Generation of pancreatic endoderm from stem cell-derived definitive endoderm

Abstract

The present invention relates to a method for efficiently generating pancreatic endoderm from human definitive endoderm derived from human pluripotent stem (PS) cells. The present invention also relates to pancreatic endoderm cells obtained by the method of the present invention. Finally, the present invention relates to a culture medium and composition comprising an RAR antagonist, and the use of the RAR antagonist in inducing pancreatic endoderm cells. The present invention provides a more homogeneous and synchronized pancreatic cell population with increased efficiency.

Description

[Technical Field]

[0001] The present invention relates to a method for efficiently generating pancreatic endoderm from human definitive endoderm derived from human pluripotent stem (PS) cells. [Background technology]

[0002] Beta cell (BC) transplantation has the potential to ultimately cure type 1 diabetes. However, the limited availability of donor beta cells limits the use of this treatment as a clinical therapy. Pluripotent stem (PS) cells can proliferate indefinitely and differentiate into many cell types. Therefore, PS cells are a promising source of beta cells; however, they must be efficiently and reproducibly differentiated into pancreatic cells before they can be used to treat diabetes.

[0003] During vertebrate embryonic development, pluripotent cells give rise to three germ layers: ectoderm, mesoderm, and endoderm. Induction of definitive endoderm (DE) is the first step toward the formation of endoderm-derived tissues. Generation of pancreatic endoderm (PE) from DE cells is required for the generation of insulin-producing beta cells. PE cells, which have the potential to become endocrine progenitor cells (EP), are characterized by the coexpression of two key transcription factors, PDX1 and NKX6.1.

[0004] Stepwise in vitro differentiation protocols have been established for generating pancreatic cells from PS cells. These protocols generally mimic key events in pancreatic development, including several stages: the DE, which co-expresses SOX17 and FOXA2, the gastrula, the posterior foregut, the PE, the EP, and ultimately the formation of mature beta cells. To date, efficient DE differentiation of hES cells has been achieved by activin A treatment. The next major step in generating pancreatic beta cells is generating PE, which co-expresses PDX1 and NKX6.1. Several groups have developed in vitro protocols capable of differentiating PS cells into DE and PE, and almost all published protocols include the addition of a retinoic acid receptor (RAR) agonist during the induction of PE.

[0005] Patients with type 1 diabetes can be treated with transplantation of pancreatic islets from human donors, and some patients achieve insulin independence. However, donor islets are scarce and of variable quality, making insulin-producing cells derived from pluripotent stem cells an attractive alternative to pancreatic islets. A key differentiation step is differentiation from definitive endoderm (DE) to pancreatic endoderm (PE), characterized by coexpression of PDX1 and NKX6.1. However, this differentiation step is less efficient than previous differentiation steps to DE, and batch-to-batch variability is often observed. Improving the efficiency of PE induction increases the number of endocrine progenitor cells (EP) capable of forming beta cells and also reduces the number of undesired cell types. Batch-to-batch variability in differentiation efficiency is a well-known problem, and improving differentiation stability becomes important when moving to large-scale production processes and clinical trials. Therefore, differentiation protocols must be optimized at every stage to obtain large numbers of functional beta cells (BCs) in the final step. Therefore, the efficiency and stability of current differentiation protocols need to be improved, which is critical for the further development of these cells into the endocrine lineage. Summary of the Invention

[0006] The present invention improves the efficiency of differentiating human PS cells into mature beta cells by providing a method for increasing the proportion of NKX6.1 / PDX1 double-positive cells, a characteristic of PE cells committed to a pancreatic fate.

[0007] Furthermore, the present invention provides a more efficient, more homogeneous and synchronized population of pancreatic cells, which is important for the further development of these cells down the endocrine lineage.

[0008] In one aspect, the present invention relates to a method for deriving pancreatic endoderm cells from human definitive endoderm derived from human pluripotent stem (PS) cells.

[0009] In one aspect, the present invention relates to a method for inducing pancreatic endoderm precursors from human definitive endoderm derived from human pluripotent stem (PS) cells, the method comprising culturing the definitive endoderm with a RAR antagonist.

[0010] In one aspect, the present invention relates to a method for inducing pancreatic endoderm cells from human definitive endoderm derived from human pluripotent stem (PS) cells, the method comprising the steps of culturing the definitive endoderm with an RAR antagonist to obtain pancreatic endoderm precursors, and then inducing pancreatic endoderm cells by culturing the pancreatic endoderm precursors with an RAR agonist.

[0011] In one aspect, the present invention relates to a method for differentiating definitive endoderm cells derived from human pluripotent stem cells into pancreatic endoderm precursors, the method comprising culturing the definitive endoderm (DE) in a culture medium comprising a retinoic acid receptor (RAR) antagonist.

[0012] In one aspect, the method of the present invention further comprises culturing pancreatic endoderm precursors in a cell culture medium containing an RAR agonist, thereby inducing pancreatic endoderm (PE), wherein the cells are PDX1+ / NKX6.1+ double positive.

[0013] In one aspect, the present invention relates to pancreatic endoderm cells or pancreatic endoderm cell precursors obtainable by the methods of the present invention.

[0014] In another aspect, the present invention relates to synchronized pancreatic endoderm cells or synchronized pancreatic endoderm cell precursors obtainable by the method of the present invention.

[0015] In one aspect, the present invention relates to a culture medium and / or composition comprising an RAR antagonist and an RAR agonist.

[0016] In one aspect, the present invention relates to pancreatic endoderm cells produced by exposing definitive endoderm derived from human pluripotent stem cells to a RAR antagonist to obtain pancreatic endoderm precursors, and subsequently exposing the pancreatic endoderm precursors to a RAR agonist to obtain pancreatic endoderm.

[0017] In one aspect, the present invention relates to the use of RAR antagonists to induce pancreatic endoderm precursors from definitive endoderm cells derived from human pluripotent stem cells.

[0018] In one aspect, the present invention relates to the use of an RAR antagonist, followed by the use of an RAR agonist, to induce pancreatic endoderm cells from definitive endoderm cells derived from human pluripotent stem cells.

[0019] In one aspect, the present invention relates to a bioreactor comprising a population of pancreatic endodermal progenitor cells or pancreatic endoderm cells obtained by the method of the present invention.

[0020] In one aspect, the present invention provides improved pancreatic endoderm cell populations, i.e., PEs with an increased percentage of NKX6.1+ / PDX1+ double positive cells.

[0021] In one aspect, the present invention provides a more homogeneous population of pancreatic endoderm cells.

[0022] In one aspect, the present invention provides a more synchronized pancreatic endoderm cell population.

