation. A lineage-specificmarker refers to a 9 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 characteristic specifically associated with the phenotype of cells of a lineage of interest and can be used to assess the differentiation of an uncommitted cell to the lineage of interest.

[0054] "Markers", as used herein, are nucleic acid or polypeptide molecules that are differentially expressed in a cell of interest. In this context, differential expressionmeans an increased level for a positivemarker and a decreased level for a negativemarker as compared to an undifferentiated cell or a cell at another stage of differentiation. The detectable level of themarker nucleic acidor polypeptide is sufficiently higher or lower in the cells of interest compared toother cells, such that the cell of interest can be identified and distinguished from other cells using any of a variety of methods known in the art.

[0055] As used herein, "cell density" and "seeding density" are used interchangeably herein and refer to the number of cells seeded per unit area of a solid or semisolid planar or curved substrate.

[0056] As used herein, "suspension culture" refers to a culture of cells, single cells or clusters, suspended in medium rather than adhering to a surface.

[0057] As used herein, a cell is "positive for" a specific marker, "positive", or "+" when the specific marker is sufficiently detected in the cell. Similarly, the cell is "negative for", "negative" or "‑" for a specificmarkerwhen the specificmarker is not sufficiently detected in the cell. In particular, positive by fluorescence activated cell sorting cytometry ("FACS") is usually greater than about 2%, whereas the negative threshold by FACS is usually less than about 1%. Positive by polymerase chain reaction cytometry ("PCR") is usually less thanor equal to about 30 cycles (Cts);whereasnegativebyPCR isusually more than about 31 cycles.

[0058] In attempts to replicate the differentiation of pluripotent stem cells into functional pancreatic endocrine cells in static in vitro cell cultures, the differentiation process is often viewed as progressing through a number of consecutive stages. In particular, the differentiation process is commonly viewed as progressing through multiple stages. In this step- wisedifferentiation, "Stage1" refers to thefirst step in thedifferentiationprocess, thedifferentiationofpluripotent stemcells into cells expressing markers characteristic of the definitive endoderm ("Stage 1 cells"). "Stage 2" refers to the second step, the differentiation of cells expressing markers characteristic of the definitive endoderm cells into cells expressing markers characteristic of primitive gut tube cells ("Stage 2 cells"). "Stage 3" refers to the third step, differentiation of cells expressing markers characteristic of primitive gut tube cells into cells expressing markers characteristic of foregut endoderm cells ("Stage 3 cells"). "Stage 4" refers to the fourth step, the differentiation of cells expressing markers characteristic of foregut endoderm cells into cells expressingmarkers characteristic of pancreatic endoderm cells ("Stage 4 cells"). "Stage 5" refers to the fifth step, the differentiation of cells expressing markers characteristic of pancreatic endoderm cells into cells expressing markers characteristic of one or both of pancreatic endoderm cells and pancreatic endocrine precursor cells (collectively referred to as "Stage 5 cells" or, alternatively, "pancreatic endoderm / endocrine precursor cells"). Stage6 refers to the sixth step, thedifferentiationof cells expressingmarkers characteristic of pancreatic endocrine precursor cells into cells expressing markers characteristic of immature beta-cells ("Stage 6 cells"). In the process of, and for purposes of producing the cells of and populations of cells of the invention, a seventh step, "Stage 7", is used and refers to differentiation of cells expressing markers characteristic of immature beta-cells (pancreatic endocrine cells) into cells expressing markers characteristic of functional beta-cells and that have a more matured phenotype as compared to Stage 6 cells. By "functional beta-cells that have a more matured phenotype" or "Stage 7 cells" is meant a pancreatic endocrine cell that, when compared to a Stage 6 cell, is not only single hormonal insulin +, MAFA+, NKX6.1+, UCN3+, SLC2A1+andPDX1+, but also expressesMAFAat a higher level than a lessmature pancreatic endocrine cell, in particular an immature beta-cell.

[0059] It is to be noted that not all cells in a particular population progress through these stages at the same rate. Consequently, it is not uncommon in in vitro cell cultures to detect the presenceof cells that haveprogressed less, ormore, down the differentiation pathway than the majority of cells present in the population, particularly at the later differentiation stages. For example, it is not uncommon to see the appearance of markers characteristic of pancreatic endocrine cells during the culture of cells at Stage 5. For purposes of illustrating the present invention, characteristics of the various cell types associated with the above-identified stages are described herein.

[0060] "Definitive endoderm" as used herein, refers to cells which bear the characteristics of cells arising from the epiblast during gastrulation andwhich form thegastrointestinal tract and its derivatives.Definitive endodermcells express at least one of the following markers: FOXA2 (also known as hepatocyte nuclear factor 3-β ("HNF3-β")), GATA4, SOX17, CXCR4, Brachyury, Cerberus, OTX2, goosecoid, C-Kit, CD99, and MIXL1. Markers characteristic of the definitive endoderm cells are CXCR4, FOXA2, and SOX17. Thus, definitive endoderm cells may be characterized by their expression of CXCR4, FOXA2, and SOX17. In addition, depending on the length of time cells are allowed to remain in Stage 1, an increase in HNF4α may be observed.

[0061] "Primitive gut tube cells", as used herein, refers to cells derived fromdefinitive endodermand that can give rise to all endodermal organs, suchas lungs, liver, pancreas, stomach, and intestine.Gut tubecellsmaybecharacterizedby their substantially increased expression of HNF4α over that expressed by definitive endoderm cells. For example, a ten‑ to forty-fold increase in mRNA expression of HNF4α may be observed during Stage 2.

[0062] "Foregut endoderm cells", as used herein, refers to cells that give rise to the esophagus, lungs, stomach, liver, pancreas, gall bladder, and a portion of the duodenum. Foregut endoderm cells express at least one of the following 10 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 markers: PDX1, FOXA2, CDX2, SOX2, and HNF4α. Foregut endoderm cells may be characterized by an increase in expression of PDX1 compared to gut tube cells. For example, greater than fifty percent of the cells in Stage 3 cultures typically express PDX1.

[0063] "Pancreatic endoderm cells", as used herein, refers to cells that express at least one of the following markers: PDX1, NKX6.1, HNF1β, PTF1α, HNF6, HNF4α, SOX9, NGN3, gastrin; HB9, or PROX1. Pancreatic endoderm cells may be characterized by their lack of substantial expression of CDX2 or SOX2.

[0064] "Pancreatic endocrineprecursor cells", as usedherein, refers to pancreatic endodermcells capable of becoming a pancreatic hormone expressing cell. Pancreatic endocrine precursor cells express at least one of the followingmarkers: NGN3;NKX2.2;NeuroD1; ISL1;PAX4;PAX6; orARX.Pancreatic endocrineprecursor cellsmaybecharacterizedby their expression of NKX2.2 and NeuroD1.

[0065] "Pancreatic endocrine cells", as used herein, refer to cells capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin, ghrelin, and pancreatic polypeptide. In addition to these hormones, markers characteristic of pancreatic endocrine cells include one or more of NeuroD1, ISL1, PDX1, NKX6.1, PAX4, ARX, NKX2.2, HB9 and PAX6.

[0066] "Beta cells" ("β cells") are pancreatic endocrine cells capable of expressing insulin, but not glucagon, soma- tostatin, ghrelin, and pancreatic polypeptide. Pancreatic endocrine cells expressing markers characteristic of β cells can be characterized by their expression of insulin and at least one of the following transcription factors: PDX1, NKX2.2, NKX6.1, NeuroD1, ISL1, HNF3β, HB9, MAFA and PAX6.

[0067] "Functional beta-cells" are pancreatic endocrine cells that display the well-established processes that ensure rapid and regulated glucose-stimulated insulin secretion ("GSIS"), specifically an increase in mitochondrial respiratio- n / activity followedby thefirst phaseandsecondphaseof insulin secretion ("biphasicGSIS"). In detail, functional beta-cells exhibit at least one of the following characteristics of biphasic GSIS: (i) coupling of mitochondrial respiration / activity with insulin secretion; (ii) rapid insulin secretion response to heighteneddemand (here definedashigh glucose concentration); (iii) ability to rapidly turn off insulin secretion after demand has subsided; (iv) ability formultiple rounds of "on-off" switching of insulin secretion; (v) ability to secrete the correct amount of insulin as dictated by demand; and (vi) ability to respond to multiple insulin secretagogues (for example, Exendin‑4, or amino acids - L-Glutamine and L-Arginine). Functional beta- cells can be characterized by their expression of insulin and at least one of the following transcription factors: PDX1, NKX2.2, NKX6.1, NeuroD1, ISL1, HNF3β, HB9, PAX6, MAFA, SLC2A1, UCN3, and GLP1R.

[0068] "Immature beta cells" are pancreatic endocrine cells that do not display glucose-dependent mitochondrial respiration / activity, and biphasic GSIS. Immature beta cells expressing markers characteristic of β cells can be char- acterized by their expression of insulin and at least one of the following transcription factors: PDX1, NKX2.2, NKX6.1, NeuroD1, ISL1, HNF3β, HB9, MAFA, and PAX6.

[0069] "Aur-liquid interface" or "ALI", as used herein, refers to the air-liquid interface that exists in an open culture vessel or a culture vessel partially filled with medium. Although referred to herein as "air" for convenience, the invention is not limited to the mixture of gases and compositions found in the ambient environment. The invention specifically con- templates and includes gaseous mixtures having compositions different from the ambient environment including, for example, mixtures enriched for a particular component or in which a particular component has been depleted or eliminated.

[0070] Used interchangeably herein are "d1", "1d", and "day 1"; "d2", "2d", and "day 2", and so on. These number letter combinations refer to a specific day of incubation in the different stages during the stepwise differentiation protocol of the instant application.

[0071] First phase (1st) insulin secretion represents the rapid exocytosis of a small pool of bound and readily releasable insulin granules upon a sudden increase in glucose concentration.

[0072] Second phase (2nd) of insulin secretion, lower in amplitude but longer in duration compared to 1st phase insulin secretion, represents the translocation of granules from a reserve pool of granules, and their binding / priming for release.

[0073] OCR is defined as the oxygen consumption rate, and is an indicator of mitochondrial respiration, specifically via the electron transport chain ("ETC"); a direct measure of mitochondrial activity.

[0074] "Effective amount" or "therapeutic amount" or equivalents therein refers to the amount of the compound that should be present to provide somedegree of differentiation of the hESCs or further differentiation of partially differentiated hESCssuchas those subjected to oneormorepreviousdifferentiation stages. In additional examples, the compoundmay be present in the culture medium of the hESC’s or may be added to the hESCs’ during some stage of growth. In some embodiments, a compound, agent, small molecule or growth factor is used to produce definitive endoderm, foregut, pancreatic foregut and pancreatic endoderm-lineage cells including pancreatic hormone secreting cells. In certain examples, the stem cells may be exposed to the compound, agent, small molecule or growth factor prior to any differentiation or during the first stageof differentiation, whereas in other examples, the stemcellmay first be differentiated to an intermediate cell type such as, for example, definitive endoderm, and then exposed to the compound, agent, small molecule or growth factor.

[0075] "Compounds", "small molecule compounds" or equivalents thereof the invention refers to compounds encom- 11 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 passed by generic formulae disclosed herein (e.g. Table XI) and includes any specific compounds within that formula whose structure is disclosed herein or analogues thereof The compounds of the inventionmay be identified either by their chemical structure or chemical name.When the chemical structure and chemical name conflict, the chemical structure is determinative of the identity of the compound. The compounds of the invention may contain one or more chiral centers or double bonds and therefore, may exist as stereoisomers, such as double-bond isomers (i.e., geometric isomers), enantiomers or diastereomers. Accordingly, the chemical structures depicted herein encompass all possible enantiomers and stereoisomers of the illustrated compounds including the stereoisomerically pure form (e.g., geometrically pure, enantiomerically pure or diastereomerically pure) and enantiomeric and stereoisomeric mixtures. Enantiomeric and stereoisomericmixtures canbe resolved into their component enantiomers or stereoisomers using separation techniques or chiral synthesis techniques well known to the skilled artisan. The compounds of the invention also include isotopically labeled compoundswhereoneormoreatomshaveanatomicmassdifferent from theatomicmass conventionally found in nature. Examples of isotopes that may be incorporated into the compounds of the invention include, but are not limited to, 2H, 3H,13C,14C,15N,180, 17O,31P, 32P, 35S,18Fand36Cl. Further, it shouldbeunderstood,whenpartial structuresof the compounds of the invention are illustrated, that brackets indicate the point of attachment of the partial structure to the rest of the molecule. B. Characterization of Pluripotent Stem Cells

[0076] Pluripotent stemcellsmayexpressoneormoreof thedesignatedTRA‑1‑60andTRA‑1‑81antibodies (Thomson et al. 1998, Science 282:1145‑1147). Differentiation of pluripotent stem cells in vitro results in the loss of TRA‑1‑60 and TRA‑1‑81 expression. Undifferentiated pluripotent stem cells typically have alkaline phosphatase activity, which can be detected by fixing the cells with 4% paraformaldehyde, and then developing with an alkaline phosphatase substrate kit sold under the trademark VECTOR® Red, as described by the manufacturer (Vector Laboratories, Inc., Burlingame, California). Undifferentiated pluripotent stem cells also typically express OCT4 and TERT, as detected by reverse transcription polymerase chain reaction ("RT-PCR").

[0077] Another desirable phenotype of propagated pluripotent stem cells is a potential to differentiate into cells of all threegerminal layers: endoderm,mesoderm, andectoderm.Pluripotency of stemcellsmaybe confirmed, for example, by injecting cells into severe combined immunodeficiency ("SCID") mice, fixing the teratomas that form using 4% paraf- ormaldehyde, and then examining histologically for evidence of cell types from these three germ layers. Alternatively, pluripotencymaybedeterminedby thecreationofembryoidbodiesandassessing theembryoidbodies for thepresenceof markers associated with the three germinal layers.

[0078] Propagated pluripotent stem cell linesmay be karyotyped using a standardG-banding technique and compared topublishedkaryotypesof the correspondingprimate species. It is desirable to obtain cells that havea "normal karyotype," which means that the cells are euploid, wherein all human chromosomes are present and not noticeably altered. C. Sources of Pluripotent Stem Cells

[0079] Anypluripotent stemcellsmaybe used in themethodsof the invention. Exemplary types of pluripotent stemcells that may be used include established lines of pluripotent cells, including pre-embryonic tissue (such as, a blastocyst), embryonic tissue, or fetal tissue taken any time during gestation, typically but not necessarily, before approximately 10 to 12 weeks gestation. Nonlimiting examples are established lines of human embryonic stem cells ("hESCs") or human embryonic germ cells, such as, the human embryonic stem cell lines H1 (NIH Code: WA01), H7 (NIH Code: WA07), H9 (NIH Code: WA09) (WiCell Research Institute, Madison, WI, USA), and SA002 (Cellartis AB Corporation, Goteburg, Sweden).

[0080] Cells taken from a pluripotent stem cell population already cultured in the absence of feeder cells are also suitable. Induced pluripotent cells (IPS), or reprogrammed pluripotent cells, derived from adult somatic cells using forced expression of a number of pluripotent related transcription factors, such as OCT4, NANOG, SOX2, KLF4, and ZFP42 (Annu Rev Genomics HumGenet 2011, 12:165‑185; see also IPS, Cell, 126(4): 663‑676) may also be used. The human embryonic stem cells used in the methods of the invention may also be prepared as described by Thomson et al. (U.S. Patent No. 5,843,780; Science, 1998, 282:1145‑1147; Curr Top Dev Biol 1998, 38:133‑165; Proc Natl Acad Sci U.S.A. 1995, 92:7844‑7848). Mutant human embryonic stem cell lines, such as, BG01v (BresaGen, Athens, Georgia.), or cells derived from adult human somatic cells, such as, cells disclosed in Takahashi et al., Cell 131: 1‑12 (2007) may also be used. In certain embodiments, pluripotent stem cells suitable for use in the present inventionmay be derived according to themethods described in: Li et al. (Cell StemCell 4: 16‑19, 2009);Maherali et al. (Cell StemCell 1: 55‑70, 2007); Stadtfeld et al. (Cell Stem Cell 2: 230‑240); Nakagawa et al. (Nature Biotechnol 26: 101‑106, 2008); Takahashi et al. (Cell 131: 861‑872, 2007); andU.S. Patent App. Pub. No. 2011 / 0104805. In certain embodiments, pluripotent stem cells suitable for use in the present invention may be considered "naive" and derived according to the methods described in: Gafni et al. (Nature, 504:282, 2013), and Ware et al. (PNAS, 111: 4484‑4489, 2014). All of these references, patents, and patent 12 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 applications are herein incorporated by reference in their entirety, in particular, as they pertain to the isolation, culture, expansion and differentiation of pluripotent cells.

[0081] Other sources of pluripotent stem cells include induced pluripotent stem cells (IPS, Cell, 126(4): 663‑676). Yet other sources of suitable cells include human umbilical cord tissue-derived cells, human amniotic fluid-derived cells, human placental-derived cells, and human parthenotes. In one embodiment, the umbilical cord tissue-derived cells may beobtainedby themethodofU.S.PatentNo. 7,510,873. Inanother embodiment, theplacental tissue-derivedcellsmaybe obtained using the methods of U.S. Patent App. Pub. No. 2005 / 0058631. In another embodiment, the amniotic fluid- derived cells may be obtained using the methods of U.S. Patent App. Pub. No. 2007 / 0122903. The disclosure of each of thesepatent applications is incorporated in its entirety herein as it pertains to the isolation and characterization of the cells. In certain embodiments, the pluripotent stem cells may be of non-embryonic origins. D. Expansion and Culture of Pluripotent Stem Cells

[0082] Many different known methods of expanding and culturing pluripotent stem cells may be used in the claimed invention. For example, the pluripotent stem cellsmay be plated onto a suitable culture substrate. In one embodiment, the suitable culture substrate is an extracellular matrix component, such as those derived from basement membrane or that may form part of adhesion molecule receptor-ligand couplings. A suitable culture substrate is a reconstituted basement membrane sold under the trademarkMATRIGEL™ (Corning Incorporated, Corning, New York). MATRIGEL™ is a soluble preparation from Engelbreth-Holm Swarm tumor cells that gels at room temperature to form a reconstituted basement membrane.

[0083] Other extracellular matrix components and component mixtures known in the art are suitable as an alternative. Dependingon the cell typebeingproliferated, thismay include laminin, fibronectin, proteoglycan, entactin, heparin sulfate, and the like, alone or in various combinations.

[0084] The pluripotent stem cells may be plated onto the substrate in a suitable distribution and in the presence of a medium,which promotes cell survival, propagation, and retention of the desirable characteristics.All these characteristics benefit from careful attention to the seeding distribution and can readily be determined by one of skill in the art. Suitable culture media may be made from the following components, Dulbecco’s Modified Eagle’s medium ("DMEM") sold under the trademark GIBCO® (Catalog No. 11965‑092) by Life Technologies Corporation, Grand Island New York; Knockout Dulbecco’s Modified Eagle’s medium ("KO DMEM") sold under the trademark GIBCO® (Catalog No. 10829‑018) by Life Technologies Corporation; Harn’s F12 / 50% DMEM basal medium: 200 mM L-glutamine sold under the trademark GIBCO® (Catalog No.25030‑081) by Life Technologies: non-essential amino acid solution sold under the trademark GIBCO® (Catalog No. 11140‑050) by Life Technologies; β-mercaptoethanol, Sigma-Aldrich Company, LLC Saint Louis, MO, (CatalogNo.M7522); human recombinant basic fibroblast growth factor ("bFGF") sold under the trademarkGIBCO® (Catalog No. 13256‑029) by Life Technologies. Large-scale expansion and controlled differentiation processes of human embryonic stem cells can also be achieved using suspension bioreactors. E. Differentiation of Pluripotest Stem Cells

[0085] Aspluripotent cellsdifferentiate towards functionalβcells, theydifferentiate throughvariousstageseachofwhich may be characterized by the presence or absence of particular markers. Differentiation of the cells into these stages is achieved by the specific culturing conditions including the presence and lack of certain factors added to the culturemedia. In general, this differentiation may involve differentiation of pluripotent stem cells into definitive endoderm lineage, and definitive endodermcells. These cellsmay thenbe further differentiated into primitive gut tube cells, which in turnmay then be differentiated into foregut endoderm cells. Foregut endoderm cells may be differentiated into pancreatic endoderrn cells, which may then be further differentiated into pancreatic endocrine precursor cells or pancreatic endoderm / pan- creatic endocrine precursor cells. These cells may be differentiated into pancreatic hormone producing or secreting cells. This application provides for the staged differentiation of pluripotent stem cells towards pancreatic endocrine cells, preferably by culturing the cells at the air-liquid interface, or in suspension, that exists within a culture vessel partially filled withmedium, specifically by culturing cells at theair-liquid interface, or in suspension, in oneormoreofStages5 through7.

[0086] One or more of the thyroid hormones triiodothyronine ("T3") and thyroxine ("’T4"), and analogues thereof, alone or in further combination with an AL.K‑5 inhibitor may be used in the cell culturing at one or more of Stages 1 through 7 of differentiation, and preferably at each of Stages 5 through 7. Alternatively, the ALK‑5 inhibitor may be used alone in one or more stages of differentiation, but preferably at each of Stages 5 through 7 and more preferably at each of Stages 5 to 6. More preferably, one or more of the thyroid hormones or their analogues and an ALK5 inhibitor is used in one or more differentiation stages, preferably at each of Stages 5 through 7 and more preferably at each of Stages 5 to 6. Suitable thyroid hormone analogues may include, without limitation: GC‑1 (Sobertirome) (available from R&D Systems, Inc. Minneapolis, Minnesota); 3,5-diiodothryopropionic acid ("DIPTA"); KB‑141 discussed in J. Steroid Biochem. Mol. Biol., 2008, 111: 262‑267 and Proc. Natl. Acad. Sci. US 2003, 100: 10067‑10072; MB07344 discussed in Proc. Natl. Acad. Sci. 13 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 US 2007, 104: 15490‑15495; T0681 discussed in J. Lipid Res., May 2009, 50:938 and Endocr. Pract. 2012, 18(6): 954‑964, the disclosures of which are incorporated herein by reference in their entireties. Useful ALK5 inhibitors include: ALK5 inhibitor II (Enzo Life Sciences, Inc., Farmingdale, New York), which is also the preferred ALK5 inhibitor; ALK5i (Axxora, Inc., San Diego, California), SD208 (R&D Systems); TGF-β inhibitor SB431542 (Xcess Biosciences, Inc., San Diego, California); ITD‑1 (Xcess Biosciences); LY2109761 (Xcess Biosciences); A83‑01 (Xcess Biosciences); LY2157299 (XcessBiosciences); TGF-β receptor inhV (EMDMilliporeChemical,Gibstown,NewJersey);TGF‑β receptor inh I (EMDMillipore); TGF-β receptor inh IV (EMDMillipore); TGF-β receptor inh VII (EMDMillipore); TGF-β receptor inh VIII (EMDMillipore); TGF-β receptor inh II (EMDMillipore): TGF-β receptor inhVI (EMDMillipore); andTGF-β receptor inh VI (EMD Millipore).

[0087] In additional preferred embodiments of the invention, the methods include treating cells at one or more stages, but preferably treating cells during Stage 7, with a differentiationmedium that includes one or both of an antioxidant, such as vitamin E, acetyl cysteine, vitaminC, antioxidant supplement (CatalogNo. A1345, Sigma-Aldrich Company, LLCSaint Louis,Missouri), glutathione, superoxidedismutase, catalaseand the likeandcombinations thereof. In stillmorepreferred embodiments, in carrying out Stage 6, a gamma secretase inhibitor is used, which can be gamma secretase inhibitor XX (EMDMillipore), gammasecretase inhibitor XXI (EMDMillipore), gammasecretase inhibitor XVI (EMDMillipore), N‑[(3,5- difluorophenyl)acetyl]‑L-alanyl‑2-phenyl]glycine‑1,1-dimethylethyl ester ("DAPT") (Catalog No. 2634, Tocris Bioscience, Bristol, United Kingdom), and the like and combinations thereof. Useful amounts of gamma secretase inhibitor may be about 50 nM to 3000 nM, preferably about 50 nM to 500 nM. The amount of antioxidant may be about 0.1 to 100 µM, alternatively about 0.1 to 20 µM, and preferably about 1 to 10 µM. Alternatively, useful amounts of antioxidant may be about 100 nM to 5 mM, about 1000 nM to 2 mM, and preferably about 0.1 to 1 mM.

[0088] In embodiments of the invention, certain small molecules are used in the medium of one or more stages of differentiation, preferably at one or both of Stages 6 and 7. The small molecules of interest are those capable of inhibiting aurora kinase, p90 ribosomal S6 kinase, or methyl transferase receptor DOT1L and preferably are used along with antioxidants that reduce oxidative stress of cultured cells. Of particular interest are aurora kinase inhibitor II and RSK inhibitor II. Aurora kinase inhibitor II is a cell permeable compound known by the name of (4‑(4’-benzatnidoatillitio)‑6,7- dimethoxyquinazoline). RSK inhibitor II is a racemic mixture of a the dihydropteridinone 2‑(3,5-difluoro‑4-hydroxy- anilino)‑8-isopentyl‑5,7-dimethyl‑7H-pteridin‑6-one. Other aurora kinase inhibitors of interest include ZM447439 and PF03814735. Also of interest are protein methyltransferase receptor inhibitors of DOT1L, particularly EPZ‑5676. EPZ‑5676 is an S-adenosyl methionine ("SAM") inhibitor of protein methyltransferase DOT1L. This compound is a DOT1L (disruptor of telomere silencing 1-like) methyltransferase inhibitor shown to inhibit cell proliferation. It is known by the chemical names 9H-Purin‑6-amine, 9‑[5-deoxy‑5‑[[cis‑3‑[2‑[6‑(1,1-dimethylethyl)‑1H-benzimidazol‑2-yl]ethyl]cyclo- butyl](1-methylethyl)amino]-β-D-ribofuranosyl]‑and (2R,3R,4S,5R)‑2‑(6-aminopurin‑9-yl)‑5‑[[[3‑[2‑(6-tert-butyl‑1H-ben- zimidazol‑2-yl)ethyl]cyclobutyl]‑propan‑2-ylamino]methyl]oxolane‑3,4-diol. Further inhibitors of interest are a DNA methyltransferase inhibitor such as 5-Azacytidine ("AZT"), histone deacetylase inhibitors, such as Pyroxamide and CI994. Additional small molecules of interest include UNC0638, UNC0646, UNC0642 and A366 (G9a and GLP histone lysine methyltransferase inhibitors), TC-E5003 (PRMT1 arginine methyltransferase inhibitor), SB747651A dihydrochlor- ide (MSK1 inhibitor; also inhibits other AGC group kinases), PFI1 (BET bromodomain inhibitor), LY303511 (BRD2, BRD3 and BRD4 inhibitor), MS436 (BRD4 bromodomain inhibitor), and MC1568 (selectively inhibits HDAC class II), and 3- Deazaneplanocin A ("DEZA").

[0089] In a preferred embodiment of the invention, the small molecule is used in the medium of one or more of Stage 6 and 7 and more preferably in Stage 7. The amount of small molecule useful may be determined by selecting the amount showing the best expression of maturation markers and which amounts are not producing toxic effects. Typically, the amounts useful will be about 500 nM to 10µM, alternatively, about 500 nM to 5µM, and preferably about 500 nM to 2µM. Differentiation of Pluripotent Cells Into Cells Expressing Markers Characteristic of Pancreatic Endocrine Cells With a Matured Phenotype (Functional beta-cells)

[0090] Characteristics of pluripotent stemcells arewell known to those skilled in the art, andadditional characteristics of pluripotent stemcells continue tobe identified.Pluripotent stemcellmarkers include, for example, theexpression of oneor more of the following: ABCG2, cripto, FOXD3, CONNEXIN43, CONNEXIN45, OCT4, SOX2, NANOG, hTERT, UTF1, ZFP42, SSEA‑3, SSEA‑4, TRA‑1‑60, TRA‑1‑81. These may be detectable by RT-PCR.

[0091] Exemplary pluripotent stemcells include the humanembryonic stemcell lineH9 (NIH code:WAO09), the human embryonic stem cell line H1 (NIH code:WA01), the human embryonic stem cell lineH7 (NIH code:WA07), and the human embryonic stem cell line SA002. Also suitable are cells that express at least one of the followingmarkers characteristic of pluripotent cells: ABCG2, cripto, CD9, FOXD3, CONNEXIN43, CONNEXIN45, OCT4, SOX2, NANOG, hTERT, UTF1, ZFP42, SSEA‑3, SSEA‑4, TRA‑1‑60, and TRA‑1‑81.

[0092] Suitable for use in the present invention is a cell that expresses at least one of the markers characteristic of the definitive endoderm lineage. In one aspect of the present invention, a cell expressing markers characteristic of the 14 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 definitive endoderm lineage is a primitive streak precursor cell. In an alternate aspect, a cell expressing markers characteristic of the definitive endoderm lineage is amesendoderm cell. In an alternate aspect, a cell expressingmarkers characteristic of the definitive endoderm lineage is a definitive endoderm cell.

[0093] Also suitable for use in the present invention is a cell that expresses at least one of the markers characteristic of the pancreatic endoderm lineage. In one aspect of the present invention, a cell expressing markers characteristic of the pancreatic endoderm lineage is apancreatic endodermcell wherein theexpressionofPDX1andNKX6.1are substantially higher than the expression of CDX2 and SOX2. In certain embodiments, more than 30% of the cells express PDX1 and NKX6.1and less than30%of the cells expressCDX2orSOX2asmeasuredbyFACS.Particularly useful are cells inwhich the expression of PDX1 and NKX6.1 is at least two-fold higher than the expression of CDX2 or SOX2.

[0094] Still also suitable for use in the present invention is a cell that expresses at least one of themarkers characteristic of the pancreatic endocrine lineage. In one aspect of the invention, a cell expressing markers characteristic of the pancreatic endocrine lineage is a pancreatic endocrine cell. The pancreatic endocrine cell may be a pancreatic hormone- expressing cell capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin, ghrelin, or pancreatic polypeptide. In a preferred embodiment, the pancreatic endocrine cell is an insulin-producing (3 cell.

[0095] In certain embodiments of the invention, to arrive at the cells expressing markers characteristic of a functional beta-cell (pancreatic endocrine beta cells of a matured phenotype), a protocol starting with pluripotent stem cells is employed. This protocol includes: Stage 1: Pluripotent stem cells, such as embryonic stem cells obtained from cell culture lines, are treated with the appropriate factors to induce formation of definitive endoderm cells. Stage 2: Cells resulting from Stage 1 are treated with the appropriate factors to induce formation of cells into mar- kers expressing characteristic of primitive gut tube cells. Stage 3: Cells resulting from Stage 2 cells are treated with the appropriate factors to induce further differentiation into cells expressing markers characteristic of foregut endoderm cells. Stage 4: Cells resulting from Stage 3 are treated with the appropriate factors to induce further differentiation into cells expressing markers characteristic of pancreatic endoderm cells. The cells are optionally further cul- tured at the air-liquid interface or in suspension culture at late Stage 4; for late Stage 4 cells are transi- tioned to the air-liquid interface or aggregated as clusters that are cultured in suspension. Stage 5: Cells resulting from Stage 4 are treated with the appropriate factors, including in certain embodiments: (i) one or more of T3, T4 or an analogue thereof; (ii) an ALK5 inhibitor; or (iii) both of (i) and (ii) and cultured, optionally at the air-liquid interface or in suspension, to induce differentiation to cells expressing markers characteristic of pancreatic endoderm / endocrine precursor cells. Stage 6: Cells resulting from Stage 5 cells are treated with the appropriate factors including in certain embodi- ments: (i) one or more of T3, T4 or an analogue thereof; (ii) an ALK5 inhibitor; (iii) one or more of a gamma secretase inhibitor, an RSK inhibitor, a bone morphogenic protein (BMG) receptor inhibitor and a protein methyltransferase inhibitor of DOT1L; (iv) both of (i) and (ii); (v) (i), (ii) and (iii); (vi) (i) and (iii); or (vii) (ii) and (iii) and cultured, optionally at the air-liquid interface or in suspension, to induce differentiation into cells expressing markers characteristic of pancreatic endocrine cells, in particular immature beta-cells. Stage 7: Cells resulting from Stage 6 cells are treated with appropriate factors including in certain embodiments: (i) one or more of T3, T4 or thereof; (ii) an ALK5 inhibitor; (iii) an anti-oxidant, (iv) one or more of an aurora kinase inhibitor, an RSK inhibitor and a protein methyltransferase inhibitor of DOT1L; (v) (i) and (ii); (vi) (i) and (iii); (vii) (i) and (iv); (viii) (ii) and (iii); (ix) (ii) and (iv); (x) (i), (ii), and (iii); (xi) (i), (iii), and (iv); (xii) (ii), (iii), and (iv); (xiii) (i), (ii) and (iv); (xiv) (iii) and (iv); or (xv) (i), (ii), (iii) and (iv) and cultured, at the air-liquid interface or in suspension, to induce formation of functional beta-cells, pancreatic endocrine cells that ex- press single hormonal insulin and are PDX1, NKX6.1, UCN3, SLC2A1 and MAFA positive and which have a higher level of expression of MAFA than the Stage 6 cells and the resulting cell population has a higher percentage of both MAFA positive and single hormonal insulin expressing cells than the Stage 6 cells.

[0096] While the invention in certain embodiments encompasses differentiating pluripotent stem cells (e.g. pre-Stage 1 cells) to Stage 7 cells, the invention also encompasses differentiating cells at other stages towards Stage 7. In particular, the inventionencompassesdifferentiationofStage4 toStage7cells. Although theprocess is described indiscrete stages, the treatment, as well as the progress of the cells through the differentiation process, may be sequential or continuous. 15 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 Differentiation of pluripotent stem cells to Stage 6 or Stage 7 cells can be carried out in suspension cultures.

[0097] The efficiency of differentiationmay be determined by exposing a treated cell population to an agent, such as an antibody, that specifically recognizes a protein marker expressed by the differentiated cells of interest. Methods for assessing expression of protein and nucleic acid markers in cultured or isolated cells are standard in the art. These methods includeRT-PCR, Northern blots, in situ hybridization (see, e.g.,Current Protocols inMolecular Biology (Ausubel et al., eds. 2001 supplement)), and immunoassays such as immunohistochemical analysis of sectionedmaterial, western blotting and for markers that are accessible in intact cells, flow cytometry analysis (FACS) (see, e.g., Harlow and Lane, Using Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press (1998)).

[0098] The differentiated cells may also be further purified. For example, after treating pluripotent stem cells with the methods of the present invention, the differentiated cellsmay be purified by exposing a treated cell population to an agent, such as an antibody, that specifically recognizes a protein marker characteristically expressed by the differentiated cells being purified. In certain embodiments, the differentiated cells are not purified.

[0099] Any suitable growth medium containing sufficient quantities of vitamins, minerals, salts, glucose, amino acids and carrier proteins desirable for cells differentiation may be used for the various Stages 1 through 7. Preferably, the following are used: Stage 1‑ MCDB‑131 (available from Life Technologies Corporation, Grand Island, NY) or RPMI (available fromSigma-Aldrich); Stage2 -MCDB‑131orDulbecco’sModifiedEagle’sMediumF12 ("DMEM-F12");Stage3 through 5-MCDB‑131, BLAR (Table 1 and Table IV), or DMEM; and Stages 6 and 7 - BLAR or CMRL (Life Technologies). Preferably, the glucose concentration of the medium is kept at or, more preferably, lower than about 10 mM for Stages 1 through 4 and greater than about 10 mM for Stages 5 through 7. Preferably for Stage 7, the medium contains sufficient quantity of vitamins, non-essential amino acid, lipids, sodium pyruvate and trace elements, for example Formulation I. Stage 1: Differentiation of pluripotent cells into cells expressing markers characteristic of definitive endo- derm cells.

