Methods for stem cell differentiation

Abstract

The present invention relates to a method for accelerated and cost-effective generation of insulin-producing beta cells from pluripotent stem cells using a fully defined, small molecule-based differentiation protocol. Further, the present invention relates to a method for obtaining pancreatic endoderm cells from posterior foregut cells. The use of the cells obtained with the method according to the invention is described as well.

Description

[0001] DESCRIPTION TITLE: METHODS FOR STEM CELL DIFFERENTIATION

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a method for accelerated and cost-effective generation of insulinproducing beta cells from pluripotent stem cells using a fully defined, small molecule-based differentiation protocol. Further, the present invention relates to a method for obtaining pancreatic endoderm cells from posterior foregut cells. The use of the cells obtained with the method according to the invention is described as well.

[0004] TECHNICAL BACKGROUND

[0005] Diabetes mellitus (DM) stands as a major global health challenge, characterized by chronic hyperglycemia due to insulin insufficiency or resistance. As a metabolic disorder, DM significantly impacts the quality of life, escalates healthcare costs, and increases mortality risk. The condition is linked to detrimental effects on various organs, including blood vessels, eyes, kidneys, heart, and nerves. Alarmingly, statistical data from 2019 estimated the global prevalence of diabetes at 463 million individuals, a number projected to rise to 578 million by 2030 and 700 million by 2045. The incidence of undiagnosed diabetes and impaired glucose tolerance further compounds the concern, emphasizing the urgent need for effective management strategies.

[0006] Type 1 diabetes (T1 D), characterized by autoimmune destruction of pancreatic beta cells, necessitates life-long administration of exogenous insulin. Current treatments, including islet transplantation, face challenges like donor shortages and immunosuppression, leading researchers to seek alternative approaches. This backdrop underscores the need for novel therapies that can overcome the limitations of lifelong insulin dependence and transplant-related complications.

[0007] Recent advancements in stem cell technology have spotlighted the potential of human pluripotent stem cells (hPSCs), including human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs), in generating pancreatic beta-like cells. This approach, which involves sophisticated techniques of differentiation, reprogramming, and genomic alterations, holds promise for producing functional insulin-secreting cells. Despite progress, challenges remain, particularly in achieving mature, functional beta-like cells capable of responding adequately to glucose challenges. Hence, there is a need for a quick, reliable, affordable, and well-defined method to differentiate PSCs into insulin-producing beta cells.

[0008] SUMMARY OF THE INVENTION

[0009] The invention is defined as follows.

[0010] Pluripotent stem cells obtained by a method which involved the destruction of a human embryo do not form part of the presently claimed invention. Posterior foregut cells obtained by a method which involved destruction of a human embryo do not form part of the presently claimed invention.

[0011] According to an embodiment, the invention concerns a method for differentiating pluripotent stem cells, the method comprising the steps of: a) culturing the pluripotent stem cells in a first culture medium comprising at least one GSK-3 inhibitor in an effective amount for a first period of time in order to obtain definitive endoderm cells, b) culturing the cells obtained in step a) in a second culture medium comprising i. at least one BMP type I receptor inhibitor mimicking the effect of growth factor noggin, ii. at least one retinoic acid receptor agonist, and iii. BRD7552 in an effective amount for a second period of time in order to obtain posterior foregut cells, c) culturing the cells obtained in step b) in a third culture medium comprising i. at least one PARP-1 inhibitor, ii. forskolin, iii. at least one anti-inflammatory glucocorticoid, and iv. at least one of ALK5 inhibitor II and / or BRD7552 in an effective amount for a third period of time in order to obtain pancreatic endoderm cells; wherein the pluripotent stem cells are not obtained by a method which involved the destruction of a human embryo and wherein the pluripotent stem cells are human inducible pluripotent stem cells.

[0012] According to an embodiment, the invention concerns a method for obtaining pancreatic endoderm cells, the method comprising the step c) of culturing posterior foregut cells in a culture medium comprising i. at least one PARP-1 inhibitor, ii. forskolin, iii. at least one anti-inflammatory glucocorticoid, iv. and / or at least one of ALK5 inhibitor II and BRD7552 in an effective amount for a period of time in order to obtain pancreatic endoderm cells; wherein the posterior foregut cells are not obtained by a method which involved destruction of a human embryo and wherein the posterior foregut cells are obtained from human inducible pluripotent stem cells.

[0013] According to an embodiment, the invention concerns a method further comprising step d), wherein step d) comprises culturing the cells obtained in step c) in a fourth culture medium comprising at least one aurora kinase inhibitor, RSKi2, and BRD7552 in an effective amount for a fourth period of time in order to obtain pancreatic endocrine cells.

[0014] According to an embodiment, the invention concerns a method wherein the cells in at least one of steps a) to d) are cultured in a 2D or 3D configuration culture system.

[0015] According to an embodiment, the invention concerns a method wherein the cells in steps a) to d) are cultured in a 3D configuration culture system.

[0016] According to an embodiment, the invention concerns a method wherein i. the definitive endoderm cells are obtained within 72 hours, and / or ii. wherein the posterior foregut cells are obtained within 144 hours, and / or iii. wherein the pancreatic endoderm cells are obtained within 240 hours, and / or iv. wherein the pancreatic endocrine cells are obtained within 408 hours after starting step a).

[0017] According to an embodiment, the invention concerns a method wherein the at least one GSK-3 inhibitor in step a) is selected from the group consisting of CHIR 99021, 3F8, BIO, and CHIR 98014.

[0018] According to an embodiment, the invention concerns a method in step a) the first culture medium additionally comprises IDE-1 and / or IDE-2.

[0019] According to an embodiment, the invention concerns a method wherein in step a) the IDE-1 and / or IDE-2 are added at the same time as the GSK-3 inhibitor and / or at a later stage within the first period of time. According to an embodiment, the invention concerns a method wherein the at least one BMP type I receptor inhibitor mimicking the effect of growth factor noggin is a combination of LDN193189 and dorsomorphin.

[0020] According to an embodiment, the invention concerns a method wherein the at least one retinoic acid receptor agonist in step b) is selected from the group consisting of retinoic acid, TTNPB and AM 580.

[0021] According to an embodiment, the invention concerns a method wherein the second culture medium additionally comprises a TGF-beta receptor inhibitor, preferably wherein the TGF-beta receptor inhibitor is selected from the group consisting of SB 431542, A 83-01 , SB 505124, SB 525334, and RepSox.

[0022] According to an embodiment, the invention concerns a method wherein the at least one PARP- 1 inhibitor in step c) is nicotinamide.

[0023] According to an embodiment, the invention concerns a method wherein the at least one antiinflammatory glucocorticoid in step c) is selected from the group consisting of dexamethasone, and prednisolone.

[0024] According to an embodiment, the invention concerns a method wherein the third culture medium additionally comprises a TGF-beta Rl kinase inhibitor, preferably wherein the TGF-beta Rl kinase inhibitor is selected from the group consisting of ALK5 inhibitor II, ALK5 inhibitor GW788388, and ALK5 inhibitor SB525334.

[0025] According to an embodiment, the invention concerns a method wherein the at least one aurora kinase inhibitor in step d) is selected from the list consisting of ZM 447439, SP-96, Aurora kinase inhibitor 2, AT9283, and JNJ-7706621.

[0026] According to an embodiment, the invention concerns a method wherein i. the first culture medium in step a) comprises CHIR 99021 in an effective amount to obtain definitive endoderm cells, and / or wherein ii. the second culture medium in step b) comprises dorsomorphin, LDN193189, retinoic acid, SB431542, and BRD7552 in an effective amount to obtain posterior foregut cells, and / or wherein iii. the third culture medium in step c) comprises nicotinamide, forskolin, ALK5 inhibitor II, dexamethasone and BRD7552 in an effective amount to obtain pancreatic endoderm cells, and / or wherein iv. the fourth culture medium in step d) comprises ZM 447439, BRD7552, and RSKi2 in an effective amount to obtain pancreatic endocrine cells.

[0027] According to an embodiment, the invention concerns a method wherein i. the first period of time is 24 to 48 hours, and / or wherein ii. the second period of time is 48 to 72 hours, and / or wherein iii. the third period of time is 48 to 96 hours, and / or wherein iv. the fourth period of time is 144 to 240 hours.

[0028] According to an embodiment, the invention concerns a method for treating diabetes in a subject in need thereof, wherein the method comprises administering to the subject pancreatic endocrine cells that have been obtained by a method described above.

[0029] According to an embodiment, the invention concerns a method wherein a therapeutically effective amount of cells is administered to the subject.

[0030] According to an embodiment, the invention concerns a method wherein administration is by transplantation of the cells to the desired location.

[0031] According to a first aspect, a method for differentiating pluripotent stem cells is provided, the method comprising the steps of: a) culturing the pluripotent stem cells in a first culture medium comprising at least one GSK-3 inhibitor in an effective amount for a first period of time in order to obtain definitive endoderm cells, b) culturing the cells obtained in step a) in a second culture medium comprising i. at least one BMP type I receptor inhibitor mimicking the effect of growth factor noggin, ii. at least one retinoic acid receptor agonist, and iii. BRD7552 in an effective amount for a second period of time in order to obtain posterior foregut cells, c) culturing the cells obtained in step b) in a third culture medium comprising i. at least one PARP-1 inhibitor, ii. forskolin, iii. at least one anti-inflammatory glucocorticoid, and iv. at least one of ALK5 inhibitor II and / or BRD7552 in an effective amount for a third period of time in order to obtain pancreatic endoderm cells; wherein the pluripotent stem cells are not obtained by a method which involved the destruction of a human embryo and wherein the pluripotent stem cells are human inducible pluripotent stem cells.