[0023] The present invention may also solve further problems that become apparent from the disclosure of the exemplary embodiments. [Brief explanation of the drawings]

[0024] [Figure 1] Figure 1 shows the effect of AGN 193109 compared to a standard PE induction protocol on Pdx1 / Nkx6.1 expression at day 10 of PE differentiation (PE10) as assessed by FACS analysis. This figure shows the average percentage of Pdx1 / Nkx6.1 double-positive cells in four independent experiments comparing standard PE with AGN 193109 treatment (1A), as well as an example FACS plot from a representative experiment (1B). Additionally, an example of the effect of AGN 193109 on PE10 induction when applied to suboptimal induction using the standard protocol is shown (1C). [Figure 2] Figure 2 shows the effect of AGN 193109 on the expression of pancreatic endoderm markers. Figure 2A shows the effect of inhibiting RA signaling with AGN 193109 or activating RA signaling with AM580 on the expression of pancreatic endoderm markers Nkx6.1, CPA1, Ptf1a, and Dlk1 on PE10, as assessed by Nanostring analysis of gene expression. Figure 2B shows a FACS plot of the expression of Nkx6.1 relative to Dlk1 on PE11 in standard protocol compared to AGN 193109 treatment. [Figure 3]Figure 3 shows the effect of AGN1931091 on immature endocrine differentiation of PE10. Figure 3A shows the effect of inhibition of RA signaling by AGN193109 on PE2-3 and activation of RA signaling by AM580 on the pancreatic endoderm stage compared to the standard protocol as assessed by Nanostring analysis of gene expression. Figure 3B shows a FACS plot of the expression of the pancreatic endocrine marker Nkx2.2 in PE10 treated with the standard protocol compared to AGN 193109 treatment on PE2-3. [Figure 4] Figure 4 shows the effect of RAR activation by AM580 and RAR inhibition by AGN 193109 on PE2-3 compared to the standard protocol on pancreatic endoderm induction as assessed by FACS analysis for Pdx1 / Nkx6.1 on PE10. [Figure 5] Figure 5 shows the dose-response effect of AGN 193109 on PE induction in two examples, as assessed by FACS analysis of Pdx1 / Nkx6.1 in PE10. In one experiment, AGN 193109 was tested at concentrations of 1, 3, and 10 μM in PE2-3 (5A). In another experiment, AGN 193109 was tested at concentrations of 0, 5, 1, 10, and 20 μM in PE2-3 (5B). [Figure 6] Figure 6 shows the effect of AGN 193109 timing, as assessed by FACS analysis of Pdx1 / Nkx6.1 on PE10. In one experiment, the exposure time to 10 μM AGN 193109 is increased from 2 days (PE2-3) to 3 days (PE1-3) or 4 days (PE0-3) during the 4-day LDN phase from PE0-3 (6A). In another experiment, the 2-day treatment with 10 μM AGN 193109 is maintained and the LDN phase is shortened from 4 days to 3 days or 2 days (6A). [Figure 7]Figure 7 shows the effect of AGN 193109 on the induction of BC stage cells, as assessed by FACS analysis on days 6-9 of beta cell differentiation (BC6-9) for C-pep, Nkx6.1, and glucagon expression. Figure 7A shows the average percentage of C-pep / Nkx6.1 double-positive cells in four independent experiments comparing AGN 193109 with the standard protocol on days PE2-3. Figure 7B shows a representative example comparing AGN 193109 with the standard protocol. [Figure 8] Figure 8 shows the functionality of the protocol with AGN 193109 in the bioreactor, as assessed by FACS analysis for Pdx1 / Nkx6.1 in PE 10. The figure shows examples of two representative experiments performed in the 1 L tank of the bioreactor (DASgip, Eppendorf), with and without the addition of 10 µM AGN 193109 in PE 2-3. [Figure 9] Figure 9 shows the BC phenotype after cryopreservation and thawing of BC3 followed by long-term culture of cells treated with either the standard PE protocol or AGN193109, as assessed by FACS analysis for C-pep / Nkx6.1 in BC6 and BC13. DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention relates to methods for generating pancreatic endoderm precursors and pancreatic endoderm from definitive endoderm derived from human pluripotent stem cells.

[0026] The present invention relates to a method for differentiating definitive endoderm cells derived from human pluripotent stem cells into pancreatic endoderm NKX6.1+ / PDX1+ double positive cells, the method comprising the steps of culturing the definitive endoderm in a culture medium containing a retinoic acid receptor (RAR) antagonist to obtain pancreatic endoderm precursors, and then culturing the pancreatic endoderm precursors in a culture medium containing a retinoic acid receptor (RAR) antagonist.

[0027] The present invention provides a more homogeneous and synchronized population of pancreatic endoderm cells with increased efficiency.

[0028] The transcription factors NKX6.1 and PDX1 are markers of the PE cell population, which is one of the cellular stages required to reach the endocrine cell population. Increasing the proportion of NKX6.1 / PDX1 double-positive cells in the PE cell population increases efficiency.

[0029] The differentiation method of the present invention results in a significantly increased proportion of NKX6.1 / PDX1+ double positive cells in the PE cell population, ie, with higher efficiency.

[0030] Furthermore, the present invention provides a more homogeneous and synchronized population of pancreatic endoderm cells, which is important for the further development of these cells into the endocrine lineage, ie, into beta cells or insulin-producing cells.

[0031] In this invention, we describe a method to increase the efficiency and stability of PE induction, which also improves the efficiency of further differentiation into insulin-producing beta cells.

[0032] Inducing PE induction with a retinoic acid receptor (RAR) antagonist (AGN 193109) during the first stage of the PE protocol (LDN stage, i.e., 4 days referred to as PE0-PE3) significantly increases PE induction in the second stage (i.e., 8 days referred to as PE4-PE11) containing a retinoic acid receptor agonist (the percentage of PDX1+ / NKX6.1+ pancreatic endoderm). Furthermore, it stabilizes PE induction by reducing batch-to-batch variability. Finally, the quality of pancreatic endoderm is improved due to increased expression of Ptf1a, an additional marker of PE.

[0033] The present invention also describes DLK1 as a marker for pancreatic endoderm, and shows that DLK1 is mainly expressed in PDX1+ / NKX6.1 high cells. DLK1 is a surface marker and can be used to enrich pancreatic endoderm at the pancreatic endoderm stage by live cell sorting using methods such as FACS or MACS.

[0034] PE cells can be differentiated into EP cells, which can further differentiate into BC cells. Finally, improvement of the PE step leads to an overall improvement in BC differentiation efficiency, resulting in a greater number of C-pep / NKX6.1 double-positive cells.

[0035] Definition: stem cells Stem cells are undifferentiated cells that are defined by their ability at the single cell level to both self-renew and differentiate to produce progeny cells, including self-renewing progenitor cells, non-renewing progenitor cells, and terminally differentiated cells.Stem cells are also characterized by their ability to differentiate in vitro into functional cells of various cell lineages from multiple germ layers (endoderm, mesoderm, and ectoderm), and to give rise to the tissues of multiple germ layers after transplantation, and to contribute to substantially most, if not all, tissues after injection into blastocysts.