[0100] Pluripotent stem cells may be differentiated into cells expressing markers characteristic of definitive endoderm cells bymethods known in the art, or bymethods proposed in the invention. Methods reported as useful for differentiating pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage are disclosed in: D’ Amour et al., NatureBiotechnology23, 1534‑1541 (2005); Shinozaki et al., Development 131, 1651‑1662 (2004):McLean et al., Stem Cells 25, 29‑38 (2007); and D’ Amour et al., Nature Biotechnology 24, 1392‑1401 (2006). Additional suitable differentiationmethods are disclosed in: U.S. Patent App. Pub. No. 2007 / 0254359; U.S. Patent App. Pub. 2009 / 0170198; U.S. Patent App. Pub. 2011 / 0091971; U.S. Patent App. Pub. 2010 / 0015711; U.S. Patent App. Pub. 2012 / 0190111; U.S. Patent App.Pub. 2012 / 0190112; andU.S. Patent App.Pub. 2012 / 0196365. Thesedisclosures are incorporated herein by reference in their entireties as they pertain to the differentiation of pluripotent stem cells into definitive endoderm cells.

[0101] In one embodiment, the pluripotent cells are treated with a suitable growth medium, preferably MCDB‑131 or RPMI. Themedium is preferably supplemented with a growth differentiation factor, such as growth differentiation factor 8 ("GDF8"), and a glycogen synthase kinase‑3 β ("GSK3β") inhibitor, such as the cyclic aniline-pyridintriazine compounds disclosed in U.S. Patent App. Pub. No. 2010 / 0015711 (incorporated herein in its entirety by reference) to induce differentiation into cells expressing markers characteristic of definitive endoderm cells. A preferred GSK3β inhibitor is 14-prop‑2-en‑1-yl‑3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1 ~2,6~.1~8,12~]heptaco- sa‑1(25),2(27),3,5,8(26),9,11,21,23-nonaen‑16-one ("MCX compound"). Treatment may involve contacting pluripotent stem cells with amedium supplementedwith about 50 ng / ml to about 150 ng / ml, alternatively about 75 ng / ml to about 125 ng / ml, preferably about 100 ng / ml of GDF8. The treatmentmay also involve contacting cells with about 0.1 to about 5µM, alternatively about 0.5 to about 2.5µM,preferably about 1µMofMCXcompound. Thepluripotent cellsmaybecultured for about two to five days, preferably about two to three days, to facilitate differentiation into cells expressing markers characteristic of the definitive endoderm cells.

[0102] In a preferred embodiment, the cells are cultured in the presence of GDF8 and MCX compound for one day, followed by culturing in the presence of GDF8 and a lower concentration of MCX compound for one day, followed by culturing in the presence of GDF8 for one day in the absence of MCX compound. In particular, the cells are cultured in the presence of GDF8 and about 1µMMCX compound for one day, followed by culturing in the presence of GDF8 and about 0.1µM MCX compound for one day, followed by culturing in the presence of GDF8 for one day in the absence of MCX compound. Alternatively, the cells may be cultured in the presence of GDF8 and about 1µMMCX compound for one day, followed by culturing in the presence of GDF8 and about 0.1µM MCX compound for one day.

[0103] Alternatively, the pluripotent stem cells may be cultured inmedium containing activin A in the absence of serum, then culturing the cells with activin A and serum, and then culturing the cells with activin A and serum of different concentration as disclosed in D’Amour et al., Nature Biotechnology 23, 1534‑1541 (2005). In another alternative, the pluripotent stem cells may be differentiated into cells expressing markers characteristic of definitive endoderm cells by culturing the pluripotent stem cells in medium containing activin A in the absence of serum, then culturing the cells with activin A with serum as disclosed in D’ Amour et al, Nature Biotechnology, 2005. Pluripotent stem cells may also be 16 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 differentiated into cells expressing markers characteristic of the definitive endoderm lineage by culturing the pluripotent stem cells in medium containing activin A and aWNT ligand in the absence of serum, then removing theWNT ligand and culturing the cells with activin A with serum as disclosed in D’ Amour et al., Nature Biotechnology 24, 1392‑1401 (2006).

[0104] In one embodiment of the invention, pluripotent stem cells are treated with activin A andWNT3A to result in the formation of cells expressing markers characteristic of definitive endoderm cells. Treatment may involve contacting pluripotent stemcellswith about 50 ng / ml to about 150 ng / ml, alternatively about 75ng / ml to about 125 ng / ml, alternatively about 100 ng / ml of activin A. The treatment may also involve contacting the cells with about 10 ng / ml to about 50 ng / ml, alternatively about 15 ng / ml to about 30 ng / ml, alternatively about 20 ng / ml of WNT3A. The pluripotent cells may be cultured for approximately threedays to arriveat thedefinitive endodermcells. In oneembodiment, thecells are cultured in the presence of activin A andWNT3A for one day followed by culturing in the presence of activin A (withoutWNT3A being present) for the remainder.

[0105] In order to detect formation of cells expressingmarkers characteristic of definitive endoderm cells, the cells may be tested for the presenceof themarkers before andafter carrying out a particular protocol. Pluripotent stemcells typically donot expresssuchmarkers. Thus, differentiationof pluripotent cells canbedetectedwhencellsbegin toexpressmarkers characteristic of definitive endoderm. Stage 2: Differentiation of cells expressing markers characteristic of definitive endoderm cells into cells ex- pressing markers characteristic of primitive gut tube cells.

[0106] The cells expressing markers characteristic of the definitive endoderm cells may be further differentiated into cells expressing markers characteristic of gut tube cells in a growth medium, such as MCDB‑131 or DMEM-F12. In one embodiment, the formation of cells expressing markers characteristic of gut tube cells includes culturing the cells expressing markers characteristic of the definitive endoderm cells with a medium containing fibroblast growth factor ("FGF"), preferablyFGF7orFGF10, todifferentiate thecells.Forexample, thecell culturemay include fromabout10ng / ml to about 75 ng / ml, alternatively from about 25 ng / ml to about 75 ng / ml, still alternatively from about 30 ng / ml to about 60 ng / ml, alternatively about 50 ng / ml of a fibroblast growth factor, preferably FGF7 or FGF10, more preferably FGF7, and most preferably about 25 ng / ml FGF7. The cells may be cultured under these conditions for about two to three days, preferably about two days.

[0107] In another embodiment, the formation of cells expressing markers characteristic of gut tube cells includes culturing the cells expressing markers characteristic of the definitive endoderm lineage with a fibroblast growth factor, preferably, FGF7orFGF10, andascorbic acid (VitaminC). The culturemediummay include fromabout 0.1mM toabout 0. 5 mM ascorbic acid, alternatively from about 0.2 mM to about 0.4 mM ascorbic acid, alternatively about 0.25 mM of ascorbic acid. The cell culturemay also include fromabout 10 ng / ml to about 35 ng / ml, alternatively fromabout 15 ng / ml to about 30 ng / ml, alternatively about 25 ng / ml of the fibroblast growth factor, preferably FGF7 or FGF10, more preferably FGF7. For example, the cell culture may include about 0.25 mM of ascorbic acid and about 25 ng / ml of FGF7. In one embodiment, the Stage 1 cells are treated for 2 days with FGF7 and ascorbic acid. Stage 3: Differentiation of cells expressing markers characteristic of primitive gut tube cells into cells expres- sing markers characteristic of foregut endoderm cells.

[0108] The primitive gut tube cells resulting from Stage 2 may be further differentiated into Stage 3 cells, or cells expressing markers characteristic of the foregut endoderm, by culturing these cells in a growth medium such as MCDB‑131, DMEM, or a custom media such as BLAR (Table I). The medium may be supplemented with: (i) a fibroblast growth factor, preferably, FGF7 or FGF10 and more preferably FGF7; (ii) retinoic acid ("RA"); (iii) a Sonic Hedgehog ("SHH") signaling pathway antagonist (such asSmoothenedAritagonist 1 ("SANT‑1")which is 1-piperazinamine, N‑[(3,5- dimethyl‑1-phenyl‑1H-pyrazol‑4-yl)methylene]‑4‑(phenylmethyl)‑ or ((E)‑4-bei,ixyl-N‑((3,5-dimethyl‑1-phenyl‑1H-pyra- zol‑4-yl),ethylene-piperazin‑1-amine), BPI ‑1 which is 2-methoxyethyl 1,4,5,6,7,8-hexahydro‑4‑(3hydroxyphenyl)‑7‑(2- methoxyphenyl)‑2-methyl‑5-oxo‑3-quinolinecarboxylate, and preferably SANT‑1; (iv) a protein kinase C ("PKC") acti- vator, such as ((2S,5S)‑(E,E)‑8‑(5‑(4‑(trifluoromethyl)phenyl)‑2,4-pentadieneoylamino)benzolactam) ("TPB"), phor- bol‑12,13-dibutyrate ("PDBu"), phorbol‑12-myristate‑13-acetate ("PMA") or indolactam V ("ILV") and preferably TPB; (v) a bone morphogenic protein ("BMP") inhibitor, such as LDN‑193189, noggin, or chordin and preferably LDN‑193189; and (vi) ascorbic acid. Alternatively, a smoothened ("SMO") receptor inhibitor (such as MRT10 (N[[[3-benzoylamino) phenyl]ammo]thioxomethyl]‑3,4,5-trimethoxybenzamide)) or cyclopaminemayalsobeused.Forexample, thecell culture may include fromabout 100 nM to about 500 nM, alternatively fromabout 100 nM to about 400 nM, alternatively about 200 nM of a PKC activator. The cells may be cultured in the presence of these growth factors, small molecule agonists and antagonists for about two to four days, preferably about two to three days, more preferably about two days.

[0109] Alternatively, Stage 3 cells may be obtained from the Stage 2 cells by culturing these cells in a culture medium supplemented with an SMO receptor inhibitor, SANT‑1, retinoic acid and noggin. The cells may be cultured for 17 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 approximately two to four days, preferably about two days.

[0110] In one embodiment, themedium is supplemented with: from about 10 ng / ml to about 35 ng / ml, alternatively from about 15 ng / ml to about 30 ng / ml, alternatively about 25 ng / ml of the fibroblast growth factor, preferably FGF7 or FGF10, morepreferablyFGF7; fromabout 0.1mMtoabout0.5mMascorbic acid, alternatively fromabout0.2mMtoabout 0.4mM, alternatively about 0.25mM of ascorbic acid; from about 0.1 µM to about 0.4 µMof SANT‑1; from about 100 to about 300 nMofTPB;and fromabout 50nM toabout200nM,andabout 100nMofLDN‑193189. Inanother embodiment, themedium is supplemented with about 25 ng / ml of FGF‑7, about 1 µM of retinoic acid, about 0.25 µM of SANT‑1, about 200 nM of TPB, about 100 nM of LDN‑193189, and about 0.25 mM of ascorbic acid.

[0111] In one embodiment, themedium is supplementedwith fromabout 0.1µM to about 0.3µMof SANT‑1, fromabout 0.5 µM to about 3 µM of retinoic acid and from about 75 ng / ml to about 125 ng / ml of noggin. Stage 4: Differentiation of cells expressing markers characteristic of foregut endoderm cells into cells ex- pressing markers characteristic of pancreatic endoderm cells.

[0112] In one embodiment, the methods of the invention include deriving Stage 4 cells by treating Stage 3 cells with a differentiation medium that may be any suitable growthmedium and preferably is MCDB‑131, DMEM, or a custommedia suchasBLAR(Table I).Themediummaybesupplementedwithoneormoreof the following: (a) anALK5 inhibitor selected from the group consisting of: TGF-β receptor inh V, TGF-β receptor inh I, TGF-β receptor inh IV, TGF-β receptor inh VII, TGF-β receptor inh VIII, TGF-β receptor inh II, TGF-β receptor inh VI, TGF-β receptor inh III, TGF‑ β inhibitor SB431542, SD‑208, ITD‑1, LY2109761, A83‑01, LY2157299, ALK5i and ALK5 inhibitor II; (b) a thyroid hormone selected from the group consisting of T3, T4, analogues of T3, analogues of T4 and mixtures thereof; (c) an SHH signaling pathway antagonist selected fromSANT‑1 orHIP‑1; (d) aBMP receptor inhibitor selected fromLDN‑193189, noggin or chordin; (e) a PKC activator selected fromTPB, PPBu, PMA and ILV; (f) a fibroblast growth factor selected fromFGF‑7 or FGF‑10; (g) retinoic acid; and (h) ascorbic acid;. For example, a growth medium such as MCDB131 or, and preferably, BLARmay be supplemented with an SHH signaling pathway antagonist (such as SANT‑1 or HPI‑1), a BMP inhibitor (such as LDN‑193189, noggin or chord in), ascorbic acid, and a PKC activator (such as TPB, PDBu, PMA or ILV), to provide a useful differentiation media.

[0113] Culturing Stage 3 cells in such medium for about two to four days, preferably about two to three days, more preferably about three days usually is sufficient to differentiate theStage 3 cells into Stage 4 cells. In another embodiment, the medium may be supplemented with an SMO inhibitor and SHH signaling pathway antagonist. In a preferred embodiment, the Stage 3 cells may be treated with a medium supplemented with about 0.25 µM SANT‑1; about 100 nMRA; about 2 ng / ml FGF7; about 100 nMLDN‑193189; about 0.25mMascorbic acid; and about 200 nMof TPB for three days.

[0114] In Stage 4, cellsmay be cultured at the air-liquid interface, either during the entire stage or after about 2 to 3 days of planar culturing. Specifically, the present invention provides an in vitro cell culture for differentiating cells derived from pluripotent stem cells at the air-liquid interface comprising: (a) a culture vessel; (b) a volume of growthmediumwithin said vessel sufficient to fill only a portion of the volume of the vessel; (c) air within the vessel that fills a portion of the vessel adjoining themedium; (d) a porous substrate locatedat the interfacebetween themediumand theair; and (e) cells derived from pluripotent stem cells disposed upon the surface of the substrate such that themedium contacts only a portion of the surface of the cells. Alternatively, Stage 4 may be carried out entirely in planar culture.

[0115] Alternatively in Stage 4, after about 2 to 3 days of planar culture, cells may be aggregated to cell clusters. Specifically, thepresent inventionprovidesan in vitro cell culture for differentiating cells preparedasaggregatedcells such as cell clusters comprising: (a) a culture vessel that facilitates aggregation of cells, or forming cell clusters; (b) a volume of growth medium (culture medium) within said vessel; and (c) cells derived from pluripotent stem cells disposed within the vessel such that the cells are induced to aggregate and form clusters. In embodiments, the vessel is a plate with wells or microwells, such as Aggrewell™ plates (microwell plates; STEMCELL Technologies Inc., Vancouver, Canada). In particular, cell clusters prepared in plates with microwells to form aggregates of cells of uniform size and shape, for example using Aggrewell™ plates. In embodiments, about 500 to 2000 cells are seeded per well or microwell to prepare aggregated cells or cell clusters. In particular, about 50 to about 3000 cells are seeded per well or microwell, preferably about 50 to about 2000 cells, about 50 to 1000 cells, about 50 to about 900 cells, about 50 to about 800 cells, about 100 to about 800 cells, about 250 to about 800 cells, or about 500 to about 800 cells are seeded. More preferably, about 700 to about 800 cells are seeded per well or microwell.

[0116] In a further embodiment, the cell clusters formed by the method of aggregating cells may be further cultured in suspension (suspension culture), comprising removing the aggregated cells or cell clusters from the wells or microwells and seeding the aggregated cells / cell clusters in a suspension culture vessel. In embodiments, aggregated cells / cell clusters are seeded in a suspension culture at a cell density of about 0.75 x 106 cells / ml to about 2.0 x 106 cells / ml, preferably about 1 x 106 cells / ml to about 2.0 x 106 cells / ml, more preferably about 1.5 x 106 cells / ml to about 2.0 x 106 cells / ml. Any suspension culture system known to those skilled in the art may be useful for suspension culturing the 18 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 aggregated cells. In particular, the suspension culture system may include seeding aggregated cells or cell clusters in a flask, such as a spinner flask or a spinner wheel flask.

[0117] In a further embodiment, the cells at the completion of Stage 4 (after 2 or 3 days of culture) may be treated with a Rho-associated kinase ("ROCK") inhibitor such as Y27632 ((1R,4r)‑4‑((R)‑1-aminoethyl)‑N‑(pyriditn‑4-yl)cyclohexane- carboxamide), GSK269962 (N‑[3‑[[2‑(4-Amino‑1,2,5-oxadiazol‑3-yl)‑1-ethyl‑1H-imidazo[4,5-c]pyridin‑6-yl]oxy]phe- nyl]‑4‑[2‑(4-morpholinyl)ethoxy]benzamide), H1152 ((S)‑(+)‑2-Methyl‑1‑[(4-methyl‑5-isoquinolinyl)sulfonyl]homopipera- zine, 2HCl,) and, SR3677 (N‑[2‑[2‑(Dimethylamino)ethoxy]‑4‑(1H-pyrazol‑4-yl)phenyl‑2,3-dihydro‑1,4-benzodioxin‑2- carboxamide dihydrochloride). In certain embodiments about 1 to 20 µM, about 1 to 15 µM, about 1 to 10 µMor about 10 µM of the ROCK inhibitor may be used.

[0118] In certain embodiments of the invention herein, only late Stage 4 cells, for example, cells that have been cultured for 1 to 2 days or 1 to 3 days in adherent planar cultures,may subsequently be cultured at the air-liquid interface or cultured toaggregatedcells, suchascell clusters, for completionofStage4. Inoneembodiment of the inventionherein, lateStage4 cells thatwere treatedwith aROCK inhibitor are culturedat the air-liquid interface. In embodiments, 0.5 to about 0.75 x 105 cells / micro liter are seeded to be cultured at the air-liquid interface; alternatively, about 2 to 6 x 106 cells are seeded to be cultured at the air-liquid interface. In another embodiment, late Stage 4 cells that were treated with a ROCK inhibitor are cultured to aggregate the cells to formaggregated cells such as cell clusters. In embodiments, cells are seeded in a vessel that facilitates aggregation of cells, or cell clusters (three dimensional aggregates of cells). In some embodiments, the vessel is a plate with wells or microwells. In particular, cell clusters are prepared in plates with microwells to form aggregates of cells of uniform size and shape, for example using Aggrewell™ plates (STEMCELL Technologies Inc., Vancouver,Canada). In embodiments, about 50 to3000 cells are seededperwell ormicrowell to prepareaggregated cells or cell clusters. In particular, about 50 to about 3000 cells are seeded per well or microwell, preferably about 50 to about 2000 cells, about 50 to 1000 cells, 50 to 900 cells, 50 to 800 cells, 100 to 800 cells, 250 to 800 cells, or about 500 to about 800cells are seeded.Morepreferably, about 700 to800cells are seededperwell ormicrowell. In another embodiment, the aggregated cells or cell clusters are further cultured in suspension. In certain embodiments, the adherent cells in planar culture may be treated with a cell detachment solution, such as a solution containing proteolytic and collagenolytic enzymes such as TrypLE™, Accutase™, or Dispase™ prior to culturing at the air-liquid interface or culturing to aggregate cells such as forming cell clusters.

[0119] In an alternate embodiment, Stage 4 cells may be obtained fromStage 3 cells by treating the Stage 3 cells with a differentiation medium comprising a growth medium supplemented with an ALK5 inhibitor, noggin, and a PKC activator, such as TPB. In certain embodiments, the medium may be supplemented with about 0.1 µM ALK5 inhibitor, about 100 ng / mLofnoggin, andabout500nMTPB.Thecell culturemaybe inamonolayer format.The treatmentmay last for a total of about three days. In certain embodiments, the cells may be treated for two days and then on the last day the cells may be treated with proteolytic enzymes, collagenolytic enzymes or both to generate a single cell suspension. The resulting cells may be seeded at the air-liquid interface or seeded to generate aggregated cells or cell clusters. The single cells may be aggregated into cell clusters having a diameter of less than about 100microns followed by culturing in the presence of an ALK5 inhibitor and LDN‑193189. In certain embodiments, the cell clusters having a diameter of less than about 100 micronsmay be cultured in amedium supplementedwith about 200 nMALK5 inhibitor and about 100 nMLDN‑193189. In an alternate embodiment, culturing Stage 4 cells at the air-liquid interface or in suspension may significantly enhance pancreatic endoderm markers along with endocrine-related markers. Stage 5: Differentiation of cells expressing markers characteristic of pancreatic endoderm cells into cells ex- pressing markers characteristic of pancreatic endocrine precursor cells.

[0120] In oneembodiment, themethodsof the invention includegenerationof Stage5 cells by treatingStage4cellswith a differentiationmedium thatmay be any suitable growthmediumand preferably isMCDB‑131,DMEMor a custommedia suchasBLAR(Table I).Themediummaybesupplementedwithoneormoreof the following: (a) anALK5 inhibitor selected from the group consisting of: TGF-β receptor inh V, TGF-β receptor inh I, TGF-β receptor inh IV, TGF-β receptor inh VII, TGF-β receptor inh VIII, TGF-β receptor inh II, TGF-β receptor inh VI, TGF-β receptor inh III, TGF-β inhibitor SB431542, SD‑208, ITD‑1, LY2109761, A83‑01, LY2157299, ALK5i and ALK5 inhibitor II; (b) a thyroid hormone selected from the group consisting of T3, T4, analogues of T3, analogues of T4 and mixtures thereof; (c) an SHH signaling pathway antagonist selected fromSANT‑1orHP‑1; (d) aBMPReceptor Inhibitor selected fromLDN‑193189, noggin or chordin: (e) retinoic acid; (f) ascorbic acid; (g) heparin; and (b) zinc sulfate, and culturing the cells, preferably at the air-liquid interface, for about two to four days, preferably about three days, to differentiate the cells into Stage 5 cells. In another embodiment, the growth medium is also supplemented with one or both of an SMO inhibitor (such as MRT10 or cyclopamine) and a fibroblast growth factor selected from FGF‑7 or FGF‑10. The treatment of the Stage 4 cells is carried out for about two to four days, preferably about three days to differentiate the cells into Stage 5 cells.

[0121] In a preferred embodiment, the Stage 4 cells are differentiated into Stage 5 cells by treating the cells with a mediumsupplementedwith fromabout0.1µMtoabout0.4µMofSANT‑1andpreferablyabout0.25µMSANF‑1,about50 19 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 nMRA, fromabout 0.1mM to about 0.5mMascorbic acid, alternatively fromabout 0.2mM to about 0.4mMand preferably about 0.25mMascorbic acid, fromabout 50 nM to about 200 nMandpreferably about 100 nMLDN‑193189, about 1µMof T3, and about 10000 nM ALK5 inhibitor, more preferably ALK 5 inhibitor II. In still another embodiment, the cells are optionally also treated with about 1 to 15 µM, alternatively about 1 to 10 µM, alternatively about 5 to 10 µM, preferably about 10µMof zinc sulfate (ZnSO4) and about 1 to 100µg / ml, preferably about 10µg / ml of heparin. The treatment of the Stage 4 cells is carried out for about two to four days, preferably about 3 days to differentiate the cells into Stage 5 cells.

[0122] In yet another embodiment, themethods of the invention include obtainingStage 5 cells by treatingStage 4 cells with amediumsupplementedwith heparin, anSMO inhibitor or anSHHsignalingpathwayantagonist,RA, aBMP receptor inhibitor and anALK5 inhibitor and culturing the cells at the air-liquid interface for about 3 days to differentiate the cells into Stage 5 cells. In an alternative embodiment, the medium may be supplemented with both an SMO inhibitor and an SHH signalingpathwayantagonist, alongwithRA,aBMPreceptor inhibitorandanALK5 inhibitor.Thus, inoneembodiment, the Stage 4 cells may be differentiated into Stage 5 cells by treating the Stage 4 cells with a medium supplemented with heparin, ZnSO4, an SMO inhibitor or an SHH signaling pathway antagonist, RA, LDN‑193189 and ALK5 inhibitor II. In an alternative embodiment, the medium may be supplemented with both an SMO inhibitor and an SHH signaling pathway antagonist. In one embodiment, the Stage 4 cells are differentiated into Stage 5 cells by treating the cells with a medium supplementedwith about 10µg / ml of heparin, about 0.25µMSANT‑1, about 50 nMRA, about 50 nMLDN‑193189, about 10 nM of T3 and about 1000 nM ALK5 inhibitor. Suitable ALK5 inhibitors include but are not limited to SD‑208, ALK5 inhibitor II, TGF-β receptor inh V, TGF-β receptor inh I, TGF-β receptor inh IV, TGF-β receptor inh VII, TGF-β receptor inh VIII, TGF-β receptor inh II, TGF-β receptor inh VI, TGF-β receptor inh III and combinations thereof. The treatment of the Stage 4 cells is carried out for about two to four days, preferably about 3 days to differentiate the cells into Stage 5 cells.

[0123] In apreferred embodiment, theALK5 inhibitor isALK5 inhibitor II. In another preferred embodiment, about 10000 nM of ALK5 inhibitor II is used. In an alternate preferred embodiment, the Stage 4 cells are treated with a medium supplemented with about 10µg / ml of heparin, about 0.25µMSANT‑1, about 50 nMRA, about 100 nM LDN‑193189, and about 10,000 nM (10 mM) of ALK5 inhibitor II. In yet another alternate embodiment, the methods of the invention include treating Stage 4 cells with a medium supplemented with an SMO inhibitor or an SHH signaling pathway antagonist, RA, andanALK5 inhibitor andculturing the cells, preferably at theair-liquid interfaceor in suspension for about twodays to four days, preferably about 3 days, differentiate the cells into Stage 5 cells. In an alternate embodiment, the medium may be supplementedwithboth anSMO inhibitor andanSHHsignalingpathwayantagonist. In oneembodiment, theStage4cells aredifferentiated intoStage5cellsby treating thecellswithamediumsupplementedwithabout0.25µMSANT‑1, about50 nM RA, about 50 nM LDN‑193189, about 1 µM T3 and about 1000 nM of an ALK5 inhibitor.

[0124] Theamount of cells seeded for culturingat theair-liquid interfacemayvary. For example, to culture thecells at the air-liquid interface, droplets of a single-cell suspension containing about 0.5 to 6 x 105 cells / µl may be seeded on a porous substrate (e.g. filter). Thesuspensionmaycontain fromabout 2 x105 cells / µl to about 6 x105 cells / µl; about 4 x105 cells / µl toabout 6x105cells / µl; about5 x105cells / µl toabout 6x105cells / µl; about5 x105cells / µl to about6 x105cells / µl; about 2 x 105 cells / µl to about 5 x 105 cells / µl; about 2 x 105 cells / µl to about 4 x 105 cells / µl; or about 3 x 105 cells / µl that may be seeded onto a porous substrate such as a filter located at the air-liquid interface. In some embodiments, droplets of a single-cell suspension containing fromabout 0.5 x 105 cells / µl to about 0.75 x 105 cells / µl; about 0.6 x 105 cells / µl to about 0.75 x 105 cells / µl; or about 0.5 x 105 cells / µl to about 0.6 x 105 cells / µl are seeded onto a porous support to be cultured at the ALI.

[0125] For suspension culture, cell clusters are generated in plates with microwells that are then seeded to suspension culture system. In embodiments, Aggrewell™ plates are used to induce aggregation in which about 50 to 3000 cells are seeded per well or microwell to prepare aggregated cells or cell clusters. In particular, about 50 to about 3000 cells are seeded per well or microwell, preferably about 50 to about 2000 cells, about 50 to 1000 cells, about 50 to about 900 cells, about 50 to about 800 cells, about 100 to about 800 cells, about 250 to about 800 cells, or about 500 to about 800 cells are seeded.More preferably, about 700 to about 800 cells are seeded perwell ormicrowell. The aggregated cells / cell clusters are seeded in a suspension culture at a cell density of about 0.75 x 106 cells / ml to about 2.0 x 106 cells / ml, preferably about 1 x 106 cells / ml to about 2.0 x 106 cells / ml, about 1.25 x 106 cells / ml to about 2.0 x 106 cells / ml, andmore preferably about 1.5 x 106 cells / ml to about 2.0 x 106 cells / ml.

[0126] In another embodiment, themethods of the invention include treatingStage 4 cellswith amediumsupplemented with a BMP receptor inhibitor (e.g. LDN‑193189, noggin or chordin) and an ALK5 inhibitor for about 1 day to differentiate Stage 4 cells into Stage 5 cells. For example, the mediummay be supplemented with about 100 nM of LDN‑1931 89 and with about 100 nM of ALK5 inhibitor and about 1 µM T3. The cells may be in adherent planar culture or in the form of clusters. In certain embodiments, the cells may be treated with a cell detachment solution, such as a solution containing proteolytic and collagenolytic enzymes prior to culturing at the air-liquid interface or culturing to form aggregated cells or cell clusters.

[0127] In accordance with the foregoing method, the invention further provides a cell culture for differentiating cells expressing markers characteristic of the pancreatic endoderm into cells expressing markers characteristic of pancreatic endocrineprecursor cells (pancreaticendoderm / pancreaticendocrineprecursor cells) comprising: (a)aculturevessel; (b) 20 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 a volume of growthmediumwithin said vessel sufficient to fill only a portion of the volume of said vessel; (c) air within said vessel that fills a portion of said vessel adjoining saidmedium; (d) a porous substrate located at the interface between said mediumandsaidair; and (e) cells expressingmarkers characteristic of pancreatic endodermcells derived frompluripotent stemcells disposed upon the surface of said substrate such that saidmediumcontacts only a portion of the surface of said cells.

[0128] In accordance with the foregoing method, alternatively the invention further provides a cell culture for differ- entiating cells expressingmarkers characteristic of the pancreatic endoderm into cells expressingmarkers characteristic of pancreatic endocrine precursor cells (pancreatic endoderm / pancreatic endocrine precursor cells) comprising: (a) a culture vessel that facilitates aggregation of cells, or forming cell clusters; (b) a volumeof growthmedium (culturemedium) within said vessel; (c) cells derived frompluripotent stemcells disposedwithin the vessel such that the cells are induced to aggregate and form clusters; and (d) generated cell clusters disposed in suspension (suspension culture). In embodi- ments, the vessel for cell aggregation is a plate with wells or microwells, such as Aggrewell™ plates (microwell plates; STEMCELL Technologies Inc., Vancouver, Canada). In embodiments, the suspension culture system may include seeding aggregated cells or cell clusters in a suspension culture vessel, such as flask, for example a spinner flask or a spinner wheel flask. Stage 6: Differentiation of cells expressing markers characteristic of pancreatic endocrine precursor cells into cells expressing markers characteristic of immature beta-cells.

[0129] In one embodiment, the methods of the invention include obtaining Stage 6 cells by treating Stage 5 cells with a differentiation medium that may be any suitable growth medium, preferably such as MCDB‑131 or CMRL, and more preferably, a custommedia such as BLAR (Table I). Themediummay be supplemented with one or more of the following: (a) anALK5 inhibitor selected from the group consisting of: TGF-β receptor inhV, TGF-β receptor inh I, TGF-β receptor inh IV, TGF-β receptor inh VII, TGF-β receptor inh VIII, TGF-β receptor inh II, TGF-β receptor inh VI, TGF-β receptor inh III, TGF-β inhibitor SB431542, SD‑208, ITD‑1, LY2109761, A83‑01, LY2157299, ALK5i and ALK5 inhibitor II; (b) a thyroid hormone selected from the group consisting of T3, T4, analogues of thereof and mixtures thereof; (c) a BMP receptor inhibitor preferably selected fromLDN‑193189,nogginor chordin; (d) agammasecretase inhibitor suchgammasecretase inhibitor XX, gamma secretase inhibitor XXI, gamma secretase inhibitor XVI, or DAPT; (e) ascorbic acid; (f) heparin; and (g) zinc sulfate. The cells may be cultured, preferably at the air-liquid interface or in suspension culture, for about two to four, preferably for about three days, to differentiate the Stage 5 cells into Stage 6 cells. Optionally, themedium can further be supplemented with one or more of an SHH signaling pathway antagonist, a smoothened receptor inhibitor, a fibroblast growth factor and retinoic acid.

[0130] In a preferred embodiment, the Stage 5 cells may be differentiated into Stage 6 cells by treatment with amedium supplemented with about 50 nMRA, about 0.25mMascorbic acid, about 100 nMLDN‑193189, about 10,000 nM of ALK5 inhibitor and preferably ALK 5 inhibitor II, 1 µM T3, about 100 nM of a gamma secretase inhibitor for about seven days. Alternatively, Stage 5 cells may be differentiated into Stage 6 cells by treatment with a medium supplemented with about 0.25 µM SANT‑1, about 50 nM RA, about 0.25 mM ascorbic acid, about 1000 nM ALK5 inhibitor and 1 µM T3 for about three days. The cells may be cultured in such media for an additional two days, or more, if desired.

[0131] Alternatively, Stage 5 cells may be differentiated into Stage 6 cells by treatment with a medium supplemented with heparin, an SMO inhibitor or an SHH signaling pathway antagonist, a BMP inhibitor, T3, T4, analogues thereof and mixtures thereof andanALK5 inhibitor andculturing, preferablyat theair-liquid interfaceor in suspensionculture, for about one to seven days, alternatively about six days, alternatively about seven days. In an alternate embodiment, the medium maybesupplementedwithbothanSMOinhibitorandanSHHsignalingpathwayantagonist. Forexample, thecellsmaybe cultured in themediumsupplementedwithabout 10µg / mlof heparin, about 0.25µMSANT‑1, about 100nMLDN‑193189, about 1000 nM of T3 and about 500 to about 10,000 nM, alternatively about 500 nM, alternatively about 1000 mM, and alternatively about 10,000 nM of an ALK5 inhibitor. Suitable ALK5 inhibitors include but are not limited to SD‑208, ALK5 inhibitor II, TGF-β receptor inh V, TGF-β receptor inh I, TGF-β receptor inh IV, TGF-β receptor inh VII, TGF-β receptor inh VIII, TGF-β receptor inh II, TGF-β receptor inh VI, TGF-β receptor inh III and combinations thereof.

[0132] In a preferred embodiment, theALK5 inhibitor is ALK5 inhibitor II. In amore preferred embodiment, about 10,000 nM (10mM) of ALK5 inhibitor II is used. Accordingly, in one embodiment, Stage 5 cells may be differentiated into Stage 6 cells by treatmentwith amediumsupplementedwith heparin, anSMO inhibitor or anSHHsignaling pathway antagonist, a BMP inhibitor, T3, T4, analogues thereof andmixtures thereof, andALK5 inhibitor and culturing, preferably at the air-liquid interface or in suspension, preferably for about seven days. In an alternate embodiment, the medium may be supple- mented with both an SMO inhibitor and an SHH signaling pathway antagonist. In certain embodiments, the cells may be treated with a cell detachment solution, such as a solution containing proteolytic and collagenolytic enzymes prior to culturing at the air-liquid interface or in suspension.

[0133] In another embodiment, Stage 5 cells may be differentiated into Stage 6 cells by treatment with a medium supplemented with heparin, an SMO inhibitor or an SHH signaling pathway antagonist, a BMP inhibitor T3, and ALK5 21 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 inhibitor II and culturing at the air-liquid interface for about 5 days to about 7 days, alternatively about 5 days, alternatively about 6 days, alternatively about 7 days In these embodiments, themediummay be supplementedwith about 10µg / ml of heparin, about 0.25µMSANT‑1, about 100 nMLDN‑193189, about 1000 nMof T3 and about 10,000 nMof ALK5 inhibitor II. In certain embodiments, themediummay be further supplementedwith zinc sulfate (ZnSO4). For example, themedium may be further supplemented with about 10 mM ZnSO4. In an alternate embodiment, the mediummay be supplemented with both an SMO inhibitor and an SHH signaling pathway antagonist.