[0032] According to a second aspect, a method for obtaining pancreatic endoderm cells is provided, the method comprising the step c) of culturing posterior foregut cells in a culture medium comprising i. at least one PARP-1 inhibitor, ii. forskolin, iii. at least one anti-inflammatory glucocorticoid, iv. and / or at least one of ALK5 inhibitor II and BRD7552 in an effective amount for a period of time in order to obtain pancreatic endoderm cells; wherein the posterior foregut cells are not obtained by a method which involved the destruction of a human embryo and wherein the posterior foregut cells are obtained from human inducible pluripotent stem cells.

[0033] According to another aspect, a method for obtaining pancreatic endoderm cells is provided, the method comprising the step c) of culturing posterior foregut cells in a culture medium comprising i. at least one PARP-1 inhibitor, ii. forskolin, iii. at least one anti-inflammatory glucocorticoid, iv. at least one of ALK5 inhibitor II and / or BRD7552 in an effective amount for a period of time in order to obtain pancreatic endoderm cells.

[0034] Suitably, the methods may further comprise step d), wherein step d) comprises culturing the cells obtained in step c) in a fourth culture medium comprising at least one aurora kinase inhibitor, RSKi2, and BRD7552 in an effective amount for a fourth period of time in order to obtain pancreatic endocrine cells.

[0035] Suitably, the cells in at least one of steps a) to c) may be cultured in a 2D or 3D configuration culture system.

[0036] Suitably, the cells in at least one of steps a) to d) may be cultured in a 2D or 3D configuration culture system.

[0037] Suitably, the cells in steps a) to d) may be cultured in a 3D configuration culture system. Suitably, i. the definitive endoderm cells may be obtained within 72 hours, and / or ii. wherein the posterior foregut cells may be obtained within 144 hours, and / or iii. wherein the pancreatic endoderm cells may be obtained within 240 hours, and / or iv. wherein the pancreatic endocrine cells may be obtained within 408 hours after starting step a).

[0038] Suitably, the at least one GSK-3 inhibitor in step a) may be selected from the group consisting of CHIR 99021, 3F8, BIO, and CHIR 98014.

[0039] Suitably, in step a) the first culture medium may additionally comprise IDE-1 and / or IDE-2.

[0040] Suitably, in step a) the IDE-1 and / or IDE-2 may be added at the same time as the GSK-3 inhibitor and / or at a later stage within the first period of time.

[0041] Suitably, the at least one BMP type I receptor inhibitor mimicking the effect of growth factor noggin may be a combination of LDN193189 and dorsomorphin.

[0042] Suitably, the at least one retinoic acid receptor agonist in step b) may be selected from the group consisting of retinoic acid, TTNPB and AM 580.

[0043] Suitably, the second culture medium may additionally comprise a TGF-beta receptor inhibitor, preferably wherein the TGF-beta receptor inhibitor is selected from the group consisting of SB 431542, A 83-01, SB 505124, SB 525334, and RepSox.

[0044] Suitably, the at least one PARP-1 inhibitor in step c) may be nicotinamide.

[0045] Suitably, the at least one anti-inflammatory glucocorticoid in step c) may be selected from the group consisting of dexamethasone, and prednisolone.

[0046] Suitably, the third culture medium may additionally comprise a TGF-beta Rl kinase inhibitor, preferably wherein the TGF-beta Rl kinase inhibitor may be selected from the group consisting of ALK5 inhibitor II, ALK5 inhibitor GW788388, and ALK5 inhibitor SB525334.

[0047] Suitably, the at least one aurora kinase inhibitor in step d) may be selected from the list consisting of ZM 447439, SP-96, Aurora kinase inhibitor 2, AT9283, and JNJ-7706621. Suitably, i. the first culture medium in step a) may comprise CHIR 99021 in an effective amount to obtain definitive endoderm cells, and / or wherein ii. the second culture medium in step b) may comprise dorsomorphin, LDN193189, retinoic acid, SB431542, and BRD7552 in an effective amount to obtain posterior foregut cells, and / or wherein iii. the third culture medium in step c) may comprise nicotinamide, forskolin, ALK5 inhibitor II, dexamethasone and BRD7552 in an effective amount to obtain pancreatic endoderm cells, and / or wherein iv. the fourth culture medium in step d) may comprise ZM 447439, BRD7552, and RSKi2 in an effective amount to obtain pancreatic endocrine cells.

[0048] Suitably, i. the first period of time may be 24 to 48 hours, and / or wherein ii. the second period of time may be 48 to 72 hours, and / or wherein iii. the third period of time may be 48 to 96 hours, and / or wherein iv. the fourth period of time may be 144 to 240 hours.

[0049] According to a further aspect a method for treating diabetes in a subject in need thereof is provided, wherein the method comprises administering to the subject pancreatic endocrine cells that have been obtained by the described method according to invention.

[0050] Suitably, a therapeutically effective amount of cells may be administered to the subject.

[0051] Suitably, administration may be by transplantation of the cells to the desired location.

[0052] Any references to methods of treatments may also refer to (i) compounds, pharmaceutical compositions and / or medicaments involving the endocrine cells of the invention and / or (ii) the pancreatic endocrine cells of the present invention, for use in a method for treatment of the human (or animal) body by therapy (or for diagnosis) as disclosed herein. Accordingly, any reference to methods of treatment may be references to the corresponding second medical uses and / or the use of the (i) compounds, pharmaceutical compositions and / or medicaments involving the endocrine cells of the present invention, and / or (ii) pancreatic endocrine cells of the present invention, for the manufacture of a medicament for the treatment of any diseases disclosed herein. It will be appreciated that, except for where the context requires otherwise, the considerations set out in this disclosure should be considered to be applicable to all aspects of the invention.

[0053] Various aspects of the invention are described in further detail below.

[0054] BRIEF DESCRIPTION OF THE FIGURES

[0055] HUES8 cells are disclosed herein as reference examples.

[0056] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0057] Figure 1 gives an overview of directed differentiation of pluripotent stem cells using a small molecule-based protocol. (A) Schematic representation of the four-stage process for directed differentiation of pluripotent stem cells. The protocol spans a duration of 15 days and involves the sequential administration of 15 small molecules. (B) Phase contrast images at different stages of the directed differentiation process. Each image corresponds to a specific time point during the 15-day protocol. These images allow for the visualization of morphological changes occurring throughout the differentiation process.

[0058] Figure 2 shows the characterization of stage 1. (A) Diagram of directed differentiation during stage 1 : definitive endoderm. (B) Small molecule screen for definitive endoderm induction. iPSC were treated for 2 days with stage 1 media containing different concentration of CHIR, as shown in A. On day 2, quantitative analysis was performed by flow cytometry used to examine the expression endodermal and pluripotency markers including SOX17,c-kit, CXCR4, OCT4 and double staining for c-kit / CXCR4, n=3. (C) samples were taken over the course of 5 days to examine the gene expression of OCT4, SOX17 and FOXA2 by qRT-PCR analysis. The Y-axis represents mRNA normalized to Actin expression. (D) Samples taken one day later on day 3 show a higher percentage of positive cells for definitive endoderm markers and lower percentage of cells positive to pluripotency markers, n=8. The error bars show the standard deviation. CHIR99021.

[0059] Figure 3 shows optimization of stage 2 and 3. (A) Diagram of directed differentiation during stage 2-3: pancreatic foregut / progenitors. (B) PSC derived definitive endoderm spheroids were treated + / - BRD7552 at S2 to examine the effect of addition of PDX1 inducer. Several markers for primitive gut tube HNF4a, HNF1 b, posterior foregut GATA4 ,GATA6 , HNF6 and PDX1 were tested by qRT-PCR analysis, n=2-4. (C). The addition of BRD7552 was further tested at S3 where gene expression during stage 3 (day 5-8) of markers specific for pancreatic progenitors / endoderm including PDX1 , NGN3, PTF1A and CHRGA is shown. The Y-axis represents mRNA normalized to Actin. The error bars show the standard deviation.

[0060] Figure 4 shows PSC cells differentiate into islet-like spheroids at day 15. End stage in vitro characterization. (A) Diagram of directed differentiation during stage 4: beta cells. (B) Quantitative analysis were performed for end stage markers including INS, GOG, NKX6.1 and PDX1 , n=3-5. (C). The S4 SC-b spheroids were subjected to staining with Dithizone, insulin containing cells stain in red. (D) immunofluorescent staining to visualize the presence of c-peptide (green) and glucagon (red) in stage 4 differentiated human pluripotent stem cells, nucleus was stained with DAPI (blue). (E) SC-speroids at S4 are shown in electron micrographs containing granules. (F) The addition of BRD7552 was further tested at S4 where gene expression during stage 4 (day 9-15) of Insulin (beta-cells) and glucagon (alpha cells) is shown. The Y-axis represents mRNA normalized to Actin. The error bars show the standard deviation, n=4-6.

[0061] Figure 5 shows both embryonic and induced stem cells differentiate into islet-like spheroids at day 15. End stage in vitro characterization. (A) shows the gene expression in both embryonic (HUES8) and induced pluripotent stem cells (iPSC L1) of end stage markers at day 15. This includes both differentiation (INS,GCG,NEUROD1 ,NKX2.2 and MAFA) and maturation markers (GLUT1 ,IAPP,GLP1 R, ERRy and ABCC8). The Y-axis represents mRNA normalized to Actin. The error bars show the standard deviation, n=4. (B) Electron micrographs of granules from SC- beta cells from embryonic stem cell line (HUES8) and induced pluripotent stem cells (IPSCL1) differentiated with the small molecule protocol at S4, day 15.