[0036] Stem cells are classified by their developmental potential as follows: (1) totipotent (meaning they can give rise to all embryonic and extraembryonic cell types), (2) pluripotent (meaning they can give rise to all embryonic cell types), (3) multipotent (meaning they can give rise to a subset of cell lineages, but all within a particular tissue, organ, or physiological system (e.g., hematopoietic stem cells (HSCs) can produce progeny that include HSCs (self-renewing), blood cell-restricted oligopotent progenitors, and all cell types and elements that are normal components of blood (e.g., platelets)), (4) oligopotent (meaning they can give rise to a more restricted subset of cell lineages than pluripotent stem cells), and (5) unipotent (meaning they can give rise to a single cell lineage (e.g., spermatogenic stem cells)).

[0037] human pluripotent stem cells As used herein, "human pluripotent stem cells" (hPSCs) refer to cells that can be derived from any source and, under appropriate conditions, can produce human progeny of distinct cell types that are derivatives of all three germ layers (endoderm, mesoderm, and ectoderm). hPSCs may have the ability to form teratomas in 8-12 week-old SCID mice and / or form identifiable cells of all three germ layers in tissue culture. The definition of human pluripotent stem cells includes various types of embryonic cells, including human blastocyst-derived stem (hBS) cells, often referred to in the literature as human embryonic stem (hES) cells (see, e.g., Thomson et al. (1998); Heins et al. (2004)), as well as induced pluripotent stem cells (see, e.g., Yu et al. (2007); Takahashi et al. (2007)). Various methods and other embodiments described herein may require or utilize hPSCs from a variety of sources. For example, hPSCs suitable for use can be obtained from developing embryos. Additionally, or alternatively, suitable hPSCs can be obtained from established cell lines and / or human induced pluripotent stem (hiPS) cells.

[0038] As used herein, "hiPSC" refers to human induced pluripotent stem cells.

[0039] ES cell lines can also be derived from single blastomeres without destroying the ex utero embryo and without affecting clinical outcome (Chung et al. (2006) and Klimanskaya et al. (2006)).

[0040] Blastocyst-derived stem cells As used herein, the term "blastocyst-derived stem cells" refers to BS cells, and the human form is called "hBS cells." In the literature, the cells are often referred to as embryonic stem cells, more specifically, human embryonic stem cells (hESCs).

[0041] Therefore, the pluripotent stem cells used in the present invention can be, for example, embryonic stem cells prepared from blastocysts, as described in WO03 / 055992 and WO2007 / 042225, or commercially available hBS cells or cell lines.However, it is further envisioned that any human pluripotent stem cells can be used in the present invention, including differentiated adult cells that are reprogrammed into pluripotent cells by treating the adult cells with certain transcription factors, such as OCT4, SOX2, NANOG, and LIN28, as disclosed in, for example, Yu, et al. (2007), Takahashi et al. (2007), and Yu et al. (2009).

[0042] In one embodiment, the cell population comprising PE cells is obtained from a somatic cell population. In another embodiment, the somatic cell population is induced to dedifferentiate into embryonic-like stem (ES, e.g., pluripotent) cells. Such dedifferentiated cells are also called induced pluripotent stem cells (iPSCs).

[0043] In one embodiment, the cell population comprising PE cells is derived from embryonic stem (ES, e.g., pluripotent) cells. In another embodiment, the cell population comprising pancreatic cells is a pluripotent cell, such as an ES-like cell.

[0044] In one embodiment, the cell population comprising PE cells is embryonic differentiated stem (ES or pluripotent) cells. Differentiation occurs in embryoid bodies and / or in monolayer cell culture, or a combination thereof.

[0045] In one embodiment, the cell population is a population of stem cells, hi another embodiment, the cell population is a population of stem cells differentiated to the pancreatic endoderm lineage.

[0046] In one embodiment, the further differentiated stem cells are human embryonic stem cells or induced pluripotent stem cells.

[0047] differentiation As used herein, "differentiating" or "differentiation" refers to the process by which cells progress from an undifferentiated state to a differentiated state, from an immature state to a less immature state, or from an immature state to a mature state. For example, early undifferentiated embryonic pancreatic cells can proliferate and express characteristic markers such as PDX1, NKX6.1, and PTF1a. Mature or differentiated pancreatic cells do not proliferate and secrete high levels of pancreatic endocrine hormones or digestive enzymes. For example, fully differentiated beta cells secrete high levels of insulin in response to glucose. Changes in cellular interactions and maturation occur when cells lose markers of undifferentiated cells or gain markers of differentiated cells. The loss or acquisition of a single marker can indicate that a cell is "mature or fully differentiated."

[0048] The term "differentiation factor" refers to a compound that is added to pancreatic cells to enhance their differentiation into mature endocrine cells, which also contain insulin-producing beta cells.

[0049] Exemplary differentiation factors include hepatocyte growth factor, keratinocyte growth factor, exendin-4, basic fibroblast growth factor, insulin-like growth factor-1, nerve growth factor, epidermal growth factor, platelet-derived growth factor, and glucagon-like peptide 1.

[0050] In one embodiment, differentiating the cells comprises culturing the cells in a medium containing one or more differentiation factors.

[0051] Definitive endoderm cells (DE cells) Definitive endoderm cells are characterized by the expression of the marker SOX17. Additional markers of DE are FOXA2 and CXCR4.

[0052] "SOX17" (SRY-box 17), as used herein, is a member of the SOX (SRY-related HMG-box) family of transcription factors involved in the control of embryonic development and cell fate determination.

[0053] "FOXA2" (Forkhead box A2), as used herein, is a member of the forkhead class of DNA-binding proteins.

[0054] "CXCR4" (CXC motif chemokine receptor 4), as used herein, is a CXC chemokine receptor specific for stromal cell-derived factor 1.

[0055] Non-limiting examples of DE induction protocols are the conventional D'Amour protocol (Novocell, Nature Biotec 2006, 2008) and the protocol described in WO2012 / 175633, which is incorporated herein by reference in its entirety.

[0056] In one embodiment, the DE cells of the methods of the present invention are SOX17+ positive.

[0057] In one embodiment, the DE cells of the methods of the present invention are SOX17+ / FOXA2 double positive.

[0058] In one embodiment, the DE cells of the methods of the present invention are SOX17+ / FOXA2+ / CXCR4+ triple positive.

[0059] Pancreatic endoderm precursors As used herein, "pancreatic endoderm precursors" or "pancreatic endoderm cell precursors" are cells obtained by culturing definitive endoderm cells derived from human pluripotent stem cells in a culture medium containing a RAR antagonist.

[0060] These cells, when further cultured in a culture medium containing an RAR agonist, result in the induction of pancreatic endoderm cells as defined below.

[0061] Pancreatic endoderm cells (PE cells) Pancreatic endoderm cells are characterized by the expression of at least 5% NKX6.1+ / PDX1+ double positive markers. Additional markers of PE are PTF1A and CPA1.

[0062] "PDX1," as used herein, refers to a homeodomain transcription factor involved in pancreatic development.

[0063] "NKX6.1," as used herein, is a member of the NKX transcription factor family.

[0064] "PTF1A," as used herein, is a component of the pancreatic transcription factor 1 complex (PTF1), a protein known to play a role in mammalian pancreatic development.