[0134] In a particularly preferred embodiment of the invention, one or more of an aurora kinase inhibitor, preferably aurora kinase inhibitor II, an RSK inhibitor, preferably RSK inhibitor II and the protein methyl transferase inhibitor of DOT1L, preferably EPZ‑5676, is added to the medium. The amount added may be from about 100 to 5000 nM, alternatively about 1000 to 5000 nM, alternatively about 2000 to 5000 nM, alternatively about 3000 to 5000 nM, and preferably about 1000 to 2000 nM for the aurora kinase and RSK inhibitors and about 100 to 1000 nM for the DOT1L inhibitor, and more preferably about 1µM to about 10 nM.

[0135] In accordance with the foregoing method, the invention further provides a cell culture for differentiating cells expressing markers characteristic of pancreatic endoderm / endocrine precursor cells into cells expressing markers characteristic of immature beta cells, comprising: (a) a culture vessel; (b) a volume of growth medium within said vessel sufficient to fill only a portion of the volumeof said vessel; (c) air within said vessel that fills a portion of said vessel adjoining said medium; (d) a porous substrate located at the interface between said medium and said air; and (e) cells expressing markers characteristic of pancreatic endoderm / endocrine precursor cells derived from pluripotent stem cells disposed upon the surface of said substrate such that said medium contacts only a portion of the surface of said cells.

[0136] In accordance with the foregoing method, alternatively the invention further provides a cell culture for differ- entiating cells expressing markers characteristic of the pancreatic endocrine precursor cells (or pancreatic endoder- m / endocrine precursor cells) into cells expressing markers characteristic of immature beta cells comprising: (a) a culture vessel that facilitates aggregation of cells, or forming cell clusters; (b) a volume of growthmedium (culturemedium)within said vessel; (c) cells derived from pluripotent stem cells disposed within the vessel such that the cells are induced to aggregate and form clusters; and (d) generated cell clusters disposed in suspension (suspension culture). In embodi- ments, the vessel for cell aggregation is a plate with wells or microwells, such as Aggrewell™ plates. In embodiments, the suspension culture systemmay include seeding aggregated cells or cell clusters in a suspension culture vessel, such as flask, for example a spinner flask or a spinner wheel flask.

[0137] In one embodiment, Stage 5 cells cultured according to embodiments of the invention are utilized and differentiated into Stage 6 cells, while in other embodiments Stage 5 cells cultured according to other protocols may be utilized in the current method to obtain Stage 6 and Stage 7 cells.

[0138] In an embodiment, the methods of the invention result in the formation of Stage 6 cells that are single-hormone positive. Thus, in one embodiment, themethods of the invention result in Stage 6 cells, which co-expressNKX6.1, insulin, chromogranin and PDX1. In another embodiment, the methods of the invention result in Stage 6 cells, which co-express NKX6.1 and insulin. In certain embodiments of the invention, themethod employs a custommedium, BLAR (see Table I), atStages4 to6or lateStage4 to6orStages5and6.Themediummaybeexchangedeverydayoralternativelyeveryother day.

[0139] In another embodiment, the invention relates to a method of forming Stage 6 cells co-expressing NKX6.1 and chromogranin comprising culturing Stage 4, preferably late Stage 4 cells to Stage 6 cells at the air-liquid interface or in suspension. In yet another embodiment, the invention relates to amethod of forming single hormone insulin positive cells expressing NKX6.1 Stage 6 cells by culturing Stage 4, preferably late Stage 4 cells, to Stage 6 cells at the air-liquid interface or in suspension. Stage 7: Differentiation of cells expressing markers characteristic of immature beta-cells to cells expressing markers characteristic of functional beta-cells capable of a two phase GSIS and mitochondrial respiration re- sponse.

[0140] In one embodiment, themethods of the invention include treating Stage 6 cells with a differentiationmedium that may be any suitable growth medium, preferably such as MCDB‑131 or CMRL or, and more preferably, a custom media such as BLAR001 (Table 1) or BLAR004 (Table IV) for seven days.

[0141] The medium is supplemented with one or more of the following: 2.7g / 1000 ml sodium bicarbonate, and supplemented with a 1:200 dilution of ITS-X; 1X GlutaMAX™; 2% FAF-BSA; 10 µg / ml of heparin ("H"); 10 nM of T3 ("low T3"); 1 mMN-Acetyl cysteine ("NAC"); 0.5 µMof ZM447439 ("ZM"); and the following components which constitute Formulation I ("FI") (Table XII) - 1:200 dilution of RPMI Vitamin Supplement; 1:200 dilution of MEM non-essential amino acid supplement; 1:2000dilution of chemically defined lipid concentrate; 1:200dilution of sodiumpyruvate; 1:2000dilution of trace elements A (Corning, Catalog No. 25‑021); 1:2000 dilution of trace elements B (Corning, Catalog No. 25‑022). Additional compounds that may be added to obtain Stage 7 include about 10 nM of T3 ("low T3"); 5 µM 5-Azacytidine ("AZT") (Sigma Aldrich, Catalog No. A2385); or about 1 µM 3-Deazaneplanocin A ("DEZA") (Biovision, Inc., Catalog No. 22 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 2060). In embodiments to obtain Stage 7 cells, the medium does not contain an ALK5 inhibitor.

[0142] In one embodiment, the Stage 6 cells may be differentiated into Stage 7 cells by treatment with a medium supplemented with, about 10 nMof T3, about 0.5µMof one ormore of aurora kinase inhibitor II, and about 1mMN-acetyl cysteine. Alternatively, Stage 6 cells may be differentiated into Stage 7 cells by treatment with a medium supplemented with heparin, T3, T4, analogues thereof ormixtures thereof, an antioxidant, and aurora kinase inhibitor, ormixtures thereof and culturing, at the air-liquid interface or suspension cultures, for about seven to twenty-one days, alternatively about seven to ten days, preferably about seven days.

[0143] In accordance with the foregoing method, the invention further provides a cell culture for differentiating cells expressing markers characteristic of pancreatic endocrine precursor cells into cells expressing markers characteristic of functional beta.‑cells, comprising: (a) aculturevessel; (b) avolumeofgrowthmediumwithinsaidvessel sufficient to fill only a portion of the volumeof said vessel; (c) air within said vessel that fills a portion of said vessel adjoining saidmedium; (d) a porous substrate located at the interface between said medium and said air; and (e) cells expressing markers characteristic of pancreatic endoderm / endocrine precursor cells derived from pluripotent stem cells disposed upon the surface of said substrate such that said medium contacts only a portion of the surface of said cells.

[0144] In one embodiment, Stage 6 cells cultured according to embodiments of the invention are utilized and differentiated into Stage 7 cells, while in other embodiments Stage 6 cells cultured according to other protocols may be utilized in the current method to obtain Stage 7 cells. In another embodiment, themethods of the invention result in the formationofStage7cells that aresingle-hormonepositive.Thus, inoneembodiment, themethodsof the invention result in Stage 7 cells, which co-express NKX6.1, chromogranin, PDX1, UCN3, SLC2A1 and MAFA. In another embodiment, the methods of the invention result in Stage 7 cells, which co-express NKX6.1, PDX1, insulin, UCN3, SLC2A1 and MAFA. In still another embodiment, a population of cells inwhich each of the cells, at least about 10%, alternatively at least about 20 %,alternatively at least about 30%,alternatively at least about 40%,alternatively at least about 50%,alternatively at least about 60%,alternatively at least about 70%,alternatively at least about 80%,or alternatively at least about 90%of the cell population express insulin, PDX1, NKX6.1, UCN3, SLC2A1 and MAFA result.

[0145] In some embodiments, at least 10 % of the cells of the resulting cell population express insulin, PDX1, NKX6.1, UCN3, SLC2A1 and MAFA. In other embodiments, at least 20 % of the cells of the population express insulin, PDX1, NKX6.1, UCN3, SLC2A1 and MAFA. In other embodiments, at least 30 % of the cells of the population express insulin, PDX1, NKX6.1, UCN3, SLC2A1 andMAFA. In still other embodiments, at least 40%of the cells of the population express insulin, PDX1, NKX6.1, UCN3, SLC2A1 andMAFA. In yet other embodiments, at least 50% of the cells of the population express insulin, PDX1,NKX6.1,UCN3,SLC2A1andMAFA. In still other embodiments, at least 60%,70%,80%or 90%of the cells express insulin, PDX1, NKX6.1, UCN3, SLC2A1 and MAFA. In alternative embodiments, at least 91, 92, 93, 94, 95, 96, 97, 98, or 99 % of the cells of the population express insulin, PDX1, NKX6.1, UCN3, SLC2A1 and MAFA.

[0146] In certain andpreferred embodiments of the invention, themethodemploys a custommedium,BLAR (Table I), at Stages 4 through 7 or late Stage 4 through 7, or Stages 5, 6 and7. Themediummaypreferably be exchangedevery day or alternatively every other day.

[0147] In another embodiment, the invention relates to amethod of formingStage 7 cells co-expressingNKX6.1, PDX1, MAFA,UCN3,SLC2A1andchromogranin comprising culturingStage4, preferably lateStage4cells toStage7cells at the air-liquid interface, or in suspension. In yet another embodiment, the invention relates to a method of forming single hormone insulin positive cells (functional beta-cells) expressing NKX6.1, PDX1, UCN3, SLC2A1 andMAFAStage 7 cells by culturing Stage 4, preferably late Stage 4 cells, to Stage 7 cells at the air-liquid interface or in suspension.

[0148] Although the present invention contemplates culturing at the air-liquid interface for all stages in the path from pluripotent cell to pancreatic endocrine cell, the invention provides for the formation of Stage 1 to Stage 4 cells in planar or submerged culture and Stage 5, 6 and 7 cells by culturing the cells at the air-liquid interface or in suspension culture. In other embodiments, the present invention relates to a stepwise method of differentiation of pluripotent cells comprising culturing Stage 4, 5 and 6 cells at the air-liquid interface. In certain embodiments, cells cultured during Stages 4 through 7 may be cultured at the air-liquid interface. In other embodiments, only late Stage 4 to Stage 6 cells, or Stage 5 and Stage 6 cells, are cultured at the air-liquid interface or in suspension In yet another alternative embodiment, Stage 1 through 4 are carriedout byculturing thecells inplanar cultures, andStage5 throughStage7, orStage6 throughStage7,or onlyStage7 are carried out by culturing in suspension culture.

[0149] Additionally, culturing during one, or all of Stages 5, 6 and7 is carried out in the presenceof oneormoreof T3, T4, their analogues and an ALK5 inhibitor, or one or more of T3, T4 and their analogues; or an ALK5 inhibitor. In preferred embodiments, culturing during one ormore, and preferably all of Stages 5, 6 and 7 is carried out in the presence of T3 and an ALK5 inhibitor and more preferably in the presence of T3 and ALK5 inhibitor II. In more preferred embodiments, culturing during Stage 7 is carried out in the presence of T3 at low concentration. In preferred embodiments, culturing during Stage 7 is carried out with no ALK5 inhibitor included.

[0150] Whencells are culturedat theair-liquid interface ("ALI"), the cellsmaybeculturedonaporoussubstrate such that the cells are in contact with air on the top side and with cell culture media at the bottom side. For example, a sufficient volume of media may be added to the bottom of a culture vessel containing the porous substrate (e.g. a filter insert) such 23 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 that themedia contacts the bottom surface of cells residing on the substrate but does not encapsulate or submerge them. Suitable porous substrates can be formed of anymaterial that will not adversely affect the growth and differentiation of the cells. Exemplary porous substrates are made of polymers such as polyethylene terephthalate ("PET"), polyester or polycarbonate. Suitable porous substrates may be coated or uncoated. In one embodiment, the coating may be MATRIGEL™. In another embodiment of the invention, the porous substrate is a porous filter insert, whichmay be coated withMATRIGEL™. Inanother embodiment of the invention, theporoussubstrate is anuncoatedfilter insert. Theporosity of the substrate should be sufficient to maintain cell viability and promote differentiation of the cells.

[0151] Culturing of the cells at the air-liquid interface includes seeding the cells on a porous substrate such as a porous filter insert. In certain embodiments, the substrate pore sizemay range fromabout 0.3 to about 3microns. Seedingmaybe accomplished by releasing cells as single cells frommonolayer cultures or clusters of cells frommonolayer cultures into a suspension and subsequently aliquoting the single cell suspension or suspended cell culture onto a porous substrate at theALI.Thecellsmaybeseededonto theporoussubstrate fromasuspensionhavingabout1000cells / µl toabout100,000 cells / µl. The cellsmay be seeded as droplets of the cell suspension containing individual cells or aggregates or clusters of cells. In certain embodiment, the cellsmaybeculturedat theair-liquid interfaceusing themethodsdisclosed inU.S.Patent App. Pub. No. 2014 / 0186305, the disclosure of which is herein incorporated as it pertains to the culturing and differentiating pluripotent stem cells at the air liquid interface.

[0152] The media may be exchanged or refreshed every other day or, preferably, daily. The cells grown on top of the poroussubstratearegenerally not single cells, but rather theyare in the formofa sheet orexist asanaggregatecell cluster. Cells cultured at the ALI may experience higher oxygen tension as compared to cells submerged in media.

[0153] In certain embodiments, themethods of the inventionmay be carried out by culturing and differentiating the cells as clusters in a suspension culture. Exemplary suitable methods for the suspension culture and differentiation of pluripotent stem cells are disclosed in U.S. Patent App. Pub. Nos. 2014 / 0242693 and 2014 / 0295552, the disclosures of which are herein incorporated as it pertains to the culturing and differentiating pluripotent stem cells using suspension clusters. In particular embodiments, the present invention provides an in vitro cell culture including differentiating cells prepared as aggregated cells such as cell clusters comprising seeding cells in a culture vessel that facilitates aggregation of cells, or forming cell clusters, with a growth medium (culture medium) such that the cells are induced to aggregate and form clusters. In embodiments, the vessel useful for inducing aggregation of cells is a plate with wells or microwells, such as Aggrewell™ plates. The resulting aggregated cells or cell clusters are further cultured in suspension (suspension culture), comprising removing the aggregated cells / cell clusters from the microwells and seeding them in a suspension culture vessel such that the aggregated cells are differentiated in suspension.

[0154] Embodiments of the present invention encompass formation of late Stage 4 to 7, preferably Stage 5 to 7 cells, at the air-liquid interface or in suspension. The cells may be formed by differentiating pluripotent stem cells or by further differentiating Stage 3, 4, 5 or 6 cells. Stage 4 cells may be cultured entirely at the air-liquid interface or the cells may be cultured in submerged planar culture during the early portion of Stage 4, (about one to two days) and then cultured at the air-liquid interface or in suspension for the latter portion of Stage 4 (about day two to day three). Preferably, Stage 4. is not carried out at the ALI, or in suspension, but rather in submerged culture.

[0155] In one embodiment, the present invention provides a method for producing cells expressing markers char- acteristic of functional beta-cells frompluripotent stemcells, comprising: culturingpluripotent stemcells; differentiating the pluripotent stemcells into cells expressingmarkers characteristic of the pancreatic endoderm; and differentiating the cells expressing markers characteristic of the pancreatic endoderm into cells expressing markers characteristic of the pancreatic endocrine cells by culturing, at the air-liquid interface or in suspension. The method may include treatment with amedium supplemented with: (i) T3, T4 or their analogues; (ii) an ALK5 inhibitor; or both (i) and (ii). Themethodmay include differentiating the cells expressing markers characteristic of foregut endoderm cells (Stage 3 cells) into cells expressingmarkers characteristic of pancreatic endodermcells (Stage4 cells) by treatmentwith amediumsupplemented with: (i) one or both of T3, T4 or their analogues; (ii) ALK5 inhibitor; or both (i) and (ii); and culturing in a planar culture. The method may also include differentiating cells expressing markers characteristic of pancreatic endoderm cells (Stage 4 cells) into cells expressing markers characteristic of the immature beta-cells (Stage 6 cells) by treatment with a medium supplementedwith: (i) oneorbothofT3,T4or their analogues; (ii)ALK5 inhibitor; or both (i) and (ii); andculturing inaplanar culture or culturing at the air-liquid interface or in suspension. Themethod further includes differentiatingStage 6 cells into cells expressingmarkers characteristic of functional beta-cells and that have amore matured phenotype as compared to Stage6 (Stage7cells) by treatmentwithamediumsupplementedwith: (i) oneorbothofT3,T4or their analogues; (ii) ALK5 inhibitor; or both (i) and (ii); along with an one or more of an aurora kinase inhibitor, an RSK inhibitor, and an inhibitor of protein methyltransferase DOT1L and, optionally an anti-oxidant such as vitamin E or acetyl cysteine. The preferred amount of acetyl cysteine that is useful is about 0.1 to about 2mM. The preferred amount of vitamin E is about 0.1 to about 10 µM. In yet another embodiment, the method further includes carrying out Stage 6 by treatment of Stage 5 cells with a mediumsupplementedwith: (i) oneor bothof T3,T4or their analogues; (ii) ALK5 inhibitor or; both (i) and (ii); alongwithone or more of a gamma secretase inhibitor, an RSK inhibitor and an inhibitor of protein methyltransferase DOT1L. In still another embodiment, Stage 6 is carried out by treatment of Stage 5 cellswith amediumsupplementedwith: (i) one or both 24 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 of T3, T4or their analogues; (ii) ALK5 inhibitor; or both (i) and (ii); alongwith oneormoreof a gammasecretase inhibitor, an RSK inhibitor and an inhibitor of protein methyltransferase DOT1L followed by carrying out Stage 7 by treatment with a medium supplementedwith: (i) one or both of T3, T4 or their analogues; (ii) ALK5 inhibitor; or both (i) and (ii); alongwith an oneormoreof anaurora kinase inhibitor, anRSK inhibitor, an inhibitor of proteinmethyltransferaseDOT1Land, optionally anti-oxidant such as vitamin E or acetyl cysteine.

[0156] One embodiment of the invention is a method of forming functional beta-cells (pancreatic endocrine cells expressing markers characteristic of a matured phenotype) (Stage 7 cells) comprising differentiating cells expressing markers characteristic of the pancreatic endoderm cells (Stage 4 cells) into cells expressing markers characteristic of Stage 7 cells by culturing at the air-liquid interface or in suspension. A cell expressingmarkers characteristic of functional beta-cells of a more matured phenotype expresses PDX1 and at least one of the following transcription factors: NKX2.2, NKX6.1, NeuroD1, ISL1, HNF3β, MAFA, UCN3, SLC2A1, PAX4, HB9 and PAX6. In one embodiment, themethods of the invention result in the formation of Stage 6 cells, which are positive for NKX6.1, PDX1,HB9andMAFA.Preferably, at least during Stages 5 through 7, the method includes treatment with a medium supplemented with T3, T4 or an analogue thereof, an ALK5 inhibitor, or both. The Stage 6 cells may be cells that are positive for NKX6.1, PDX1, HB9 and MAFA. In other embodiments, the Stage 6 or 7 cells are single hormone positive cells. For example, the Stage 6 and 7 cells may be cells that: (a) co-expressNKX6.1and chromogranin; (b) co-expressNKX6.1and insulin; or (c) co-expressNKX6.1,PDX1, MAFA and single hormonal insulin. The Stage 7 cells express single hormonal insulin and MAFA at increased levels and increased numbers of cells within a cell population as compared to Stage 6 cells.

[0157] In another embodiment, the invention provides a method of enhancing the number of single hormone positive cells (e.g. cells that co-express NKX6.1 and insulin or cells that co-express NKX6.1 and chromogranin) by culturing and differentiating a population of PDX1andNKX6.1 co-expressing cells, preferably at an air-liquid interface or in suspension. In another embodiment, pancreatic endoderm cells cultured at the air-liquid interface or in suspension are further differentiated to functional beta-cells by treatment with a compound selected from the following: ALK5 inhibitor, BMP inhibitor, gamma-secretase inhibitor, Ephrin ligands, EphB inhibitor, PKC inhibitor, EGFr inhibitor, retinoic acid, vitamin C, T3 / T4, glucose, cell cycle regulators, WNT regulators, SHH inhibitor, aurora inhibitor, anti-oxidants, vitamin E, acetyl- cysteine, or combinations thereof.

[0158] In further embodiments, the present invention relates to a stepwisemethod of differentiating pluripotent cells that includes culturing Stage 4 through Stage 6 cells in a media containing sufficient amounts of: (i) one or more of T3, T4 and their analogues; (ii) an ALK5 inhibitor; or both (i) and (ii); and further culturing the Stage 6 cells in a media that optionally contains one ormore of an aurora kinase inhibitor, an RSK inhibitor, and an inhibitor of proteinmethyltransferase DOT1L, and an antioxidant to generate functional beta-cells (pancreatic endocrine cells of amatured phenotype) and populations of functional beta-cells that express insulin, PDX1, NKX6.1, UCN3, SLC2A1 and MAFA.

[0159] Stage6and7cells generatedaccording to themethodsdescribedhereinarealsowell-suited for use in screening compounds for their effect on the secretion of pancreatic hormones and endocrinemarkers. In particular, Stage 4 through Stage 7 cells cultured at ALI, or in suspension culture, can be tested in different culture formats from384 to 6-well formats. Such formats allow for evaluation of a variety of small molecules or biologics at various doses and time intervals on subsequent expression of pancreatic endoderm, pancreatic endocrine precursor, pancreatic endocrine, and pancreatic betacellmarkers.SuchanevaluationmaybeaccomplishedbymeasuringgeneexpressionbyPCR,proteinexpressionby FACS or immune staining, or by ELISA for secretion of factors by cells affected by addition of small molecules / biologics. F Cells obtainable by the methods of the invention.

[0160] The inventionprovides forStage7cells or apopulationofStage7cells obtainable byamethodof the invention. In certain embodiments, the cells or cell populations are not purified after differentiation. In certain embodiments, these Stage 7 cells express single hormonal insulin and are PDX1, NKX6.1, UCN3, SLC2A1 and MAFA positive; in addition these cells havea higher level of expression ofMAFA than theStage 6 cells. The cells expressUCN3at a higher level than Stage6cells (immaturebeta-cells). The resultingcell populationhasahigher percentageof bothMAFApositiveandsingle hormonal insulin-expressing cells than the Stage 6 cells. The invention also provides an insulin positive cell or population of insulin positive cells, expressingmarkers characteristic of functional beta-cells (pancreatic endocrine cells of amatured phenotype), characterized by NKX6.1 expression (preferably greater than about 30 %), PDX1 expression (preferably greater than about 30%), UCN3expression (preferably greater than about 10%), SLC2A1expression (preferably greater than about 10 %), and MAFA expression (preferably greater than about 10 %).

[0161] In embodiments of the invention, theStage7cells or cell populations are insulin producing cells or a population of insulin producing cells. The insulin producing cells are functionally mature beta cells that exhibit mitochondrial respir- ation / activity and GSIS response to glucose similar to human islet cells. In embodiments of the invention, functionally mature beta cells are generated in suspension culture.

[0162] In embodiments, the functional beta-cell exhibits glucose-stimulated insulin secretion and glucose-dependent mitochondrial respiration. In embodiments, the glucose-stimulated insulin secretion and glucose-dependent mitochon- 25 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 drial respiration is similar to that of human islet cells. In embodiments of the invention, the functional beta-cell secretes insulin in multiple phases.

[0163] In theembodiments, theglucose-dependentmitochondrial respiration hasamaximumoxygenconsumption rate response following glucose stimulation in the range of about 20% to about 80% over basal oxygen consumption rate, preferablyabout20%toabout70%,20%toabout toabout 60%,morepreferablyabout 20%,about25%,about30%,about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or about 80%. In embodiments, the oxygen consumption rate response occurs at least 10 minutes to about 15 minutes following glucose stimulation, preferably about 10minutes, about 11minutes, about 12minutes, about 13minutes, about 14minutes, about 15minutes. In embodiments, the higher oxygen consumption rate over basal OCR ismaintained for at least 60minutes to at least 80minutes, preferably at least 70minutes to at least 80minutes,more preferably at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 minutes

[0164] In the embodiments above, the glucose-stimulated insulin secretion comprises a rapid, bi-phasic insulin secretion in response to glucose stimulation. In embodiments, a first phase of the bi-phasic insulin secretion has at least a four-fold increase toat least aneight-fold increaseover baseline secretion, preferably at least a four-fold increase, a five-fold increase, a six-fold increase, a seven-fold increase, or an eight-fold increase over baseline secretion. In embodiments, a second phase of the bi-phasic insulin secretion has at least a two-fold to at least a four-fold increase over baseline secretion, preferably at least a two-fold increase, a three-fold increase or a four-fold increase over baseline secretion. In embodiments, the insulin secretion occurs at least five minutes to at least ten minutes following glucose stimulation, preferably at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes or at least 10 minutes following glucose stimulation.

[0165] Tables A and B illustrate exemplary culture conditions suitable for use in embodiments of methods of the invention. As used in TablesAandBbelow, "MCX" isMCXcompound, "AA" is activin, "ALK5 inh." is ALK5 inhibitor, "RA" is retinoic acid, "Vit. C" is ascorbic acid, "inh." is inhibitor, and "act." is activator. In certain embodiments, any one of the treatments at one stage (e.g. any one of Stage 1, 2, 3, 4, 5, 6 or 7) may be combined with any one of the treatments at another stage (e.g. anyoneofStage1, 2, 3, 4, 5, 6 or 7). In other embodiments, Stage4cells obtainedbymethodsdifferent from those in Table A may be differentiated into Stage 5 to Stage 7 cells using the culture conditions shown in Table B. In other embodiments, Stage 5 cells obtained by methods different from those in Tables A and B may be differentiated into Stage 6 orStage 7 cells using the culture conditions shown in TableB. In yet other embodiments, Stage 6 cells obtained by methods different from those in Tables A and Bmay be differentiated into Stage 7 cells using the culture conditions shown in Table B. Table A: Exemplary culture conditions to obtain Stage 1 to Stage 4 cells according to embodiments of the invention Stage 1 Stage 2 Stage 3 Stage 4 Treatment of Pluripotent stem cells Stage 1 cells Stage 2 cells Stage 3 cells With at least AA & Wnt3 A GDF8 & MCX FGF7 FGF7 & Vit. C SANT‑1, RA & noggin FGF7, retinoic acid, SANT‑1, a PKC act.(e.g. TPB), a BMP inh. (e.g. LDN‑193189), & Vit. C FGF7, retinoic acid, SANT‑1, a PKC act.(e.g. TPB) & Vit. C 26 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Table A: Exemplary culture conditions to obtain Stage 1 to Stage 4 cells according to embodiments of the invention Stage 1 Stage 2 Stage 3 Stage 4 Treatment of Pluripotent stem cells Stage 1 cells Stage 2 cells Stage 3 cells FGF7, retinoic acid, SANT‑1, a PKC act. (e.g. TPB), a BMP inh. (e.g. LDN‑193189), & Vit. C Other op- tional com- ponents 0.5% FAF-BSA 0.5% FAF-BSA BLAR medium 2% FAF-BSA Duration of Treatment Approximately 2‑5 days; pre- ferably about 3‑4 days Approximately 2‑3 days; preferably about 2 days; prefer- ably about 3 days when AA & Wnt3A at Stage 1 Approximately 2‑4 days, preferably about 2 days Approximately 2‑4 days, preferably about 3 days Stage 1 Stage 2 Stage 3 Stage 4 Culture con- dition Planar Culture or cell clusters (suspension culture) (roller bottles) Planar Culture or cell clusters (roller bot- tles) Planar Culture or cell clusters (roller bottles) Planar Culture optionally cells at end of Stage 4 are seeded at the ALI using Y-compound alternatively cells are transitioned to clusters; cell clusters or roller bottles Table B: Exemplary culture conditions to obtain Stage 5 to Stage 7 cells according to embodiments of the invention Stage 5 Stage 6 Stage 7 Treatment of Stage 4 cells Stage 5 cells Stage 6 cells With at least SANT‑1, a PKC act.(e.g. TPB), a BMP inh. (e.g. LDN‑193189), Vit C, an ALK5 inh. (e.g. ALK5 inh. II), T3 SANT‑1, a PKC act.(e.g. TPB), a BMP inh. (e.g. LDN‑193189), an ALK5, inh. (e.g. ALK5 inh. II), T3 BMP inh. (e.g. LDN‑193189), an ALK5 inh. (e.g.ALK5 inh. II), T3, gamma secretase inh., heparin BMP inh. (e.g. LDN‑193189), Vit C, an ALK5 inh (e.g.ALK5 inh. II), T3, gamma secretase inh., heparin T3, N-Acetyl Cysteine, ZM447439, hepar- in, ± ALK5 27 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Table B: Exemplary culture conditions to obtain Stage 5 to Stage 7 cells according to embodiments of the invention Stage 5 Stage 6 Stage 7 T3, N-Acetyl Cysteine, ZM447439, hepar- in, ALK5 + one of: UNC0638, UNC0642, TC-E5003, A366, PF03814735 SB747651, PFI1, LY303511, MS436 or MC1568 T3, N-Acetyl Cysteine, ZM447439, hepar- in, no ALK5 + one of: UNC0638, UNC0642, TC-E5003, A366, PF03814735 SB747651, PFI1, LY303511, MS436 or MC1568 no ALK5, low T3, ZM, H, NAC no ALK5, low T3, ZM, H, NAC, AZT & DEZA Other op- tional com- ponents Replace T3 with T4 or thyroid hormone analo- gue ZnSO4 ZnSO4 ZnSO4 an ALK5 inh. Replace T3 with T4 or thyroid hormone analogue AZT DEZA Duration of Treatment Approximately 2‑4 days, preferably about 3 days Approximately 6‑8 days, preferably about 7 days Approximately 6‑7 days; alternatively ap- proximately 7‑14 days; alternatively about 7‑23 days Culture con- dition ALI or cell clusters (sus- pension culture) (roller bottles) ALI or cell clusters (roller bottles) ALI or cell clusters (roller bottles)

[0166] Publications cited throughout this document are hereby incorporated by reference in their entirety. The present invention is further illustrated, but not limited, by the following non-limiting examples. EXAMPLES

[0167] The suppliers of the materials and compounds used in the following examples are identified in Table X. Example 1 Screening and identification of small molecules that upregulate MAFA or UCN3 expression.

[0168] The following example is directed to identification of small molecules that can enhance thematuration status of a pancreatic beta cell, via increasing the gene expression of mature beta cell markers including MAFA (v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog A) or UCN3 (urocortin 3). Cells of the human embryonic stem cell lineH1 ("H1-hESC")withEZ8mediaat passage28wereseededassingle cells at 0.094x106 cells / cm2onMATRIGEL™at a 1:30 dilution on coated dishes in amedia of Dulbecco’sModified Eagle’sMediumNutrient mixture F‑12 ("DMEM-F12") , GlutaMAX™ina1:100dilution ("1Xconcentration"), 0.25mMascorbicacid ,100ng / mlfibroblastgrowth factor2 ("FGF2") ,1 ng / mlof transforminggrowth factorbeta ("TGFβ") , insulin-transferrin-selenium-ethanolamine ("ITS-X") at a1:100dilution, 2% fatty-acid free bovine serum albumin ("FAF-BSA"), and 20 ng / ml of insulin-like growth factor‑1 ("IGF‑1"), supple- mented with 10µM of Rock Inhibitor Y‑27632 ("Y-compound"). Y-compound was added to only during the first 24 hours post-seeding. Forty-eight hours post-seeding, the cultures were washed in incomplete PBS (phosphate buffered saline without magnesium or calcium).

[0169] For FIGS. 1A to 1H the cultureswere differentiated using the following protocol. During Stages 1 through 4 of the protocol, cultures were maintained on planar adherent cultures. a. Stage 1 (3 days): Cells were cultured for one day in the following Stage 1 media: MCDB‑131 medium containing 2000ml sodium bicarbonate (Sigma-Aldrich Co. LLC, St. Louis, Missouri, Catalog No. 5761), and supplemented with 28 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 0.5% FAF-BSA, GlutaMAX™ in a 1:100 dilution ("1X concentration"), 4.5mM D-glucose to obtain a concentration of 10mM of D-glucose, 100 ng / ml growth differentiation factor 8 ("GDF8"), and 1.5 µM of a 14-Prop‑2-en‑1- yl‑3,5,7,14,17,23,27-heptaazatetracyclo [19.3.1.1~2,6~.1~8,12~]heptacosa‑1(25),2(27),3 5,8(26),9,11,21,23-non- aen‑16-one ("MCX compound"). Cells were then cultured for an additional day in MCDB‑131 medium containing 2.7g / 1000ml sodium bicarbonate, and supplemented with 0.5% FAF-BSA, 1X concentration of GlutaMAX™, 4.5mM D-glucose to obtain a concentration of 10mM D-glucose, 100 ng / ml GDF8, and 0.1 µMMCX compound. Cells were thencultured for anadditional day inMCDB‑131containing2.7g / 1000ml sodiumbicarbonate, andsupplementedwith 0.5% FAF-BSA, 1X concentration of GlutaMAX™, 4.5mM D-glucose to obtain a concentration of 10mM D-glucose, and 100ng / ml GDF8. b. Stage 2 (2 days): Cells were treated for two days with MCDB‑131 medium containing 2.7g / 1000ml sodium bicarbonate, and supplemented with 0.5%FAF-BSA, 1XGlutaMAX™, 4.5mMD-glucose to obtain a concentration of 10nM D-glucose, 0.25 mM Ascorbic acid, and 50 ng / ml fibroblast growth factor 7 ("FGF7"). c. Stage 3 (2 days): Cells were treated for two dayswith BLAR001 custommedium (seeTable I) containing 3.6g / 1000 ml sodium bicarbonate, and supplemented with a 1:200 dilution of ITS-X; 4.5mMD-glucose to obtain a concentration of 10mMD-glucose; 1XGlutaMAX™;2%FAF-BSA; 25ng / ml FGF7; 0.25µMSANT‑1 (N‑[(3,5-dimethyl‑1-phenyl‑1H- pyrazol‑4-yl)methylene]‑4‑(phenylmethyl)‑1-piperazineamine); 1µMretinoicacid ("RA"); 0.25mMascorbic acid, 300 nMof thePKCactivator ((2S, 5S‑(E,E)‑8‑(5‑(4-trifluoromethyl)phenyl‑2,4,‑pentadienoylamino)benzolactam ("TPB"); and the bone morphogenic protein ("BMP") receptor inhibitor LDN‑193189-HCl ("LDN-HCl") for two days. The concentration of LDN-HCl used for the first day of stage 3 was 100 nM, and for the second day of stage 3 was 10 nM. d. Stage 4 (3 days): Cells were treated with BLAR001 medium containing 3.6g / 1000 ml sodium bicarbonate, and supplemented with a 1:200 dilution of ITS-X; 4.5mM D-glucose to obtain a concentration of 10mM D-glucose; 1X concentration of GlutaMAX™; 2% FAF-BSA; 0.25 µM SANT‑1; 50 nM RA; 2 ng / ml FGF7; 50 nM LDN-HCl; 0.25 mM ascorbic acid; and 200 nM TPB for three days. At the end of Stage 4 (3 days), cells cultured on planar dishes were seededonan air-liquid-interface ("ALI"). Specifically, cellswere treated for 4 hourswith 10µMofY‑27632, rinsedwith PBS and treated for approximately 2 minutes with the enzyme TrypLE™ Express Enzyme at a concentration of 1X followed by removal of the enzyme, and removal of the cells from the onMATRIGEL™surface by gentle tapping of the flask. The resulting suspension of cells were seeded at a density of 0.5‑1.0 x 106 cells (in 5 µl aliquots) on either 0.4 micron or 3.0micron porous cell culture filter inserts on 10 cmplates. 8.0ml ofmediawas added to the bottomof each insert and no further media was added to the apical, or top, side of the filter. The media was replaced daily for the duration of Stages 5, 6, and 7. e.Stage5 (3days):Cellswere treatedon theALIwithBLAR001mediumcontaining2.7g / 1000mlsodiumbicarbonate, and supplemented with a 1:200 dilution of TTS-X; 14.5 mM D-glucose to achieve a final concentration of 20 mM D- glucose; 1XGlutaMAX™;2%FAF-BSA;10µg / mlof heparin ("H"); 10µMZnSO4; 0.25µMSANT‑1; 50nMRA;100nM LDN-HCl; 1 µM of T3 in the form of 3,3’, 5-triiodo-L-thryonine sodium salt; 10 µM of 2‑(3‑(6-Methylpyridin‑2-yl)‑1H- pyrazol‑4-yl)‑1,5-naphthyridine ("ALK5 inhibitor II" or "ALK5") for three days. f. Stage6 (7days): Cellswere treated on theALIwithBLAR001mediumcontaining 2.7g / 1000ml sodiumbicarbonate, and supplemented with a 1:200 dilution of TTS-X; 14.5 mM D-glucose to achieve a final concentration of 20 mM D- glucose; 1X GlutaMAX™; 2°% FAF-BSA; 10 µg / ml of heparin; 10 µM ZnSO4; 100 nM LDN-HCl; 1 µM of T3; 10 µM ALK5 inhibitor II; and 100nM (S,S)‑2‑[2‑(3,5-Difluorophenyl)acetylamino]‑N‑(5-methyl‑6-oxo‑6,7-dihydro‑5H-diben- zo[b,d]azepin‑7-yl)propionamide ("gamma secretase inhibitor XX") for seven days. g.Stage7 (7days): Cellswere treatedon theALIwithBLAR001mediumcontaining 27g / 1000ml sodiumbicarbonate, and supplemented with a 1:200 dilution of ITS-X; 1X GlutaMAX™; 2% FAF-BSA; 10 µg / ml of heparin ("H"); 10 µM ZnSO4; 1 µM of T3; 10 µM of ALK5 inhibitor II; 1 mM N-Acetyl cysteine ("NAC"). In addition, during Stage 7 (S7), for FIGS. 1A‑1H, the cells were conditioned at 20 mM D-glucose plus BME Vitamin supplement (1:100 dilution of 100X) and exposed to all 79 small molecules as listed and described in Table XI. Of the 79 small molecules, the following compounds induced expression of theMAFA or UCN3: (i) Zebularine; (ii) Lomeguatrib; (iii) 5-Azacytidine; (iv) Mitoxantrone dihydrochloride; (v) EGCG; (vi) Fisetin; (vii) SGI 1027; (viii) Temozolomide; (ix) L002; (x) C646; and (xi) SGC0946.