[0062] Figure 6 shows in vitro and in vivo characterization functional assays. (A) Glucose stimulated c- peptide secretion of S4 derived iPSC L1 and HUES8 spheroids, n=3. (B) C-peptide content compared between HUES8 and iPSCLI S4 spheroids, n=3. (C) Stimulation index compared between S4 SC-b spheroids and human islet samples, n=3-7 (D) Change in OCR in response to 16.8 mM glucose (G17), oligomycin (Olig.) (2 M), FCCP (2 pM) and rotenone (Rot.) (1 pM) in S7w3 SC-islets (n = 5) and adult islets (n = 5). (E) Mitochondrial properties of SC-B spheroids compared to human islets n=1. (F) schematic representation of the animal experiments transplanted into mice. (G) Human C-peptide levels were measured after an overnight fast and 60 min following an i.p. glucose bolus at 2 (n = 9), 4 (n = 9) and 8 (n = 5) weeks post-transplant of S4 day 15 cells and in mice engrafted with 2-2, 5xE6 cells under the kidney capsule. (H) immunofluorescence image of stained graft tissue after 12 weeks post-transplantation. The error bars show the standard deviation.

[0063] DETAILED DESCRIPTION OF THE INVENTION Pluripotent stem cells obtained by a method which involved the destruction of a human embryo do not form part of the presently claimed invention. Posterior foregut cells obtained by a method which involved the destruction of a human embryo do not form part of the presently claimed invention.

[0064] The present invention provides a novel, efficient, and cost-effective method for guiding pluripotent stem cells (PSCs) into insulin-producing beta cells, addressing the challenges of traditional approaches. Utilizing small molecules, the method expedites the differentiation process and enhances scalability, potentially revolutionizing diabetes treatment and regenerative medicine. The proposed 3D culture system mimics the pancreatic environment, improving the functionality of the derived beta cells. This study contributes significantly to diabetes research, providing a foundation for a scalable, efficient beta cell production platform with therapeutic potential.

[0065] The invention is based upon the inventors’ surprising finding that when using a certain combination of small molecules at certain time points the differentiation of PSC into insulinproducing beta cells can be accelerated.

[0066] General definitions

[0067] The term “differentiate” as used herein refers to a cellular process in which an unspecialised cell type (such as a pluripotent cell) becomes a partially specialised (multipotent or unipotent stem cells) or terminally specialised (i.e. terminally differentiated) cell.

[0068] “Pluripotent stem cell” (PSC) as used herein refers to stem cells with the ability to form all three of the basic body layers (ectoderm / endoderm / mesoderm) and germ cells. In vivo pluripotent stem cells can potentially become or produce any cell or tissue the body needs to repair itself. The term encompasses induced pluripotent stem cells (iPSCs; made from skin or blood cells by a process called ‘reprogramming’), embryonic PSCs (ePSCs; derived from embryos), nuclear transfer ePSCs (ntePSCs), and parthogenic ePSCs (pePSCs).

[0069] The terms “first culture medium”, “second culture medium”, “third culture medium”, and “fourth culture medium” as used herein refer to the culture medium used in the corresponding “first period of time”, “second period of time”, “third period of time” and “fourth period of time” at the corresponding stage of the differentiation process of the PSC, i.e. at stage 1 (S1), stage 2 (S2), stage 3 (S3), and stage 4 (S4), respectively.

[0070] The “culture medium” as used herein comprises a base medium to which small molecules may be added. The base medium can vary from stage to stage and can vary from day to day even if the stage of the differentiation process is still the same. The term “base medium” as used herein refers to any medium known to a person skilled in the art to be suitable to culture cells (for example PSCs). The base medium can be anima-free. In one example the base medium is anima-free. The base medium itself can be supplemented with various supplements known to a person skilled in the art. Examples for base media with and without supplements are B1-RPMI, B1.2-RPMI with or without at least one of B27 and Glutamax as supplements / additives, B2.1 - DMEM F12 with or without at least one of Glutamax, B27, trace elements B, trace elements A, lipid concentrate, and antibiotics (e.g. penicillin-streptomycin) as supplements / additives.

[0071] The term “stage” as used herein refers to the stages of cell differentiation whereby the various stages are defined based on various (types of) markers. Directed differentiation as disclosed herein was executed in four stages, with media changes every two days, except for the first stage. However, it will be appreciated that media change may be performed as and when it is necessary. The frequency at which media changes may be desirable may depend on factors such as cell confluency and / or volume of media used. Merely by way of example, lower cell confluency may require less frequent media changes. By the same token, a greater volume of media used may allow the media change frequency to be reduced.

[0072] The term “therapeutically effective amount” means an amount of a therapeutic agent or product (e.g., cells) according to the present invention effective in producing the desired therapeutic effect.

[0073] In some cases, the term “beta cells” may be used interchangeably with “beta-like cells” in connection with the invention. In other cases, it may be clear from the context as to if “beta cells” in respect of endogenous / native insulin producing beta cells or if “beta-like cells” in connection with the generation of insulin producing beta-like cells from pluripotent stem cells or other cells is being referenced.

[0074] Detailed media compositions are provided in [Table 1],

[0075] The differentiation of PSCs in accordance with the invention (e.g., to insulin producing beta cells or insulin producing beta-like cells) may include the following stages together with the respective markers characteristic for the stages:

[0076] 1) Stage 0 (SO): at this stage spheroids made up of PSCs (iPSCs or ePSCs) are formed and express markers like OCT4, SOX2, and NANOG; the spheroids form the starting point for the actual differentiation; 2) Stage 1 : at the end of differentiation stage 1 the cells express the markers FOXA2, S0X17, and optionally CXCR4 characterising them as definitive endoderm cells;

[0077] 3) Stage 2: at the end of differentiation stage 2 the cells express the markers HNF4a, HNF1 b and NKX6.1 characterising them as posterior foregut cells;

[0078] 4) Stage 3: at the end of differentiation stage 3 the cells express the markers PDX1 , NKX6.1 , and SOX9 characterising them as pancreatic progenitor cells;

[0079] 5) Stage 4: at the end of differentiation stage 4 the cells express the markers INS, MAFA, NKX2.2, MAFB, and GCG characterising them as pancreatic endocrine cells (e.g., corresponding to insulin producing beta-like cells or insulin producing beta cells).

[0080] Some or all of the stages may be of the same or different lengths. Exemplary lengths of each of the stages are discussed elsewhere herein.

[0081] Suitably, the addition of small molecules to the base medium during each of the stages can be done at any time independently of each other (e.g., all at once at the beginning of a stage or some molecules might be added at the beginning, some after a certain time within that stage, etc).

[0082] The term “small molecule” as used herein refers to a compound which has a low molecular weight (< 1000 daltons), and which may regulate a biological process. Suitably, the compound may be an organic compound. It does not include polynucleotides or (poly)peptides or other genetic material.

[0083] The small molecules are added in an “effective amount” which means that the amount added is sufficient to achieve the desired differentiation to the next stage either by that small molecule alone or in combination with one or more other small molecules. In contrast thereto, a too small amount would not lead to the desired differentiation, and a too high amount would not lead to the differentiation, and might even have a negative impact on cell development.

[0084] As used herein the term “3D configuration culture system” refers to an artificially created environment in which biological cells are permitted to grow or interact with their surroundings in all three dimensions. Unlike “2D configuration culture systems” (e.g. a Petri dish), a 3D cell culture allows cells in vitro to grow in all directions, similar to how they would in vivo. These three- dimensional cultures are usually grown in bioreactors (e.g. in small capsules) in which the cells can grow into spheroids, or 3D cell colonies.

[0085] A method for obtaining pancreatic endoderm cells and / or pancreatic endocrine cells According to one aspect of the invention, a method for differentiating pluripotent stem cells is provided, the method comprising the steps of: a) culturing the pluripotent stem cells in a first culture medium comprising at least one GSK-3 inhibitor in an effective amount for a first period of time in order to obtain definitive endoderm cells, b) culturing the cells obtained in step a) in a second culture medium comprising i. at least one BMP type I receptor inhibitor mimicking the effect of growth factor noggin, ii. at least one retinoic acid receptor agonist, and iii. BRD7552 in an effective amount for a second period of time in order to obtain posterior foregut cells, c) culturing the cells obtained in step b) in a third culture medium comprising i. at least one PARP-1 inhibitor, ii. forskolin, iii. at least one anti-inflammatory glucocorticoid, iv. and at least one ofALK5 inhibitor II and BRD7552 in an effective amount for a third period of time in order to obtain pancreatic endoderm cells.

[0086] According to another aspect of the invention, a method for differentiating pluripotent stem cells is provided, the method comprising the steps of: a) culturing the pluripotent stem cells in a first culture medium comprising at least one GSK-3 inhibitor in an effective amount for a first period of time in order to obtain definitive endoderm cells, b) culturing the cells obtained in step a) in a second culture medium comprising i. at least one BMP type I receptor inhibitor, ii. at least one retinoic acid receptor agonist, and iii. BRD7552 in an effective amount for a second period of time in order to obtain posterior foregut cells, c) culturing the cells obtained in step b) in a third culture medium comprising i. at least one PARP-1 inhibitor, ii. forskolin, iii. at least one anti-inflammatory glucocorticoid, iv. and at least one ofALKk5 inhibitor II and BRD7552 in an effective amount for a third period of time in order to obtain pancreatic endoderm cells.