[0065] "CPA1," as used herein, is a member of the carboxypeptidase A family of zinc metalloproteases. This enzyme is produced in the pancreas.

[0066] In one embodiment, the present invention relates to a method for differentiating DE into PE, wherein the cells are NKX6.1+ / PDX1+ double positive.

[0067] In one embodiment, the present invention relates to a method of differentiating DE into PE, wherein at least 5% of the PE cells co-express PDX1 and NKX6.1.

[0068] In one embodiment, the present invention relates to a method of differentiating DE into PE, wherein at least 5%, at least 10%, 10-30%, 10-40%, 5-70%, 10-80%, or 5-100% of the PE cells are PDX1+ / NKX6.1+ double positive.

[0069] In one embodiment, the present invention relates to a method for differentiating DE into PE, wherein at least 70% of the PE cells are PDX1+ / NKX6.1+ double positive.

[0070] In one embodiment, the present invention relates to a method for differentiating DE into PE, wherein at least 80% of the PE cells are PDX1+ / NKX6.1+ double positive.

[0071] In one embodiment, the present invention relates to a method for differentiating DE into PE, wherein at least 90% of the PE cells are PDX1+ / NKX6.1+ double positive.

[0072] In one embodiment, the present invention relates to a method for differentiating DE into PE, wherein at least 95% of the PE cells are PDX1+ / NKX6.1+ double positive.

[0073] In one embodiment, the present invention relates to a method for differentiating DE into PE, wherein at least 98% of the PE cells are PDX1+ / NKX6.1+ double positive.

[0074] In one embodiment, the present invention relates to a method for differentiating DE into PE, wherein 80-100% of the PE cells are PDX1+ / NKX6.1+ double positive.

[0075] In one embodiment, the present invention relates to a method for differentiating DE into PE, wherein 90-100% of the PE cells are PDX1+ / NKX6.1+ double positive.

[0076] In one embodiment, the present invention relates to an in vitro or in vivo pancreatic endoderm cell population obtainable by the method of the present invention.

[0077] In one embodiment, the present invention relates to an in vitro pancreatic endoderm cell population obtainable by the method of the present invention.

[0078] In one embodiment, the present invention relates to an in vivo pancreatic endoderm cell population obtainable by the method of the present invention.

[0079] In one embodiment, the present invention provides a pancreatic endoderm cell population having increased co-expression of PDX1 and NKX6.1, ie, increased expression of PDX1+ / NKX6.1+ double positive cells.

[0080] In one embodiment, the present invention relates to a pancreatic endoderm cell population obtainable by the method of the present invention, which has increased expression of PDX1+ / NKX6.1+ double positive cells.

[0081] In one embodiment, the present invention relates to a pancreatic endoderm cell population obtainable by the method of the present invention, wherein the pancreatic endoderm cells are at least 70% PDX1+ / NKX6.1+ double positive.

[0082] In one embodiment, the present invention relates to a pancreatic endoderm cell population obtainable by the method of the present invention, wherein the pancreatic endoderm cells are 80 to 100% PDX1+ / NKX6.1+ double positive.

[0083] In one embodiment, the present invention relates to a pancreatic endoderm cell population obtainable by the method of the present invention, wherein the pancreatic endoderm cells are 90 to 100% PDX1+ / NKX6.1+ double positive.

[0084] In one embodiment, the present invention provides a method that shortens the LDN step and has a shorter LDN step.

[0085] In one embodiment, the present invention provides a method with a shorter LDN phase, shortening the LDN phase in half, to two days.

[0086] In one embodiment, the present invention relates to a method that shortens the LDN phase to 2 days, a shorter duration than standard methods.

[0087] An additional approach to increasing the yield of functional beta cells involves sorting live cells at certain stages during the differentiation process. Sorting at the pancreatic endoderm stage is attractive as undesired cells commit to the pancreatic lineage at this step. This approach requires the use of antibodies that recognize surface antigens and thus enable live cell sorting. Therefore, we describe the discovery of Delta-like non-canonical Notch ligand 1 (DLK1) as a surface marker for human ES cell-derived pancreatic endoderm.

[0088] Synchronized PE populations are populations in which no immature further differentiation into endocrine progenitor cells (EP) occurs until signals to induce endocrine differentiation are applied to the cell culture. Immature endocrine differentiation at the PE stage is a common feature of many published protocols. Synchronized PE populations are advantageous in that they allow for better control over the induction of endocrine lineages and beta cells.

[0089] Obtaining synchronized cells is an advantage as it results in a more homogenous cell population to continue further differentiation into beta cells or insulin-producing cells.

[0090] Transplantation of differentiated cells at the beta cell stage showed a trend for the addition of AGN193109 to improve the beta-to-alpha cell ratio, meaning that the ratio of insulin-positive cells to glucagon-positive cells was higher with AGN193109 treatment 8 weeks after transplantation, as assessed by immunohistochemistry and quantitative image analysis.

[0091] "DLK1" as used herein is a transmembrane protein containing multiple epidermal growth factor repeats that functions as a regulator of cell growth. The encoded protein is involved in the differentiation of several cell types. DLK1 is a non-canonical Notch ligand.

[0092] "NKX2.2" as used herein is a homeodomain transcription factor. Nkx2.2 is a key regulator of proper islet cell lineage specification during pancreas development.

[0093] In one embodiment, the present invention provides PE endoderm cell populations that are more selectively, more homogeneously, and more efficiently induced into EP and beta cell populations.

[0094] In one embodiment, the pancreatic endoderm cell populations obtained by the methods of the present invention are more homogeneous and more synchronized, and more efficiently induce EP and beta cell populations.

[0095] Furthermore, DLK1 is a surface marker that is highly expressed in PE cell populations with improved PDX1 / NKX6.1 expression.

[0096] In one embodiment, the PE cells obtainable by the method of the present invention are DLK1 positive.

[0097] In one embodiment, the PE cells obtainable by the method of the invention express at least 5% DLK1.

[0098] In one embodiment, PE cells obtainable by the method of the present invention express 5 to 30% DLK1.

[0099] In one embodiment, the present invention relates to a population of PE cells, wherein the cells are DLK1 positive.

[0100] In one embodiment, the present invention relates to the surface marker DLK1 of PE, and the cells are NKX6.1+ / PDX1+ double positive.

[0101] In one embodiment, the present invention relates to the surface marker DLK1 of PE, and the cells are about 80% NKX6.1+ / PDX1+ double positive.

[0102] In one embodiment, the invention relates to the surface marker DLK1 of PE, wherein the cells are more than 80% NKX6.1+ / PDX1+ double positive.

[0103] In one embodiment, the present invention provides an improved PE cell population having at least 5% expression of DLK1.

[0104] In one embodiment, the present invention provides an improved PE cell population having at least 10% expression of DLK1.

[0105] In one embodiment, the present invention provides an improved PE cell population having at least 20% expression of DLK1.

[0106] In one embodiment, the present invention provides an improved PE cell population having at least 30% expression of DLK1.

[0107] In one embodiment, the present invention provides an improved PE cell population having 30-80% expression of DLK1.