[0170] For FIGS. 2A to 2B the cultures were differentiated using the following protocol: during Stages 1 through 4 of the protocol, cultures were maintained on planar adherent cultures. 29 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 a. Stage 1 (3 days): Cells were cultured for one day in the following Stage 1 media: MCDB‑131 medium containing 2.7g / 1000ml sodium bicarbonate, and supplemented with 0.5% FAF-BSA, GlutaMAX™ in a 1:100 dilution ("1X concentration"), 4‑5mM D-to obtain a concentration of 10mM of D-glucose, 100 ng / ml growth differentiation factor 8 ("GDF8"), and 1.5 µM of a 14--Prop‑2-en‑1-yl‑3,5,7,14,17,23,27-heptaazatetracycle [19.31.1~2,6~.1~8,12~]hepta- cosa‑1(25),2(27),3,5,8(26),9,11,21,23-nonaen‑16-one ("MCXcompound").Cellswere thencultured for anadditional day in MCDB‑131medium containing 2.7g / 1000ml sodium bicarbonate, and supplemented with 0.5% FAF-BSA, 1X concentration of GlutaMAX™, 4.5mMD-glucose to obtain a concentration of 10mMD-glucose, 100 ng / ml GDF8, and 0.1µMMCX compound. Cells were then cultured for an additional day inMCDB‑131 containing 2.7g / 1000ml sodium bicarbonate, and supplemented with 0.5%FAF-BSA, 1X concentration of GlutaMAX™, 4. 5mMD-glucose to obtain a concentration of 10mM D-glucose, and 100ng / ml GDF8. b. Stage 2 (2 days): Cells were treated for two days with MCDB‑131 medium containing 2.7g / 1000ml sodium bicarbonate, and supplemented with 0.5%FAF-BSA, 1XGlutaMAX™, 4.5mMD-glucose to obtain a concentration of 10mM D-glucose, 0.25 mM Ascorbic acid, and 50 ng / ml Fibroblast growth factor 7 ("FGF7"). c. Stage 3 (2 days): Cells were treated for two days with BLAR001 custommedium containing 3.6g / 1000 ml sodium bicarbonate, andsupplementedwith a1:200dilutionof ITS-X; 4.5mMD-glucose toobtain a concentrationof 10mMD- glucose; 1X GlutaMAX™; 1% FAF-BSA; 25 ng / ml FGF7; 0.25 µMSANT‑1 (N‑[(3,5-dimethyl‑1-phenyl‑1H-pyrazol‑4- yl)methylene]‑4‑(phenylmethyl)‑1-piperazineamine); 1µM retinoic acid ("RA"); 0.25mMascorbic acid; 300 nMof the PKC activator ((2S, 5S‑(E,E)‑8‑(5‑(‑4-trifluoromethyl)phenyl‑2,4,‑pentadienoylamino)benzolactam ("TPB"); and the bonemorphogenic protein ("BMP") receptor inhibitor LDN‑193189-HCl ("LDN-HCl") for two days. The concentration of LDN-HCl used for the first day of stage 3 was 100nM, and for the second day of stage 3 was 50nM. d. Stage 4 (3 days): Cells were treated with BLAR001 medium containing 3.6g / 1000 ml sodium bicarbonate, and supplemented with a 1:200 dilution of ITS-X; 4.5mM D-glucose to obtain a concentration of 10mM D-glucose, 1X concentration of GlutaMAX™; 1% FAF-BSA; 0.25 µM SANT‑1; 50 nM RA; 2 ng / ml FGF7; 70 nM LDN-HCl; 0.25 mM ascorbic acid; and 200 nM TPB for three days. At the end of Stage 4 (3 days), cells cultured on planar dishes were seeded on an air-liquid-interface ("ALI"). Specifically, the cells were treated for 4 hours with 10 µMof Y27632, rinsed withPBSand treated for approximately 2minuteswith theenzymeTrypLE™ExpressEnzymeat a concentrationof 1X followed by removal of the enzyme, and removal of the cells from the onMATRIGEL™surface by gentle tapping of the flask. The resulting suspension of cells were seeded at a density of 0.5‑1.0 x 106 cells (in 5 µl aliquots) on either 0.4 micron or 3.0micron porous cell culture filter inserts in 10 cm plates. 8.0ml of media was added to the bottom of each insert and no further media was added to the apical, or top, side of the filter. The media was replaced daily for the duration of Stages 5, 6, and 7. e.Stage5 (3days):Cellswere treatedon theALIwithBLAR001mediumcontaining2.7g / 1000mlsodiumbicarbonate, and supplemented with a 1:200 dilution of ITS-X; 14.5 mM D-glucose to achieve a final concentration of 20 mM D- glucose; 1X GlutaMAX™; 2% FAF-BSA; ‑10 µg / ml of heparin ("H"); 10 µMZnSO4; 0.25 µMSANT‑1; 50 nMRA; 100 nMLDN-HCl; 1µMofT3 in the formof 3,3’, 5-triiodo-L-thryonine sodiumsalt; 10µMof2‑(3‑(6-Methylpyridin‑2-yl)‑1H- pyrazol‑4-yl)‑1,5-naphthyridine ("ALK5 inhibitor II" or "ALK5") for three days. f. Stage6 (7days): Cellswere treated on theALIwithBLAR001mediumcontaining 2.7g / 1000ml sodiumbicarbonate, and supplemented with a 1:200 dilution of ITS-X; 14.5 mM D-glucose to achieve a final concentration of 20 mM D- glucose; 1XGlutaMAX™;2%FAF-BSA; 10µg / mlof heparin ("H"); 10µMZnSO4; 100nMLDN-HCl; 1µMofT3; 10µM ALK5 inhibitor II; and 100nM (S,S)‑2‑[2‑(3,5-Difluorophenyl)acetylamino]‑N‑(5-methyl‑6-oxo‑6 7-dihydro‑5H-diben- zo[b,d]azepin‑7-yl)propionamide ("gamma secretase inhibitor XX") for seven days. g.Stage7 (7days):Cellswere treatedon theALIwithBLAR001mediumcontaining2.7g / 1000mlsodiumbicarbonate, andsupplementedwitha1:200dilutionof ITS-X;1XGlutaMAX™;2%FAF-BSA; 10µg / ml of heparin ("H"); 10nMofT3 ("low T3"); 1 mM N-Acetyl cysteine ("NAC"). Further for FIGS. 2A and 2B, 0.5 µM of ZM447439 ("ZM"); and the following components which constitute Formulation I ("FI") (TableXII) - 1:200dilution ofRPMI vitamin supplement; 1:200dilution ofMEMnon-essential aminoacid supplement; 1:2000dilution of chemically defined lipid concentrate; 1:200dilution of sodiumpyruvate; 1:2000dilution of traceelements A; and 1:2000 dilution of trace elements B were added for seven days. Additional compounds added during Stage 7 included either 5 µM 5-Azacytidine ("AZT"); or 1 µM or 10 µM 3-Deazaneplanocin A ("DEZA").

[0171] Theembodiments described herein useApplicant’s proprietary BLARmedia, specifically BLAR001 orBLAR004 media. BLARmedia was first described in PCT / US13 / 75939 and U.S. Serial No. 13 / 998,884, both filed on December 18, 30 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 2013, which claim the benefit of 61 / 747,662 filed on December 31, 2012; and then again in Rezania et al (2014) Nature Biotech, 32 (11) 1124‑1134, Supplemental Table 3 (published online, September 11, 2014), which references are incorporated herein in their entireties. BLAR001 and BLAR004 are different in the concentrations or levels of the listed agent or excipient. Table I: List of components of BLAR001 medium Amino Acids Concentration (mM) Glycine 3.0E‑02 Alanine 3.0E‑02 Arginine 3.0E‑01 Aspargine 1.0E‑01 Aspartic Acid 1.0E‑01 Cysteine 2.0E‑01 Glutamic acid 3.0E‑02 Histidine 1.1E‑01 Isoleucine 1.0E‑02 Leucine 9.0E‑02 Lysine hydrochloride 1.5E‑01 Methiane 3.0E‑02 Phenylalanine 3.0E‑02 Proline 1.0E‑01 Serine 1.0E‑01 Theronine 3.0E‑02 Tryptophan 2.0E‑03 Tyrosine disodium 1.0E‑02 Valine 3.0E‑02 Vitamins Biotin 3.0E‑05 Choline chloride 5.0E‑03 D-Calcium pantothenate 1.5E‑03 Folinic Acid Calcium salt 2.3E‑03 Niacinamide 4.9E‑03 Pyridoxine hydrochloride 9.7E‑04 Riboflavin 1.0E‑05 Thiamine hydrochloride 3.0E‑03 Vitamin B12 3.7E‑06 i-Inositol 2.8E‑03 Salts / Minerals Calcium Chloride (CaCl2‑2H2O) 3.0E‑01 Cupric sulfate (CuSO4‑5H2O) 4.8E‑06 Ferric sulfate (FeSO4+‑7H2O) 1.0E‑03 Magnesium Sulfate (MgSO4‑7H2O) 4.1E‑01 31 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Salts / Minerals Potassium Chloride (KCl) 3.8E+00 Sodium Bicarbonate (NaHCO3) 1.4E+01 Sodium Chloride (NaCl) 1.1E+02 Sodium Phosphate dibasic (Na2HPO4‑7H2O) 5.0E‑01 Zinc Sulfate (ZnSO4‑H2O) 1.0E‑04 Other Adenine 1.0E‑03 D-glucose (Dextrose) 5.0E+00 Lipoic Acid 1.2E‑05 Phenol Red 1.0E‑02 Table I: List of components of BLAR001 medium Sodium Pyruvate 1.0E+00 Thymidine 9.8E‑05

[0172] In general, the embodiments herein differentiate human pluripotent cells substantially as described above in Stages1‑7.ForStage7, varioussmallmoleculeswereaddedand their effectwasevaluatedbyquantitative real-timePCR. Table II lists the small molecules and their targets. These small molecules were evaluated and 2µMof eachwas added at Stage7. The chemical names and structures of the compounds tested in this Example are shown in Table XI. Table II: Small molecules evaluated Molecule Name Target Tocris Biosciences Catalog No. Zebularine DNA methyltransferase and cytidine deami- nase inhibitor 2293 Decitabine DNA methyltransferase inhibitor 2624 RG 108 Non-nucleoside DNA methyltransferase inhi- bitor 3295 Lomeguatrib O6-methylguanine-DNA methyltransferase (MGMT) inhibitor 4359 5-Azacytidine DNA methyltransferase inhibitor 3842 mitoxantrone dihydrochloride Topoisomerase II inhibitor; immunosuppres- sive and antineoplastic agent 4250 EGCG DNMT1 inhibitor 4524 Fisetin DNMT1 inhibitor 5016 SGI 1027 DNA methyltransferase inhibitor 5155 Temozolomide DNA-methylating antitumor agent 2706 L002 p300 inhibitor 5045 C 646 Selective p300 / CBP HAT inhibitor 4200 SGC 0946 Highly potent and selective DOTIL inhibitor; cell permeable 4541 UNC 0224 Potent G9a histone lysine methyltransferase inhibitor 3861 32 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Table II: Small molecules evaluated Molecule Name Target Tocris Biosciences Catalog No. UNC 0638 Selective G9a and GLP histone lysine methyl- transferase inhibitor 4343 BIX 01294 G9a-like protein and G9a histone lysine methyltransferase inhibitor 3364 UNC 0646 Potent and selective G9a / GLP inhibitor 4342 UNC 0642 Potent and selective G9a and GLP histone ly- sine methyltransferase inhibitor 5132 TC-E 5003 Selective PRMT1 arginine methyltransferase inhibitor 5099 A 366 Potent and selective G9a / GLP histone lysine methyltransferase inhibitor 5163 KU 55933 Potent and selective ATM kinase inhibitor 3544 KU 60019 Potent ATM kinase inhibitor 4176 PF 03814735 Aurora kinase A and B inhibitor 4821 ZM 447439 Inhibits Aurora kinase B 2458 U0126 Potent, selective inhibitor of MEK1 and 2 1144 SL 327 Selective inhibitor of MEK1 and MEK2; brain penetrant 1969 H 89 dihydrochloride Protein kinase A inhibitor 2910 SB 747651 A dihydrochloride Potent MSK1 inhibitor; also inhibits other AGC group kinases 4630 SNS 314 mesylate Potent pan-Aurora kinase inhibitor 4584 Kaempferol RSK2 inhibitor; blocks histone H3Ser10 phos- phorylation 3603 PRT 4165 Inhibitor of Bmi1 / Ring1A; blocks histone H2A ubiquitination 5047 P 22077 USP7 inhibitor; blocks deubiquitination of Tip60 (KAT5) histone lysine acetyltransferase 4485 PFI 1 BET bromodomain inhibitor 4445 I-BET 151 hydrochloride BET bromodomain inhibitor 4650 LY 303511 BRD2, BRD3 and BRD4 inhibitor 2418 MS 436 Potent and selective BRD4 bromodomain in- hibitor 5173 SGC-CBP 30 Potent CREBBP / EP300 bromodomain inhibi- tor 4889 1-CBP 112 Selective CREBBP / EP300 bromodomain inhi- bitor 4891 Bromosporine Broad spectrum bromodomain inhibitor 4758 PFI 3 Potent and selective SMARCA4 and polybro- mo 1 inhibitor; also inhibits SMARCA2 5072 UNC 926 hydrochloride L3MBTL1 domain inhibitor 4516 33 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Table II: Small molecules evaluated Molecule Name Target Tocris Biosciences Catalog No. UNC 1215 Potent inhibitor of L3MBTL3 Kme reader do- main; cell permeable 4666 TC-H 106 Class I histone deacetylase inhibitor 4270 MC 1568 Selectively inhibits HDAC class II (IIa) 4077 Pyroxamide Histone deacetylase inhibitor 4403 CI 994 Histone deacetylase inhibitor 20952 SBHA Histone deacetylase inhibitor 3810 KD 5170 Histone deacetylase inhibitor 4001 LMK 235 Selective HDAC4 / HDAC5 inhibitor 4830 TCS HDAC6 20b Selective HDAC6 inhibitor 4805 PCI 34051 Potent and selective histone deacetylase 8 (HDAC8) inhibitor; induces apoptosis in T cell- derived cell lines 4643 Scriptaid Histone deacetylase inhibitor 2421 NSC 3852 Histone deacetylase inhibitor 2521 Sodium 4-Phenylbutyrate Histone deacetylase inhibitor 2682 M 344 Histone deacetylase inhibitor 2771 Valproic acid, sodium salt Histone deacetylase inhibitor 2815 SAHA Class I and II HDAC inhibitor 4652 GSK J2 Inactive isomer of GSK-J1 (Cat. No. 4593) 4688 GSK J5 Inactive isomer of GSK J4 (Cat. No. 4594);l cell permeable 4689 GSK J1 Potent histone demethylase JMJD3 / UTX inhi- bitor 4593 GSK J4 Histone demethylase JMJD3 / UTX inhibitor; cell permeable 4594 Daminozide Selective histone demethylase KDM2 / 7 sub- family inhibitor 4684 Tranylcypromine hydrochloride Irreversible inhibitor of lysine-specific de- methylase 1 (LSD1); also inhibits MAO 3852 RN 1 dihydrochloride LSD1 inhibitor 4977 IOX 1 Histone demethylase inhibitor; cell permeable 4464 JIB 04 Pan Jumonji histone demethylase inhibitor; active in vivo 4972 Sirtinol Selective sirtuin family deacetylase inhibitor 3521 Splitomicin Sir2p inhibitor 1542 Resveratrol SIRT1 activator 1418 EX 527 Selective SIRT1 inhibitor 2780 Tenovin‑1 SIRT1 and SIRT2 inhibitor; activates p53 3365 Salermide SIRT1 and SIRT2 inhibitor 4127 34 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Table II: Small molecules evaluated Molecule Name Target Tocris Biosciences Catalog No. AK 7 Selective SIRT2 inhibitor; brain penetrant 4754 3-Aminobenzamide PARP inhibitor; demethylates DNA 0788 PJ 34 hydrochloride Potent PARP inhibitor; alters epigenetic marks in thyroid cancer cells 3255 IOX 2 Potent, selective HIF‑1α prolyl hydroxylase‑2 (PHD2) inhibitor 4451 Forskolin PKA activator; blocks nuclear export of HDAC5 1099 Retinoic acid Endogenous retinoid; alters HDAC-mediated gene repression 0695

[0173] Quantification and characterization of differentiated cells: For quantification of gene expression at various stages, human islets, H1-hESC, Stage 6 day 7 (S6D7), and Stage 7 day 7 to Stage 7 day 14 (S7D7-S7D14) cells were harvested as ALI clusters, substantially as described in Rezania et al. (2014), supra. Gene expression was assessed in cells using custom Taqman Arrays (Applied Biosystems, Foster City, California). Data were analyzed using Sequence Detection Software (Applied Biosystems, Foster City, California), and normalized usingGAPDHas a housekeeping gene to undifferentiated H1-hESC using the ΔΔCt method. Primer details are outlined in Table III. Table III: List of RT-qPCR primers. Gene Assay ID 1 MAFA Hs04419861_sH 2 UCN3 Hs00846499_s1 3 GAPDH Hs99999905_m1

[0174] FIGS. 1A to 1D are graphs depicting data from quantitative real-time PCR analysis of the expression of MAFA after treatment with small molecules, in which various small molecules increase expression of MAFA compared to S6D7, or untreated or DMSO treated cultures at Stage 7 (S7D7). Addition of UNC0638 (FIG. 1A; selective G9a andGLP histone lysine methyltransferase inhibitor), UNC0646 (FIG. 1A; potent and selective G9a / GLP inhibitor), UNC0642 (FIG. 1A: potent and selective G9a and GLP histone lysine methyltransferase inhibitor), TC-E5003 (FIG. 1B; selective PRMT1 arginine methyltransferase inhibitor), A366 (FIG. 1B; potent and selective G9a / GLP histone lysine methyltransferase inhibitor), PF03814735 (FIG. 1B; Aurora kinase A and B inhibitor), ZM447439 (FIG. 1B; inhibits Aurora kinase B), SB747651A dihydrochloride (FIG. 1B; potent MSK1 inhibitor; also inhibits other AGC group kinases), PFI1 (FIG. 1B; BET bromodomain inhibitor), LY303511 (FIG. 1B; BRD2, BRD3 and BRD4 inhibitor), MS436 (FIG. 1B; Potent and selective BRD4bromodomain inhibitor), andMC1568 (FIG. 1C; selectively inhibitsHDACclass II (IIa)) significantly upregulated the expression of MAFA as compared to S6D7, or untreated or DMSO treated cultures at Stage 7. FIGS. 1E to 1H are graphs depicting data from quantitative real-time PCR analysis of the expression of UCN3 after treatment with small molecules, addition of 5-Azacytidine ("AZT") (FIG. 1E; DNAmethyltransferase inhibitor), Pyroxamide (FIG. 1G; histone deacetylase inhibitor), and C1994 (FIG. 1G; histone deacetylase inhibitor) significantly upregulated the expression of UCN3 as compared to S6D7, or untreated or DMSO treated cultures at Stage 7.

[0175] FIGS. 2A and 2B demonstrate the robustness of selected small molecules as either MAFA or UCN3 up- regulators, as their effect is maintained across different Stage 7 conditioning protocols.

[0176] The culturing conditions shown in FIGS. 2A and 2B differ fromFIG. 1 by the following changes in Stage 7 (S7): (i) removal of ALK5 inhibitor II ("ALK5"); (ii) lowering of T3 concentration ("lowT3"); (iii) addition of ZM447439 ("ZM"); and (iv) the addition of a cocktail of vitamins, trace elements, lipids, and amino acids (Formulation I ‑Table XII). Specifically, the basal condition testedwas: (1) noALK5 inhibitor II ("ALK5"); (2) lowT3; (3) lowZM; (4) lowH; (5) lowNAC; (6)FI; andBLAR 001 . AZTwas confirmed as an UCN3 (FIG. 2B), but not MAFA (FIG. 2A), up-regulator during Stage 7. In addition to the small molecules tested, 3-Deazaneplanocin A ("DEZA"; FIG. 2A) was found to be an effective up-regulator of MAFA, but not UCN3 (FIG. 2B), expression, even in the presence of ZM447439.

[0177] In summary, this example demonstrates the identification of small molecules that either up-regulate maturation 35 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 markers MAFA, or UCN3 expression during Stage 7. Example 2 Generation of endocrine cells with improved maturation marker expression, and with human-islet-similar glucose-de- pendent mitochondrial respiration kinetics on an air-to-liquid interface.

[0178] The followingexample demonstrates thegeneration ofC-PEPTIDE (short 31 aminoacid polypeptide connecting insulin A‑ and B-chain in pro-insulin molecule) cells that co-express the followingmaturation markers, PIDX1 (Pancreatic and duodenal homeobox 1); NKX6.1 (NK6 homeobox 1); MAFA; UCN3, SLC2A1 (Solute carrier family 2 member 1; also called GLUT1 / Glucose transporter 1); and show glucose-dependent mitochondrial respiration kinetics similar to human islet cells. Cells of the human embryonic stem cell line H1 ("H1-hESC") (with EZ8 media at passage 28 were seeded as single cells at 0.094 x 106 cells / cm2 on MATRIGEL™ at a 1.30 dilution coated dishes in a media of Dulbecco’s Modified Eagle’s Medium Nutrient mixture F‑12 ("DMEM-F12"), GlutaMAX™ in a 1:100 dilution ("1X concentration"), 0.25mM ascorbic acid, 100ng / ml fibroblast growth factor 2 ("FGF2"), 1 ng / ml of transforming growth factor beta ("TGFβ"), insulin- transferrin-selenium-ethanolamine ("ITS-X") at a 1:100 dilution, 2% fatty-acid free bovine serum albumin ("FAF-BSA"), and20ng / ml of insulin-like growth factor‑1 ("IGF‑1"), supplementedwith10µMofRock InhibitorY‑27632 ("Y-compound"). Forty-eight hours post-seeding, the cultures were washed in incomplete PBS (phosphate buffered saline without magnesium or calcium).

[0179] ForFIGS. 3A‑3M,4A‑4Eand5A‑5F the culturesweredifferentiated using the followingprotocol. DuringStages 1 through 4 of the protocol, cultures were maintained on planar adherent cultures. a. Stage 1 (3 days): Cells were cultured for one day in the following Stage 1 media: MCDB‑131 medium containing 2.7g / 1000ml sodium bicarbonate, and supplemented with 0. 5% FAF-BSA, GlutaMAX™ in a 1:100 dilution ("1X concentration"), 4.5mM D-glucose to obtain a concentration of 10mM of D-glucose, 100 ng / ml growth differentiation factor 8 ("GDF8"), and 1.5 µM of a 14-Prop‑2-en‑1-yl‑3,5,7,14,17,23,27-heptaazatetracyclo [19.3.1.1~2,6~.1~8,12~] heptacosa‑1(25),2(273,3,5,8(26),9,11,21,23-nonaen‑16-one ("MCX compound"). Cells were then cultured for an addi- tional day inMCDB‑131medium containing 27g / 1000ml sodiumbicarbonate, and supplementedwith 0.5%FAF-BSA, 1X concentration of GlutaMAX™, 4.5mMD-glucose to obtain a concentration of 10mMD-glucose, 100 ng / ml GDF8, and 0.1 µM MCX compound. Cells were then cultured for an additional day in MCDB‑131 containing 2.7g / 1000ml sodium bicarbonate, and supplemented with 0.5% FAF-BSA, 1X concentration of GlutaMAX™, 4.5mM D-glucose to obtain a concentration of 10mM D-glucose, and 100ng / ml GDF8. b. Stage 2 (2 days): Cells were treated for two dayswithMCDB‑131mediumcontaining 2.7g / 1000ml sodiumbicarbonate, and supplementedwith 0.5%FAF-BSA, 1XGlutaMAX™, 4.5mMD-glucose to obtain a concentration of 10mMD-glucose, 0.25 mM ascorbic acid, and 50 ng / ml fibroblast growth factor 7 ("FGF7"). c. Stage 3 (2 days): Cells were treated for two days with BLAR001 custom medium containing 3.6g / 1000 ml sodium bicarbonate, and supplemented with a 1:200 dilution of ITS-X; 4.5mM D-glucose to obtain a concentration of 10mM D- glucose; 1X GlutaMAX™; 1% FAF-BSA; 25 ng / ml FGF7; 0.25 µM SANT‑1 (N‑[(3,5-dimethyl‑1-phenyl‑1H-pyrazol‑4-yl) methylene]‑4‑(phenylmethyl)‑1-piperazineamine); 1 µM retinoic acid ("RA"); 0.25 mM ascorbic acid; 300 nM of the PKC activator ((2S, 5S‑(E,E)‑8‑(5‑(4-trifluoromethyl)phenyl‑2,4,‑pentadienoylamino)benzolactam ("TPB"); and the bonemor- phogenic protein ("BMP") receptor inhibitor LDN‑193189-HCl ("LDN-HCl") for two days. The concentration of LDN-HCl used for the first day of stage 3 was 100nM, and for the second day of stage 3 was 50nM. d. Stage 4 (3 days) andALI-transition at S4D3:Cells were treatedwith BLAR001mediumcontaining 3.6g / 1000ml sodium bicarbonate, and supplemented with a 1:200 dilution of ITS-X; 4. 5mM D-glucose to obtain a concentration of 10mM D- glucose;1XconcentrationofGlutaMAX™;1%FAF-BSA;0.25µMSANT‑1;50nMRA;2ng / mlFGF7;70nMLDN-HCl; 0.25 mM ascorbic acid; and 200 nM TPB for three days. At the end of Stage 4 (3 days), cells cultured on planar dishes were seeded on an air-liquid-interface ("ALI"). For the ALI transition, cells were treated for 4 hours with 10 µM of Y-compound, rinsedwith PBSand treated for approximately 2minuteswith the enzymeTrypLE™Express Enzymeat a concentration of 1X followed by removal of the enzyme, and removal of the cells from the onMATRIGEL™ surface by gentle tapping of the flask. The resulting suspension of cells were seededat a density of 0.5‑1.0 x 106 cells (in 5µl aliquots) on either 0.4micron or 3.0micron porous cell culture filter inserts in 10 cmplates. 8.0ml ofmediawasadded to the bottomof each insert andno furthermediawas added to the apical, or top, side of the filter. Themediawas replaced daily for the duration of Stages 5, 6, and 7. e.Stage5 (3days):Cellswere treatedon theALIwithBLAR001mediumcontaining2.7g / 1000mlsodiumbicarbonate, and supplemented with a 1:200 dilution of ITS-X; 14.5mMD-glucose to achieve a final concentration of 20mMD-glucose; 1X GlutaMAX™; 2%FAF-BSA; 10µg / ml of heparin ("H"); 10µMZnSO4; 0.25µMSANT‑1; 50nMRA; 100nMLDN-HCl; 1µM of T3 in the form of 3,3’, 5-triiodo-L-thryonine sodium salt (Sigma Aldrich, Catalog No. T6397); 10 µM of 2‑(3‑(6- Methylpyridin‑2-yl)‑1H-pyrazol‑4-yl)‑1,5-naphthyridine ("ALK5 inhibitor II" or "ALK5") for three days. f. Stage6 (7days): Cellswere treated on theALIwithBLAR001mediumcontaining 2.7g / 1000ml sodiumbicarbonate, and 36 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 supplemented with a 1:200 dilution of ITS-X; 14.5mMD-glucose to achieve a final concentration of 20mMD-glucose; 1X GlutaMAX™;2%FAF-BSA;10µg / mlof heparin ("H"); 10µMZnSO4;100nMLDN-HCl; 1µMofT3; 10µMALK5 inhibitor II; and 100nM (S,S)‑2‑[2‑(3,5-Difluorophenyl)acetylamino]‑N‑(5-methyl‑6-oxo‑6,7-dihydro‑5H-dibenzo[b,d]azepin‑7-yl) propionamide ("gamma secretase inhibitor XX") (for seven days. g. Stage 7 (7 days): Cells on theALIwere treatedwith two kinds of customBLARmedia: BLAR001andBLAR004. The first medium is BLAR001medium (list of components is outlined on Table I) containing 2.7g / 1000ml sodium bicarbonate, and supplemented with a 1:200 dilution of ITS-X; 1X GlutaMAX™; 2% FAF-BSA; 10 µg / ml of heparin ("H"); 10 nM of T3 ("low T3"); 1 mM N-Acetyl cysteine ("NAC"); 0.5 µM of ZM447439 ("ZM"); and the following components which constitute Formulation I ("FI") - 1:200 dilution of RPMI vitamin supplement; 1:200 dilution of MEM non-essential amino acid supplement; 1:2000 dilution of chemically defined lipid concentrate; 1:200 dilution of sodium pyruvate; 1:2000 dilution of trace elements A; 1:2000 dilution of trace elements B for seven days. Additional compounds added during Stage 7 included either 1µMof T3 or 10 nMof T3 ("lowT3"); 10µMALK5 inhibitor II; 5µM5-Azacytidine ("AZT"); or 1µMor 10µM 3-Deazaneplanocin A ("DEZA"). The second medium was BLAR004 (list of components is outlined in Table IV) medium containing 2.7g / 1000ml sodium bicarbonate, and supplemented with a 1:200 dilution of ITS-X; 1XGlutaMAX™; 2% FAF- BSA; 10 µg / ml of heparin ("H"); 1 mM N-Acetyl cysteine ("NAC"); 0.5 µM of ZM447439 ("ZM"); and the following components which constitute Formulation I ("FI") - 1:200 dilution of RPMI vitamin supplement; 1:200 dilution ofMEMnon- essential amino acid supplement; 1:2000 dilution of chemically defined lipid concentrate; 1:200 dilution of sodium pyruvate; 1:2000 dilution of trace elements A; 1:2000 dilution of trace elements B for seven days. Additional compounds added during Stage 7 included either 1 µM of T3 or 10 nM of T3 ("low T3"); 10 µM ALK5 inhibitor II; 5 µM 5-Azacytidine ("AZT"); or 1 µM or 10 µM 3-Deazaneplanocin A ("DEZA"). Table IV: List of components of BLAR004 medium Amino Acids Concentration (mM) Glycine 2.0E‑01 Alanine 2.0E‑01 Arginine 5.0E‑02 Aspargine 2.0E‑02 Aspartic Acid 2.0E‑02 Cysteine 3.0E‑02 Glutamic acid 5.0E‑02 Histidine 8.0E‑02 Isoleucine 7.0E‑02 Leucine 1.5E‑01 Lysine hydrochloride 2.0E‑01 Methionine 2.0E‑02 Phenylalanine 6.0E‑02 Proline 1.0E‑01 Serine 1.0E‑01 Theronine 1.4E‑01 Tryptophan 5.0E‑02 Tyrosinedisodium 6.0E‑02 Valine 2.0E‑01 Vitamins Biotin 3.0E‑05 Choline chloride 5.0E‑03 D-Calcium pantothenate 1.5E‑03 Folinic Acid Calcium salt 2.3E‑03 37 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Vitamins Niacinamide 4.9E‑03 Pyridoxine hydrochloride 9.7E‑04 Riboflavin 1.0E‑05 Thiamine hydrochloride 3.0E‑03 Vitamin B12 3.7E‑06 i-Inositol 2.8E‑03 DL-alpha-Tocopherol Acetate 1.0E‑03 Asocrbic acid 5.0E‑02 Retinol, all trans (vit. A) 1.0E‑04 25-hydroxyvitamin D 3.0E‑05 Vitamin K1‑ (phylloquinone) 1.0E‑09 Para-Aminobenzoic Acid 3.0E‑04 Cholesterol 1.0E‑04 Salts Calcium Chloride (CaCl2‑2H2O) 3.0E‑01 Cupric sulfate (CuSO4‑5H2O) 4.8E‑06 Ferric sulfate (FeSO4‑7H2O) 1.0E‑03 Magnesium Sulfate (MgSO4‑7H2O) 4.1E‑01 Potassium Chloride (KCl) 3.8E+00 Sodium Bicarbonate (NaHCO3) 1.4E+01 Table IV: List of components of BLAR004 medium Sodium Chloride (NaCl) 1.1E+02 Sodium Phosphate dibasic (Na2HPO4‑7H2O) 5.0E‑01 Zinc Sulfate (ZnSO4‑H2O) 1.0E‑04 Other Adenine 1.0E‑03 D-glucose (Dextrose) 5.0E+00 Lipoic Acid 1.2E‑05 Phenol Red 1.0E‑02 Sodium Pyruvate 1.0E‑01 Thymidine 9.8E‑05 Putrescine 1.0E‑02 Glutathione 1.0E‑03

[0180] Characterization and quantification of differentiated cells: For quantification of gene expression at various stages, human islets, H1-hESC, Stage 6 day 7 (S6D7), and Stage 7 day 7 to Stage 7 day 14 (S7D7-S7D14) cells were harvested as ALI clusters, as described in Rezania et al., Nature Biotechnology, 2014; 32(11): 1121‑1133. Gene expression was assessed in cells using custom Taqman Arrays (Applied Biosystems, Foster City, California). Data were analyzed using Sequence Detection Software (Applied Biosystems, Foster City, California), and normalized using GAPDH as a housekeeping gene to undifferentiated H1-hESC using the ΔΔCt. method. Primer details are outlined in Table V. 38 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 Table V: List of qRT-PCR primers. Gene Assay ID 1 GJD2 Hs00950432_m1 2 GLP1R Hs00157705_m1 3 INHA Hs00171410_m1 4 INHBB Hs00173582_m1 5 PDK1 Hs00176853_m1 6 SIX2 Hs00195590_m1 7 UCP2 Hs01075227_m1 8 SLC2A1 Hs00892681_m1 9 G6PC2 Hs01549773_m1 10 INS Hs00355773_m1 11 NKX6.1 Hs0023235_m1 12 PDX1 Hs00236830_m1 13 NEUROD1 Hs00159598_1 14 CHGA Hs00154441_m1 15 MAFA Hs04419861_sH 16 UCN3 Hs00846499_s1 17 GAPDH Hs99999905_m1

[0181] For quantification of protein co-localization at various stages, human islets, and S7D7 cells were harvested as a ALI cell clusters and analyzed by immunofluorescence ("IF"). H1-hESC-derived cells were prepared and stained as substantially described in Rezania et al. (2014) above, and using the antibodies listed in Table VI herein. For cryosection- ing, cells were rinsed with PBS followed by overnight fixation in 4%PFA at 4°C. Following fixation, 4%PFAwas removed, the cells were rinsed twice with PBS, and incubated overnight at 4°C in 30% sucrose solution. The samples were cryopreserved inOCTsolution, and5µmsectionswereplacedonSuperfrost plusslides (VWRInternational, LLC,Radnor, PA, Catalog No. 48311‑703).