[0087] According to another aspect of the invention, a method for obtaining pancreatic endoderm cells is provided, the method comprising the step c) of culturing posterior foregut cells in a culture medium comprising i. at least one PARP-1 inhibitor, ii. forskolin, iii. at least one anti-inflammatory glucocorticoid, iv. and at least one of ALK5 inhibitor II and BRD7552 in an effective amount for a period of time in order to obtain pancreatic endoderm cells.

[0088] By this method pancreatic endoderm cells according to S3 can be obtained within 96 hours after starting step c) with posterior foregut cells.

[0089] In other words, in one aspect, the present invention provides a method for obtaining pancreatic endoderm cells, the method comprising culturing posterior foregut cells in a culture medium comprising i. at least one PARP-1 inhibitor, ii. forskolin, iii. at least one anti-inflammatory glucocorticoid, iv. and at least one of ALK5 inhibitor II and BRD7552.

[0090] In one example the methods may further comprise step d), wherein step d) comprises culturing the cells obtained in step c) in a fourth culture medium comprising at least one aurora kinase inhibitor, RSKi2, and BRD7552, in an effective amount for a fourth period of time in order to obtain pancreatic endocrine cells.

[0091] With these methods the invention provides a step wise, completely small molecule-based, differentiation method for the directed differentiation of pluripotent stem cells (PSC) towards stem cell-islet clusters that contain insulin producing beta-like cells. With the method according to the invention it is possible to obtain pancreatic endoderm cells within about 9 days and pancreatic endocrine cells within about 15 days. The methods provided herein therefore provide a mechanism by which it is possible to reduce the time and costs associated with stem cell differentiation into pancreatic endoderm / endocrine cells, thereby facilitating simpler upscaling of these differentiation methods.

[0092] Additionally, the protocol allows a medium change every other day instead of every day. In one example the culture medium is changed every second day.

[0093] The reduced time and the reduced requirement for a medium change decrease the costs of the method.

[0094] Further, by only using already approved small molecules upscaling of the method is facilitated.

[0095] In one example, the cells in at least one of steps a) to c) are cultured in a 2D or 3D configuration culture system.

[0096] In one example, the cells in at least one of steps a) to d) are cultured in a 2D or 3D configuration culture system.

[0097] The 2D culture system involves growing cells on a flat surface, such as tissue culture-treated plastic dishes or plates coated with extracellular matrix (ECM) components like laminin, fibronectin, or collagen. This system allows for easy observation, manipulation, and access to the cells.

[0098] 1. Cells were seeded in feeder-free conditions on ECM coated plates laminin or vitronectin. Cell concentration is 10.000-20.000 cells / cm2 and E8 medium supplemented with Y- 27632 (10 pM) for the first 24h and E8 media only for the remaining period.

[0099] 2. Differentiation is started when cells reach 60-70% confluency (2-4 days).

[0100] In another example the cells in steps a) to c) are cultured in a 3D configuration culture system.

[0101] In another example the cells in steps a) to d) are cultured in a 3D configuration culture system.

[0102] The 3D culture system involves growing cells in a three-dimensional environment, which better mimics the in vivo conditions, allowing cells to interact with each other in a more physiologically relevant manner.

[0103] 1. Cells grown in 2D flasks (80% confluency) are dissociated using accutase, passed through 40um filter and seeded at 0.5 million cells per ml in E8 medium supplemented with Y- 27632 (10 pM) for the first 24h and E8 media only for the next 24h. 2. Differentiation is started after 48h following the described protocol

[0104] The differentiation methods described herein may be performed completely in a dynamically agitated 3D culture without requiring a cell sorting step. This is in contrast with most current methods, which use a combination of 2D / 3D culture.

[0105] Suitably the culturing in step a) is performed for at least about 48 hours, for at least about 60 hours, for at least about 72 hours, for at least about 84 hours, or more. Suitably the culturing in step a) may be performed for at least about 72 hours. Suitably the culturing in step a) may be performed for at least about 48 hours.

[0106] Suitably the culturing in step a) may be performed for about 48 hours, for about 60 hours, for about 72 hours, for about 84 hours, or more. Suitably the culturing in step a) may be performed for about 72 hours. Suitably the culturing in step a) may be performed for about 48 hours.

[0107] Suitably the culturing in step a) may be performed for up to about 48 hours, for up to about 60 hours, for up to about 72 hours, or for up to about 84 hours, or more. Suitably the culturing in step a) may be performed for up to about 72 hours. Suitably the culturing in step a) may be performed for up to about 48 hours.

[0108] Suitably the culturing in step b) may be performed for at least about 48 hours, for at least about 60 hours, for at least about 72 hours, for at least about 84 hours, or more. Suitably the culturing in step b) may be performed for at least about 72 hours. Suitably the culturing in step b) may be performed for at least about 48 hours.

[0109] Suitably the culturing in step b) may be performed for about 48 hours, for about 60 hours, for about 72 hours, for about 84 hours, or more. Suitably the culturing in step b) may be performed for about 72 hours. Suitably the culturing in step b) may be performed for about 48 hours.

[0110] Suitably the culturing in step b) may be performed for up to about 48 hours, for up to about 60 hours, for up to about 72 hours, or for up to about 84 hours, or more. Suitably the culturing in step b) may be performed for up to about 72 hours. Suitably the culturing in step b) may be performed for up to about 48 hours.

[0111] Suitably the culturing in step c) be performed for at least about 48 hours, for at least about 60 hours, for at least about 72 hours, for at least about 84 hours, for at least about 96 hours or more. Suitably the culturing in step b) may be performed for at least about 96 hours. Suitably the culturing in step b) may be performed for at least about 72 hours. Suitably the culturing in step c) may be performed for about 48 hours, for about 60 hours, for about 72 hours, for about 84 hours, for about 96 hours, or more. Suitably the culturing in step c) may be performed for about 96 hours. Suitably the culturing in step c) may be performed for about 72 hours.

[0112] Suitably the culturing in step c) be performed for up to about 48 hours, for up to about 60 hours, for up to about 72 hours, for up to about 84 hours, for up to about 96 hours, or more. Suitably the culturing in step c) may be performed for up to about 72 hours. Suitably the culturing in step c) may be performed for up to about 48 hours.

[0113] Suitably the culturing in step d) may be performed for at least about 96 hours, for at least about 108 hours, for at least about 120 hours, for at least about 132 hours, for at least about 144 hours, for at least about 156, for at least about 168 hours or more. Suitably the culturing in step d) may be performed for at least about 168 hours. Suitably the culturing in step b) may be performed for at least about 120 hours.

[0114] Suitably the culturing in step d) be performed for about 96 hours, for about 108 hours, for about 120 hours, for about 132 hours, for about 144 hours, for about 156, for about 168 hours or more. Suitably the culturing in step d) may be performed for about 168 hours. Suitably the culturing in step b) may be performed for about 120 hours.

[0115] Suitably the culturing in step d) may be performed for up to about 96 hours, for up to about 108 hours, for up to about 120 hours, for up to about 132 hours, for up to about 144 hours, for up to about 156, for up to about 168 hours or more. Suitably the culturing in step d) may be performed up to about 168 hours. Suitably the culturing in step b) may be performed up to about 120 hours.

[0116] In one example i. the definitive endoderm cells are obtained within 72 hours and / or ii. wherein the posterior foregut cells are obtained within 144 hours, and / or iii. wherein the pancreatic endoderm cells are obtained within 240 hours, and / or iv. wherein the pancreatic endocrine cells are obtained within 408 hours after starting step a.

[0117] In one example i. the definitive endoderm cells are obtained within 48 hours, and / or ii. wherein the posterior foregut cells are obtained within 120 hours, and / or iii. wherein the pancreatic endoderm cells are obtained within 216 hours, and / or iv. wherein the pancreatic endocrine cells are obtained within 336 hours after starting step a.

[0118] In one example the pluripotent stem cells are human inducible pluripotent stem cells or human embryonic stem cells.

[0119] In one example the PSCs are cultured in a way to form spheroids before step a) starts. For example, the PSCs may be cultured in E8 Flex medium with Rocki as supplement. Accordingly, the pluripotent stem cells of step a) may be pluripotent stem cell spheroids.

[0120] The methods according to the invention provide an optimized method which may result in an output of pancreatic endoderm cells, and optionally pancreatic endocrine cells (i.e. insulin producing beta-like cells) at a fraction of the price and time spent using conventional methods.

[0121] Step a)

[0122] According to the method of the invention for differentiating pluripotent stem cells, at least one GSK-3 inhibitor (glycogen synthase kinase 3 inhibitor) is added to the base medium on day 0 in order to obtain definitive endoderm cells. Appropriate GSK-3 inhibitors that can be used for the method herein are well known to a person killed in the art. The GSK-3 inhibitor can be selected from the non-limiting list consisting of CHIR 99021 , 3F8, A 1070722, AR-A 014418, AZD 2858, BIO, CHIR 98014, CHIR 99021 trihydrochloride, lndirubin-3'-oxime, Kenpaullone, MeBIO, SB 216763, SB 415286, TC-G 24, TCS 2002, TCS 21311 , TDZD 8, TWS 119.

[0123] In one example the at least one GSK-3 inhibitor in step a) is selected from the group consisting of CHIR 99021 , 3F8, BIO, and CHIR 98014. In another example the at least one GSK-3 inhibitor is CHIR 99021.

[0124] In one example the GSK-3 inhibitor in step a) is CHIR 99021.