[0108] In one embodiment, the present invention provides an improved PE cell population having 20-80% expression of DLK1.

[0109] In one embodiment, the present invention relates to a synchronized cell population derived from human pluripotent stem cells, which exhibits at least 70% pancreatic endoderm cells expressing PDX1+ / NKX6.1+ double positive.

[0110] In one embodiment, the present invention relates to a synchronized cell population derived from human pluripotent stem cells, which presents at least 70% of pancreatic endoderm cells that express PDX1+ / NKX6.1+ double positive, and which further expresses PTF1a.

[0111] In one embodiment, the present invention relates to a synchronized cell population derived from human pluripotent stem cells exhibiting at least 70% pancreatic endoderm cells expressing PDX1+ / NKX6.1+ double positive, wherein the cell population further expresses 5-30% DLK1.

[0112] In one embodiment, the present invention relates to a synchronized cell population derived from human pluripotent stem cells, which exhibits at least 70% pancreatic endoderm cells expressing PDX1+ / NKX6.1+ double positive and expresses less than 1% NKX2.2.

[0113] In one embodiment, the present invention relates to a synchronized cell population derived from stem cells that exhibits less than 1% NKX2.2 positivity.

[0114] In one embodiment, the PE cells obtainable by the method of the present invention are less than 1% NKX2.2 positive.

[0115] In one embodiment, the present invention relates to a method for differentiating DE into PE, wherein the PE cells are less than 1% NKX2.2 positive.

[0116] In one embodiment, the present invention provides a pancreatic endoderm cell population that is more homogeneous and synchronized, i.e., has a lower induction of immature endocrine differentiation at the PE stage, characterized by reduced expression of NGN3 and NeuroD.

[0117] In one embodiment, the present invention relates to a pancreatic endoderm cell population obtainable by the method of the present invention, which has reduced expression of NGN3 and NeuroD.

[0118] In one embodiment, the present invention relates to a pancreatic endoderm cell population obtainable by the method of the present invention, in which the expression of NGN3 and NeuroD is reduced by at least two-fold.

[0119] In one embodiment, the present invention relates to a pancreatic endoderm cell population obtainable by the method of the present invention, wherein the PE population is further differentiated into beta cells with at least 40% C-PEP+ / NKX6.1+ double positivity.

[0120] Endocrine precursor cells (EP cells) Endocrine precursor cells are characterized by the expression of markers NGN3, NeuroD, and NKX2.2, which are characteristic of EP cells committed to an endocrine cell fate.

[0121] As used herein, an "EP cell population" is a population of pancreatic beta cell precursors, wherein at least 5% of the cell population is NKX6.1 / NKX2.2 double positive.

[0122] "NGN3," as used herein, is a member of the neurogenin family of basic loop-helix-loop transcription factors.

[0123] "NKX2.2" and "NKX6.1," as used herein, are members of the NKX transcription factor family.

[0124] "NeuroD," as used herein, is a member of the NeuroD family of basic helix-loop-helix (bHLH) transcription factors.

[0125] beta cells As used herein, the term "beta cells" refers to cells that reside within small cell clusters called the islets of Langerhans within the pancreas.

[0126] Beta cells are characterized by the co-expression of INS / NKX6.1 and C-PEP / NKX6.1.

[0127] Beta cells respond to high blood glucose levels by secreting the peptide hormone insulin (INS), which acts on other tissues to promote glucose uptake from the blood, for example in the liver, which promotes energy storage by glycogen synthesis.

[0128] In one embodiment, the EP cells obtainable by further derivation of the PE cells obtained by the method according to the invention are further differentiated into insulin-producing cells, optionally together with cells differentiated into glucagon-, somatostatin-, pancreatic polypeptide-, and / or ghrelin-producing cells.

[0129] As used herein, "insulin-producing cells" refers to cells that produce and store or secrete detectable amounts of insulin. An "insulin-producing cell" can be an individual cell or a collection of cells.

[0130] In one embodiment, the present invention provides an improved PE cell population that results in the induction of a beta-like cell population with increased expression of C-PEP+ / NKX6.1+.

[0131] Retinoic acid receptor (RAR) antagonists Retinoic acid receptor antagonists selectively counteract the effects of retinoids on one or more of the retinoic acid receptor subtypes, RARα, RARAβ, and RARγ.

[0132] AGN 193109 is an orally active retinoic acid receptor (RAR) antagonist that targets all three RAR subtypes with higher affinity (RARα / β / γ Kd = 2 nM) than all-trans retinoic acid / ATRA (RARα / β / γ Kd = 9 / 12 / 19 nM). AGN 193109 potently antagonizes ATRA-induced transcription in RARα, RARβ, and RARγ-transfected CV-1 cells (85%, 62%, and 100%, respectively, at equimolar concentrations of AGN 193109 to ATRA). AGN 193109 has also been widely employed to block RAR-mediated physiological and pathological processes in vivo in mice and rats via oral (1–10 mg / kg) or topical (0.3–36 μmol / kg) administration.

[0133] Non-limiting examples of RAR antagonists include AGN193109, AGN 194431, AGN 194301, SR 11335, BMS 453, BMS 195614, LE 135, LG 100815, MM11253, CD 2665, ER 50891.

[0134] In one embodiment, the RAR antagonist is selected from the group including but not limited to AGN193109, AGN 194431, AGN 194301, SR 11335, BMS 453, BMS 195614, LE 135, LG 100815, MM11253, CD 2665, and ER 50891.

[0135] In one embodiment, the RAR antagonist is AGN193109.

[0136] In one embodiment, the present invention relates to a method for differentiating definitive endoderm cells derived from human pluripotent stem cells into pancreatic endoderm progenitor cells, the method comprising culturing the definitive endoderm in a culture medium comprising a retinoic acid receptor (RAR) antagonist.

[0137] In one embodiment, the present invention relates to a method for differentiating definitive endoderm cells derived from human pluripotent stem cells into pancreatic endoderm progenitor cells, the method comprising culturing the definitive endoderm in a culture medium comprising AGN 193109.

[0138] In one embodiment, the present invention relates to a method for differentiating DE into PE, the method comprising a first step of culturing DE in a culture medium containing an RAR antagonist to obtain pancreatic endoderm precursors, and a subsequent second step of culturing the pancreatic endoderm precursors in a cell culture medium containing an RAR agonist.

[0139] In one embodiment, the present invention relates to a method for differentiating DE into PE, the method comprising a first step of culturing DE in a culture medium containing AGN 193109 to obtain pancreatic endoderm precursors, and a subsequent second step of culturing the pancreatic endoderm precursors in a cell culture medium containing AM580.

[0140] In one embodiment, the present invention relates to a method for differentiating DE into PE, the method comprising a first step of culturing DE in a culture medium containing an RAR antagonist to obtain pancreatic endoderm precursors, and a subsequent second step of culturing the pancreatic endoderm precursors in a cell culture medium containing an RAR agonist, a fibroblast growth factor, and a Rock inhibitor, and optionally a BMP inhibitor.