[0182] For IF-staining, primary antibodies were added at appropriate dilutions overnight at 4 °C, while secondary antibodies were added for 30 min at room temperature followed by rinsing with PBS and adding Vectastain mounting reagent with DAPI (Vector Laboratories Inc., Burlingame, California, Catalog No. H‑1200). The sections were visualized using a Nikon Ti fluorescence microscope (Nikon Instruments, Inc., Melville, NY). Table VI: List of antibodies used for IF analysis. Antigen Species Source Dilution C-PEPTIDE Mouse Abcam (Catalog No. ab8297) 1:100 GLUCAGON Rabbit Abcam (Catalog No. ab92517) 1:100 PDX1 Goat R&D Systems (Catalog No. AF2419) 1:33 MAFA Rabbit Life Span Biosciences (Catalog No. LP9872) 1: 100 (Antigen retrieval) GLUT1 Rabbit Abcam (Catalog No. ab115730) 1:300 NKX6.1 Rabbit Novus Biologicals (Catalog No. NBP1‑49672) 1:100 UCN3 Rabbit Novus Biologicals (Catalog No. NBP1‑80732) 1:100 (Antigen retrieval) 39 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Table VI: List of antibodies used for IF analysis. Antigen Species Source Dilution NEUROD1 Rabbit Abcam (Catalog No. 16508) 1:100 KI67 Rabbit A beam (Catalog No. ab16667) 1:100 (Antigen retrieval) SOX2 Goat Sigma (Catalog No. sc‑17320) 1:50 CDX2 Mouse BioGenex (Catalog No. MU392A-UC) 1:50 Donkey anti-mouse IgG (H+L) Second- ary antibody, Alexa Fluor 488 Mouse Life Technologies (Catalog No. A21202) 1:50 Donkey anti-rabbit IgG (H+L) Second- ary Antibody, Alexa Fluor 546 Rabbit Life Technologies (Catalog No. A10040) 1:100‑1:200 Donkey anti-goat IgG (H+L) Secondary Antibody, Alexa Fluor 546 Goat Life Technologies (Catalog No. A11056) 1:50

[0183] For quantification of glucose-dependent mitochondrial activity at various stages, matured human islets, S6D7 ALI clusters, and S7D7ALI clusters were harvested, and their oxygen consumption rate ("OCR")measured on the XFe24 Extracellular Flux Analyzer (Seahorse Bioscience, Catalog No. 102238‑100) before and after injection of 20 mM D- glucose. ALI clusters were removed from their Stage 7 conditioning and incubated for 2 hours in both a 37°C non-CO2 environment andmedium designed to achieve a baseline OCR. The pre-incubation medium contains 1 mMD-glucose, 1 mML-glutamine, and 1mM sodium pyruvate in XF Basemedium. After pre-incubation, ALI clusters were loaded onto the Seahorse machine in which the following measurements were made: (i) 3X baseline OCR (pre-D-glucose injection); and (ii) 5X post-D-glucose (post-injection incubation time: 72 minutes). All OCR measurements were normalized to DNA content of individual samples of ALI or Aggrewell clusters in spinners. DNA was isolated by the QIAamp DNA MicroKit (Qiagen, Catalog No. 56304), and DNA content measured by the NanoDrop 8000 UV-Vis Spectrophotometer (Thermo Scientific, Catalog No. ND8000).

[0184] FIGS. 3A‑3Mdemonstrate an increase in the gene expression, after seven days of Stage 7 conditioning, of a set ofmaturationmarkers inALLcell clusters to levelsobserved inhuman islets.Amaturationmarker is definedhereasagene involved in positively stimulating a rapid glucose-stimulated insulin secretion (GSIS). The following list details the changes in theStage7differentiationprotocol thatwereobserved to improve thegeneexpressionofmaturationmarkersbyS7D7 in ALI clusters: (i) removal of ALK5 inhibitor II; (ii) lowering of T3 concentration; (iii) addition of DEZA; (iv) addition of AZT; (v) addition of ZM; (vi) lowering glucose concentration to 5.56mM; and (vii) the addition of a defined cocktail of vitamins, non- essential amino acids, lipids, sodium pyruvate, and trace elements (Formulation I - "FI"). Similar observations and results were seen regarding the gene expression signature of maturation genes for both BLAR001‑ and BLAR004-based conditioning during Stage 7. FIG. 3A shows that INHBB was enriched above the expression seen in human islets in conditions utilizing ALK5 inhibitor II. The observation suggests that in S7-cells, the inhibition of TGFB1 (Transforming growth factor beta 1) by ALKS inhibitor II elicits a negative TGF-beta signaling profile that inhibits GSIS processes. Removal of ALKS inhibitor in the absence or presence of AZT / DEZA was obsered to decrease INHBB to levels seen in human islets, thus eliminating the negative TGF-beta signaling influences on GSIS during Stage 7. In addition, FIG. 3B shows that INHAexpressionwasobserved to increase tohuman islet levels inALI clustersby the removal ofALK5 inhibitor II.

[0185] While MAFA and SLC2A1 expression, shown in FIG. 3C and 3D respectively, was observed to have decreased by the removal ofALK5 inhibitor II, theadditionofAZT / DEZA rescuesMAFAandSLC2A1expression tohuman islet levels. UCN3 (FIG. 3E), G6PC2 (Glucose‑6-phosphatase catalytic subunit 2; FIG. 3F), and PDK1 (Pyruvate dehydrogenase kinase 1; FIG. 3G) expression was increased to levels at or above human islets by the addition of AZT / DEZA and removal of ALK5 inhibitor II in ALI clusters. The expression of INS (Insulin; FIG. 3H), GJD2 (Gap junction protein, delta 2; also CX36 / connexin 36; FIG. 3I), SIX2 (SIX homeobox 2; FIG. 3J), and PDX1 (FIG. 3K) was observed to increase in a gradual step-wisemanner, first by removal of ALK5 inhibitor II and second by the addition of AZT / DEZA. No changewas observed in expression of NKX6.1 (FIG. 3L), and GLP1R (Glucagon like peptide 1 receptor; FIG. 3M) across conditions, but their expression in ALI clusters were consistently at or above levels observed in human islets.

[0186] FIG4A‑4Edemonstrate thegenerationofC-PEPTIDEcells inALI cell clusters that co-express, in termsof protein presence, the followingmaturationmarkers: PDX1 (FIG. 4A), NKX6.1 (FIG. 4B),MAFA (FIG. 4C), SLC2A1 (FIG. 4D), and UCN3 (FIG. 4E) at S7D7. For each of FIG4A‑4E, IF-staining is shown as single channels with C-PEPTIDE on the top row and maturation protein of interest in the bottom row. Representative human islet staining is shown in the left column, No 40 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 ALK5, low T3, ZM, H, NAC, AZT / DEZA , FI, BLAR001 condition in themiddle column, and No ALK5, low T3, ZM, H, NAC, AZT / DEZA,FI, BLAR004condition in the right column.Most, if not all, C-PEPTIDEpositive cellswere co-positive forPDX1 (FIG. 4A) and NK.X6.1 (FIG. 4B), as seen in human islets. A significant portion of C-PEPTIDE positive cells were also co- positive for MAFA (FIG. 4C), SLC2A1 (FIG. 4D), and UCN3 (FIG. 4E) across both BLAR001 and BLAR004 conditions. However, the expression of MAFA, SLC2A1 and UCN3 was not restricted to C-PEPTIDE co-positive cells.

[0187] FIGS. 5A‑5E show the generation of C-PEPTIDE cells at S7D7 that exhibit human-islet-similar glucose- dependent mitochondrial respiration kinetics on air-to-liquid interface ("ALI"), specifically by Stage 7 specific ’No ALK5, low T3, ZM, H, NAC, AZT, DEZA, FI, BLAR001’ conditioning. Mitochondrial respiration or activity, represented by the oxidative consumption rate ("OCR"), was measured at baseline and post‑20 mM D-glucose injection. Five OCR measurements over a period of 72minutes weremade post‑20mMD-glucose injection, andmeasurements are depicted as thepercentageover baselineofOCRnormalized byDNAcontent of each individual sample. FIG. 5A shows that human islets (black circle line) rapidly responded tohighD-glucose, demonstratedbyanOCR123.3%±12.92over baselineafter 15 minutes ("min") post-injection ("ip"), and maintained a high OCR over time (131.5%± 11.32; 72 min ip). Conversely, S6D7ALI clusters (grey triangle line),whichwereenriched for immatureC-PEPTIDEpositive cellswereobserved to lacka rapid OCR response to high D-glucose (104.4%± 3.37; 15 min ip) and exhibit a relatively weak OCR response over time (113.3%± 4.51; 72min ip). FIG. 5D depicts that within the S7D7ALI cluster group only theNoALK5, low T3, ZM, H, NAC, AZT / DEZA, FI, BLAR001 condition (grey square line) were observed to exhibit human-islet-similar glucose-dependent mitochondrial respiration kinetics (112.4%± 3.25 - 15 min ip; 129.5%± 3.78 - 72 min ip). The following S7D7 ALI cluster conditions (black square line) were seen to exhibit glucose-dependent mitochondrial kinetics indistinguishable from immature S6D7ALI clusters: ALK5, T3, ZM, H, NAC, FI, BLAR001 (100.3%± 4.04 - 15min ip; 107.8%± 6.51 - 72min ip) (FIG. 5B); NoALK5, lowT3, ZM,H,NAC,FI, BLAR001 (96.9%±3.06 - 15min ip; 109.0%± 4.58 - 72min ip) (FIG. 5C); No ALK5, low T3, ZM, H, NAC, FI, BLAR004 (103.4%± 4.76 - 15min ip; 113.6%± 6.72 - 72min ip) (FIG. 5E); and No ALK5, low T3, ZM, H, NAC, AZT / DEZA, FI, BLAR004 (102.1% ± 4.04 - 15 min ip; 112.7% ± 3.38 - 72 min ip) (FIG. 5F).

[0188] In summary, this example demonstrates that incorporating improvements in Stage 7 conditioning enhances the maturation status of hESC-derivedmaturing beta-cells on the ALI. Specifically, this example shows the generation on the ALI of C-PEPTIDE cells that co-express a multitude of beta-cell maturation markers, and exhibit beta-cell-specific functionality similar to human islets, such as glucose-dependent mitochondrial respiration. Example 3 Generation of robust pancreatic endoderm and immature beta-cells in cell cluster format amenable to scal- able suspension culture

[0189] The followingexample demonstrates thegenerationof either robust pancreatic endodermor immaturebeta cells in Aggrewell™ cell cluster format amenable to scalable suspension culture. The suspension culture used in this Example wasAggrewell™clusters cultured inspinner flasks ("Aggrewell™clusters in spinners").Cellsof thehumanembryonic stem cell line H1 ("H1-hESC") with EZ8 media at passage 28 were seeded as single cells at 0.094 x 106 cells / cm2 on MATRIGEL™ at a 1:30 dilution coated dishes in a media of Dulbecco’s Modified Eagle’s Medium Nutrient mixture F‑12 ("DMEM-F12"), GlutaMAX™ in a 1:100 dilution ("1X concentration"), 0.25mM ascorbic acid, 100ng / ml fibroblast growth factor 2 ("FGF2"), 1 ng / ml of transforming growth factor beta ("TGFβ"), insulin-transferrin-selenium-ethanolamine ("ITS- X") at a 1:100 dilution, 2% fatty-acid free bovine serum albumin ("FAF-BSA"), and 20 ng / ml of insulin-like growth factor‑1 ("IGF‑1"), supplemented with 10µM of Rock Inhibitor Y‑27632 ("Y-compound"). Y-compound was added only during the first 24 hours post-seeding. Forty-eight hours post-seeding, the cultures were washed in incomplete PBS (designated as "‑ / ‑" meaning phosphate buffered saline without magnesium or calcium).

[0190] For FIGS. 6A to 6L, the cultures were differentiated using the following protocol. S6D6 ALI cell clusters shown in FIGS. 6I to 6L were cultured as described above, with the exception that at Stage 4 day 3, Aggrewell™ cell clusters were prepared either from fresh S4D3 monolayer or cryopreserved S4D3 cells (as described in more detail below) by being cultured for an additional 48 hours (Stage 4 day 5 or S4D5). Media was exchanged daily throughout the differentiation protocol. a. Stage 1 (3 days): Cells were cultured for one day in the following Stage 1 media: MCDB‑131 medium containing 2.7g / 1000ml sodium bicarbonate, and supplemented with 0. 5% FAF-BSA, GlutaMAX™ in a 1:100 dilution ("1X concentration"), 4.5mM D-glucose to obtain a concentration of 10mM of D-glucose, 100 ng / ml growth differentiation factor 8 ("GDF8") , and 1.5 µM of a 14-Prop‑2-en‑1-yl‑3,5,7,14,17,23,27-heptaazatetracyclo [l 9.3.1.1~2,6~.1~8,12~] heptacosa‑1(25),2(27),3,5,8(26),9,11,21,23-nonaen‑16-one ("MCX compound") (a GSK‑3β inhibitor). Cells were then cultured for an additional day inMCDB‑131medium containing 2.7g / 1000ml sodiumbicarbonate, and supplementedwith 0.5% FAF-BSA, 1X concentration of GlutaMAX™, 4. 5mM D-glucose to obtain a concentration of 10mM D-glucose, 100 ng / ml GDF8, and 0.1 µM MCX compound. Cells were then cultured for an additional day in MCDB‑131 containing 2.7g / 1000ml sodium bicarbonate, and supplemented with 0.5% FAF-BSA, 1X concentration of GlutaMAX™, 4.5mM D- 41 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 glucose to obtain a concentration of 10mM D-glucose, and 100ng / ml GDF8. b. Stage 2 (2 days): Cells were treated for two dayswithMCDB‑131mediumcontaining 2.7g / 1000ml sodiumbicarbonate, and supplementedwith 0.5%FAF-BSA, 1XGlutaMAX™, 4.5mMD-glucose to obtain a concentration of 10mMD-glucose, 0.25 mM Ascorbic acid, and 50 ng / ml Fibroblast growth factor 7 ("FGF7"). c. Stage 3 (2 days): Cells were treated for two days with BLAR001 custommedium (see Table I) containing 3.6g / 1000 ml sodium bicarbonate, and supplemented with a 1:200 dilution of ITS-X; 4. 5mM D-glucose to obtain a concentration of 10mM D-glucose; 1X GlutaMAX™; 1% FAF-BSA; 25 ng / ml FGF7; 0.25 µM SANT‑1 (N‑[(3,5-dimethyl‑1-phenyl‑1H- pyrazol‑4-yl)methylene]‑4‑(phenylmethyl)‑1-piperazineamine); 1 µM retinoic acid ("RA") (Sigma Aldrich, Catalog No. R2625); 0.25 mM ascorbic acid; 300 nM of the PKC activator (2S,5S‑(E,E)‑8‑(5‑(4-trifluoromethyl)phenyl‑2,4,‑penta- dienoylamino)benzolactam ("TPB"); and the bone morphogenic protein ("BMP") receptor inhibitor LDN‑193189-HCl ("LDN-HCl") for twodays.TheconcentrationsofLDN-HCHused for thefirst dayof stage3were100nM,and for thesecond day of stage 3 was 10 nM. d. Stage 4 (3 days): Cells were treated with BLAR001 medium containing 3.6g / 1000 ml sodium bicarbonate, and supplemented with a 1:200 dilution of ITS-X; 4.5mM D-glucose to obtain a concentration of 10mM D-glucose; 1X concentration of GlutaMAX™; 1% FAF-BSA; 0.25 µM SANT‑1; 50 nM RA; 2 ng / ml FGF7; 70 nM LDN-HCl; 0.25 mM ascorbic acid; and 200 nM TPB for three days. e.Cryopreservation ofStage4Day3monolayer:Cryopreserved cell banks fromS4D3monolayerwereestablishedby the following procedure. Briefly, aS4D3monolayerwas treated for 4 hourswith 10µMY-compound. Cellswere then released with TrypLE™ Express Enzyme as a single cell suspension, followed by a neutralization of the enzyme with a ’release’ medium (Stage 4 complete medium, as detailed in "section k" supplemented with 4 kU / ml DNase I and 10 µM Y- compound). Single cells were spun down, resuspended in a cold ’release’ medium, and counted by the Nucleocounter® NC‑100. Cold ’cryopreservation’ media (60% KSR; 15% BLAR001; 5% HEPES (1M concentration); 20% DMSO) was added in a 1-to‑1 ratio add to the single cell suspension in cold ’release’ medium. 5.0 x 106 cells in 4.5ml of 1-to‑1 ’cryoperservation / release’ mediumwere added to a single 5ml cryopreservation vial. Vial(s) were transferred to the CRF (Controlled rate Freezer) (Planar PLC, Catalog No. Kryo 360) in which the cells were frozen by the following freezing profile, and stored long-term in liquid nitrogen. The CRF freezing procedure is shown in Table VII. f. Thawing of cryopreserved S4D3 monolayer cells: Frozen 5 ml vials containing 5.0 x 106 S4D3 monolayer cells were thawed in a 37 °Cwater bath for 2minutes. Cells were collected in post-S4D3medium (detailed below in section "g"), and added at a density of approximately 787 cells per Aggrewell™ well of an Aggrewell™ 400EX plate. g. Stage 4 (2 days) for Aggrewell™ cluster transition: For Aggrewell™ cluster generation, Stage 4 day 3monolayer cells, or thawed cryopreserved Stage 4 day 3 cells, were treated with Y-compound for 4 hours, rinsed with PBS, and treated for 3 minuteswith AccutaseCell Detachment Solution, followed by removal of the enzyme, and removal of the cells from the on MATRIGEL™ surface by gentle tapping of the flask. The resulting suspension of cells were added at a density of approximately 787 cells per Aggrewell™ well of an Aggrewell™ 400EX plate, and the plate gently spun down at 100 x g to generateAggrewell™ clusters. Theamount of cells per cluster can range from50 to 3000cells. Themediumused to transit Stage 4 day 3 monolayer cells to Aggrewell™ clusters, and 48 hour post-aggregation culturing is as follows ("post-S4D3 medium"): Cells were treated with BLAR001 medium containing 3.6g / 1000 ml sodium bicarbonate, and supplemented with a 1:200 dilution of ITS-X; 4.5mM D-glucose to obtain a concentration of 10mM D-glucose; 1X concentration of GlutaMAX™; 1%FAF-BSA; 0.25µMSANT‑1; 50 nMRA; 2 ng / ml FGF7; 70 nMLDN-HCl; 0.25mMascorbic acid; and 200 nM TPB for two days. Ten (10) µMY-compound and 2 µg / ml human recombinant laminin was added to the medium only during the first 24 hours. After two days, here referred to asS4D5, theAggrewell™ clusterswere removed from thewells of the Aggrewell™ plates, and transferred to a PBS0.1MAG spinners ("Aggrewell clusters in suspension"). h. Stage 5 (3 days): S4D5 Aggrewell™ clusters were retrieved from Aggrewell™ 400EX plates and transferred to PBS0.1MAG spinners at a cell density of 1.5‑2.0 million cells / ml with the rotation speed of 27 rpm (rounds per minute) inStage5medium for three days.Cellswere treatedwithBLAR001mediumcontaining 2.7g / 1000ml sodiumbicarbonate, supplemented with a 1:200 dilution of ITS-X; 14.5mMD-glucose to achieve a final concentration of 20mMD-glucose; 1X GlutaMAX™; 2%FAF-BSA; 10µg / ml of heparin ("H"); 10µMZnSO4; 0.25µMSANT‑1; 50nMRA; 100nMLDN-HCl; 1µM of T3 in the form of 3,3’, 5-triiodo-L-thryonine sodium salt; and 10 µM of 2‑(3‑(6-Methylpyridin‑2-yl)‑1H-pyrazol‑4-yl)‑1,5- naphthyridine ("ALK5 inhibitor II" or "ALK5"). 4 kU / ml DNaseI and 5µMY-compoundwere supplemented only on Stage 5 day 1. i. Stage 6 (6 days to 8 days): Aggrewell™ clusters were treated in BLAR001 medium containing 2.7g / 1000 ml sodium bicarbonate, supplementedwith a 1:200 dilution of ITS-X; 14.5mMD-glucose to achieve a final concentration of 20mMD- glucose; 1XGlutaMAX™; 2% FAF-BSA; 10µg / ml of heparin ("H"); 10µMZnSO4; 100 nM LDN-HCl; 1µMof T3; 10µMof ALK5 inhibitor II"), and 100 nMof (S,S)‑2‑[2‑(3,5-Difluorophenyl)acetylamino]‑N‑(5-methyl‑6-oxo‑6,7-dihydro‑5H-ibenzo [b,d]azepin‑7-yl)propionamide ("gamma secretase inhibitor "XX"). 42 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 Table VII: CRF Freezing procedure. S4D3 monolayer Freezing Profile Step Cooling Rate (°C / min) Target Temp (°C) Cool To 1 WAIT 4 N / A 2 WAIT for TEMPERATURE 4 Chamber 6 Sample 3 1 ‑7 Sample 4 25 ‑45 Chamber 5 10 ‑25 Chamber 6 0.2 ‑45 Sample 7 25 ‑160 Chamber 8 HOLD (20 min) ‑160 N / A

[0191] Quantification and characterization of differentiated cells: For quantification of protein co-localization at various stages, S4D5 Aggrewell™ clusters, and S6D6 Aggrewell™ clusters were harvested and analyzed by immuno- fluorescence ("IF"). The characterization procedure and reagents used were as shown Table VI of Example 2.

[0192] For quantification of gene expression at various stages, Stage 4 Day 5 Aggrewell™ clusters, and Stage 6 Day 6 Aggrewell™clusterswereharvestedandanalyzedby reverse transcriptasequantitativepolymerase chain reaction ("qRT- PCR"), as described in Nature Biotechnology, (32) 11, 1121‑1133. The characterization procedure and reagents used were as shown in Table V of Example 2.

[0193] For quantification of protein presence co-localization, Stage 4 Day 5 Aggrewell™ clusters, and Stage 6 D6 Aggrewell™clusterswereharvestedandanalyzedbyfluorescence-activatedflowcytometry ("FACS").FACSstainingwas conducted as described in Nature Biotechnology, 2014 (32) 11, 1121‑1133, and used the antibodies listed in Table VII. Differentiated cells were incubated in TrypLE™ Express for 5‑10 minutes at 37°C, released into a single-cell suspension, after which theywerewashed twicewith a staining buffer of PBS containing 0.2%BSA. Intracellular antibody stainingwas accomplishedbyutilizing theLIVE / DEADVioletFluorescent reactivedyeat4°C for30minutes followedbyasinglewash in coldPBS. Fixing of cells was in 300µl of Cytofix / CytopermBuffer followed by twowashes inPerm / WashBuffer. Cells were then incubated with the appropriate antibodies at 4°C for 30 minutes (for unconjugated antibodies) or 1 hour (for conjugated antibodies), and then washed twice prior to analysis on the BD FACS Canto II using BD FACS Diva Software with at least 30,000 events being acquired. Non-viable cells were excluded during FACS analysis, and gating was determined by using isotype antibodies ("IgG"). Table VIII: List of antibodies used for FACS analysis. Antigen Species Source / Catalog Number Dilution Alexa Fluor 647 anti-KI67 Mouse BD, Catalog No. 561126 Neat PE anti-PDX1 Mouse BD, Catalog No. 562161 Neat Alexa Fluor 647 anti-INSULIN Rabbit Cell Signaling, Catalog No. 9008S 1:80 INSULIN Rabbit Cell Signaling, Catalog No. 3014S 1:10 PE anti-NKX6.1 Mouse BD, Catalog No. 563023 1:40 CHGA Rabbit DAKO, Catalog No. IS502 1:10 PE anti-NEUROD1 Mouse BD, Catalog No. 563001 1:40 GLUCAGON Mouse Sigma-Aldrich, Catalog No. G2654 1:250 Anti-Mouse IgG(H+L) Secondary Antibody, Alexa Fluor 647 conjugate Goat Life technology, Catalog No. A21235 1:4000 43 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Table VIII: List of antibodies used for FACS analysis. Antigen Species Source / Catalog Number Dilution F(ab’)2 anti-rabbit IgG(H+L) Secondary Antibody, RPE conjugate Goat Life technology, Catalog No. A10542 Neat

[0194] FIG. 6A depicts the procedure through which a (i) fresh S4D3 monolayer or S4D3 cryopreserved cells was assembled (ii) via the Aggrewell™ method into (iii) cell clusters. FIG. 6B shows that high protein presence of pancreatic endodermTFPDX1 (top, left), andNKX6.1 (top,middle), but low protein presence of endocrine TFNEUROD1 (top, right), alternate non-pancreatic endoderm lineage allocating TFSOX2 (bottom,middle), andCDX2 (bottom, right) was detected in S4D5 Aggrewell™ clusters. FACS analysis (FIG. 6C) shows that 99.3±0.1% of the cells were PDX1+ (left), 84.4±0.1% NKX6.1+ (middle), but 2.2±0.4%NKX6.1+ NEUROD1+ (right) or 1.35±0.55%NKX6.1+ CHGA+ (middle). FIG. 6D shows that the robust pancreatic endoderm characteristics were maintained in S4D4 Aggrewell™ clusters derived from S4D3 cryopreserved cells, as thehighgeneexpression ofPDX1 (top, left),NKX6.1 (top, right),and lowexpressionofNEUROD1 (bottom, left), and CHGA (bottom, right) wasmaintained as compared to a S4D3monolayer. Overall, S4D5 Aggerewell™ clusters exhibited a greater degree of non-endocrine pancreatic endoderm characteristics than previous Stage 4 cells reported in Nature Biotechnology, 2014 (32) 11, 1121‑1133. The results are significant as Aggrewell™ clusters provide a scalable starting point with which to develop suspension culture based of differentiation towards the beta cell.

[0195] FIG. 6E depicts the procedure through which a (i) fresh S4D3 monolayer or S4D3 cryopreserved cells was assembled (ii) via the Aggrewel™ method into cell clusters, and (iii) differentiated in suspension culture to (iv) immature beta-cells by S6D6. FACS analysis of S6D6 Aggrewell™ clusters shows an immature beta-cell protein profile, as 78.5 ±1.87% of cells were NKX6.1+ CHGA+ (left, FIG. 6F), 73.6±4.34% NKX6.1+ NEUROD1+ (right, FIG. 6F), and 38.4 ±5.96%NKX6.1+ INSULI+ (left,FIG.6G).Themajority of the INSULIN-positivepopulation (50.2±6.92%of total cellswere INSULIN+)wasNKX6.1+ (left,FIG. 6G) andnotGLUCAGON-positive (7.43±1.49% INSULIN+GLUCAGON+; (right,FIG. 6G). FIG. 6H shows by IF that at S6D6 themajority of C-PEPTIDE-positive cells in Aggrewell™ clusters were both PDX1+ (left) andNKX6.1÷ (middle).Surprisingly, the protein presence of thematuration gatekeeper TFMAFA (right) was already easily detected at S6D6, and was expressed at a higher level than previous ALI clusters at S6D6-S6D7 (Nature Biotechnology, 2014 (32) 11, 1121‑1133) (FIG. 6I; see also FIG. 3C for ALI S7D7 comparison). Similarly, the expression of INSULIN (FIG. 6J), PDX1 (FIG. 6K), and NKX6.1 (FIG. 6L) was higher than previous ALI clusters at S6D6-S6D7. The results prove that S4D5 Aggrewell™ clusters can be cultured in suspension culture towards an immature beta-cell state, providing a scalable and primed starting point towards suspension culture based differentiation to a functional, mature beta-cell.

[0196] In summary, this example demonstrates that Stage 4 cells can be made to aggregate into cell clusters, while maintaining their phenotype of pancreatic endoderm. Moreover, these Stage 4 cell clusters can be further differentiated towards a robust immature beta-cell phenotype (Stage 6) by scalable suspension culture. Example 4 Suspension culture generation of endocrine cells with enhanced beta-cell maturation marker expression and protein presence similar to matured human islets

[0197] The following example demonstrates the generation by suspension culture of C-PEPTIDE cells that exhibit co- expression and protein presence of the following maturation markers, PDX1, NKX6.1, MAFA, UCN3, and SLC2A1. Cells of the H1-hESC cell line cultured with EZ8 media at passage 28 were seeded as single cells at 0.094 x 106 cells / cm2 on MATRIGEL™ at a 1:30 dilution coated dishes in a media of DMEM-F12, GlutaMAX™ in a 1:100 dilution ("1X concentra- tion"), 0.25mM ascorbic acid, 100ng / ml FGF2, 1 ng / ml of TGFβ, ITS-X at a 1:100 dilution, 2% FAF-BSA, and 20 ng / ml of IGF‑1", supplemented with 10µM of Y-compound. Y-compound was added only during the first 24 hours post-seeding. Forty-eight hours post-seeding, the cultures were washed in PBS (‑ / ‑).

[0198] ForFIGS. 7A to7M, the cultureswere differentiatedusing the followingprotocol. DuringStages 1 through4of the protocol, cultures were maintained on planar adherent cultures. Beginning from the S4D3 monolayer, or cryopreserved S4D3cells,Aggrewell™cell clusterswereprepared,andcultured foranadditional 48hours (Stage4day5orS4D5).Media was exchanged daily throughout the differentiation protocol. During stages 5, 6, and 7 Aggrewell™ clusters were cultured in suspension culture as described in Example 3, except that Stage 6 occurs over 7 days as compared to 6 days. a. Stage 1 (3 days): Cells were cultured for one day in the following Stage 1 media: MCDB‑131 medium containing 2.7g / l000ml sodium bicarbonate, and supplemented with 0.5% FAF-BSA, GlutaMAX™ in a 1:100 dilution ("1X 44 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 concentration"), 4.5mM D-glucose to obtain a concentration of 10mM of D-glucose, 100 ng / ml GDF8, and 1.5 µM of MCXcompound.Cellswere thencultured for anadditional day inMCDB‑131mediumcontaining2.7g / 1000ml sodium bicarbonate, and supplemented with 0.5% FAF-BSA, 1X concentration of GlutaMAX™, 4.5mMD-glucose to obtain a concentration of 10mM D-glucose, 100 ng / ml GDF8, and 0.1 µM GSK‑3β inhibitor. Cells were then cultured for an additional day inMCDB‑131containing2.7g / 1000ml sodiumbicarbonate, andsupplementedwith0.5%FAF-BSA, 1X concentration of GlutaMAX™, 4.5mMD-glucose to obtain a concentration of 10mMD-glucose, and 100ng / ml GDF8. b. Stage 2 (2 days): Cells were treated for two days with MCDB‑131 medium containing 2.7g / 1000ml sodium bicarbonate, and supplemented with 0.5%FAF-BSA, 1XGlutaMAX™, 4.5mMD-glucose to obtain a concentration of 10mM D-glucose, 0.25 mM Ascorbic acid, and 50 ng / ml FGF7. c. Stage 3 (2 days): Cells were treated for two days with BLAR001 custommedium containing 3.6g / 1000 ml sodium bicarbonate, andsupplementedwith a1:200dilutionof ITS-X; 4.5mMD-glucose toobtain a concentrationof 10mMD- glucose;1XGlutaMAX™;1%FAF-BSA;25ng / mlFGF7;0.25µMSANT‑1; 1µMRA;0.25mMascorbicacid; 300nMof TPB"); and LDN-HCl for two days. The concentration of LDN-HCl used for the first day of stage 3were 100 nM, and for the second day of stage 3 was 10 nM. d. Stage 4 (3 days): Cells were treated with BLAR001 medium containing 3.6g / 1000 ml sodium bicarbonate, and supplemented with a 1:200 dilution of ITS-X; 4.5mM D-glucose to obtain a concentration of 10mM D-glucose; 1X concentration of GlutaMAX™; 1% FAF-BSA; 0.25 µM SANT‑1; 50 nM RA; 2 ng / ml FGF7; 70 nM LDN-HCl; 0.25 mM ascorbic acid; and 200 nM TPB for three days. Crvopreservation of Stage 4 Dav 3 monolayer: Cryopreserved cell banks from S4D3 monolayer were established by the procedure outlined in Example 3 and Table VII. e. Thawingof cryopreservedS4D3monolayer cells: Frozen5ml vials containing5.0X106S4D3monolayer cellswere thawed as described in Example 3. f. Stage 4 (2 days) for Aggrewell™ cluster transition: Aggrewell™ clusters were generated from Stage 4 day 3 monolayer cells, or thawed cryopreserved Stage 4 day 3 cells, as described in Example 3. g. Stage 5 (3 days): S4D5 Aggrewell™ clusters were retrieved from Aggrewell™ 400EX plates and transferred to PBS0.1MAGspinners (at a cell density of 1.5‑2.0million cells / mlwith the rotation speedof 27 rpm (rounds perminute) in Stage 5 medium for three days. Cells were treated with BLAR001 medium containing 2.7g / 1000 ml sodium bicarbonate, supplemented with a 1:200 dilution of ITS-X; 14.5 mM D-glucose to achieve a final concentration of 20 mMD-glucose; 1XGlutaMAX™; 2%FAF-BSA; 10µg / ml of heparin ("H"); 10µMZnSO4; 0.25µMSANT‑1; 50 nMRA; 100 nM LDN-HCl; 1 µM of T3; and 10 µM of ALK5 inhibitor II. 4 kU / ml DNasel and 5 µM Y-compound were supplemented only on Stage 5 day 1. h. Stage 6 (7 days): Aggrewell™ clusters were treated in BLAR001 medium containing 2.7g / 1000 ml sodium bicarbonate, supplemented with a 1:200 dilution of ITS-X; 14.5 mM D-glucose to achieve a final concentration of 20mMD-glucose; 1XGlutaMAX™; 2%FAF-BSA; 10µg / ml of heparin ("H"); 10µMZnSO4; 100 nMLDN-HCl; 1µMof T3; 10 µM of ALK5 inhibitor II, and 100 nM of gamma secretase inhibitor XX. i. Stage 7 (6 days to 7 days): Aggrewell™ clusters were treated in either BLAR001 or BLAR004 medium containing 2.7g / 1000ml sodiumbicarbonate, andsupplementedwitha1:200dilutionof ITS-X;1XGlutaMAX™;2%FAF-BSA;10 µg / mlof heparin ("H"), 10nMofT3 ("lowT3"); 1mMNAC:0.5µMZM"); and the followingcomponentswhichconstitute Formulation I ("FI") - 1:200 dilution of RPMI vitamin supplement; 1:200 dilution of MEM non-essential amino acid supplement; 1:2000dilution of chemically defined lipid concentrate; 1:200dilution of sodiumpyruvate; 1:2000dilution of trace elements A; 1:2000 dilution of trace elements B for seven days. Additional compounds added during Stage 7 included either 4µMAZT; or 1µMDEZA. For clarity, Aggrewell™ clusters in suspensionwere cultured during Stage 7 in two conditions utilizing the concentrations described above (in either BLAR001 or BLAR004 base medium): (i) no AL.K5, low T3, ZM, H, NAC; (ii) no ALK5, low T3, ZM, H, NAC, AZT, DEZA.