[0125] In one example GSK-3 inhibitor is added at the beginning of step a), which also initiates the method according to the invention until S1 is terminated, i.e. when the cells can be characterized as definitive endoderm cells. In this example, GSK-3 inhibitor is present within the first culture medium. The minimum amount of time that the cells need to be cultured in a medium comprising at least one GSK-3 inhibitor is 24 hours. In one example no further small molecule is added in addition to the GSK-3 inhibitor, neither within the first 24 hours nor within the second 24 hours of S1 . This provides some pulse effect to the cells with 24 hours GSK-3 inhibitor followed by 24 hours without any additional small molecule.

[0126] In one example in step a) the first culture medium additionally comprises IDE-1 and / or IDE-2.

[0127] In another example in step a) the IDE-1 and / or IDE-2 are added at the same time as the GSK-3 inhibitor and / or at a later stage within the first period of time.

[0128] In one example the GSK-3 inhibitor is combined with IDE-1 and / or IDE-2. Either IDE1 and / or IDE2 may be added at the same time as the GSK-3 inhibitor at the beginning of step a) followed by a second addition of IDE1 and / or IDE2 after 24 hours or without any addition of IDE1 / IDE2 after 24 hours.

[0129] In one example CHIR 99021 is added at the beginning of stage 1 (at the beginning of step a)) and no other small molecule is added for the next 48 hours.

[0130] In another example CHIR99021 is added at the beginning of stage 1 (at the beginning of step a)) together with IDE1 or IDE2 followed by a second addition of IDE1 or IDE2, respectively, after 24 for hours.

[0131] In the context of the present disclosure, step a) of the method may be referred to as the step of “culturing pluripotent stem cells”.

[0132] Suitable concentration ranges of the small molecules are provided in Table 1.

[0133] Step b)

[0134] According to step b) of the method according to the invention at least one BMP type I receptor inhibitor mimicking the effect of growth factor noggin is added. In one example a combination of at least two BMP type I receptor inhibitors that mimic the effect of growth factor noggin is added.

[0135] In one example a combination of LDN193189 and / or LDN193189 dihydrochloride and dorsomorphin is added, which in combination mimic the effect of growth factor noggin. Other combinations of molecules mimicking the effect of noggin may be used as well, for example DMH2 and K 02288.

[0136] LDN193189 is a potent and selective ALK2 and ALK3 inhibitor and inhibits BMP4-mdiated Smad1 / 5 / 8 activation. Dorsomorphin is an AMP-activated protein kinase (AMPK) and (BMP) type I receptor (ALK2, ALK3, ALK6) inhibitor.

[0137] Other combinations of (BMP) type I receptor inhibitors that may be used are DMH2 with LDN193189, DMH2 with dorsomorphin, DMH2 with K 02288, K 02288 with dorsomorphin, K 02288 with LDN193189. In one example, a combination of LDN193189 and dorsomorphin may be used.

[0138] Further, at least one retinoic acid receptor agonist is added in step b).

[0139] In one example the at least one retinoic acid receptor agonist in step b) is selected from the group consisting of retinoic acid, TTNPB, AM 580, AC 261066, Adapalene, AM 80, BMS 753, BMS 961 , CD1530, CD2314, CD437, Ch55, and DC 271.

[0140] In one example the at least one retinoic acid receptor agonist in step b) is selected from the group consisting of retinoic acid, TTNPB, and AM 580.

[0141] In one example the at least one retinoic acid receptor agonist is retinoic acid.

[0142] Further, in step b) BRD7552 is added as a PDX-1 inducer.

[0143] In one example the second culture medium (step b)) additionally comprises a TGF-beta receptor inhibitor, preferably wherein the TGF-beta receptor inhibitor is selected from the group consisting of SB 431542, A 83-01 , SB 505124, SB 525334, RepSox, A 77-01 , AZ 12799734, D 4476, Disitertide, Galunisertib, GW 788388, IN 1130, LY 2109761 , R 268712, SD 208, and SM 16.

[0144] Suitable concentration ranges of the small molecules are provided in [Table 1], In the context of the present disclosure, step b) of the method may be referred to as the step of “culturing definitive endoderm cells”.

[0145] Step c)

[0146] Step c) comprises the addition of i. at least one PARP-1 inhibitor, ii. forskolin, iii. at least one anti-inflammatory glucocorticoid, iv. and at least one of ALK5 inhibitor II and BRD7552. The at least one PARP-1 inhibitor (poly(ADP-ribose) polymerase 1 inhibitor) promotes differentiation of mesenchymal stem cells to insulin producing cells when used in combination with other growth factors (and high glucose concentration, e.g. 20mM).

[0147] Appropriate PARP-1 inhibitors that can be used for the method herein are well known to a person skilled in the art. A non-limiting list of PARP-1 inhibitors comprises AZD 2461 , BGP 15, EB 47, GeA-69, H10, nicotinamide, Olaparib, OUL 35, PARPi-FL, PARPYnD, PJ 34 hydrochloride, Rucabarib camsylate, Veliparib dihydrochloride.

[0148] In one example the PARP-1 inhibitor is nicotinamide.

[0149] Forskolin or coleonol is a cell-permeable, potent, reversible and rapid activator of adenylyl cyclase, an enzyme that converts ATP to cAMP and pyrophosphate.

[0150] In one example the at least one anti-inflammatory glucocorticoid in step c) is selected from the group consisting of dexamethasone, and prednisolone. In one example, the anti-inflammatory glucocorticoid in step c) is dexamethasone. Alternatively, a person skilled in the art would be aware of suitable anti-inflammatory glucocorticoids that can be used.

[0151] Further, the third culture medium comprises at least one of ALK5 inhibitor II and BRD7552. The third culture medium may comprise both an ALK5 inhibitor II and BRD7552, or just an ALK5 inhibitor II, or just BRD7552; in addition to the other components described herein for the third culture medium.

[0152] ALK5 inhibitor II (ALK5i) is a cell permeable, potent, selective and ATP-competitive inhibitor of TGF-p Rl kinase (IC50=23nM, 4nM and 18nM for binding, auto-phosphorylation, and cellular assay in HepG2 cells of TGF-p Rl kinase, respectively).

[0153] In one example the third culture medium additionally comprises a TGF-beta Rl kinase inhibitor, preferably wherein the TGF-beta Rl kinase inhibitor is selected from the group consisting of ALK5 inhibitor II, ALK5 inhibitor GW788388, and ALK5 inhibitor SB525334.

[0154] Suitable concentration ranges of the small molecules are provided in [Table 1], In the context of the present disclosure, step c) may be referred to as the step of “culturing posterior foregut cells”. It will be clear that the step of culturing posterior foregut cells may be as part of the method for differentiating pluripotent stem cells, or may be as part of the method for obtaining pancreatic endoderm cells. Herein, when reference is made to step c), it will be appreciated that this may be in the context of step c) as defined in the method for differentiating pluripotent stem cells and / or in the context of the step of culturing posterior foregut cells in a method for obtaining pancreatic endoderm cells.

[0155] Step d)

[0156] According to step d) the method may further comprise culturing the cells obtained in step c) in a fourth culture medium comprising at least one aurora kinase inhibitor, RSKi2 (RSK inhibitor II), and BRD7552 in an effective amount for a fourth period of time in order to obtain pancreatic endocrine cells.

[0157] In one example the at least one aurora kinase inhibitor in step d) is selected from the list consisting of ZM 447439, SP-96, Aurora kinase inhibitor 2, AT9283, and JNJ-7706621.

[0158] ZM 447439 is a selective and ATP-competitive inhibitor for Aurora A and Aurora B with IC50 of 110 nM and 130 nM, respectively.

[0159] RSKi2 also referenced under CAS 501437-28-1 , controls the biological activity of RSK.

[0160] In the context of the present disclosure, step d) may be referred to as the step of “culturing posterior foregut cells”.

[0161] Steps a) to d)

[0162] In one example for the method according to the invention i. the first culture medium in step a) comprises CHIR 99021 in an effective amount to obtain definitive endoderm cells, and / or wherein ii. the second culture medium in step b) comprises dorsomorphin, LDN193189, retinoic acid, SB431542, and BRD7552 in an effective amount to obtain posterior foregut cells, and / or wherein iii. the third culture medium in step c) comprises nicotinamide, forskolin, ALK5 inhibitor II, dexamethasone and BRD7552 in an effective amount to obtain pancreatic endoderm cells, and / or wherein iv. the fourth culture medium in step d) comprises ZM 447439, BRD7552, and RSKi2 in an effective amount to obtain pancreatic endocrine cells.

[0163] In one example i. the first period of time is 24 to 48 hours, and / or ii. the second period of time is 48 to 72 hours, and / or 24 iii. the third period of time is 48 to 96 hours, and / or iv. the fourth period of time is 144 to 240 hours.

[0164] The duration of the four time periods might be independent of each other and a stage can be considered as finished once the respective markers are expressed. Accordingly, the duration of each of the four periods may be dictated by the length of time required for the cells to express the relevant markers.

[0165] In one example, the pluripotent stem cells may be cultured in the first culture medium until they express FOXA2, SOX17, and optionally CXCR4 characterising them as definitive endoderm cells. In one example, the definitive endoderm cells may be cultured in the second culture medium until they express HNF4a and HNF6 characterising them as posterior foregut cells.

[0166] In one example, the posterior foregut cells may be cultured in the third culture medium until they express markers PDX1 , NKX6.1 , and S0X9 characterising them as pancreatic endoderm cells.