[0141] In one embodiment, the present invention relates to a method for differentiating DE into PE, the method comprising a first step of culturing DE in a culture medium containing an RAR antagonist to obtain pancreatic endoderm precursors, and a subsequent second step of culturing the pancreatic endoderm precursors in a cell culture medium containing an RAR agonist, FGF2, and Tiger or Y27632, and optionally LDN193189.

[0142] In one embodiment, the present invention relates to the use of RAR antagonists to induce pancreatic endoderm progenitor cells from definitive endoderm derived from human pluripotent stem cells.

[0143] In one embodiment, the present invention relates to the use of an RAR antagonist to induce pancreatic endoderm progenitor cells from definitive endoderm derived from human pluripotent stem cells, followed by treatment of the pancreatic endoderm progenitor cells with an RAR agonist to induce pancreatic endoderm.

[0144] In one embodiment, the method of the present invention includes a concentration of the RAR antagonist in the range of 0, 5-100 μM, 1-50 μM, 1-300 μM, 1-250 μM, 1-20 μM, 3-17 μM, 5-15 μM, or 7-12 μM.

[0145] In one embodiment, the method of the present invention comprises a concentration of the RAR antagonist in the range of 0.5 to 100 μM.

[0146] In one embodiment, the method of the present invention comprises a concentration of the RAR antagonist in the range of 0.5 to 50 μM.

[0147] In one embodiment, the method of the present invention comprises a concentration of the RAR antagonist in the range of 0.5 to 30 μM.

[0148] In one embodiment, the method of the present invention comprises a concentration of the RAR antagonist in the range of 0.5 to 25 μM.

[0149] In one embodiment, the method of the present invention comprises a concentration of the RAR antagonist in the range of 0.5 to 20 μM.

[0150] In one embodiment, the method of the present invention comprises a concentration of the RAR antagonist in the range of 1-20 μM.

[0151] In one embodiment, the method of the present invention comprises a concentration of the RAR antagonist in the range of 10-20 μM.

[0152] In one embodiment, the method of the present invention comprises a concentration of the RAR antagonist in the range of 7-12 μM.

[0153] In one embodiment, the method of the present invention comprises a concentration of the RAR antagonist of about 10 μM.

[0154] In one embodiment, the method of the present invention comprises a concentration of the RAR antagonist of about 15 μM.

[0155] In one embodiment, the method of the present invention comprises a concentration of the RAR antagonist of about 20 μM.

[0156] In one embodiment, the present invention relates to the use of an RAR antagonist, followed by the use of an RAR agonist, to induce pancreatic endoderm cells from definitive endoderm cells derived from human pluripotent stem cells.

[0157] In one embodiment, the present invention relates to the use of a RAR antagonist at a concentration in the range of 0.5 to 100 μM, followed by the use of a RAR agonist, to induce pancreatic endoderm cells from definitive endoderm cells derived from human pluripotent stem cells.

[0158] In one embodiment, the present invention relates to the use of a RAR antagonist at a concentration of 10 μM to induce pancreatic endoderm progenitor cells from definitive endoderm cells derived from human pluripotent stem cells.

[0159] In one embodiment, the present invention relates to the use of a RAR antagonist at a concentration of 10 μM, followed by the use of a RAR agonist, to induce pancreatic endoderm cells from definitive endoderm cells derived from human pluripotent stem cells.

[0160] In one embodiment, the present invention relates to the use of a RAR antagonist at a concentration of 20 μM to induce pancreatic endoderm progenitor cells from definitive endoderm cells derived from human pluripotent stem cells.

[0161] In one embodiment, the present invention relates to the use of a RAR antagonist at a concentration of 20 μM, followed by the use of a RAR agonist, to induce pancreatic endoderm cells from definitive endoderm cells derived from human pluripotent stem cells.

[0162] In one embodiment, the present invention relates to the use of a RAR antagonist in combination with LDN followed by AM580 to induce pancreatic endoderm cells from definitive endoderm cells derived from human pluripotent stem cells.

[0163] In one embodiment, the present invention relates to the use of an RAR antagonist to improve synchronization of pancreatic endodermal progenitor cells prior to inducing pancreatic endodermal cells by treating the pancreatic endodermal progenitor cells with an RAR agonist.

[0164] In one embodiment, the present invention relates to a method for differentiating DE into PE, the method comprising a first step of culturing DE in a culture medium containing an RAR antagonist to obtain pancreatic endoderm precursors, followed by a second step of culturing the pancreatic endoderm precursors in a cell culture medium containing an RAR agonist, wherein the first step has a duration of 1 hour to 6 days or 1 to 4 days.

[0165] In one embodiment, the present invention relates to a method for differentiating DE into PE, the method comprising a first step of culturing DE in a culture medium containing an RAR antagonist to obtain pancreatic endoderm precursors, followed by a second step of culturing the pancreatic endoderm precursors in a cell culture medium containing an RAR agonist, the first step having a duration of 2 days.

[0166] In one embodiment, the present invention relates to a method for differentiating DE into PE, the method comprising a first step of culturing DE in a culture medium containing an RAR antagonist to obtain pancreatic endoderm precursors, and a subsequent second step of culturing the pancreatic endoderm precursors in a cell culture medium containing an RAR agonist, wherein the first step has a duration of less than 2 days.

[0167] Retinoic acid receptor (RAR) agonists Retinoic receptor agonists selectively bind to and activate one or more of the retinoic acid receptor subtypes, RARα, RARAβ, and RARγ.

[0168] Non-limiting examples of RAR agonists include AM580, all-trans retinoic acid, 9-cis retinoic acid, AC 261066, AC 55649, adapalene, AM 80, BMS 753, BMS 961, CD 1530, CD 2314, CD 437, Ch 55, isotretinoin, tazarotene, TTNTB, and EC19.

[0169] In one embodiment, the RAR agonist is AM580.

[0170] In one embodiment, the present invention relates to the use of a RAR agonist at a concentration in the range of 0.05 to 10 μM, preceded by a RAR antagonist, for inducing pancreatic endoderm cells from human pluripotent stem cells.

[0171] In one embodiment, the present invention relates to the use of a RAR agonist at a concentration of 1 μM preceded by a RAR antagonist to induce pancreatic endoderm cells from human pluripotent stem cells.

[0172] In one embodiment, the present invention relates to the use of a RAR agonist at a concentration of 10 μM preceded by a RAR antagonist to induce pancreatic endoderm cells from human pluripotent stem cells.

[0173] BMP inhibitors Bone morphogenetic proteins (BMPs) are signaling molecules that act locally on target cells to affect cell survival, proliferation, and differentiation. Although initially identified as osteoinductive agents, BMPs are now known to affect the formation and function of many organ systems. BMP receptor antagonists or BMP inhibitors inhibit BMP signaling, specifically by inhibiting Smad1 / 5 / 8 phosphorylation by ALK1, ALK2, ALK3, and ALK6.

[0174] Non-limiting examples of BMP inhibitors include LDN 193189, dorsomorphin, noggin, chordin, LDN 212854, LDN 214117, ML 347, DMH1, DMH2, and K 02288.