[0199] For Stages 5, 6 and 7, the cell cultures were conditioned by starting with either S4D5 AggrewellTM clusters or S4D3-transitioned ALI clusters. S4D5 AggrewellTM clusters were retrieved from AggrewellTM 400EX plates and transferred to PBS0.1MAG spinners at a cell density of 1.5‑2.0 million cells / rnl with the rotation speed of 27 rpm (rounds per minute).

[0200] Quantification and characterization of differentiated cells: Stage 7 Aggrewell™ clusters and human islets were characterized by IF, FACS, and qRT-PCR as described in Examples 2 and 3. 45 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55

[0201] FIG. 7A depicts the procedure through which a (i) fresh S4D3 monolayer or S4D3 cryopreserved cells were assembled into Aggrewell™ clusters, conditioned by suspension culture through (ii & iii) Stages 5, 6 and 7 to generate (iv) S7D7Aggrewell™clusters.Asobserved inS6D6Aggrewell™clusters (FIGS.6F -6G),S7D7Aggrewell™clusters cultured by ’NO ALK5 LOW T3 ZM HNAC DEZA AZT FI BLAR001’ during Stage 7 maintained a baseline beta-cell protein profile (FIGS.7B‑7C). 89%of cellswereNKX6.1+CHGA+ (left,FIG. 7B), 77.7%NKX6.1+NEUROD1+ (right,FIG.7B), and40.8% NKX6.1+ INSULIN+ (left, FIG. 7C). The majority of the INSULIN-positive population (46.4% of total cells were INSULIN+) was NKX6.1+ (left, FIG. 7C) and not GLUCAGON-positive (3.9% INSULIN+ GLUCAGON+; (right, FIG. 7C).

[0202] In addition to the baseline beta-cell profile, the gene expression of maturation markers MAFA (FIG. 7D), UCN3 (FIG. 7E), SLC2A1 (FIG. 7F), G6PC2 (FIG. 7G), INSULIN (FIG. 7H), and NKX6.1 (FIG. 7I), were at or above human islet levels in Stage 7 ’NO ALK5 LOW T3 ZM H NAC DEZA AZT FI BLAR001 or BLAR004’ conditioned S7D7 Aggrewell™ clusters. Theexpression levelsatS7D7ofmaturationmarkers suchas:MAFA (FIG. 3C);G2PC2 (FIG. 3F); INSULIN (FIG. 3H); and NKX6.1 (FIG. 3L) were much greater in Aggrewell™ (’NO ALK5 LOW T3 ZM H NAC DEZA AZT FI BLAR001 or BLAR004’) then in ALI clusters.

[0203] Indeed, FIGS. 7J‑7M demonstrate that the addition of DEZA and AZT to ’NO ALK5 LOW T3 ZM H NAC FI BLAR004’ Stage 7 conditioning generated a significant number of non-GLUCAGON (FIG. 7J; bottom, right)C-PEPTIDE cells that co-expressed at S7D7, in terms of protein presence, PDX1 (FIG. 7J; bottom, left), NKX6.1 (FIG. 7J; bottom, middle),MAFA (FIG. 7K; bottom, left), UCN3 (FIG. 7K; bottom, middle), and SLC2A1 (FIG. 7K; bottom, right). For each figure, IF-staining is shown as single channels with C-PEPTIDE on the top row. While the protein presence of MAFA, SLC2A1andUCN3 isnot restricted toC-PEPTIDEpositive cells, it is expected that at least about10%of thecell population would show the co-expression of C-PEPTIDE, PDX1, NKX6.1, MAFA, SLC2A1, and UCN3. In contrast, FIGS. 7L‑7M, demonstrates thegeneration, by ’NOALK5LOWT3ZMHNACFIBLAR004’-specificStage7conditioning, ofC-PEPTIDE cells that co-expressed, in terms of protein presence, byS7D7apartial set ofmaturationmarkers: PDX1 (FIG. 7L; bottom, left), NKX6.1 (FIG. 7L; bottom, middle), MAFA (FIG. 7M; bottom, left), SLC2A1 (FIG. 7M; bottom, right), but not GLUCAGON (FIG. 7L; bottom, right) and UCN3 (FIG. 7M; bottom, middle). Stage 7 ’NO ALK5 LOW T3 ZM H NAC DEZAAZTFI BLAR004’ conditioning of Aggrewell™ clusters, generated a significant number ofmature beta-cells, similar to matured adult human islets, as assessed by gene expression and protein presence.

[0204] In summary, this example demonstrates that Stage 4 cell clusters can be further differentiated in suspension culture towards functional beta-cells, by changes in Stage 7 conditioning. Specifically, example 4 shows the generation of C-PEPTIDE cells, within cell clusters in suspension culture, that co-express beta-cell maturation markers essential for proper functionality of the beta-cell Example 5 Suspension culture generation of endocrine cells with mature human-islet-similar glucose-dependent mito- chondrial respiration and glucose stimulated insulin secretion kinetics

[0205] The following example demonstrates the generation of functionallymature beta-cells by suspension culture with human-islet-similar glucose-dependent mitochondrial respiration and glucose stimulated insulin secretion ("GSIS") kinetics. Culturing conditions are the same as in Example 4. Cells of the H1-hESC cell line cultured with EZ8 media at passage 28 were seeded as single cells at 0.094 x 106 cells / cm2 on MATRIGEL™ at a 1:30 dilution coated dishes in a mediaofDMEM-F12,GlutaMAX™ ina1:100dilution ("1Xconcentration"), 0.25mMascorbic acid, 100ng / mlFGF2, 1ng / ml ofTGFβ, ITS-Xata1:100dilution, 2%FAF-BSA,20ng / mlof IGF‑1supplementedwith10µMofY-compound.Y-compound was added only during the first 24 hours post-seeding. Forty-eight hours post-seeding, the cultures were washed in PBS (‑ / ‑).

[0206] For FIGS 8A to 8I, the cultures were differentiated using the following protocol. During Stages 1 through 4 of the protocol, cultures were maintained on planar adherent cultures. Beginning from the S4D3 monolayer, or cryopreserved S4D3cells,Aggrewell™cell clusterswereprepared,andcultured foranadditional 48hours (Stage4day5orS4D5).Media was exchanged daily throughout the differentiation protocol. During stages 5, 6, and 7 Aggrewell™ clusters were cultured in suspension culture, as described in Example 3. ALI clusters, shown in FIG. 8E to 8F, were cultured as described in Example 2. Briefly: a. Stage 1 (3 days): Cells were cultured for one day in the following Stage 1 media: MCDB‑131 medium containing 2.7g / 1000ml sodium bicarbonate, and supplemented with 0.5% FAF-BSA, GlutaMAX™ in a 1:100 dilution ("1X concentration"), 4.5mMD-glucose to obtain a concentration of 10mM of D-glucose, 1 00 ng / ml GDF8, and 1.5 µM of MCXcompound.Cellswere thencultured for anadditional day inMCDB‑131mediumcontaining2.7g / 1000ml sodium bicarbonate, and supplemented with 0.5% FAF-BSA, 1X concentration of GlutaMAX™, 4.5mMD-glucose to obtain a concentration of 10mM D-glucose, 100 ng / ml GDF8, and 0. 1 µM GSK‑3β inhibitor. Cells were then cultured for an additional day inMCDB‑131containing2.7g / 1000ml sodiumbicarbonate, andsupplementedwith0.5%FAF-BSA, 1X 46 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 concentration of GlutaMAX™, 4. 5mMD-glucose to obtain a concentration of 10mMD-glucose, and 100ng / ml GDF8. b. Stage 2_(2_days): Cells were treated for two days with MCDB‑131 medium containing 2.7g / 1000ml sodium bicarbonate, and supplemented with 0.5%FAF-BSA, 1XGlutaMAX™, 4.5mMD-glucose to obtain a concentration of 10mM D-glucose, 0.25 mM Ascorbic acid, and 50 ng / ml FGF7. c. Stage_3_(2_days):Cellswere treated for twodayswithBLAR001 custommediumcontaining 3.6g / 1000ml sodium bicarbonate, andsupplementedwith a1:200dilutionof ITS-X; 4.5mMD-glucose toobtain a concentrationof 10mMD- glucose;1XGlutaMAX™;1%FAF-BSA;25ng / mlFGF7;0.25µMSANT‑1; 1µMRA;0.25mMascorbicacid; 300nMof TPB"); and LDN-HCl for two days. The concentration of LDN-HCl used for the first day of stage 3were 100 nM, and for the second day of stage 3 was 10 nM. d. Stage 4 (3 days): Cells were treated with BLAR001 medium containing 3.6g / 1000 ml sodium bicarbonate, and supplemented with a 1:200 dilution of ITS-X; 4.5mM D-glucose to obtain a concentration of 10mM D-glucose; 1X concentration of GlutaMAX™; 1% FAF-BSA; 0.25 µM SANT‑1; 50 nM RA; 2 ng / ml FGF7; 70 nM LDN-HCl; 0.25 mM ascorbic acid; and 200 nM TPB for three days. e. Cryopreservation of Stage 4 Day 3 monolaver: Cryopreserved cell banks from S4D3monolayer were established using the procedure outlined in Example 3 and Table VII. f. Thawing of cryopreservedS4D3monolayer cells: Frozen 5ml vials containing 5.0 X106S4D3monolayer cells were thawed as described in Example 3. The same procedure can be applied to transition to either ALI or Aggrewell™ clusters. g. Stage 4 (2 days) for Aggrewell™ cluster transition or S4D3 ALI cluster transition: Aggrewell™ clusters were generated fromStage 4day 3monolayer cells, or thawed cryopreservedStage 4day 3 cells, as described inExample 3. ALI clusters were generated at Stage 4 day 3 cells, as described in Example 2. Similar toExample 4, for Stages 5, 6 and 7, the cell cultureswere conditioned by startingwith either S4D5Aggrewell™ clusters or S4D3-transitioned ALI clusters. S4D5 Aggrewell™ clusters were retrieved fromAggrewell™ 400EX plates and transferred to PBS0.1MAG spinners at a cell density of 1.5‑2.0 million cells / ml with the rotation speed of 27 rpm (rounds per minute). h. Stage 5 (3 days): Aggrewell™ or ALI clusters were treated with BLAR001medium containing 2.7g / 1000ml sodium bicarbonate, supplemented with a 1:200 dilution of ITS-X; 14.5 mM D-glucose to achieve a final concentration of 20 mMD-glucose; 1XGlutaMAX™; 2%FAF-BSA; 10µg / ml of heparin ("H"); 10µMZnSO4; 0.25µMSANT‑1; 50 nMRA; 100 nM LDN-HCl; 1 µM of T3; and 10 µM of ALK5 inhibitor II for three days. 4 kU / ml DNaseI and 5 µM Y-compound were supplemented only on Stage 5 day 1 for Aggrewell™ clusters. i. Stage 6 (7 days): Aggrewell™ or ALI clusters were treated in BLAR001 medium containing 2.7g / 1000 ml sodium bicarbonate, supplemented with a 1:200 dilution of ITS-X; 14.5 mM D-glucose to achieve a final concentration of 20mMD-glucose; 1XGlutaMAX™; 2%FAF-BSA; 10µg / ml of heparin ("H"); 10µMZnSO4; 100 nMLDN-HCl; 1µMof T3; 1 0 µM of ALK5 inhibitor II, and 100 nM of gamma secretase XX. j. Stage 7 (7 days to 14 days): Aggrewell™ or ALI clusters were treated in either BLAR001 or BLAR004 medium containing 2.7g / 1000 ml sodium bicarbonate, and supplemented with a 1:200 dilution of ITS-X; 1X GlutaMAX™; 2% FAF-BSA; 10 µg / ml of heparin ("H"), 10 nM of T3 ("low T3"); 1 mMNAC; 0.5 µMZM"); and the following components which constitute Formulation I ("FI") - 1:200dilution ofRPMI vitamin supplement; 1:200 dilution ofMEMnon-essential amino acid supplement; 1:2000 dilution of chemically defined lipid concentrate; 1:200 dilution of sodium pyruvate; 1:2000 dilution of trace elements A; 1:2000 dilution of trace elements B for seven days. Additional compounds added during Stage 7 included either 4 µM AZT; or 1 µMDEZA. For clarity, Aggrewell™ or ALI clusters in suspension were culturedduringStage7 in twoconditionsutilizing theconcentrationsdescribedabove (ineitherBLAR001orBLAR004 basemedium): (i) No ALK5, Low T3, ZM, H, NAC; (ii) No ALK5, Low T3, ZM, H, NAC, AZT, DEZA. FIG 8A, 8B, 8H, 8I: During Stage 7, Aggrewell clusters in suspension were cultured to S7D313 or S7D14 in two conditions. (i) No ALK5, LowT3, 0.5µMZM,10µg / mlH,1mMNAC; (ii) S7D1 toS7D5 -NoALK5, LowT3, 0.5µMZM,10µg / mlH,1mMNAC;5 µM AZT; 1µM DEZA; S7D6 to S7D13 or S7D14 - No ALK5, Low T3, 0.5µM ZM, 10 µg / ml H, 1 mM NAC.

[0207] Quantification and characterization of differentiated cells. For quantification of glucose-dependent mito- chondrial activity of S6D7ALI clusters, Stage 7Aggrewell™ clusters, andmatured human isletswere characterized by the 47 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 Seahorse XFe24 device as described in Example 2. ALI / Aggrewell™ clusters, or human islets were harvested, and their oxygen consumption rate ("OCR") measured on the XFeC24 Extracellular Flux Analyzer before and after injection of 20 mMD-glucose. ALI / Aggrewell™ clusters, or human isletswere removed from their Stage 7 conditioning and incubated for 2 hours in both a 37°C non-CO2 environment, and medium designed to achieve a baseline OCR. The pre-incubation medium contains 1 mM D-glucose, 1 mM L-Glutamine, and 1 mM sodium pyruvate in XF Base medium. After pre- incubation, ALI / Aggrewell™ clusters, or human islets were loaded onto the Seahorse machine in which the following measurements were made: (i) 3X baseline OCR (pre-D-glucose injection); (ii) 5X post-D-glucose (post-injection incuba- tion time: 72minutes). All OCRmeasurements were normalized to DNA content of individual samples. DNAwas isolated by the QIAamp DNA MicroKit, and DNA content measured by the NanoDrop 8000 UV-Vis Spectrophotometer.

[0208] For quantification of glucose-dependent insulin secretion, Stage 7 ALI clusters, Aggrewell™ clusters and matured human islets were characterized by a cell perifusion system (BioRep Technologies, Miami, Florida, Catalog No. PERI4‑02). Briefly, to normalize insulin secretion to a baseline, ALI / Aggrewell™ clusters or human islets were transferred into warm (37°C) Kreb’s Buffer (Table IX). Cells were loaded into perifusion chambers (BioRep Technologies, Catalog No. PERI-CHAMBER), and were continuously perfused (flow rate of 100 µl / min) with four sequential Kreb’s buffers supplemented with either: (i) 3 mMD-glucose; (ii) 16.7 mMD-glucose± 100 ng / ml Exendin‑4 ("Ex4"); and (iii) 25 mMKClwith 3mMD-glucose.Specific perifusion protocols are indicated in each figure.Perfusate sampleswere collected every minute, and C-PEPTIDE protein levels (units in ng / ml) detected by a C-PEPTIDE ELISA (Mercodia, Uppsala, Sweden, Catalog No. 10‑1136‑01). Table IX: Components of Kreb’s Buffer (pH 7.4) Catalog No. Concentration NaCl Sigma-Aldrich S7653 7.5 g / 1000ml KCl Sigma-Aldrich B9333 0.357 g / 1000ml CaCl2 Sigma-Aldrich C1016 0.277 g / 1000ml MgSO4 Sigma-Aldrich M7506 0.144 g / 1000ml Na2HPO4 Sigma-Aldrich 71643 0.144 g / 1000ml KH2PO4 Sigma-Aldrich P5655 0.16g / 1000ml NaHCO3 Sigma-Aldrich S5761 0.42 g / 1000ml HEPES Thermo Fisher Scientific15630080 10 mM

[0209] FIGS. 8A and 8B show the mitochondrial response of S7D13 Aggrewell™ clusters in suspension to 20mM D- glucose. FIGS. 8A and 8B both show that human islets (black circle line) rapidly responded to high D-glucose, demonstrated by an OCR 123.3% ± 12.92 over baseline after 15 minutes ("min") post-injection ("ip"), and maintained ahighOCRover time (131.5%±11.32; 72min ip). Conversely, S6D7ALI clusters (grey triangle line),whichwereenriched for immature C-PEPTIDE positive cells, lacked a rapid OCR response to high D-glucose (104.4%± 3.37; 15 min ip) and exhibit a relatively weak OCR response over time (113.3% ± 4.51; 72 min ip). Human islet similar glucose-dependent mitochondrial respiration kineticswereobserved in the ’NoALK5, lowT3,ZM,H,NAC,FI, BLAR004’ conditionatS7D13 in the context of Aggrewell™ clusters in suspension (110.7%± 2.46 - 15min ip; 125.9%± 2.27 - 72min ip) (grey square line) (FIG. 8A). In contrast, ’NoALK5, lowT3, ZM,H, NAC, FI BLAR001 or BLAR004’ S7D7ALI clusters did not display human- islet level glucose-dependent mitochondrial respiration kinetics (FIG. 5C and 5E). In addition, ’No ALKS, low T3, ZM, H, NAC, AZT, DEZA, FI, BLAR004’ Aggrewell™ clusters in suspension consumed oxygen in response to high glucose stimulationsignificantly abovehuman islet levels (162.0%±11.51 -15min ip; 177.1%±0.99 -72min ip) (greysquare line) (FIG. 8B). DEZA and AZT were only administered during the first four days of Stage 7 conditioning, implying that the functional mitochondrial responsiveness gained by AZTandDEZAwere stable for at least 10 days in Stage 7Aggrewell™ clusters.

[0210] FIG. 8Cdemonstrates thatmaturedbeta-cellswithin human islets exhibited theability formultiple rounds of rapid bi-phasic insulin secretion in response to glucose stimuli. The second bi-phasic GSIS responsewas blunted compared to the first (~7‑8 fold first phaseof firstGSIS response). Also, human islets demonstrated theability formultiple rounds of "on- off" switching of insulin secretion, and the ability for massive insulin granule release upon KCl-mediated membrane depolarization. All conditions tested exhibited a strong insulin secretion response to KCl (FIG. 8C‑8I). The addition of Exendin‑4 ("Ex4") did not increase the amplitude of the GSIS response in human islets shown, but was included for comparison to ALI or Aggrewell™ GSIS profiles (FIG. 8D). S7D8-S7D10 ALI clusters conditioned during Stage 7 in ’No ALKS, low T3, ZM, H, NAC FI,’ whether in BLAR001 (FIG. 8E) or BLAR004 (FIG. 8F), did show a single (~4‑10 fold first 48 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 phase of first GSIS response), but not a second, and a relatively slow bi-phasic GSIS response. Also, ALI clusters did not have the ability to shut off the second phase of insulin secretion upon re-perifusion of 3mM D-glucose after stimulus. In contrast,S7D14Aggrewell™clusters conditionedduringStage7 in ’NoALK5, lowT3,ZM,H,NAC,FIBLAR004,’ exhibited a strong first bi-phasic GSIS (~5 fold first phase of first GSIS response) followed by an ability to completely shut-down of GSIS, and aweak secondmono-phasic response (FIG. 8G).With the addition of DEZA andAZT to ’NoALK5, low T3, ZM, H,NAC, FI BLAR004’ conditioning, S7D14Aggrewell™ clusters exhibitedmultiple rounds of human-islet-similar bi-phasic GSIS (∼5‑7 fold first phase of first GSIS response), and the ability to completely shut-downGSIS in between high glucose pulses (FIGS.8H‑8I). FIGS.8Hand8I represent twobiological replicatesof the ’NoALK5, lowT3,ZM,H,NAC,DEZA,AZT BLAR004’ S7D14Aggrewell™ cluster condition. In FIGS. 8H‑8I DEZA andAZTwas only administered during the first four days of Stage 7 conditioning, implying that the robust bi-phasic GSIS responses gained by AZTandDEZAwere stable for at least 10 days in Stage 7 Aggrewell™ clusters.

[0211] In summary, Example 5 demonstrates the generation of hESC-derived functional beta-cells (Stage 7) by cellular aggregation and suspension culture with human-islet-similar glucose-dependent mitochondrial respiration and GSIS kinetics. Table X: Suppliers of the materials and compounds used in Examples 1 to 5 Material / Compound Supplier (S,S)‑2‑[2‑(3,5-Difluorophenyl)acetylamino]‑N‑(5-methyl‑6- oxo‑6,7-dihydro‑5H-dibenzo[b,d]azepin‑7-yl)propionamide ("gamma secretase inhibitor XX") EMD Millipore, Catalog No. 565789 0.2% BSA BD Biosciences, San Jose, California, Catalog No. 554657 2‑(3‑(6-Methylpyridin‑2-yl)‑1H-pyrazol‑4-yl)‑1,5-naphthyridine ("ALK5 inhibitor II" or "ALK5") Enzo Life Sciences, Inc., Farmingdale, New York, Catalog No. ALX‑270‑445 3,3’, 5-triiodo-L-thryonine sodium Sigma Aldrich, Catalog No. T6397 30% sucrose solution Amresco, Solon, Ohio, Catalog No. 0335 4% PFA Sigma Aldrich, St. Louis, Missouri, Catalog No. 158127 5 ml cryopreservation vial Thermo Fisher Scientific, Catalog No. 5000‑0050 5-Azacytidine ("AZT") Sigma Aldrich, Catalog No. A2385 Accutase Cell Detachment Solution Stem Cell Technologies, Catalog No. 07920 Aggrewell™ 400EX plate STEMCELL Technologies Inc.; Vancouver, Ca- nada; Catalog No. 27840 ascorbic acid Sigma Aldrich Co. LLC, St. Louis, Missouri, Catalog No. A4544 BME Vitamin supplement Sigma Aldrich, Catalog No. B6891 chemically defined Lipid concentrate Gibco by Life Technologies, Catalog No. 11905 Cytofix / Cytoperm Buffer BD Biosciences, San Jose, California, Catalog No. 554723 Deazaneplanocin A ("DEZA") Biovision, Inc., Catalog No. 2060 D-glucose Sigma-Aldrich Co. LLC, St. Louis, Missouri, Catalog No. G8769 DMEM-F12 Life Technologies Corporation, Carlsbad, Cali- fornia, Catalog No. 11330‑032 DMSO Sigma-Aldrich, Catalog No. D2650 DNaseI Sigma-Aldrich, Catalog No. 9003‑98‑9 Exendin‑4 ("Ex4") Sigma-Aldrich, Catalog No. E7144 49 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Table X: Suppliers of the materials and compounds used in Examples 1 to 5 Material / Compound Supplier EZ8 media Gibco by Thermo Fisher Scientific, Catalog No. A151690 fatty-acid free bovine serum albumin Proliant, Inc., Boone, Idaho, Catalog No. 68700 fibroblast growth factor 2 R & D Systems Inc., Minneapolis, Minnesota, Catalog No. 233-FB‑025 fibroblast growth factor 7 ("FGF7") R&D Systems, Inc., Minneapolis, Minnesota, Catalog No. 251-KG Table X: Suppliers of the materials and compounds used in Examples 1 to 5 Material / Compound Supplier GlutaMAX™ Life Technologies Corporation, Carlsbad, Cali- fornia, Catalog No. 35050‑079 growth differentiation factor 8 ("GDF8") Peprotech, Rocky Hill, New Jersey, Catalog No. 120‑00 H1-hESC WA01 cells, WiCell Research Institute, Madi- son, Wisconsin heparin ("H") Sigma Aldrich, Catalog No. H3149 HEPES Thermo Fisher Scientific, Catalog No. 15630080 human recombinant laminin 521 BioLamina, Catalog No. LN‑521‑03 - Human Recombinant Laminin 521 incomplete PBS (phosphate buffered saline without magnesium or calcium) Life Technologies, Carlsbad, California, Catalog No. 14190 insulin-like growth factor‑1 ("IGF‑1") R & D Systems Inc., Minneapolis, Minnesota, Catalog No. 291-G1‑200 insulin-transferrin-selenium-ethanolamine Life Technologies, Carlsbad, California, Catalog No. 51500056 KSR Thermo Fisher Scientific, Catalog No. 10828028 LDN‑193189=HCl ("LDN-HCl") (6‑(4‑(2‑(piperidin‑1-yl)ethoxy) phenyl)‑3‑(pyridin‑4-yl)pyrazolo[1,5-a]pyrimidine, hydrochloride, a BMP receptor) Shanghai ChemPartners Co Ltd., Shanghai, China, Catalog No. CG468‑141007, Lot / Batch 20141212 L-Glutamine Gibco by Life Technologies, Catalog No. 25030‑081 LIVE / DEAD Violet Fluorescent reactive dye Life Technologies, Carlsbad, California, Catalog No. L34955 MATRIGEL™ Corning Incorporated, Corning, New York, Cat- alog No. 356231 MCDB‑131 medium Life Technologies, Carlsbad, California, Catalog No. ME120219L2 MEM Non-essential Amino Acid supplement Gibco by Life Technologies, Catalog No. 111400 N-Acetyl cysteine ("NAC") Sigma Aldrich, Catalog No. A9165 Nucleocounter® NC‑100 Chemometec, Alleroed, Denmark , Catalog No. 900‑004 50 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Table X: Suppliers of the materials and compounds used in Examples 1 to 5 Material / Compound Supplier OCT solution Sakura Finetek USA Inc., Torrance, California, Catalog No. 4583 PBS0.1MAG spinners PBS Biotech Inc., Camarillo, CA, Catalog No. 1A‑0.1-D‑001 Perm / Wash Buffer BD Biosciences, San Jose, California, Catalog No. 554722 porous cell culture filter insert, 0.4 micron Corning, Catalog No. 3419 porous cell culture filter insert, 3.0 micron Corning, Catalog No. 3420 retinoic acid ("RA") Sigma Aldrich, Catalog No. R2625 RPMI vitamin supplement Sigma Aldrich, Catalog No. R7256 SANT‑1 (N‑[(3,5-dimethyl‑1-phenyl‑1H-pyrazol‑4-yl)methyle- ne]‑4‑(phenylmethyl)‑1-piperazineamine) Sigma Aldrich, Catalog No. S4572 sodium bicarbonate Sigma-Aldrich Co. LLC, St. Louis, Missouri, Catalog No. 5761 sodium pyruvate Gibco by Life Technologies, Catalog No. 11360 TPB Shanghai ChemPartners Co Ltd., Shanghai, China, Catalog No. Custom, Lot / Batch CP‑0007242‑101 trace elements A Corning, Catalog No. 25‑021 trace elements B Corning, Catalog No. 25‑022 transforming growth factor beta ("TGF-β") R & D Systems Inc., Minneapolis, Minnesota, Catalog No. 240-B‑002 TrypLE™ Express Enzyme Life Technologies Corporation, Catalog No. 12604‑013 XF Base medium Seahorse Bioscience, Catalog No. 102340‑100 Y‑27632 ("Y-compound") Sigma Aldrich Co. LLC, St. Louis, Missouri, Catalog No. Y‑0503 ZM447439 ("ZM") Selleckchem.com, Catalog No. S1103 ZnSO4 Sigma Aldrich, Catalog No. Z0251 Table XI: Chemical names and structures of compounds tested in Example 1 Zebularine Decitabine Cat. No. 2293 Cat. No. 2624 Alternative Name: NSC 309132 Chemical Name: 1-β-O-Rlbofuranosyl‑2(1H)‑pyrimidinona Alternative Names: 2’-Deoxy‑6-azacytidine 5- Aza‑2’-deoxycytidine, NSC 127716 51 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Table XI: Chemical names and structures of compounds tested in Example 1 Chemical Name: 4-Amino‑1‑(2-deoxy-β-D-arythro- panto furanosyl)‑1,3,5-triazin‑2(1H)‑one RG 108 Lomeguatrib Cat. No. 3295 Cat. No. 4359 Alternative Name: PaTrin‑2 Chemical Name: 6‑[(4-Bromo‑2-thienyl)methoxy] 9H-purin‑2-amine Chemical Name: N‑Phthalyl-L-tryplophan 5-Azacytidine Mitoxantrone dihydrochloride Cat. No. 3842 Cat. No. 4250 Chemical Name: 4-Amino‑1-β-D-ribofuranosyl‑1,3,5-tria- zin‑2(1H)-one Chemical Name: 1.4-Dihydroxy‑5,8‑bis[[2‑[(2-hy- drox yethyl)amino)ethyl]amino]‑9,10-anthracene- dione dihydrochloride EGCG Fisetin Cat. No. 4524 Cat. No. 5016 Chemical Name: 2‑(3,4-Dihydroxyphenyl)‑3,7-dihy- droxy‑4H‑1-benzopyran‑4-one Alternative Name: Epigallocatechin gallate Chemical Name: 3.4,5-Trihydroxybenzoic acid (2R,3R)‑3,4-dihydro‑5,7-dihydroxy‑2‑(3,4,5-trihydmxyphe- nyl)‑2H‑1-berizopyran‑3-yl estar SGI 1027 Temozolomide Cat. No. 5155 Cat. No. 2706 52 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Table XI: Chemical names and structures of compounds tested in Example 1 Alternative Names: NSC 382858, CCRG 81045 Chemical Name: 3,4-Dihydro‑3-methyl‑4-oxoimida- zo[5,1‑d]‑1,2,3,5-tetrezine‑8-carboxamide Chamical Name: N‑[4‑[(2-Amino‑6-methyl‑4--pyrimidin yl) amino]phenyl]‑4‑(4-quinolinylamino)banzamide L002 C 646 Cat. No. 5045 Cat. No. 4200 Chemical Name: 4‑[O‑[(4-Methoxyphenyl)sulfonyl]oxi me]‑2,6-dimethyl‑2,5-cyclohexadiene‑1,4-dione Chemical Name: 4‑[4‑[[5‑(4,5-Dimethyl‑2-nitrophe- nyl)‑2-furanyl]methylenel‑4,5-dihydro‑3-methyl‑5- oxo‑1H-pyrazol‑1-yl]benzoic acid SGC 0946 UNC 0224 Cat. No. 4541 Cat. No. 3861 Chemical Name: 1[3[[[(2R,3S,4R,5R)‑5‑(4-Amino‑5-bro- mo‑7H-pyrrolo[2,3‑d]pyrimidin‑7-yl)‑3,4-dihydro xyletrahy- drofuran‑2-yl]methyl](isopropyl)amino]propyl]‑3‑[4‑(2,2-di- methylethyl)phenyl]urea Chemical Name: 7‑[3‑(Dimethylamino)propox- y]‑2‑(hex ahydro‑4-methyl‑1H‑1,4-diazepin‑1-yl}‑6- methoxy‑N{1-methyl‑4-piperidinyl)‑4-quinazolina- mine UNC 0638 BIX 01294 Cat. No. 4343 Cat. No. 3364 53 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Table XI: Chemical names and structures of compounds tested in Example 1 Chemical Name: 2-Cyclohexyl‑6-methoxy-N‑[1‑(1-meth ylethyl)‑4-piperidinyl]‑7‑[3‑(1-pyrrolidinyl)propoxyl‑4-quina- zolinamine Chemical Name: 2‑(Hexahydro‑4-methyl‑1H‑1,4- diazepin‑1-yl)‑6,7-dimethoxy-N‑[1‑(phenylmethyl)‑4- piperidinyl]‑4-quinazolinamine trihydrochloride UNC 0646 UNC 0642 Cat. No. 4342 Cat. No. 5132 Chemical Name: 2‑(4,4-Difluoro‑1-piperidinyl)‑6- methoxy-N‑[1‑(1-melhylethyl)‑4-piperidinyl]‑7‑[3‑(1- pyrrolidinyl)propoxy]‑4-quinazolinamine Chamical Name: N‑(1-Cyclohexyl‑4-piperdinyl)‑2‑[h exa- hydro‑4‑(1-methylethyl)‑1H‑1,4-diazepin‑1-yl]‑6-meth- oxy‑7‑[3‑(1-piperidinyl)propoxy]‑4-quinazolinamine TC-E 5003 A 366 Cat. No. 5099 Cat. No. 5163 Chemical Nama: N,N’‑(Sulfonyldl‑4,1-phenylene)bla(2- chloroacetamide) Chemical Name: 5’-Methoxy‑6’‑[3‑(1-pyrrolidinyl) prop oxy]spiro[cyclobutane‑1,3’‑[3H]indol]‑2’-amine KU 55933 KU 60019 Cat. No. 3544 Cat. No. 4176 54 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Table XI: Chemical names and structures of compounds tested in Example 1 Chemical Name: 2‑(4-Morpholiniyl)‑6‑(1-thlanthrenyl)‑4H- pyren‑4-one Chemical Name: (2R,6S-rel)‑2,6-Dimethyl- N‑[5‑[6‑(4-morpholinyl)‑4-oxo‑4H-pyran‑2-yl]‑9H- thioxanthen‑2-yl‑4-morpholineacetamide PF 03814735 ZM 447439 Cat. No. 4821 Cat. No. 2458 Chamical Name: N‑[2‑[(1S.4R)‑6‑[[4‑(Cyclobutylami- no)‑5‑(trifluoromethyl)‑2-pyrimidinyl]amino)‑1,2,3,4-tetrahy- dronaphthalen‑1,4-min‑9-yl]‑2-oxoethyl]‑acetamide Chemical Name: N‑[4‑[[6-Methoxy‑7‑[3‑(4-morpho- liny l)propoxy]‑4-quinazolinyl]amino]phenyl)benza- mide U0126 SL 327 Cat. No. 1144 Cat. No. 1969 Chemical Name: α‑[Amino[(4-aminophenyl)thio] methyle ne]‑2‑(trifluoromethyl)benzeneacetonitrile Chemical Name: 1,4-Diamino‑2,3-dicyano‑1,4‑bis[2-a min- ophenylthio]butadiene H 89 dihydrochloride SB 747651A dihydrochloride Cat. No. 2910 Cat. No. 4630 55 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Table XI: Chemical names and structures of compounds tested in Example 1 Chemical Name: N‑[2‑[[3‑(4-Bromophenyl)‑2-propenyl ] amino]ethyl]‑5-isoquinolinesulfonamide dihydrochloride Chemical Name: 2‑(4-Amino‑1,2,5-oxadiazol‑3- yl)‑1-ethyl-N‑4-piperidinyl‑1H-imidazo[4,5‑c]pyri- dine‑7-methanamine dihydrochloride SNS 314 mesylate Kaempferol Cat. No. 4584 Cat. No. 3603 Chomical Name: N‑(3-Chlorophenyl)‑N’‑[5‑[2‑(thiano [3,2‑d]pyrimidin‑4-ylamino)ethyl]‑2-thiazolyl]urea mathane- sulfonate Alternative Name: Kempferol Chemical Name: 3,5,7-Trihydroxy‑2‑(4-hydroxy- phenyl)‑4H-1-benzopyran‑4-one PRT 4165 P 22077 Cat. No. 5047 Cat. No. 4485 Chemical Name: 2‑(3-Pyridinylmethylene)‑1H-lnde- ne‑1,3(2H)‑diona Chemical Name: 1‑[5‑[(2,4-Difluorophenyl)thio]4- nitro‑2-thienyl]‑ethanone PFI 1 I-BET 151 dihydrochloride Cat. No. 4445 Cat. No. 4650 Chemical Name: 2-Methoxy-N‑(3-methyl‑2-oxo‑1,2,3,4-tet- rahydroquinazolin‑6-yl)benzanesulfonamide 56 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Table XI: Chemical names and structures of compounds tested in Example 1 Chemical Name: 7‑(3,5-Dimethyl‑4-Isoxazolyl)‑1,3- d ihydroxy‑8-methoxy‑1‑[(1R)‑1‑(2-pyridinyl) ethyl]‑2H-imidazo[4,5‑c]quinolin‑2-one dihydrochlor- ide LY 303511 MS 436 Cat. No. 2418 Cat. No. 5173 Chemical Name: 2‑(1-Piperazinyl)‑8-phenyl‑4H‑1-ben zo- pyran‑4-one Chemical Name: (E)4‑[2‑(2-Amino‑4-hydroxy‑5- methy lphenyl)diazenyl]‑N‑2-pyridinylbenzenesulfo- namide SGC-CBP30 I-CBP 112 Cat. No. 4889 Cat. No. 4891 Chemical Name: 2‑[2‑(3-Chloro‑4-methoxyphenyl) ethyl]‑5‑(dimethyl‑1,2-oxazol‑4-yl)‑1‑[{2S)‑2‑(morpholin‑4- yl)propyl]‑1H‑1,3-benzodiazole Chemical Name: 1‑[7‑(3,4-Dimethoxyphe- nyl)‑9‑[[(3S)‑1-methylpiperidin‑3-yl]meth- oxy]‑2,3,4,5-tetrahydro‑1,4-benzoxazepin‑4-yl]pro- pan‑1-one Bromosporine PF| 3 Cat. No. 4758 Cat. No. 5072 Chemical Name: N‑[(6‑3-Methanesulfonamido‑4-methyl phenyl)‑3-methyl[1,2,4]triazolo[4,3‑b]pyridazin‑8-yl]carba- mate Chemical Name: (2E)‑1‑(2-Hydronphe- nyl)‑3‑[(1R,4R)‑5‑(pyridin‑2-yl)‑2,5-diazabicyclo [2.2.1]heptan‑2-yl]prop‑2-en‑1-one UNC 926 hydrochloride UNC 1215 Cat. No. 4516 Cat. No. 4666 57 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Table XI: Chemical names and structures of compounds tested in Example 1 Chamical Name: (3-Bromophenyl)[4‑(1-prrolidinyl)‑1-pi- peridinyl]methanone hydrochloride Chemical Name: 2-Phenylamino‑1,4‑[4‑(pyrrolidi- nyl) piperidinyl)benzamide TC-H 106 MC 1568 Cat. No. 4270 Cat. No. 4077 Chamical Name: N1‑(2-Aminophenyl)‑N7‑(4-methylphen yl)heptanediamide Chemical Name: 3‑[5‑(3‑(3-Fluorophenyl)‑3-oxo- propen‑1-yl)‑1-methyl‑1H-pyrrol‑2-yl]‑N-hydroxy‑2- propenamide Pyroxamide CI 994 Cat. No. 4403 Cat. No. 2952 Chemical Name: N‑Hydroxy-N’‑3-pyridinyloctanediamide Alternative Name: N-acetyldinaline Chemical Name: 4‑(Acetylamino)‑N‑(2-aminophe- nyl)be nzamide SBHA KD 5170 Cat. No. 3810 Cat. No. 4001 Chemical Name: N,N’-Dihydroxyoctanediamide Chemical Name: S‑[2‑[6‑[[[4‑[3‑(Dimethylamino) prop oxy]phenyl]sulfonyl]amino]3-pyridinyl]‑2-ox- oethyl ]ethanethioc acid ester LMK 235 TCS HDAC6 20b Cat. No. 4830 Cat. No. 4805 58 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Table XI: Chemical names and structures of compounds tested in Example 1 Chemical Name: N‑[[6‑(Hydroxyamino)‑6-oxohexyl] oxy ]‑3,5-dimethylbenzamide Chemical Name: 2-Methylpropanethioic acid- S‑[(6S)‑6‑[[(1,1-dimethylethoxy)carbonyl]amino]‑7- oxo‑7‑[tricyclo[3.3.1.13.7]dec‑1-ylamino)heptyl] es- ter PCI 34051 Scriptaid Cat. No. 4643 Cat. No. 2421 Chemical Name: N‑Hydroxy‑1‑[(4-methoxyphenyl)methy l]‑1H-indole‑6-carboxamide Chemical Name: N‑Hydroxy‑1,3-dioxo‑1H-benz[do] Isoq uinoline‑2(3H)‑hexanamide NSC 3852 Sodium 4-Phenylbutyrate Cat. No. 2521 Cat. No. 2682 Altarnative Names: 4-PB, Sodium phenylbutyrata Chemical Narne: 5-Nitroso‑8-quinolinol Chemical Nume: 4-Phenylbulyric acid, sodium salt M 344 Valproic acid, sodium salt Cat. No. 2771 Cat. No. 2815 Chemical Name: 4‑(Diethylamino)‑N‑[7‑(hydroxyamino)‑7- Alternative Names: VPA, Sodium Valproate oxoheptyl]benzamide Chemical Name: Sodium 2-propylpentanoate SAHA GSK J2 Cat. No. 4652 Cat. No. 4688 Alternative Name: Vorinostat Chemical Name: N‑Hydroxy-N’-phenyloctanediamide 59 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Table XI: Chemical names and structures of compounds tested in Example 1 Chemical Name: N‑[2‑(3-Pyridinyl)‑6‑(1,2,4,5-tetra- hydro‑3H‑3-benzazepin‑3-yl)‑4-pyrimidinyl]-β-ala- nine ethyl ester GSK J5 GSK J1 Cat. No. 4689 Cat. No. 4593 Chemical Name: N‑[2‑(3-Pyridinyl)‑6‑(1,2,4,5-tetrahy- dro‑3H‑3-benzazepin‑3-yl)‑4-pyrimidinyl]-β-alanine ethyl ester Chemical Name: N‑[2‑(2-Pyridinyl)‑6‑(1,2,4,5-tetra- hydro‑3H‑3-benzazepin‑3-yl)‑4-pyrimidinyl]-β-ala- nine GSK J4 Daminozide Cat. No. 4594 Cat, No. 4684 Chamical Name: 1‑(2,2-Dimethylhydrazide)butane- dioic acid Chemical Name: N‑[2‑(2-Pyridinyl)‑6‑(1,2,4,5-tetrahy- dro‑3H-3-benzazepin‑3-yl)‑4-pyrimidinyl]-β-alanine ethyl ester Tranylcypromine hydrochloride RN 1 dihydrochloride Cat. No. 3852 Cat. No. 4977 Alternative Name: 2-PCPA Chemical Name: (±)‑trans‑2-Phenylcyclopropylamine hy- drochloride Chemical Name: 1‑(4-methyl‑1-piperezi- nyl)‑2‑[[1R’,2S’)‑2‑[4-phenylmethoxy)phenyl)cyclo- propyl]amino]elhanone dihydrochloride IOX 1 JIB 04 Cat. No. 4464 Cat. No. 4972 Alternative Name: 5-carboxy‑8HQ 60 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Table XI: Chemical names and structures of compounds tested in Example 1 Chemical Name: 8-Hydroxy‑5-quinolinecarboxylic acid Chemical Name: 5-Chloro‑2‑[(E)‑2‑[phonyl(pyri- din‑2-yl)mathylidene]hydrazin‑1-yl]pyridina Sirtinol Splitomicin Cat. No. 3521 Cat. No. 1542 Chemical Name: 1,2-Dihydro‑3H-nephtho[2,1‑b] pyran‑3-one Chemical Name: 2‑[[(2-Hydroxy‑1-naphthalenyl)methy lene]amino]‑N‑(1-phanylethyl)benzamide Resveratrol EX 527 Cat. No. 1418 Cat. No. 2780 Chemical Name: 5‑[(1E)‑2‑(4-Hydroxyphenyl)ethenyl] ‑1,3,benzanediol Chemical Name: 6-Chloro‑2,3,4,9-tetrahydro‑1H- carb azole‑1-carboxamide Tenovin‑1 Salermide Cat. No. 3365 Cat. No. 4127 Chemical Name: N‑[[[4‑(Acetylamino)pheyl}amino)thioxo- methyl]‑4‑(1,1-dimethylethyl)benzamide Chemical Name: N‑[3[[(2-Hydroxy‑1-naphthalenyl) me thylene]amino]phenyl]-α-methylbanzeneaceta- mide AK 7 3-Aminobenzamide Cat. No. 4754 Cat. No. 0788 Chemical Name: N‑(3-Bromophenyl)‑3‑[(hexahydro‑1H- azepin‑1-yl)sulfonyl]benzamide Alternative Name: 3-ABA 61 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Table XI: Chemical names and structures of compounds tested in Example 1 PJ 34 hydrochloride IOX 2 Cat. No. 3255 Cat. No. 4451 Chemical Name: N‑(5,6-Dihydro‑6-oxo‑2-phenanthridi nyl)‑2-acetamide hydrochloride Chamical Name: N‑[[1,2-Dihydro‑4-hydroxy‑2- oxo‑1‑( phenylmethyl)‑3-quinolinyl]carbonyl]glycine Forskolin Cat. No. 1099 Retinoic acid Cat. No. 0695 Chemical Name: [3R‑(3α,4aβ,5β,6β,6aα,10α,10aβ,10bα)]‑5‑(Acetyloxy)‑3- ethenyldodecahydro‑6,10,10b-trihydroxy‑3,4a,7,7,10a-pen- tamethyl‑1H-naphtho[2,1‑b]pyran‑1-one Alternative Names: Trotinoln, ATRA Chamical Name: 3,7-Dimethyl-‑(2,6,6-trimethyl‑1-c yclohexan‑1yl)2E,4E,6E,8E,‑ acid Table XII Component of Formulation I Component Concentration Supplier RPMI Vitamin Supplement 1:200 dilution Sigma Aldrich, Catalog No. R7256 MEM non-essential amino acid supplement 1:200 dilution Life Technologies, Catalog No. 111400 Chemically defined lipid concentrate 1:2000 dilution Gibco by Life Technologies, Catalog No. 11905 sodium pyruvate 1:200 dilution Life Technologies, Catalog No. 111360 Trace elements A 1:2000 dilution Corning, Catalog No. 25‑021 Trace elements B 1:2000 dilution Corning, Catalog No. 25‑022