[0167] In one example, pancreatic endoderm cells may be cultured in the fourth culture medium until they express INS, MAFA, NKX2.2, MAFB, and GCG characterising them as pancreatic endocrine cells. Methods for determining cell markers are well known in the art. Merely by way of example such methods include immunohistochemical analysis or flow cytometry.

[0168] [Table 1] indicates an example of the method according to the present invention: Cells with underlined text are the days where the media may be exchanged.

[0169] Definitions for the cells, media, small molecules, periods of time, differentiation stages etc. might be provided elsewhere herein and equally apply here. Definitions, embodiments, examples etc. herein for one aspect of the invention equally apply to all the other aspects of the invention. Unless it is apparent from the context, each of the embodiments and examples listed herein can be applied for use in any of the aspects of the invention.

[0170] Uses

[0171] According to another aspect of the invention the cells obtained by a method according the invention can be used. In one example the obtained pancreatic endocrine cells are used as transplants.

[0172] In one example the obtained pancreatic endocrine cells are used for the treatment of diabetes by cell therapy, for disease modelling, and / or for drug screening.

[0173] The cell therapy is based on the differentiation of stem cells into pancreatic beta cell precursors that can produce therapeutically relevant levels of insulin as a response to blood glucose. The beta cell precursors may then be subcutaneously implanted in patients using, for example, an encapsulation device. In a further aspect, the present invention provides a method for treating diabetes in a subject in need thereof, wherein the method comprises administering to the subject pancreatic endocrine cells that have been obtained by a method of the invention. Suitably, a therapeutically effective amount of cells may be administered. Suitably, administration may be by transplantation of the cells to the suitable location like the portal vein on the liver or under a muscle tissue.

[0174] Kits

[0175] According to another aspect of the invention a kit for use in the method of the invention is provided.

[0176] According to another aspect of the invention a kit for preparation of the culture mediums according to the method of then invention is provided.

[0177] The kits described herein can take on a variety of forms. Typically, the kits will include reagents suitable for carrying out the methods according to the invention (i.e., the small molecules disclosed herein, like for example CHIR 99021 , LDN193189, retinoic acid SB31542, BRD7552, nicotinamide, forskolin, ALK5i2, dexamethasone, ZM 447439, and RSKi2).

[0178] The kit may comprise instructions for preparing the media and for carrying out the methods according to the inventions.

[0179] Optionally, the kits may contain one or more control samples or references.

[0180] The individual components may be housed in separate containers, e.g. tubes.

[0181] Said kit optionally comprises instructions regarding the use of the contained components.

[0182] The components of the kit may be housed in a container that is suitable for transportation.

[0183] Definitions for the cells, media, small molecules, periods of time, differentiation stages etc. might be provided elsewhere herein and equally apply here. Definitions, embodiments, examples etc herein for one aspect of the invention equally apply for all the other aspects of the invention. Unless it is apparent from the context, each of the embodiments and examples listed herein can be applied for use in any of the aspects of the invention.

[0184] Aspects of the invention are demonstrated by the following non-limiting examples.

[0185] Examples

[0186] HUES8 cells are disclosed herein as reference examples. Materials and Methods

[0187] Human Pluripotent Stem Cell (hPSC) Culture and In Vitro Differentiation

[0188] Human embryonic stem cell lines

[0189] HLIES8, sourced from the Roslin Institute and MTA with Harvard, and the human induced pluripotent stem cell (iPSC) line Lumc-Gmp-ipsc_donor-02 Line 1 , developed by Leiden University Medical Center's GMP facility, were utilized in this study. HUES8 cells were maintained on Biolamina laminin-coated plates (catalog no. LN521) in Essential 8 (E8) media (Thermo Fisher, catalog no. A1517001) and passaged with Versene (Gibco, catalog no. 15040-033). For differentiation experiments, cells at 70-80% confluency were dissociated using Accutase (Stemcell Technologies, catalog no. 07920), seeded in E8 media with 10 pM ROCK inhibitor (Y- 27632; Stemcell Technologies, catalog no. 72304), and cultured in spinner flasks on a stir plate at 37 °C, 5% CO2, and 100% humidity. Directed differentiation was executed in four stages, with media changes every two days, except for the first stage. Detailed media compositions are provided in Table 1.

[0190] Human Islet Procurement and Culture

[0191] Donor islets, extracted from deceased human organ donors, were isolated following Dutch national regulations at our institute's Good Manufacturing Practice facility. Islets with a minimum of 80% purity were cultured in CMRL 1066 medium (5.5 mmol / L glucose) supplemented with 10%vhuman serum, HEPES 100 mmol / L, L-glutamine (2mmol / L), Nicotinamide, Ciproxin (2ug / mL) and Gentamicin (5ug / mL).

[0192] RNA Extraction and Quantitative PCR

[0193] RNA extraction was conducted using the RNeasy Mini kit (Qiagen, catalog no. 74106) with on- column DNase digestion. The synthesis of cDNA involved incubating RNA with Oligo(dT)15 Primer and dNTP MIX (Promega), and quantitative PCR was performed using SYBR-green master mix (BioRad, catalog no. 1708886) on Real Time PCR equipment (Biorad CFX384, C1000 Touch Thermalcycler), employing ACTB as a reference gene. Primer sequences:

[0194] Image Analysis and Immunohistochemistry

[0195] SC-islet samples underwent fixation, embedding, and sectioning for immunohistochemistry. The sections were deparaffinized, autoclave-boiled, blocked, and treated with primary and secondary antibodies. Confocal SP8 WLL and LAS X software (Leica Application Suite X) were used for imaging.

[0196] Flow Cytometry SC-islets were dispersed, fixed, and stained as detailed, then analyzed for stage-specific marker expression using Aurora spectral flow cytometers (Cytek, Aurora 3 laser) and FCS express software.

[0197] Antibodies used:

[0198] Glucose-Induced C-Peptide Secretion

[0199] Static insulin secretion assays involved equilibrating SC-islets in Krebs buffer, followed by glucose and KCI Krebs treatments. C-peptide levels in samples were measured using Human Ultrasensitive C-peptide ELISA (Mercodia) and normalized to DNA content determined by Picogreen test (Thermo Fisher).

[0200] Seahorse Assay

[0201] The Seahorse Bioscience XFe96 Extracellular Flux Analyzer was employed to monitor respiration rates of SC-islet and donor islet. The assay protocol, including medium replacement, OCR measurement, and nutrient / additive application, is described in detail. Protein content standardization was done using the BCA test (Thermo Fisher).

[0202] Transmission Electron Microscopy

[0203] SC-islets were processed for electron microscopy as described, involving fixation, post-fixation, dehydration, embedding, and ultrathin sectioning. Sections were examined using a Fei Tecnai Twin transmission electron microscope, and image analysis was performed with Aperio Imagescope (Leica). Detailed procedures for sample preparation, including immunogold staining and subsequent analysis, are outlined.

[0204] Single-Cell RNA Sequencing Sample Preparation

[0205] SC-islets were prepared for scRNA sequencing by dispersing into single-cell solutions, filtering, and counting. Cells were frozen using Cryostor (Sigma) for subsequent analysis using 10X Genomics 3' V3 chemistry. Sequencing was performed on an Illumina platform.

[0206] Single Cell RNA Sequencing Data Analysis

[0207] Sequencing data were aligned using Cell Ranger count and mapped to the GRCh38 human reference genome. Seurat software (version 3.1.1) was employed for data processing, clustering, visualization, and annotation of cell types. Insulin-expressing cells were identified, subset, and combined for analysis, including comparison with mature beta cells.

[0208] Transplantation Research in Animal Models

[0209] Animal studies were conducted in compliance with European Parliament regulations and approved by the animal welfare committee at Leiden University Medical Center. Immunodeficient NSG-RIP-DTR mice were used, with detailed descriptions of housing, monitoring, surgery, and post-operative care provided. The functional capacity of SC-derived islets was assessed using intraperitoneal glucose tolerance tests at specified intervals post-surgery.

[0210] Dithizone (DTZ) Staining

[0211] DTZ staining was performed on SC-islets using a prepared solution of the zinc-chelating dye, with the procedure involving staining, rinsing, and imaging with an Olympus CKX53 light microscope.

[0212] Data Collection and Statistical Analysis

[0213] Data were statistically analyzed using GraphPad Prism software. Mean and standard deviation values are presented, and the number of independent experiments is denoted by "n."

[0214] Data Accessibility

[0215] Data from single-cell RNA-seq will be deposited in the Gene Expression Omnibus (GEO), and an accession number will be assigned accordingly.

[0216] Results

[0217] Directed Differentiation of Pluripotent Stem Cells via a Small Molecule-Based Protocol

[0218] Small molecules that support the differentiation and maintenance of pancreatic cells have been identified in the past, and remarkable progress has been made in identifying a wide range of chemical candidates. However, a fully small molecule based approach has not been developed so far. The present invention presents a novel approach to the directed differentiation of pluripotent stem cells (PSCs) into a specific lineage, utilizing a meticulously designed protocol that incorporates the sequential administration of small molecules over a 15-day period. This method represents a significant advancement in stem cell research, offering a more controlled and efficient pathway towards differentiation compared to traditional methods. As depicted in Figure 1A, the differentiation protocol is divided into four distinct stages, each characterized by the administration of specific small molecules that are critical for the transition of PSCs through various stages of differentiation. The protocol initiates with the activation of signaling pathways that prime the pluripotent stem cells for differentiation. Subsequent stages involve the stepwise administration of small molecules that guide the cells towards the desired lineage, culminating in the final differentiation stage. Figure 1 B presents a series of phase contrast images that correspond to each stage of the differentiation protocol, providing a visual representation of the morphological changes occurring in 3D cell clusters.