[0175] In one embodiment, the BMP inhibitor is LDN193189.

[0176] In one embodiment, the invention relates to the use of a RAR antagonist in combination with the BMP inhibitor LDN, wherein the BMP inhibitor is at a concentration range of 25-200 nM.

[0177] In one embodiment, the invention relates to the use of a RAR antagonist in combination with the BMP inhibitor LDN, wherein the BMP inhibitor is at a concentration of 50 nM.

[0178] In one embodiment, the invention relates to the use of a RAR antagonist in combination with the BMP inhibitor LDN, wherein the BMP inhibitor is at a concentration of about 50 nM.

[0179] ROCK inhibitors Rho-associated coiled-coil-containing kinase (ROCK) is an effector of the RhoA small GTPase and belongs to the AGC family of serine / threonine kinases. ROCK kinases have many functions, including cell contraction, migration, apoptosis, survival, and proliferation. Rho-associated, coiled-coil-containing protein kinase ROCK inhibitors are a class of compounds that target and inhibit rho kinase.

[0180] In one embodiment, the Rock inhibitor is Tiger or Y27632.

[0181] In one embodiment, the present invention relates to the use of a RAR antagonist in combination with a Rock inhibitor, wherein the Rock inhibitor is at a concentration ranging from 1 to 20 μM.

[0182] In one embodiment, the present invention relates to the use of a RAR antagonist in combination with a Rock inhibitor, wherein the Rock inhibitor is at a concentration of 5 μM.

[0183] bFGF Basic fibroblast growth factor (FGF), also known as FGF2, is a growth factor and signaling protein encoded by the FGF2 gene.

[0184] In one embodiment, the growth factor is bFGF.

[0185] In one embodiment, the invention relates to the use of a RAR antagonist in combination with bFGF, wherein the bFGF is at a concentration ranging from 25 to 200 nM.

[0186] bioreactor Suspension culture bioreactors allow for the large-scale expansion and differentiation of stem cells and / or their progeny in a controlled and reproducible culture system. These systems provide a homogenous culture environment in which conditions such as temperature, pH, and oxygen concentration can be monitored and controlled. Furthermore, these systems allow for the production of large numbers of cells under consistent culture conditions and with minimal culture variability.

[0187] Culture media / compositions A solid, liquid, or semi-solid medium designed to support the growth of microorganisms or cells. Different types of commercial media are used for the growth of different types of cells.

[0188] In one embodiment, the present invention relates to a culture medium comprising a retinoic acid receptor (RAR) antagonist for generating pancreatic endoderm precursors from definitive endoderm derived from human pluripotent stem cells.

[0189] In one embodiment, the present invention relates to a culture medium comprising AGN 193109 for generating pancreatic endoderm precursors from definitive endoderm derived from human pluripotent stem cells.

[0190] In one embodiment, the present invention relates to a culture medium comprising a retinoic acid receptor (RAR) antagonist followed by a RAR agonist for generating pancreatic endoderm / PDX1+ / NKX6.1+ from definitive endoderm derived from human pluripotent stem cells.

[0191] In one embodiment, the present invention relates to a culture medium comprising AGN 193109 followed by AM580 for generating pancreatic endoderm / PDX1+ / NKX6.1+ from definitive endoderm derived from human pluripotent stem cells.

[0192] In one embodiment, the present invention relates to a culture medium comprising a retinoic acid receptor (RAR) antagonist.

[0193] In one embodiment, the present invention relates to a culture medium comprising a retinoic acid receptor (RAR) antagonist, to which a retinoic acid receptor (RAR) agonist is subsequently added.

[0194] In another embodiment, the present invention relates to a culture medium comprising a retinoic acid receptor (RAR) antagonist and a BMP inhibitor, to which a retinoic acid receptor (RAR) agonist is subsequently added.

[0195] In another embodiment, the present invention relates to a culture medium comprising a retinoic acid receptor (RAR) antagonist, a BMP inhibitor, and a Rock inhibitor, to which a retinoic acid receptor (RAR) agonist and a Rock inhibitor are subsequently added.

[0196] In another embodiment, the present invention relates to a culture medium comprising a retinoic acid receptor (RAR) antagonist, a BMP inhibitor, and a Rock inhibitor, followed by the addition of a retinoic acid receptor (RAR) agonist, a Rock inhibitor, and FGF2.

[0197] In one embodiment, the present invention relates to a culture medium comprising a retinoic acid receptor (RAR) antagonist, wherein the RAR antagonist is AGN 193109, followed by the addition of a retinoic acid receptor (RAR) agonist, wherein the RAR agonist is AM580.

[0198] In one embodiment, the present invention relates to a culture medium comprising a retinoic acid receptor (RAR) antagonist, wherein the RAR antagonist is AGN 193109.

[0199] In one embodiment, the present invention relates to a culture medium further comprising the BMP inhibitor LDN193189.

[0200] In one embodiment, the present invention relates to a culture medium further comprising FGF2.

[0201] In one embodiment, the present invention relates to a culture medium further comprising the Rock inhibitor Tiger or Y27632.

[0202] In one embodiment, the present invention relates to a composition comprising: a) culture medium containing a retinoic acid receptor (RAR) antagonist, such as AGN 193109; b) Definitive endoderm cells and / or pancreatic endoderm cells derived from human pluripotent stem cells.

[0203] protocol As used herein, the term "LDN phase" refers to the 4 days from PE0 to PE3, where LDN is added for the first 2 days, i.e., PE0 to PE1, and LDN and the RAR antagonist are added for the last 2 days, i.e., PE2 to PE3.

[0204] Cells differentiated to the DE stage in suspension culture in shake flasks or bioreactors are followed by PE differentiation (WO2012 / 175633, incorporated herein by reference in its entirety). AGN193109 is added to the culture medium during the 2-day LDN stage. PE induction, the second stage of the PE differentiation protocol, is performed using standard protocols (WO2014 / 033322, incorporated herein by reference in its entirety) without modification.

[0205] Protocols for obtaining pancreatic cells from stem cells are exemplified by, but not limited to, those described in D'Amour, KA et al. (2006), Jiang, J. et al. (2007), and Kroon, E. et al. (2008).

[0206] Protocols for obtaining pancreatic cells from somatic cells or somatic cells induced to dedifferentiate into pluripotent cells such as ES-like cells are exemplified by, but not limited to, those described in Aoi, T. et al. (2008), D'Amour, K. A. et al. (2006), Jiang, J. et al. (2007), Kroon, E. et al. (2008), Takahashi, K. et al. (2007), Takahashi, K., and Yamanaka, S. (2006), and Wernig, M. et al. (2007).

[0207] Unless otherwise indicated herein, terms provided in the singular also include plural references.