[0212] The above examples describe various small molecules, which typically function as cell cycle inhibitors, but unexpectedly until described in the invention herein, they induce expression of key mature beta-cell markers including PDX1, NKX6.1, MAFA, UCN3 and SLC2A1. Example 6: Generation of pancreatic endoderm derived from CyT49 hESC in multiple types of suspension culture.

[0213] The followingexample demonstrates the improvedgenerationof pancreatic endodermcells derived fromhuman pluripotent stem cells, in particular CyT49 human embryonic stem cell line ("hESC"), utilizing a differentiation protocol in multiple suspension culture formats (roller bottle and the PBS Biotech MagLift™ vertical wheel bioreactor). Table XIII lists the particular enhancements made to CyT49 hESC-derived pancreatic endoderm. Specially, the pancreatic endoderm 62 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 generated from CyT49 hESC exhibits a lower prevalence of both alternative endoderm lineages and endocrine differentiation, both important requirements for further efficient generation of insulin-producing cells, and more akin to H1 hESC-derived pancreatic endoderm (Example 3). Moreover, the time required to generate CyT49 hESC-derived pancreatic endoderm is reduced by 5 days. The newCyT49 hESC-derived pancreatic endoderm protocol can be applied to roller bottle and bioreactor formats allowing for both scale-out and scale up manufacturing. Table XIII: Improvements to generation of pancreatic endoderm. CyT49 hESC-derived PEC‑01 H1 hESC-derived (Example 3) New CyT49 hESC- derived (Example 6) Protocol duration 15 days 10 days 10 days Prevalence of other en- doderm lineages Intermediate Low Low Prevalence of endocrine differentiation Intermediate Low Low Format Roller bottle, bioreactors (8,895,300; 8,445,273) Scale-up; bioreactor (Current disclosure) Roller bottle, bioreactor (Current disclosure)

[0214] Table XIV outlines major adjustments made to the differentiation protocol for generation of improved CyT49 hESC-derived pancreatic endoderm. Major differences include the lack of BMP-inhibition during Stages 3‑4, and the use of MCDB131 (Stages 1‑2) and BLAR001 (Stages 3‑4) as base mediums. Table XIV: hESC-derived differentiation protocol to pancreatic endoderm. Stage Stage 1 Stage 2 Stage 3 Stage 4 CyT49 hESC-de- rived PEC‑01 Wnt3A, AA, ITS; RPMI1 640 FGF7, TGF-beta inh IV, ITS; RPMI1640 TTNPB, KAAD-Cyclopa- mine, Noggin, B27, ITS; DMEM / HI Glucose EGF, FGF7, Noggin, B27; DMEM / HI Glu- cose H1-derived (Ex- ample 3) MCX, GDF8; MCDB131 FGF7; MCDB131 FGF7, SANT, TPPB, LDN, RA, ITSX; BLAR001 FGF7, SANT, TPPB, LDN, RA, ITSX; BLAR001 New CyT49 hESC-derived (Example 6) Wnt3A, AA, ITSX; MCDB131 FGF7; MCDB131 FGF7, SANT, TPPB, RA, ITSX; BLAR001 FGF7, SANT, TPPB, RA, ITSX; BLAR001

[0215] Cells of the human embryonic stem cell line CyT49 ("CyT49-hESC"; NIH registration 0041) at passage 27 of working cell bank 4A ("WCB4A") were seeded as single cells at 0.033 x 106 cells / cm2 on tissue culture treated dishes in a growth media of Dulbecco’s Modified Eagle’s Medium Nutrient (DMEM) mixture F‑12 with 1:100X GlutaMAX ("DMEM- F12"; Life Technologies, Carlsbad, California, Catalog No. 10565), XetioFree ("XF") Knockout Serum Replacement ("KSR"; LifeTechnologies,CatalogNo. 12618) ina1:10dilution ("10%concentration"),MEMNon-essential aminoacids in a 1:100 dilution ("1X concentration") Penicillin-Streptomycin in a 1:100 dilution ("1X concentration"), 10ng / ml of human Activin A (R&D Systems, Minneapolis, Minnesota, Catalog No. 338-AC), and 10ng / ml human Heregulin-beta 1 (Pepro- Tech, Rocky Hill, New Jersey, Catalog No. 100‑03). Human serum AB in a 1:10 dilution ("10% concentration"; Valley Biomedical, Winchester, Virginia, Catalog No. HP‑1022) was used only during the first 24 hours post-seeding. Ninety-six hours post-seeding, the confluent CyT49 hESC cultures were passaged in the growth media mentioned above. Alternatively, confluent CyT49 hESC were released as a single cell suspension by Accumax (Innovative Cell Technol- ogies, San Diego, California, Catalog No. AM105) treatment (3 minutes at 37 °C), and aggregated at a speed of 31 rpm, andataconcentrationofonemillioncellsperml in roller bottles (Corning,Corning,NewYork,CatalogNo.CLS431644) ina media of DMFM-F12 with 1:100X GlutaMAX (Life Technologies, Catalog No. 10565), 1X concentration of Penicillin- Streptomycin, StemPro® Supplement in a 1:50 dilution (Life Technologies, Catalog No. ME130070L1), 10ng / ml human Activin A, 10ng / ml human Heregulin-beta 1, and 200ng / ml LR3-IGF1 (Cell Sciences, Newburyport, Massachusetts, Catalog No. LRM001).

[0216] For FIGS. 9A‑9Q the cultures were differentiated using the following protocol (depicted in Fig. 9A) in suspension culture format.DuringStages1 through4of theprotocol, suspensionculturesweremaintained ineither 2-liter roller bottles 63 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 ("RB") at 31 rpm, 0.1PBSMini or 0.5PBSMiniMagLift™ verticalwheel bioreactors (PBSBiotech,Camarillo,Ca) at 45 rpm. "0.1PBS" refers to the 100ml Mini MagLift™ vertical wheel bioreactor, and "0.5PBS" refers to the 500ml Mini MagLift™ vertical wheel bioreactor. CyT49 hESC aggregates were input at approximately 0.18‑0.36 million cells per ml in 500ml media volumeof a2-liter roller bottle, 100mlmedia volumeof a0.1PBSMini, or 500mlmedia volumeof a0.5PBSMini.Cell culture media was exchanged daily. a. Stage 1 (2 days): Undifferentiated ES aggregates were cultured for one day in the following Stage 1 media: MCDB‑131 medium containing 2.7g / 1000ml sodium bicarbonate, and supplemented with 0.5% FAF-BSA, Gluta- MAX™ ina1:100dilution ("1Xconcentration"), 4.5mMD-glucose toobtainafinal concentrationof 10mMofD-glucose, 100ng / ml human Activin A, 50ng / ml mouse Wnt3A (R and D Systems, Catalog No. 1324-WN), and ITS-X ("Insulin- Transferrin-Selenium-Ethanolamine"; ThermoFisher Scientific,Waltham,Massachusetts, CatalogNo. 51500056) in a 1:10000 dilution. Cells were then cultured for an additional day in MCDB‑131 medium containing 2.7g / 1000ml sodium bicarbonate, and supplemented with 0.5% FAF-BSA, 1X concentration of GlutaMAX™, 4.5mMD-glucose to obtain a concentration of 10mM D-glucose, 100ng / ml human Activin A, and ITS-X in a 1:10000 dilution. b. Stage 2 (3 days): Aggregates were treated for two days with MCDB‑131 medium containing 2.7g / 1000ml sodium bicarbonate, and supplemented with 0.5%FAF-BSA, 1XGlutaMAX™, 4.5mMD-glucose to obtain a concentration of 10mM D-glucose, 0.25 mM ascorbic acid, and 50 ng / ml fibroblast growth factor 7 ("FGF7"; PeproTech, Catalog No. AF‑100‑19). c. Stage 3 (2 days): Aggregates were treated for two days with BLAR001 custom medium containing 3.6g / 1000 ml sodium bicarbonate, and supplemented with a 1:200 dilution of ITS-X; 4. 5mMD-glucose to obtain a concentration of 10mM D-glucose; 1X GlutaMAX™; 0.25% FAF-BSA; 25 ng / ml FGF7; 0.25 µM SANT‑1 (N‑[(3,5-dimethyl‑1-phe- nyl‑1H-pyrazol‑4-yl)methylene]‑4‑(phenylmethyl)‑1-piperazineamine); 1 µM retinoic acid ("RA"); 0.25 mM ascorbic acid; and 300 nM of the PKC activator ((2S, 5S‑(E,E)‑8‑(5‑(4-trifluorometyl)phenyl‑2,4,‑pentadienoylamino)benzo- lactam ("TPB"); for two days. d. Stage 4 (3 days): Aggregates were treated with BLAR001 medium containing 3.6g / 1000 ml sodium bicarbonate, and supplemented with a 1:200 dilution of ITS-X; 4.5mMD-glucose to obtain a concentration of 10mMD-glucose; 1X concentration of GlutaMAX™; 0.25% FAF-BSA; 0.25 µMSANT‑1; 50 nMRA; 2 ng / ml FGF7; 0.25 mM ascorbic acid; and 300 nM TPB for three days.

[0217] U.S. Patent Nos. 8,445,273; 8,895,300 incorporated herein by reference in their entireties describe PEC‑01 ("Pancreatic endoderm cell - 01") d15 generated by at least the protocol listed below. CyT49 hESCaggregateswere input at 1.0 µl to 2.0 µl aggregate pellet volume ("APV") per ml. Stages 1 through 4 of the protocol below, were maintained as suspension cultures in 2-liter roller bottles in a 500ml volume rotating at 31 rpm. a. Stage 1 (2 days; d1-d2): Undifferentiated ES aggregates were treated for one day with RPMI1640 medium (Life Technologies, Catalog No. 21870‑076), supplemented with 1X concentration of GlutaMAX™; 0.2% Fetal Bovine Serum ("FBS";HyClone, LoganUtah,CatalogNo.SH30070.03); 1Xconcentration of Penicillin-Streptomycin; 1:5000 dilution of ITS (Invitrogen, Waltham, Massachusetts, Catalog No. 41400); 100ng / ml Activin A; and 50ng / ml Wnt3A. Cells were then cultured for an additional day in RPMI1640 medium, supplemented with 1X concentration of GlutaMAX™; 0.2%Fetal BovineSerum; 1X concentration of Penicillin-Streptomycin; 1:5000 dilution of ITS; 100ng / ml Activin A; and 50ng / ml Wnt3A. b. Stage 2 (3 days: d3-d5): Aggregates were treated for one day with RPMI1640 medium, supplemented with 1X concentration of GlutaMAX™; 0.2%Fetal Bovine Serum; 1X concentration of Penicillin-Streptomycin; 1:1000 dilution of ITS; 25ng / ml human FGF7; and 2.5uM Transforming Growth Factor-beta inhibitor IV ("TGF-β"; EMD Bioscience, Billerica, Massachusetts, Catalog No. 616454). Cells were then cultured for an additional two days in RPMI11640 medium, supplemented with 1X concentration of GlutaMAX™; 0.2% Fetal Bovine Serum; 1X concentration of Penicillin-Streptomycin; 1:1000 dilution of ITS; and 25ng / ml human FGF7. c. Stage 3 (3 days; d6-d8): Aggregates were treated for three days with DMEM-MI Glucose medium (Invitrogen, CatalogNo. 11960‑051), supplementedwith B‑27Supplement in a 1:50 dilution (Invitrogen,CatalogNo. 17504‑044); 1X concentration of Penicillin-Streptomycin; 1X concentration of GlutaMAX™; 1:1000 dilution of ITS; 3nM TTNPB ("4‑[(E)‑2‑(5,6,7,8-Tetrahydro‑5,5,8,8-tetramethyl‑2-naphthalenyl)‑1-propenyl] benzoic acid"; Sigma-Aldrich, St. Louis, Missouri, Catalog No. T3757); 0.25uM KAAD-Cyclopamine ("3-Keto-N-aminoethyl-N’-aminocaproyldihydro- cinnamoyl Cyclopamine"; Toronto Research Chemicals, North York, Canada, Catalog No. K171000); and 50ng / ml 64 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 human Noggin (R and D Systems, Catalog No. 3344-NG). d.Stage4 (3days; d9-d12: d12 is referred toas "PEC‑01d12"):Aggregateswere treated for threedayswithDMEM-HI Glucosemedium, supplementedwithB‑27Supplement ina1:50dilution; 1XconcentrationofPenicillin-Streptomycin; 1X concentration of GlutaMAX™; 50ng / ml human epidermal growth factor ("EGF"; PeproTech, Catalog No. AF‑100‑15); 50ng / ml FGF7; and 50ng / ml human Noggin. e. Cryopreservation at d12: Aggregates were resuspended in DMEM-HI Glucose medium, supplemented with 1X concentration of GlutaMAX™; 15% concentration DMSO ("Dimethyl sulfoxide"; Origen Biomedical, Austin, Texas, Catalog No. CP‑10); 60% concentration XF-free KSR; and 25mM HEPES buffer solution ("N‑2-hydroxyethylpiper- azine-N‑2-ethane sulfonic acid"; ThermoFisher Scientific, Catalog No. 15630080). The d12 aggregates were cryopreserved in a controlled rate freezer ("CRF"; Planer PLC, Shepperton Sunbury-on-Thames, United Kingdom, CatalogNo.Kryo 560‑16) using the following protocol: (1) start temperature ‑2.0C; (2) - 2.0C / min to ‑9.0C; (3) at ‑9.0C for 10 minutes and manually initiate freezing; (4) - 0.2C / min to ‑40C; (5) ‑25C / min to ‑150C; (6) transferred to liquid nitrogen storage. f.Post-cryopreservation toPEC‑01 (3days;d13-d15):Aggregateswere treated for threedayswithDMEM-HIGlucose medium, supplemented with B‑27 Supplement in a 1:50 dilution; 1X concentration of Penicillin-Streptomycin; 1X concentration of GlutaMAX™; 50ng / ml human epidermal growth factor ("EGF"; PeproTech, Catalog No. AF‑100‑15); 50ng / mlFGF7;and50ng / ml humanNoggin.On thefirst dayonly 10U / mlDNase I (Roche,Basel, Switzerland,Catalog No. 04536282001) is supplemented to the media.

[0218] Characterization of differentiated cells: For quantification of gene expression utilizing 2-liter roller bottle, 0.1 PBS, or 0.5 PBS suspension culture methods, CyT49-hESC, Stage 4 day 3 ("S4D3"), and PEC‑01 d15 aggregates were harvested, as described inNatureBiotechnology, 2014, (32) 11, 1121‑1133.Gene expressionwas assessed in cells using custom Taqman Arrays (Applied Biosystems, Foster City, California). Data were analyzed using Sequence Detection Software (Applied Biosystems, Foster City, California), and normalized using GAPDH as a housekeeping gene to undifferentiated CyT49-hESC using the ΔΔCt method. Primer details are outlined in Table XV. Table XV: List of qRT-PCR primers. Gene Assay ID 1 PTF1A Hs00603586_g1 2 CDX2 Hs00230919_m1 3 SOX2 Hs01053049_s1 4 NKX6.1 Hs00232355_m1 5 PDX1 Hs00236830_m1 6 NEUROD1 Hs00159598_m1 7 CHGA Hs00154441_m1 8 GAPDH Hs99999905_m1

[0219] For quantification of protein presence co-localization utilizing 2-liter roller bottle, 0.1PBS, or 0.5PBSsuspension culture methods, CyT49-hESC, Stage 4 day 3 ("S4D3"), and PEC‑01 d15 aggregates were harvested and analyzed by fluorescence-activated flow cytometry ("FACS"). FACS staining was conducted as described in Nature Biotechnology, 2014 (32) 11, 1121‑1133, and used the antibodies listed in Table XVI. Differentiated cells were incubated in Accumax (Innovative Cell Technologies, San Diego, California, Catalog No. AM105) for 20 minutes at room temperature, released into a single-cell suspension, after which they were washed twice with autoMACS Running Buffer (Bergisch, Germany, Miltenyl Biotec, Catalog No. 120‑091‑221), fixed in 4% Paraformaldehyde ("4% PFA"; Sigma-Aldrich, Catalog No. 158127) for 30 minutes at room temperature followed by two washes in the autoMACS Running buffer. Cells were then incubated with the appropriate antibodies (Table XVI) prior to analysis on the BD FACS Canto II using BD FACS Diva Software with at least 30,000 events being acquired. Non-viable cells were excluded during FACS analysis, and gating was determined by using isotype antibodies ("IgG"). 65 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 Table XVI: List of antibodies used for FACS analysis. Antigen Species Source / Catalog Number Dilution Alexa Fluor 647 anti-NKX6.1 Mouse BD, Catalog No. 563338 1:3 PE anti-PDX1 Mouse BD, Catalog No. 562161 1:2 Alexa Fluor 405 anti-CHGA Mouse Novus Biologicals, Catalog No. N-BP2‑33198AF405 1:100 PerCP-Cy™5.5 anti-SOX2 Mouse BD, Catalog No. 561506 1:2 PE anti-CDX2 Mouse BD, Catalog No. 563428 1:4

[0220] For quantification of cell yields utilizing 2-liter roller bottle, 0.1 PBS, or 0.5 PBS suspension culture methods, CyT49-hESC and Stage 4 day 3 ("S4D3") aggregates were harvested, and counted by the Nucleocounter® NC‑100 (Chemometec, Allerod, Denmark, Catalog No. NC‑100).

[0221] FIG. 9A depicts howCyT49 hESCwere differentiated utilizing a new protocol towards the pancreatic endoderm (e.g.S4D3).PEC‑01d12andPEC‑01d15arehighlightedhereasexamplesof prior art. Suspension culturemethodswere utilized based on their compatibility for commercial scale generation of insulin-producing cells via a pancreatic endoderm intermediate, namely 2-liter roller bottle, or the 0.1 PBS, or 0.5PBS vertical wheel bioreactors.While the 2-liter roller bottle format is a useful tool for the scale-out of pancreatic endoderm cells, it is not ideal for the eventual commercial scale of insulin-producing cells. ThePBSBiotechMagLift™ vertical wheel bioreactor system is an alternative scale-up system that would provide the ability to progressively improve insulin-producing cell production while increasing volume, and maintaining small / compact aggregate size over time. FIG. 9B demonstrates that, as expected, cell yields at S4D3 reflectedasignificantexpansionof cell number relative tohESC input.The ratioofS4D3cells tohESC inputwasconsistent across all suspension culturing methods utilizing the new protocol; 4.30±0.782 for 2-liter roller bottle, 3.27±0.585 for 0.1PBS, and 4.08±0.854 for 0.5PBS, and markedly increased from prior art (2.23±0.090 PEC‑01 d12 cell per hESC input). Furthermore, wedemonstrated in FIG. 9C that the disclosed differentiation protocol can be effectively scaled-up using thePBSMagLift™ vertical wheel bioreactor system.When themedia volumewas increased from100ml (0.1PBS) to 500ml (0.5PBS) an approximately 6-fold increase in S4D3 yield (FIG. 9C) was observed. In addition, utilizing either the 0.1PBS (~150±33.2 microns) or 0.5PBS (~153±37.6 microns) suspension format resulted in uniformly smaller S4D3 aggregates relative to roller bottle based S4D3 (~274±92.5 microns), and PEC‑01 d12 (~259±55.3 microns). Cryopre- servation, thaw and further three-day culture of PEC‑01 d12 was required to reduce PEC‑01 aggregates size to the scale seen with 0.1PBS and 0.5PBS (PEC‑01 d15; ~191±45.4 microns) (FIG. 9D‑9E). It was demonstrated that a robust pancreatic endoderm phenotype was achieved utilizing the new CyT49 hESC-derived differentiation protocol across all suspension culture methods.NKX6.1 (FIG. 9F), PTF1A (FIG. 9G), PDXI (FIG. 9H) were all significantly induced in 2-liter roller bottle, 0.1PBS, 0.5PBS methods, similarly to PEC‑01 d15. Unlike the PEC‑01 d15 differentiation protocol, which resulted in the heightened expression of SOX2 (alternative endoderm lineage; FIG. 9I), NEUROD1 (early endocrine differentiation; FIG. 9K), and CHGA (early endocrine differentiation; FIG. 9L), the new differentiation protocol reduced these non-pancreatic endodermmarkers in all suspension culturemethods tested in CyT49. Indeed, such an enrichment of non-pancreatic endoderm, decreases the efficiency of pancreatic endoderm production as assessed on the protein- level. Relative to PEC‑01 d12 (~38.0%; FIG. 9M), the protein co-localization between PDX1 and NKX6.1 was enhanced across all new S4D3 suspension culture methods utilizing the new CyT49 hESC-derived differentiation protocol; 2-liter roller bottle ~64.6% (FIG. 9N), 0.1PBS ~48.9% (FIG. 9O), and 0.5PBS ~65.3% (FIG. 9P). Moreover, ~49% of PDX1+ NKX6.1+ pancreatic endoderm observed in PEC‑01 d12 also expressed SOX2+ (FIG. 9M), unlike all new S4D3 suspension culture methods utilizing the new CyT49 hESC-derived differentiation protocol; 2-liter roller bottle ~2.0% (FIG. 9N), 0.1PBS~10.0%(FIG. 9O), and0.5PBS~6.2% (FIG. 9P). The relatively lowpercentageof pancreatic endoderm seen in PEC‑01 d12 was also reflected by the increased prevalence of early endocrine differentiation (~49.6%; FIG. 9M) relative to all newS4D3suspension culturemethods; 2-liter roller bottle ~26.9% (FIG. 9N), 0.1PBS~26.6% (FIG. 9O), and 0.5PBS ~30.2% (FIG. 9P). Cryopreservation, thaw, and 3 days additional cell culture of PEC‑01 d12 was required to slightly increase the co-localization of PDX1 and NKX6.1 (PEC‑01 d15; ~47.5%; FIG. 9Q) by the reduction of SOX2 (~22.5%; FIG. 9Q) andCHGA (~42.5%; FIG. 9Q). The protein co-localization betweenNKX6.1 andCDX2was lowacross all conditions tested (FIG. 9M‑9Q), as mirrored by low CDX2 gene expression at S4D3 across all types of suspension culture (FIG. 9J). Example 7: 66 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 Generation of insulin producing cells derived from CyT49 hESC in multiple types of suspension culture.

[0222] The following example demonstrates the improved generation of pancreatic insulin-producing cells derived from the CyT49 human embryonic stem cell line ("hESC") utilizing a new differentiation protocol in multiple suspension culture formats (roller bottle, and both the 0.1PBS and 0.5PBS Biotech MagLift™ vertical wheel bioreactors). Table XVII lists the particular enhancements made to CyT49 hESC-derived insulin-producing cells. While, similar in duration to PEC‑01- derived and PEC‑01-similar-derived insulin producing cells (prior art is Stem Cells Transl Med, 2015 (10) 4, 1214‑1222), the new differentiation protocol used to produce insulin-positives cells fromCyT49 hESCexhibit the enhanced production of mono-hormonal insulin-producing cells (INS+ only), via a significant reduction of poly-hormonal insulin-producing cells (both INS+ GCG+), and the protein presence of PDX1, NKX6.1, CHGA, MAFA in mono-hormonal insulin-producing cells. The lack of GCG-presence, and induction of NKX6.1, MAFA-presence in mono-hormonal insulin-positive cells is a requirement for eventual acquisition of human-islet-similar functionality (Examples 2‑5).Moreover, the newCyT49hESC- derived pancreatic insulin-producing cell protocol exhibits, for the first time, scale-up ability in a bioreactor, which is better able to handle, than scale-out by roller bottle, the eventual commercial cell manufacturing of insulin-producing cells (Example 7). Table XVII: Improvements to generation of insulin-producing cells. PEC‑01-similar derived insulin- producing cells (CyT49) H1 hESC-derived (Example 2‑4) New CyT49 hESC- derived (Example 7) Protocol duration 12‑21 days (Stage 7) 17 days (Stage 7) 17‑25 days (Stage 7) Prevalence of total INS+ cells ~45% ~50% ~50% Prevalence of INS+ GCG+ cells Intermediate; ~15% Low; ~4% Low; ~6.6% MAFApresence in INS+ NKX6.1+ cells? Not assessed but very low gene expression Yes Yes Format Rotating 6-well plates bioreactor (Current disclosure) Roller bottle, bioreactor (Current disclosure)

[0223] Table XVIII outlines major adjustments made to the differentiation protocol for generation of improved CyT49 hESC-derived pancreatic endoderm. Major differences include small molecules and base mediums used. Abbreviations include: Nic - Nicotinamide; MG - Matrigel; GSI - gamma-secretase inhibitor; ROCKi - Rho-associated protein kinase inhibitor. Table VIII: hESC-derived differentiation protocol to insulin-producing cells. Stage Stage 5 Stage 6 Stage 7 PEC‑01-derived insulin-produ- cing cells (CyT49) (Stem Cell Trans Med, 2015) Noggin, KGF, EGF, GSI, ROCKi, B‑27; DMEM / HI Glucose Nic, ROCKi, B‑27; CMRL1066 Nic, MG, ROCKi, T3, Nic, B‑27; CMRL1066 H1 hESC-derived (Example 2‑4) SANT, RA, LDN, ALK5 inh, T3, Heparin; BLAR001 LDN, ALK5 inh, T3, XX, Heparin; BLAR001 T3, Heparin, NAC, Formulation I; BLAR001 New CyT49 hESC-derived (Ex- ample 7) SANT, RA, LDN, ALK5 inh, T3, Heparin; BLAR001 LDN, ALK5 inh, T3, XX, Heparin; BLAR001 T3, Heparin, NAC, Formulation I; BLAR001

[0224] Cells of the human embryonic stem cell line CyT49 ("CyT49-hESC"; NIH registration 0041) at passage 27 of workingcell bank4A ("WCB4A")wereseededandmaintainedasdescribed inExample6.Also,CyT49hESCaggregation, and differentiation through Stages 1‑4 was performed as described in Example 6.