[0219] Stage 1 : Definitive Endoderm (DE) Induction in 3D

[0220] For the induction of DE a small molecule approach was used by omitting Activn A and replacing it with a short (24h) pulse of CHIR99021 (CHIR), a potent Wnt pathway activator, to induce definitive endoderm formation (Figure 2A), as described by Siller et al1. First, a concentration optimization was performed where 3D PSC clusters were treated with 3 or 4 uM CHIR99021 according to Siller et al., 2016. The effectiveness of this induction was quantitatively assessed on day 2 using flow cytometry to examine the expression of key endodermal markers (SOX17, c-kit, CXCR4) and pluripotency markers (OCT4), including a double staining for c-kit / CXCR4. A slightly higher increase in endodermal marker expression was observed, when cell clusters are treated with 4uM of CHIR however this wasn’t significant increase compared to the 3uM treatment. A higher decrease in the pluripotency marker OCT4 (Figure 2B) was observed when clusters are treated with 4uM even though there was still a high expression of OCT4 at day 2. However, the OCT4 expression decreases over time which can be seen both by qPCR and FACS (Figure 2C and 2D).

[0221] To further investigate the dynamics of gene expression during definitive endoderm induction, samples were collected over a 5-day period for quantitative real-time PCR (qRT-PCR) analysis. This analysis focused on the mRNA levels, SOX17, and FOXA2, normalized to Actin expression. The results, as shown in Figure 2D, indicate a progressive increase in SOX17 and FOXA2 expression, markers indicative of definitive endoderm. These findings underscore the effective transition from a pluripotent to a definitive endoderm state over the course of the induction period.

[0222] Stage 2: Pancreatic foregut induction

[0223] It has been reported that treatment with Noggin, SB431542, Retinoic Acid (RA) are necessary for the induction of PDX1+ cells3. However, to have a completely small molecule protocol Noggin was substituted with LDN-193189 and Dorsomorphin which are effective inhibitors of BMP signaling pathways especially BM P type I receptors such as ALK2, ALK3, and ALK6. At this stage, the addition of BRD7552, a PDX1 inducer, was evaluated for its efficacy in enhancing the expression of key markers associated with primitive gut tube (HNF4a, HNF1P) and posterior foregut (GATA4, GATA6, HNF6), alongside PDX1. Quantitative RT-PCR analysis revealed upregulation of only PDX1 in the presence of BRD7552 during stage 2, indicating its pivotal role in directing the differentiation towards a pancreatic lineage (Figure 2B). Stage 3: Pancreatic progenitor / endocrine cells

[0224] During stage 3 a cocktail of 5 chemicals was used to promote the endocrine progenitor differentiation , nicotinamide, dexamethasone, forskolin and ALK5 inhibitor II (Kondo et al., 2017; Li et al., 2023) and additionally BRD7552 was further tested. The addition of BRD7552 was further tested during stage 3 (days 5-8) to evaluate its impact on the expression of markers specific to pancreatic progenitors and endoderm, including PDX1 , NGN3, PTF1A, and CHRGA (Figure 30). The gene expression analysis revealed several key findings as there was a notable increase in PDX1 expression at day 6, indicating the successful induction of pancreatic progenitors. NGN3 expression peaked at day 6 and subsequently decreased, reflecting its transient role in endocrine progenitor specification. Both PTF1A and CHRGA showed a progressive increase in expression over the course of stage 3, suggesting ongoing differentiation towards pancreatic endoderm and the maturation of endocrine cells. While the addition of BRD7552 resulted in slightly increased expression levels of these markers, the differences were not statistically significant (Figure 3C).

[0225] Stage 4: Pancreatic endoderm -Directed Differentiation into Beta Cells

[0226] Figure 4 presents the culmination of the directed differentiation process at stage 4, where PSC- derived cells form islet-like spheroids, characterized by the expression of end-stage markers. During stage 4 cells were treated with a different combination of small molecule cocktails however the addition of BRD7552 clearly shows improvement in the expression of INS and GOG, markers for the beta and alpha cells respectively (Figure 4F). Additional molecules including RSK inhibitor and ZM 44743967were added to aid the maturation of the endocrine cells and improve safety after in vivo transplantation experiments. Quantitative analysis were performed using flow cytometry for end stage markers including INS,GCG,NKX6.1 and PDX1 and what was observed were percentages of differentiated cells similar to growth factor based approaches tested in house (data not shown). Only difference were the double positive cells INS+ / GCG+ which were higher percentage at 20% nevertheless the scientific consensus is currently presumed that these double positive hormonal cells are precursors of the alpha cells6. (Figure 4B)

[0227] Additional, immunofluorescent staining and electron microscopy provided further evidence of differentiation, with the presence of c-peptide and glucagon detected, and the visualization of characteristic hormone containing granules characteristic for endocrine cells. (Figure 4C-E).

[0228] Comparative Analysis of Embryonic and Induced Pluripotent Stem Cells

[0229] Current protocols are usually developed for either hESC or hiPSC and have limited robustness. That is why it was wanted to show the small molecule protocol robustness which is summarized in Figure 5. Both embryonic (HLIES8) and induced pluripotent stem cells (iPSC L1), were differentiated and characterized demonstrating their capacity to differentiate into islet-like spheroids by day 15. Gene expression analysis highlighted the presence of both differentiation and maturation markers, affirming the robustness of the differentiation protocol across different stem cell lines (Figure 5A). However, hiPSC cells routinely had higher expression of all markers when compared to hESC cells. Electron micrographs provided visual confirmation of the granular structures characteristic of mature beta cells, further validating the successful differentiation of pluripotent stem cells PSC (Figure 5B).

[0230] In vitro and In vivo Functional Characterization of Differentiated Cells

[0231] Figure 6 delves into the functional characterization of the differentiated cells, both in vitro and in vivo. Glucose-stimulated c-peptide secretion assays confirmed the functional responsiveness of the SC-beta spheroids derived from both iPSC L1 and HLIES8 lines, with a reproducible stimulation index, nevertheless human islet samples had a higher SI but increased variability due to donor differences (Figure 6A-C) .

[0232] Oxygen consumption rate (OCR) assays and mitochondrial property analyses showed similarity except for the glucose responsiveness indicating immaturity in vitro (Figure 6D-E) .

[0233] The in vivo efficacy of the differentiated cells was assessed through transplantation into mice under the kidney capsule, with human C-peptide levels measured post-transplantation, showing sustained function over an 8-week period. Immunofluorescence imaging of the graft tissue posttransplantation confirmed the survival and function of the transplanted cells (Figure 6F-H) .

[0234] 4. Discussion

[0235] The development of efficient protocols for the directed differentiation of pluripotent stem cells (PSCs) into specific lineages is a crucial aspect of stem cell research, with significant implications for regenerative medicine, disease modeling and drug discovery. The present invention presents a novel approach to the directed differentiation of pluripotent stem cells (PSCs) into a pancreatic lineage, utilizing protocol that incorporates the sequential administration of small molecules over a 15-day period8. This method represents a significant advancement in stem cell research, offering a more controlled and efficient pathway towards differentiation compared to traditional methods that often rely on the use of complex growth factor cocktails or co-culture systems. The use of small molecules in directing the differentiation of PSCs has gained significant attention in recent years due to their numerous advantages8. Small molecules offer advantages such as low cost, ease of administration, and the ability to target specific pathways or signaling networks involved in cell fate determination9.

[0236] The 15-day protocol demonstrated remarkable efficiency in generating insulin-producing cells. Traditional methods often require longer durations and multiple growth factors to achieve similar outcomes. For instance, protocols using growth factors typically span over 30-40 days10 11to produce functional beta cells. The present protocol, by contrast, achieves this in just 15 days, significantly reducing the time required for differentiation. This expedited process is crucial for potential clinical applications where time and cost are critical factors12 13.

[0237] Additionally, by applying a specific small molecule, BRD7552, a PDX1 inducer in stages 2 till 4 it was possible to promote the differentiation and increase the efficiency of the differentiation when compared with preexisting small molecule based protocols14 15.

[0238] Notably, both embryonic (HLIES8) and induced pluripotent stem cells (iPSC L1) demonstrated robust differentiation into islet-like spheroids, with hiPSC cells showing higher expression of differentiation markers. This highlights the protocol's robustness across different stem cell lines and suggests that hiPSCs may have a higher differentiation potential under the given conditions.

[0239] The study shows that while the in vitro assays indicate the differentiated cells respond to glucose, there is more lower stimulation index when compared to human islet samples16 17. This could indicate immaturity of the differentiated cells. This also can be seen when looking at the oxygen consumption rate. The assays suggest that these cells are not fully mature in their glucose responsiveness, implying in vitro immaturity.

[0240] Despite the in vitro immaturity, the in vivo mouse model demonstrated that the differentiated cells mature further18and do not need 2-3 months post transplantation to secrete insulin1920. They also function for an extended period post- transplantation.

[0241] 5. Conclusions

[0242] In conclusion, a four-step 3D approach for direct differentiation of various human PSCs into functional beta-like cells using fully small-molecule-based cocktails is provided. This differentiation procedure is an efficient, reproducible, stable, cost-effective, and time-saving way for producing 3D healthy and disease models of the pancreas and also in the future a scalable therapy for Type 1 diabetes.

[0243] References:

[0244] 1. Siller, R., Greenhough, S., Naumovska, E. & Sullivan, G. J. Small-molecule-driven hepatocyte differentiation of human pluripotent stem cells. Stem Cell Reports 4, (2015).