[0225] For FIGS. 10A‑10AB, the cultures were further differentiated during Stages 5‑7 using the following protocol (depicted in Fig. 10A) in suspension culture format. During Stages 5 through 7 of the protocol, suspension cultures were maintained in either roller bottles ("RB") at 31 rpm, 0.1 PBSMini or 0.5 PBSMini MagLift™ vertical wheel bioreactors at 45 67 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 rpm. "0.1PBS" refers to the100mlMiniMagLift™verticalwheel bioreactor, and "0.5PBS" refers to the500mlMiniMagLift™ vertical wheel bioreactor. Note, that the Stage 7 differentiation protocol is referred to as basal conditioning, as the cocktail for inducing functionality of the insulin-producing cells is not included and is subject of another disclosure. During Stage 5‑6, media exchanges occurred daily, while at Stage 7, media was exchanged every other day. a.Stage5 (3days):S4D3CyT49 (Example6 -newCyT49hESC-derivedprotocol) orPEC‑01d15derivedaggregates were treatedwithBLAR001mediumcontaining2.7g / 1000ml sodiumbicarbonate, supplementedwitha1:200dilution of ITS-X; 14.5mMD-glucose to achieve a final concentration of 20mMD-glucose; 1XGlutaMAX™; 2%FAF-BSA; 10 µg / ml of heparin ("H"); 10µMZnSO4; 0.25µMSANT‑1; 50 nMRA; 100 nMLDN-HCl; 1µMof T3; and 10µMof ALK5 inhibitor II. b. Stage 6 (7 days): Aggregates were treated in BLAR001 medium containing 2.7g / 1000 ml sodium bicarbonate, supplementedwith a 1:200 dilution of ITS-X; 14.5mMD-glucose to achieve a final concentration of 20mMD-glucose; 1XGlutaMAX™; 2%FAF-BSA; 10µg / ml of heparin ("H"); 10µMZnSO4; 100 nMLDN-HCl; 1µMofT3; 10µMofALK5 inhibitor 11, and 100 nM of gamma secretase inhibitor XX. S5D3 aggregates in 0.5 PBS suspension culture were transferred to 0.1 PBS suspension culture for Stages 6‑7. c. Stage 7 (7 to 23days): Aggregateswere treated inBLAR001mediumcontaining 2.7g / 1000ml sodiumbicarbonate, andsupplementedwitha1:200dilutionof ITS-X;1XGlutaMAX™;2%FAF-BSA; 10µg / ml of heparin ("H"), 10nMofT3 ("low T3"); 1 mM NAC"); and the following components which constitute Formulation I ("FI") - 1:200 dilution of RPMI vitamin supplement; 1:200 dilution of MEM non-essential amino acid supplement; 1:2000 dilution of chemically defined lipid concentrate; 1:200 dilution of sodium pyruvate; 1::2000 dilution of trace elements A; 1:2000 dilution of trace elements B.

[0226] Characterizationofdifferentiatedcells:For quantification of geneexpression utilizing roller bottle, 0.1PBS, or 0.5 PBS suspension culture methods, CyT49-hESC, Stage 4 day 3 ("S4D3"), Stage 5 day 3 ("S5D3"), Stage 6 day 7 ("S6D7"), and Stage 7 day 7 ("S7D7") aggregates were harvested, as described in Nature Biotechnology, 2014, (32) 11, 1121‑1133, and Example 1. Primer details are outlined in Table XIX. Table XIX: List of qRT-PCR primers. Gene Assay ID 1 NGN3 Hs00360700_g1 2 INSULIN Hs00355773_m1 3 MAFA Hs01651425_s1 4 NKX6.1 Hs00232355_m1 5 PDX1 Hs00236830_m1 6 NEUROD1 Hs00159598_m1 7 CHGA Hs00154441_m1 8 GAPDH Hs99999905_m1

[0227] For quantification of protein presence co-localization utilizing roller bottle, 0.1 PBS, or 0.5 PBS suspension culture methods CyT49-hESC, Stage 5 day 3 ("S5D3"), Stage 6 day 7 ("S6D7"), Stage 7 day 13 ("S7D13"), and Stage 7 day 14 ("S7D14") aggregates were harvested and analyzed by fluorescence-activated flow cytometry ("FACS") as described in Example 1. List of antibodies used for FACS analysis are indicated in Table XX. Table XX: List of antibodies used for FACS analysis. Antigen Species Source / Catalog Number Dilution Alexa Fluor 647 anti-NKX6.1 Mouse BD, Catalog No. 563338 1:3 anti-PAX4 (unconjugated) Goat R and D Systems, Catalog No. AF2614 1:40 Alexa Fluor 405 anti-CHGA Mouse Novus Biologicals, Catalog No. NBP2‑33198AF405 1:100 Alexa Fluor 488 anti-INSULIN Mouse Novus Biologicals, Catalog No. NBP2‑34738AF488 1:10 68 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Table XX: List of antibodies used for FACS analysis. Antigen Species Source / Catalog Number Dilution BV421 anti-GLUCAGON Mouse BD, Catalog No. 565891 1:10 Alexa Fluor 488 Anti-goat IgG Goat Jackson Laboratories, Catalog No. 705‑546‑147 1:400

[0228] For quantification of cell yields utilizing roller bottle, 0.1 PBS, or 0.5 PBS suspension culture methods, CyT49- hESC, and Stage 6 day 7 ("S6D7") aggregates were harvested, and counted by the Nucleocounter® NC‑100 (Chemo- metec, Allerod, Denmark, Catalog No. NC‑100).

[0229] Forquantificationof protein co-localizationutilizing roller bottle, 0.1PBS,or 0.5PBSsuspensionculturemethods Stage 6 day 7 ("S6D7"), Stage 7 day 16 ("S7D16"), and Stage 7 day 23 ("S7D23") aggregates were harvested and analyzed by immunofluorescence ("IF"). CyT49-hESC-derived aggregates were prepared as described in both Nature Biotechnology, 2014, (32) 11, 1121‑1133 and in previous examples, and using the antibodies listed in Table XXI herein. Table XXI: List of antibodies used for IF analysis. Antigen Species Source Dilution INSULIN Mouse Cell Signaling (Catalog No. 8138BF) 1:250 MAFA Rabbit Life Span Biosciences (Catalog No. LP9872) 1:100 (Antigen retrieval) SYNAPTOPHYSIN Rabbit Novus Biologicals (Catalog No. NB120‑16659) 1:50 NKX6.1 Rabbit Life Span Biosciences (Catalog No. LP9878) 1:1000

[0230] FIG. 10A depicts the new protocol used to differentiate S4D3 CyT49-derived hESC through Stages 5‑7 towards an insulin-producing cell. Specifically, the insulin-producing cells derived here exhibited the co-expression and protein presence of PDX1, NKX6.1, CHGA, INSULIN andMAFA, but not GLUCAGON. FIG. 10B demonstrates that cell yields at S6D7wereapproximatelyequal to the initial hESC input for 2-liter roller bottle ("RB") (0.95±0.31S6D7-to-hESC input)and 0.1 PBS (0.92±0.23 S6D7-to-hESC input) suspension culture methods. Strikingly, suspension culturing using the 0.5 PBS format exhibited the highest cell yields at S6D7; for one hESC cell input 2.4±0.169 S6D7 cells were generated. Moreover, it was demonstrated (see FIG. 10C) that the Stages 5‑7 differentiation protocol can be effectively scaled-up using the MagLift™ vertical wheel bioreactor system. When the media volume was increased from 100ml (0.1 PBS) to 500ml (0.5PBS) an approximately 7.5-fold increase inS6D7yield (FIG. 10C)wasobserved. In addition, utilizing either the 0.1PBSor0.5PBSsuspension format resulted inuniformly smallerS5D3 (FIG.10D),S6D7 (FIG.10E)andS7D13-S7D14 (FIG. 10F) aggregates relative to 2-liter roller bottle based aggregates. Briefly, S6D7aggregates suspension culturedwith the disclosed protocol using roller bottles exhibited the following diameters; RB ~265±79.0 microns (FIG. 10E left), 0.1PBS ~190±59.9 microns (FIG. 10Emiddle), and 0.5PBS ~227±59.0 microns (FIG. 10E right). Also, utilizing the PBS Mini MagLift™ vertical wheel bioreactors maintained tight aggregate size, and architecture through S7D14 (FIG. 10F), required for human-islet level functionality (Example 7). Conversely, S7D13 aggregates in 2-liter roller bottle suspension eventually lost their tight aggregate architecture, and formed loose cellular sheets (FIG. 10F). It was demonstrated that all suspensionculturemethodsusing thenewprotocol induceda robust pancreatic insulin-producingcell genesignature;RB, 0.1 PBS and 0.5 PBS induce the maintain / increase the expression of beta-cell transcription factors PDX1 (FIG. 10G), NKX6.1 (FIG. 10H); endocrine markers / transcription factorsCHGA (FIG. 10I,NEUROD1 (FIG. 10J),NGN3 (FIG. 10K); beta-cell hormone INSULIN (FIG. 10L); and early beta-cell maturation transcription factorMAFA (FIG. 10M). Conversely, whenPEC‑01 d15 aggregateswere differentiated to insulin-producing cells using the newdifferentiation protocol in 2-liter roller bottles ("PEC‑01-derived"), a significant induction of GLUCAGON together with INSULIN expression (FIG. 10L, 10N) was observed. Indeed, protein presence analysis demonstrated that, as described previously inNatureBiotechnol- ogy, 2014, (32) 11, 1121‑1133, the new differentiation protocol induced robust pancreatic endocrine pre-cursors (Stage 5) leading to production of mono-hormonal, insulin producing cells (Stages 6‑7) in the CyT49 hESC line. FIG. 10O showed that byS5D3approximately 54.1%of aggregates cultured in 0.5PBSwereCHGA+NKX6.1+ (increased from~1%CHGA+ NKX6.1+ atS4D3 for all suspension formats; FIG. 9N‑9P), a result of thehigh rate of endocrinedifferentiation - indicatedby enrichment forPAX4+CHGA+cells at S5D3.PAX4 is adirect target geneofNGN3, thedriver of endocrinedifferentiation in the pancreas. By S7D13-S7D14 the total number of NKX6.1+ INSULIN+ cells increased dramatically in all suspension formats; from approximately 15.4% at S5D3 (FIG. 10P) to ~40.3% in RB at S6D7 (FIG. 10Q); ~44.7% in 0.1PBS at S6D7 (FIG. 10R); ~51.8% in RB at S7D13 (FIG. 10S); and ~48.9% in 0.1PBS at S7D14 (FIG. 10T). Similarly, by S7D13-S7D13 the percentage of NKX6.1+ CHGA+ cells increased from ~1% at S4D3 (FIG. 9N‑9P) to ~81.0% in RB at S6D7 (FIG. 10Q); 69 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 ~88.7% in 0.1PBSat S6D7 (FIG. 10R); ~79.6% inRBat S7D13 (FIG. 10S); and ~80.9% in 0.1PBS at S7D14 (FIG. 10T). It was demonstrated that the majority of NKX6.1+ INSULIN cells produced by the new protocol at Stage 7 were mono- hormonal, and were not enriched for the alpha-cell hormone GLUCAGON; ~18.8% INSULIN+ GLUCAGON+ in RB at S6D7 (FIG. 10Q); ~14.4% in 0.1PBS at S6D7 (FIG. 10R); ~7.7% in RB at S7D13 (FIG. 10S); and ~6.5% in 0.1PBS at S7D14 (FIG. 10T). Moreover, it was demonstrated that the poly-hormonal cells constituted only a small fraction of total cells by Stage 7 (FIG. 10S‑10T), likely originating from the CHGA+NKX6.1- population, inherently higher in number in the CyT49-derivedS4D3 cells relative toH1-derivedS4D3 cells (Example 3). Strikingly, PEC‑01-derived aggregates in 2-liter roller bottles at S6D7 exhibited ~41.9% co-localization between INSULIN and GLUCAGON. In all conditions tested, the GLUCGAON+ INSULIN+ cells were not NKX6.1+, as shown for PEC‑01-derived aggregates in 2-liter roller bottle at S6D7, and newprotocol S4D3-derived 0.1PBSat S7D14, 2-liter roller bottle at S7D13 (FIG. 10V). The robust induction of insulin- producing endocrine cells was mirrored in the IF-analysis. Aggregates whether cultured in either RB or 0.1 PBS formats showed a great enrichment for SYNAPTOPHYSIN+ NKX6.1+ INSULIN+ cells at both S6D7 (0.1 PBS ‑ FIG. 10W) and S7D16-D23 (RB ‑ FIG. 10AA; 0.1 PBS ‑ FIG. 10Y). Furthermore, the early maturation transcription factor MAFA was already induced in 0.1 PBS at S6D7 (FIG. 10X), and became co-present with INSULIN-positive cells at Stage 7 in RB at S7D23 (FIG. 10AB), and 0.1 PB S at S7D16 (FIG. 10Z).

[0231] While the invention has been described and illustrated herein by references to various specific materials, procedures andexamples, it is understood that the invention is not restricted to theparticular combinations ofmaterial and procedures selected for that purpose. Numerous variations of such details can be implied as will be appreciated by those skilled in the art. It is intended that the specification and examples be considered as exemplary, only, with the true scope and spirit of the invention being indicated by the following claims. All references, patents, and patent applications referred to in this application are herein incorporated by reference in their entirety. The invention is further described by way of the following items: 1. Amethod of differentiating pancreatic endoderm cells into functional beta cells expressing PDX1, NKX6.1, MAFA, UCN3 and SLC2A1 comprising culturing pancreatic endocrine cells in a medium supplemented with one or more of UNC0638,UNC0642,UCN0646,TC-E5003,A366,PF03814735,ZM447439,SB747651A,PFI1, LY303511,MS436, AZT, DEZA, pyroxamide, CI9994 or MC1568. 2. The method of item 1, wherein the culture medium is further supplemented with heparin, N-Acetyl Cysteine, Formulation I and one or more of T3, T4 or analogue thereof. 3. The method of item 1 or 2, wherein the culture medium lacks an ALK5 inhibitor. 4. The method of item 1 or 2, wherein the culture rnedium is supplemented with an ALK5 inhibitor. 5. Themethodof item1,wherein themedium is supplementedwithZM447439, heparin,N-AcetylCysteineandoneor more of T3, T4 or analogue thereof and wherein the medium contains no ALK5 inhibitor. 6. The method of item 5, wherein the medium is supplemented with T3. 7. The method of item 6, wherein the medium is supplemented with AZT. 8. The method of item 7, wherein the medium is supplemented with DEZA. 9. The method of item 1, wherein the functional beta-cells are obtained by step-wise differentiation of one or more of cells expressingmarkerscharacteristic of thedefinitiveendoderm, cells expressingmarkerscharacteristic of primitive gut tube cells, cells expressing markers characteristic of foregut endoderm cells, cells expressing markers char- acteristic of pancreatic endoderm cells, cells expressing markers characteristic of pancreatic endocrine precursor cells, cells expressing markers characteristic of immature beta-cells, and cells expressing markers characteristic of functional beta-cells. 10. The method of item 1, wherein the method comprises culturing the cells at the air-liquid interface. 11. The method of item 1, wherein the method comprises culturing the cells in suspension clusters. 12. An in vitro population of functional beta cells expressing single hormonal insulin, PDX1, NKX6.1 and MAFA obtained by in vitro differentiation of pancreatic endocrine cells. 70 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 13. The population of item 12, wherein the cells also express UCN3 and SLC2A1. 14. The population of item 12, wherein the cells exhibit glucose-stimulated insulin secretion and glucose-dependent mitochondrial respiration. 15. Thepopulationof item14,wherein theglucose-stimulated insulin secretionandglucose-dependentmitochondrial respiration is similar to that of human islet cells. 16. An in vitro population of pancreatic beta cells that express single hormonal insulin, PDX1, NKX6.1 and MAFA obtainedby culturingpancreatic endocrine cells in amediumsupplementedwithoneormoreofUNC0638,UNC0642, UCN0646, TC-E5003, A366, PF03814735, ZM447439, SB747651A, PFI1, LY303511, MS436, AZT, DEZA, pyrox- amide, CI9994 or MC1568. 17. The population of item 16, wherein the culture medium is further supplemented with heparin, N‑ Acetyl Cysteine, Formulation I and one or more of T3, T4 or analogue thereof. 18. The population of item 16 or 17, wherein the culture medium lacks an ALK5 inhibitor. 19. The population of item 16 or 17, wherein the culture medium is supplemented with T3. 20. The population of item 16, wherein the medium is supplemented with ZM447439, heparin, N‑ Acetyl Cysteine, Formulation I and one or more of T3, T4 or analogue thereof and wherein the medium contains no ALK5 inhibitor. 21. The population of item 16, wherein the medium is supplemented with T3. 22. The population of item 21, wherein the medium is supplemented with AZT. 23. The population of item 22, wherein the medium is supplemented with DEZA. 24. The population of item 16, wherein the cells also express UCN3 and SLC2A1. 25. The population of item 16, wherein the cells exhibit glucose-stimulated insulin secretion and glucose-dependent mitochondrial respiration. 26. Thepopulationof item16,wherein theglucose-stimulated insulin secretionandglucose-dependentmitochondrial respiration is similar to that of islet cells. 27. A method of differentiating pluripotent stem cells into functional beta cells expressing PDX1, NKX6.1, MAFA, UCN3 and SLC2A: a. Differentiating pluripotent stem cells into immature beta-cells; and b. Differentiating the immature beta-cells into functional beta-cells expressing PDX1, NKX6.1,MAFA,UCN3 and SLC2A1 by culturing immature beta-cells in a medium supplemented with one or more of UNC0638, UNC0642, UCN0646, TC-E5003, A366, PF03814735, ZM447439, SB747651A, PFT1, LY303511, MS436, AZT, DEZA, pyroxamide, CI9994 or MC1568. 28.Themethodof item27,wherein the functional beta cells haveglucose-dependentmitochondrial respirationsimilar to human islet cells. 29. The method of item 27, wherein the functional beta cells have glucose-stimulated insulin secretion similar to human islet cells. 30. The method of item 27, wherein the functional beta cells secrete insulin in multiple phases. 31. The method of item 27, wherein the culture medium is further supplemented with heparin, N‑ Acetyl Cysteine, Formulation I and one or more of T3, T4 or analogue thereof. 71 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 32. The method of item 27 or 31, wherein the culture medium lacks an ALK5 inhibitor. 33. The method of item 27 or 31, wherein the culture medium is supplemented with T3. 34. Themethodof item27,wherein themedium is supplementedwithZM447439, heparin,N-AcetylCysteineandone or more of T3, T4 or analogue thereof and wherein the medium contains no ALK5 inhibitor. 35. The method of item 34, wherein the medium is supplemented with T3. 36. The method of item 35, wherein the medium is supplemented with AZT. 37. The method of item 36, wherein the medium is supplemented with DEZA. 38. Themethod of item 27, wherein themethod comprises culturing the immature beta cells at the air-liquid interface. 39. The method of item 27, wherein the method comprises culturing the the immature beta cells in suspension clusters. 40. The method of item 27, wherein the step of differentiating pluripotent stem cells comprises: a. Differentiating the pluripotent stemcells into cells expressingmarkers characteristic of the definitive endoderm ("Stage 1 cells"); b. Differentiating the Stage 1 cells into cells expressingmarkers characteristic of primitive gut tube cells ("Stage 2 cells"); c.Differentiating theStage2cells into cells expressingmarkers characteristic of foregut endodermcells ("Stage3 cells"); d. Differentiating the Stage 3 cells into cells expressing markers characteristic of pancreatic endoderm cells ("Stage 4 cells"); e. Differentiating the Stage 4 cells into pancreatic endocrine precursor cells ("Stage 5"); and f. Differentiating the Stage 5 cells into the immature beta-cells. 41. The method of item 40, wherein steps e. and f. comprise culturing at the air-liquid interface. 42. The method of item 40, wherein the steps e. and f. comprise culturing the cells in suspension clusters. 43. The method of item 40, wherein the method comprises differentiating pluripotent stem cells into Stage 1 cells by culturing the pluripotent stem cells in a medium supplemented with MCX compound and GDF‑8. 44. The method of item 40, wherein the method comprises differentiating the Stage 1 cells into Stage 2 cells by culturing the Stage 1 cells in a medium supplemented with FGF7 and ascorbic acid. 45. The method of item 40, wherein the method comprises differentiating the Stage 2 cells into Stage 3 cells by culturing the Stage 2 cells in a medium supplemented with FGF7, retinoic acid, SANT‑1, a PKC activator, a BMP inhibitor and ascorbic acid. 46. The method of item 40, wherein the method comprises differentiating the Stage 3 cells into Stage 4 cells by culturing the Stage 3 cells in a medium supplemented with FGF7, retinoic acid, SANT‑1, a PKC activator, a BMP inhibitor and ascorbic acid. 47. The method of item 40, wherein the method comprises differentiating the Stage 4 cells into Stage 5 cells by culturing the Stage 4 cells in a medium supplemented with SANT‑1, a PKC activator, a BMP inhibitor, and ascorbic acid. 72 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 48. The method of item 47, wherein the medium is further supplemented one or more of T3, T4 or analogue thereof. 49. The method of item 40, wherein the method comprises culturing the Stage 5 cells into immature beta-cells by culturing the Stage 5 cells in a medium supplemented with a BMP inhibitor, ascorbic acid, one or more of T3, T4 or analogue thereof. 50. Themethodof item49,wherein themedium is further supplementedwith anALK5 inhibitor or a gammasecretase inhibitor. 51. Themethod of item 27, wherein themethod comprises differentiating the immature beta-cells into functional beta cells expressing PDX1, NKX6.1, MAFA, UCN3 and SLC2A by culturing immature beta-cells in a medium lacking an ALK5 inhibitorandsupplementedwithZM447439,AZT,N-acetyl cysteine,DEZA,Formulation I andoneormoreofT3, T4 or analogue thereof. 52. The method of items 14, 25 and 28, wherein the glucose-dependent mitochondrial respiration has an oxygen consumption rate response following glucose stimulation in the range of about 20% to about 80% over basal oxygen consumption rate. 53. The method of item 52, wherein the oxygen consumption rate response occurred at least 15 minutes following glucose stimulation. 54. Themethod of items 14, 25 and 28, wherein the glucose-stimulated insulin secretion comprises a rapid, bi-phase insulin secretion in response to glucose stimulation. 55. Themethod of item54,wherein a first phase of the bi-phasic insulin secretion has at least a four-fold increase to at least an eight-fold increase over baseline secretion, and a second phase has at least a two-fold increase to at least a four-fold increase over the baseline secretion. 56. Themethod of item 54 , wherein the insulin secretion occurs at least fiveminutes to at least ten minutes following the glucose stimulation. 57.Themethodof item51,wherein the functional beta cells haveglucose-dependentmitochondrial respirationsimilar to human islet cells. 58. The method of item 51, wherein the functional beta cells have glucose-stimulated insulin secretion similar to islet cells. 59.Themethodof item57,wherein theglucose-dependentmitochondrial respirationhasanoxygenconsumption rate response following glucose stimulation in the range of about 20% to about 80% over basal oxygen consumption rate. 60. The method of item 59, wherein the oxygen consumption rate response occurred at least 15 minutes following glucose stimulation. 61. The method of item 58, wherein the glucose-stimulated insulin secretion comprises a rapid, bi-phasic insulin secretion in response to glucose stimulation. 62. Themethod of item61,wherein a first phase of the bi-phasic insulin secretion has at least a four-fold increase to at least an eight-fold increase over baseline secretion, and a second phase has at least a two-fold increase to at least a four-fold increase over the baseline secretion. 63. The method of item 61, wherein the insulin secretion occurs at least five minutes to at least ten minutes following the glucose stimulation. 64. The method of item 40, wherein the Stage 4 cells are cryopreserved. 65. The method of item 40, wherein step e comprises culturing Stage 4 cryopreserved cells. 66. The method of items 6, 21, 35 and 51 wherein the T3 is in the range of 1 nM to 100 nM. 73 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 67. Themethodof items48 to 50,wherein themedium is supplementedwithT3, andwherein theT3 is in the rangeof 1 nM to 1 µM. 68. A method of differentiating pluripotent stem cells into functional beta cells expressing PDX1, NKX6.1, MAFA, UCN3 and SLC2A: a. Differentiating pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm obtained by culturing pluripotent stem cells in a medium supplemented with activin A and WNT3A; b. Differentiating the cells expressingmarkers characteristic of the definitive endoderm into immature beta-cells; and c. Differentiating the immature beta-cells into functional beta-cells expressing PDX1, NKX6.1,MAFA, UCN3 and SLC2A1 by culturing immature beta-cells in a medium supplemented with one or more of UNC0638, UNC0642, UCN0646, TC-E5003, A366, PF03814735, ZM447439, SB747651A, PFI1, LY303511, MS436, AZT, DEZA, pyroxamide, C19994 or MC1568. 69.Themethodof item68,wherein the functional beta cells haveglucose-dependentmitochondrial respirationsimilar to human islet cells. 70. The method of item 68, wherein the functional beta cells have glucose-stimulated insulin secretion similar to human islet cells. 71. The method of item 68, wherein the functional beta cells secrete insulin in multiple phases. 72. The method of item 68, wherein the culture medium is further supplemented with heparin, N‑ Acetyl Cysteine, Formulation I and one or more of T3, T4 or analogue thereof. 73. The method of item 68 or 72, wherein the culture medium lacks an ALK5 inhibitor. 74. The method of item 68 or 72, wherein the culture medium is supplemented with T3. 75. Themethodof item68,wherein themedium is supplementedwithZM447439, heparin,N-AcetylCysteineandone or more of T3, T4 or analogue thereof and wherein the medium contains no ALK5 inhibitor. 76. The method of item 75, wherein the medium is supplemented with T3. 77. The method of item 76, wherein the medium is supplemented with AZT. 78. The method of item 77, wherein the medium is supplemented with DEZA. 79. Themethod of item 68, wherein themethod comprises culturing the immature beta cells at the air-liquid interface, in suspension clusters, in roller bottles or on microcarriers. 80. The method of item 68, wherein the method comprises culturing the immature beta cells in roller bottles. 81. The method of item 80, wherein the method comprises culturing the immature beta cells in roller bottles on microcarriers. 82. The method of item 68, wherein the step of differentiating pluripotent stem cells comprises: a. Differentiating the pluripotent stemcells into cells expressingmarkers characteristic of the definitive endoderm ("Stage 1 cells") by culturing the pluripotent stem cells in a medium supplemented with activin A and WNT3A; b. Differentiating the Stage 1 cells into cells expressingmarkers characteristic of primitive gut tube cells ("Stage 2 cells"); c.Differentiating theStage2cells into cells expressingmarkers characteristic of foregut endodermcells ("Stage3 74 EP 4 691 479 A2 5 10 15 20 25 30 35 40 45 50 55 cells"); d. Differentiating the Stage 3 cells into cells expressing markers characteristic of pancreatic endoderm cells ("Stage 4 cells"); e. Differentiating the Stage 4 cells into pancreatic endocrine precursor cells ("Stage 5"); and f. Differentiating the Stage 5 cells into the immature beta-cells. 83. Themethodof item82,wherein steps e. and f. comprise culturing at the air-liquid interface, in suspension clusters, in roller bottles or on micocarriers. 84. The method of item 82, wherein the steps e. and f. comprise culturing the cells in roller bottles. 85. The method of item 82, wherein the steps e. and f. comprise culturing the immature beta cells in roller bottles on microcarriers. 86. The method of item 82, wherein the method comprises differentiating the Stage 1 cells into Stage 2 cells by culturing the Stage 1 cells in a medium supplemented with FGF7 and ascorbic acid. 87. The method of item 82, wherein the method comprises differentiating the Stage 2 cells into Stage 3 cells by culturing theStage 2 cells in amediumsupplementedwith FGF7, retinoic acid, SANT‑1, aPKCactivator and ascorbic acid. 88. The method of item 87, wherein the medium lacks a BMP inhibitor. 89. The method of item 82, wherein the method comprises differentiating the Stage 3 cells into Stage 4 cells by culturing theStage 3 cells in amediumsupplementedwith FGF7, retinoic acid, SANT‑1, aPKCactivator and ascor...

Claims

1. A cell culture comprising immature beta cells and a medium supplemented with a G9a and GLP histone lysine methyltransferase inhibitor, wherein said immature beta cells express insulin and at least one of the following transcription factors: PDX1, NKX2.2, NKX6.1, NeuroD1, ISL1, HNF3ß, HB9, MAFA, and PAX6.

2. The cell culture of claim 1, wherein the immature beta cells are pancreatic endocrine cells that do not display glucose-dependent mitochondrial respiration or activity, and the first phase and second phase of insulin secretion (biphasic GSIS).

3. The cell culture of claim 1 or claim 2, further comprising functional beta cells expressing PDX1, NKX6.1, MAFA, UCN3 and SLC2A1.

4. The cell culture of any one of claims 1-3, wherein the medium is further supplemented with an ALK5 inhibitor.

5. The cell culture of any one of claims 1-4, wherein the immature beta cells are obtained by step-wise differentiation of definitive endoderm cells, primitive gut tube cells, foregut endoderm cells, pancreatic endoderm cells, or pancreatic endocrine precursor cells.

6. The cell culture of any one of claims 1-5, further comprising functional beta cells expressing PDX1, NKX6.1, MAFA, UCN3 and SLC2A1, wherein said functional beta cells are produced by a method comprising: a) differentiating pluripotent stem cells into definitive endoderm cells; b) differentiating the definitive endoderm cells of step a) into primitive gut tube cells; c) differentiating the primitive gut tube cells of step b) into foregut endoderm cells; d) differentiating the foregut endoderm cells of step c) into pancreatic endoderm cells; e) differentiating the pancreatic endoderm cells of step d) into pancreatic endocrine precursor cells; f) differentiating the pancreatic endocrine precursor cells of step e) into immature beta cells; and g) culturing the immature beta-cells of step f) in a medium supplemented with a G9a and GLP histone lysine methyltransferase inhibitor to mature the immature beta cells into the functional beta cells expressing PDX1, NKX6.1, MAFA, UCN3 and SLC2A1, preferably, wherein said functional beta cells are pancreatic endocrine cells that are capable of rapid and regulated glucose-stimulated insulin secretion.

7. The cell culture of any one of claims 3-6, wherein i) the functional beta cells exhibit glucose-dependent mitochondrial respiration; ii) the functional beta cells exhibit glucose-stimulated insulin secretion; and / or iii) the functional beta cells secrete insulin in multiple phases.

8. The cell culture of any one of claims 1-7, wherein the medium is further supplemented with one or more of: i) heparin; ii) N-Acetyl Cysteine; and iii) one or more of T3, T4 and an analogue thereof.

9. The cell culture any one of claims 1-3 or 5-8, wherein the medium lacks an ALK5 inhibitor.

10. The cell culture of any one of claims 6-9, wherein the method comprises: i) culturing the immature beta cells at the air-liquid interface; or ii) culturing the immature beta cells in suspension clusters.

11. The cell culture of any one of claims 6-10, wherein i) step a) comprises differentiating the pluripotent stem cells into the definitive endoderm cells by culturing the pluripotent stem cells in a medium supplemented with MCX compound and GDF-8; ii) step b) comprises differentiating the definitive endoderm cells into the primitive gut tube cells by culturing the definitive endoderm cells in a medium supplemented with FGF7 and ascorbic acid; iii) step c) comprises differentiating the primitive gut tube cells into the foregut endoderm cells by culturing the primitive gut tube cells in a medium supplemented with FGF7, retinoic acid, SANT-1, a PKC activator, a BMP inhibitor and ascorbic acid; iv) step d) comprises differentiating the foregut endoderm cells into the pancreatic endoderm cells by culturing the foregut endoderm cells in a medium supplemented with FGF7, retinoic acid, SANT-1, a PKC activator, a BMP inhibitor and ascorbic acid; v) step e) comprises differentiating the pancreatic endoderm cells into pancreatic endocrine precursor cells by culturing the pancreatic endoderm cells in a medium supplemented with SANT-1, a PKC activator, a BMP inhibitor, and ascorbic acid, optionally, wherein the medium is further supplemented with one or more of T3, T4 and an analogue thereof, optionally wherein the medium is supplemented 1 nM to 1 µM T3; vi) step f) comprises culturing the pancreatic endocrine precursor cells into immature beta cells by culturing the pancreatic endocrine precursor cells in a medium supplemented with a i) a BMP inhibitor, ii) ascorbic acid, and iii) one or more of T3, T4 and an analogue thereof, optionally, wherein the medium is further supplemented with an ALK 5 inhibitor or a gamma secretase inhibitor; and / or vii) step g) comprises culturing immature beta cells in a medium lacking an ALK5 inhibitor and supplemented with a G9a histone lysine methyltransferase inhibitor, Formulation I and one or more of T3, T4 and an analogue thereof, optionally, wherein the functional beta cells exhibit glucose-dependent mitochondrial respiration, and / or optionally, wherein the mature beta cells exhibit glucose-stimulated insulin secretion.

12. The cell culture of claim 11, wherein the glucose-dependent mitochondrial respiration has an oxygen consumption rate response following glucose stimulation in the range of about 20% to about 80% over basal oxygen consumption rate.

13. The cell culture of claim 12, wherein the oxygen consumption rate response occurred at least 15 minutes following glucose stimulation.

14. The cell culture of claim 11, wherein the glucose-stimulated insulin secretion comprises a bi-phasic insulin secretion in response to glucose stimulation.

15. The cell culture of claim 14, wherein a first phase of the bi-phasic insulin secretion has at least a four-fold increase to at least an eight-fold increase over baseline secretion, and a second phase has at least a two-fold increase to at least a four-fold increase over the baseline secretion.

16. The cell culture of claim 15, wherein the insulin secretion occurs at least five minutes to at least ten minutes following the glucose stimulation.

17. The cell culture of any one of claims 1 to 5 and 7 to 9, wherein the medium comprised in said cell culture comprising immature beta cells is supplemented with one or more of TC-E5003, PF03814735, ZM447439, SB747651A, PFI1, LY303511, MS436, AZT, DEZA, pyroxamide, CI994 and MC1568.

18. The cell culture of any one of claims 1 to 5 and 7 to 9, wherein said supplemented G9a and GLP histone lysine methyltransferase inhibitor in the medium comprised in said cell culture comprising immature beta cells is one or more of UNC0638, UNC0642, UNC0646, and A366.

Images