[0245] 2. Siller, R. et al. Development of a rapid screen for the endodermal differentiation potential of human pluripotent stem cell lines. Sci Rep 6, (2016).

[0246] 3. Takeuchi, H., Nakatsuji, N. & Suemori, H. Endodermal differentiation of human pluripotent stem cells to insulin-producing cells in 3D culture. Sci Rep 4, 1-9 (2014).

[0247] 4. Kondo, Y. et al. Identification of a small molecule that facilitates the differentiation of human iPSCs / ESCs and mouse embryonic pancreatic explants into pancreatic endocrine cells. Diabetologia 60, 1454-1466 (2017).

[0248] 5. Li, J., Chen, J., Luo, X., Lu, G. & Lin, G. Single-cell transcriptome analysis of NEUROG3+ cells during pancreatic endocrine differentiation with small molecules. Stem Cell Res Ther 4, 1-5 (2023).

[0249] 6. Balboa, D. et al. Functional, metabolic and transcriptional maturation of human pancreatic islets derived from stem cells. Nature Biotechnology 202240:740, 1042-1055 (2022).

[0250] 7. Fomina-Yadlin, D. et al. Small-molecule inducers of insulin expression in pancreatic a- cells. Proc Natl Acad Sci U S A 107, 15099-15104 (2010).

[0251] 8. Thakur, G., Lee, H. J., Jeon, R. H., Lee, S. L. & Rho, G. J. Small Molecule-Induced Pancreatic p-Like Cell Development: Mechanistic Approaches and Available Strategies. International Journal of Molecular Sciences 2020, Vol. 21, Page 238821 , 2388 (2020).

[0252] 9. Zhang, Y., Li, W., Laurent, T. & Ding, S. Small molecules, big roles - the chemical manipulation of stem cell fate and somatic cell reprogramming. J Cell Sci 125, 5609 (2012).

[0253] 10. Hogrebe, N. J., Maxwell, K. G., Augsornworawat, P. & Millman, J. R. Generation of insulin-producing pancreatic cells from multiple human stem cell lines. Nat Protoc 16, 4109 (2021).

[0254] 11. Rezania, A. et al. Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells. Nat Biotechnol 32, 1121-1133 (2014).

[0255] 12. Bandeiras, C. et al. Economics of Beta-Cell Replacement Therapy. CurrDiab Rep 19, 1-8 (2019).

[0256] 13. Iworima, D. G., Rieck, S. & Kieffer, T. J. Process parameter development for the scaled generation of stem cell-derived pancreatic endocrine cells. Stem Cells Transl Med 10, 1459 (2021).

[0257] 14. Kunisada, Y., Tsubooka-Yamazoe, N., Shoji, M. & Hosoya, M. Small molecules induce efficient differentiation into insulin-producing cells from human induced pluripotent stem cells. Stem Cell Res 8, 274-284 (2012).

[0258] 15. Takeuchi, H., Nakatsuji, N. & Suemori, H. Endodermal differentiation of human pluripotent stem cells to insulin-producing cells in 3D culture. Sci Rep 4, (2014). 16. Davis, J. C. et al. Glucose Response by Stem Cell-Derived Cells In Vitro Is Inhibited by a Bottleneck in Glycolysis. Cell Rep 31, 107623 (2020).

[0259] 17. Ameri, J. et al. Efficient Generation of Glucose-Responsive Beta Cells from Isolated GP2+ Human Pancreatic Progenitors. Cell Rep 19, 36-49 (2017). 18. Bruin, J. E. et al. Accelerated Maturation of Human Stem Cell-Derived Pancreatic

[0260] Progenitor Cells into Insulin-Secreting Cells in Immunodeficient Rats Relative to Mice. Stem Cell Reports 5, 1081-1096 (2015).

[0261] 19. Bruin, J. E. et al. Maturation and function of human embryonic stem cell-derived pancreatic progenitors in macroencapsulation devices following transplant into mice. Diabetologia 56, 1987-1998 (2013).

[0262] 20. Iworima, D. G., Rieck, S. & Kieffer, T. J. Process parameter development for the scaled generation of stem cell-derived pancreatic endocrine cells. Stem Cells Transl Med 10, 1459 (2021).

[0263] Sequence Listing

Claims

Claims1. A method for differentiating pluripotent stem cells, the method comprising the steps of: a) culturing the pluripotent stem cells in a first culture medium comprising at least one GSK-3 inhibitor in an effective amount for a first period of time in order to obtain definitive endoderm cells, b) culturing the cells obtained in step a) in a second culture medium comprising i. at least one BMP type I receptor inhibitor mimicking the effect of growth factor noggin, ii. at least one retinoic acid receptor agonist, and iii. BRD7552 in an effective amount for a second period of time in order to obtain posterior foregut cells, c) culturing the cells obtained in step b) in a third culture medium comprising i. at least one PARP-1 inhibitor, ii. forskolin, iii. at least one anti-inflammatory glucocorticoid, and iv. at least one of ALK5 inhibitor II and / or BRD7552 in an effective amount for a third period of time in order to obtain pancreatic endoderm cells; wherein the pluripotent stem cells are not obtained by a method which involved the destruction of a human embryo and wherein the pluripotent stem cells are human inducible pluripotent stem cells2. A method for obtaining pancreatic endoderm cells, the method comprising the step c) of culturing posterior foregut cells in a culture medium comprising i. at least one PARP-1 inhibitor, ii. forskolin, iii. at least one anti-inflammatory glucocorticoid, iv. and / or at least one of ALK5 inhibitor II and BRD7552 in an effective amount for a period of time in order to obtain pancreatic endoderm cells; wherein the posterior foregut cells are not obtained by a method which involved the destruction of a human embryo and wherein the posterior foregut cells are obtained from human inducible pluripotent stem cells.

3. The method according to claim 1 or claim 2 further comprising step d), wherein step d) comprises culturing the cells obtained in step c) in a fourth culture medium comprising atleast one aurora kinase inhibitor, RSKi2, and BRD7552 in an effective amount for a fourth period of time in order to obtain pancreatic endocrine cells.

4. The method according to any of the previous claims, wherein the cells in at least one of steps a) to d) are cultured in a 2D or 3D configuration culture system.

5. The method according to any of the previous claims, wherein the cells in steps a) to d) are cultured in a 3D configuration culture system.

6. The method according to any of the previous claims, wherein i. the definitive endoderm cells are obtained within 72 hours, and / or ii. wherein the posterior foregut cells are obtained within 144 hours, and / or iii. wherein the pancreatic endoderm cells are obtained within 240 hours, and / or iv. wherein the pancreatic endocrine cells are obtained within 408 hours after starting step a).

7. The method according to any of the previous claims, wherein the at least one GSK-3 inhibitor in step a) is selected from the group consisting of CHIR 99021 , 3F8, BIO, and CHIR 98014.

8. The method according to any of the previous claims, wherein in step a) the first culture medium additionally comprises IDE-1 and / or IDE-2.

9. The method according to claim 8, wherein in step a) the IDE-1 and / or IDE-2 are added at the same time as the GSK-3 inhibitor and / or at a later stage within the first period of time.

10. The method according to any of the previous claims, wherein the at least one BMP type I receptor inhibitor mimicking the effect of growth factor noggin is a combination of LDN193189 and dorsomorphin.11 . The method according to any of the previous claims, wherein the at least one retinoic acid receptor agonist in step b) is selected from the group consisting of retinoic acid, TTNPB and AM 580.

12. The method according to any of the previous claims, wherein the second culture medium additionally comprises a TGF-beta receptor inhibitor, preferably wherein the TGF-betareceptor inhibitor is selected from the group consisting of SB 431542, A 83-01 , SB 505124, SB 525334, and RepSox.

13. The method according to any of the previous claims, wherein the at least one PARP-1 inhibitor in step c) is nicotinamide.

14. The method according to any of the previous claims, wherein the at least one antiinflammatory glucocorticoid in step c) is selected from the group consisting of dexamethasone, and prednisolone.

15. The method according to any of the previous claims, wherein the third culture medium additionally comprises a TGF-beta Rl kinase inhibitor, preferably wherein the TGF-beta Rl kinase inhibitor is selected from the group consisting of ALK5 inhibitor II, ALK5 inhibitor GW788388, and ALK5 inhibitor SB525334.

16. The method according to any of the claims 3 to 15, wherein the at least one aurora kinase inhibitor in step d) is selected from the list consisting of ZM 447439, SP-96, Aurora kinase inhibitor 2, AT9283, and JNJ-7706621.

17. The method according to any of the previous claims, wherein i. the first culture medium in step a) comprises CHIR 99021 in an effective amount to obtain definitive endoderm cells, and / or wherein ii. the second culture medium in step b) comprises dorsomorphin, LDN193189, retinoic acid, SB431542, and BRD7552 in an effective amount to obtain posterior foregut cells, and / or wherein iii. the third culture medium in step c) comprises nicotinamide, forskolin, ALK5 inhibitor II, dexamethasone and BRD7552 in an effective amount to obtain pancreatic endoderm cells, and / or wherein iv. the fourth culture medium in step d) comprises ZM 447439, BRD7552, and RSKi2 in an effective amount to obtain pancreatic endocrine cells.

18. The method according to any of the previous claims, wherein i. the first period of time is 24 to 48 hours, and / or wherein ii. the second period of time is 48 to 72 hours, and / or wherein iii. the third period of time is 48 to 96 hours, and / or wherein iv. the fourth period of time is 144 to 240 hours.

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