[0208] The present invention is further illustrated by the following non-limiting embodiments: 1. A method for differentiating definitive endoderm cells derived from human pluripotent stem cells into pancreatic endoderm precursors, the method comprising culturing the definitive endoderm (DE) in a culture medium containing a retinoic acid receptor (RAR) antagonist. 2. The method of embodiment 1, wherein the method further comprises culturing pancreatic endoderm precursors in a cell culture medium containing an RAR agonist, thereby inducing pancreatic endoderm (PE), and the cells are NKX6.1+ / PDX1+ double positive. 3. The method of embodiment 2, wherein the culture medium optionally further comprises a BMP inhibitor, a growth factor, and / or a Rock inhibitor. 4. The method of any one of the preceding embodiments, wherein the retinoic acid receptor (RAR) antagonist is AGN 193109. 5. The method of any one of the preceding embodiments, wherein said RAR agonist is AM580. 6. The method of embodiments 3-5, wherein the optional BMP inhibitor is LDN193189. 7. The method of embodiments 3-5, wherein the optional growth factor is bFGF2. 8. The method of embodiments 3-5, wherein the optional lock inhibitor is Tiger or Y27632. 9. The method of any one of the preceding embodiments, wherein said human pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells. 10. The method of any one of the preceding embodiments, wherein the concentration of the retinoic acid receptor (RAR) antagonist is 0, 5 to 100 μM, 1 to 50 μM, 1 to 300 μM, 1 to 250 μM, 1 to 20 μM, 3 to 17 μM, 5 to 15 μM, or 7 to 12 μM. 11. The method of any one of the preceding embodiments, wherein the concentration of the retinoic acid receptor (RAR) antagonist is 0.5 to 100 μM. 12. The method of any one of the preceding embodiments, wherein the concentration of the retinoic acid receptor (RAR) antagonist is 0.5 to 50 μM. 13. The method of any one of the preceding embodiments, wherein the concentration of the retinoic acid receptor (RAR) antagonist is 0.5 to 30 μM. 14. The method of any one of the preceding embodiments, wherein the concentration of the retinoic acid receptor (RAR) antagonist is 0.5 to 25 μM. 15. The method of any one of the preceding embodiments, wherein the concentration of the retinoic acid receptor (RAR) antagonist is 0.5 to 20 μM. 16. The method of any one of the preceding embodiments, wherein the concentration of the retinoic acid receptor (RAR) antagonist is 1-20 μM. 17. The method of any one of the preceding embodiments, wherein the concentration of the retinoic acid receptor (RAR) antagonist is 10-20 μM. 18. The method of any one of the preceding embodiments, wherein the concentration of the retinoic acid receptor (RAR) antagonist is 7-12 μM. 19. The method of any one of the preceding embodiments, wherein the concentration of the retinoic acid receptor (RAR) antagonist is 10 μM. 20. The method of any one of the preceding embodiments, wherein the concentration of the retinoic acid receptor (RAR) antagonist is 15 μM. 21. The method of any one of the preceding embodiments, wherein the concentration of the retinoic acid receptor (RAR) antagonist is 20 μM. 22. The method of any one of the preceding embodiments, wherein the step of embodiment 1 has a duration of 1 hour to 6 days or 1 to 4 days. 23. The method of any one of the preceding embodiments, wherein the step of embodiment 1 has a duration of 2 days. 24. The method of any one of the preceding embodiments, wherein the step of embodiment 1 has a duration of less than 2 days. 25. The method of any one of the preceding embodiments, wherein at least 5% of the pancreatic endoderm cells co-express PDX1 and NKX6.1. 26. The method of any one of the preceding embodiments, wherein the pancreatic endoderm cells are at least 5%, at least 10%, 10-30%, 10-40%, 5-70%, 10-80%, or 5-100% PDX1+ / NKX6.1+ double positive. 27. The method of any one of the preceding embodiments, wherein said pancreatic endoderm cells are at least 70% PDX1+ / NKX6.1+ double positive. 28. The method of any one of the preceding embodiments, wherein said pancreatic endoderm cells are at least 80% PDX1+ / NKX6.1+ double positive. 29. The method of any one of the preceding embodiments, wherein said pancreatic endoderm cells are at least 90% PDX1+ / NKX6.1+ double positive. 30. The method of any one of the preceding embodiments, wherein said pancreatic endoderm cells are at least 95% PDX1+ / NKX6.1+ double positive. 31. The method of any one of the preceding embodiments, wherein said pancreatic endoderm cells are at least 98% PDX1+ / NKX6.1+ double positive. 32. The method of any one of the preceding embodiments, wherein said definitive endoderm cells are SOX17+ / FOXA2+ / CXCR4+ triple positive. 33. The method of any one of the preceding embodiments, wherein the pancreatic endoderm cells are less than 1% NKX2.2 positive. 34. The method of any one of the preceding embodiments, wherein the method can aid in suboptimal PE differentiation. 35. The method of any one of the preceding embodiments, wherein the method includes an LDN step. 36. The method of any one of the preceding embodiments, wherein the method is shorter than the standard method, the LDN stage being 2 days. 37. The method of any one of the preceding embodiments, comprising incubating DE cells in vitro. 38. The method of any one of the preceding embodiments 1-35, comprising incubating DE cells in vivo. 39. The method of any one of the preceding embodiments 1-35, further comprising isolating the differentiated cells. 40. The method of any one of the preceding embodiments 1-35, further comprising storing the differentiated cells. 41. A pancreatic endoderm cell, which is a product of the method according to any one of the preceding embodiments. 42. A pancreatic endoderm cell culture or pancreatic endoderm cell population comprising a plurality of cells according to embodiment 41. 43. Pancreatic endoderm cells obtainable by the methods described in embodiments 1 to 40. 44. Pancreatic endoderm cells according to embodiment 43, having increased expression of Nkx6.1+ / Pdx1+ double positives. 45. The pancreatic endoderm cells of embodiment 43, wherein the pancreatic endoderm cells are at least 70% PDX1+ / NKX6.1+ double positive. 46. ​​The pancreatic endoderm cells of embodiment 43, wherein the pancreatic endoderm cells are at least 80% PDX1+ / NKX6.1+ double positive. 47. The pancreatic endoderm cells of embodiment 43, wherein the pancreatic endoderm cells are at least 90% PDX1+ / NKX6.1+ double positive. 48. The pancreatic endoderm cells of embodiment 43, wherein the pancreatic endoderm cells are at least 95% PDX1+ / NKX6.1+ double positive. 49. The pancreatic endoderm cells of embodiment 43, wherein the pancreatic endoderm cells are at least 98% PDX1+ / NKX6.1+ double positive. 50. The pancreatic endoderm cells of embodiment 43, wherein the pancreatic endoderm cells are 80-100% PDX1+ / NKX6.1+ double positive. 51. The pancreatic endoderm cells of embodiment 43, wherein the pancreatic endoderm cells are 90-100% PDX1+ / NKX6.1+ double positive. 52. The pancreatic endoderm cells of embodiment 43, wherein the pancreatic endoderm cells are less than 1% NKX2.2 positive. 53. The pancreatic endoderm cells of embodiment 43, wherein the expression of NGN3 and NeuroD is reduced. 54. The pancreatic endoderm cells of embodiment 43, wherein the expression of NGN3 and NeuroD is reduced by 2-fold. 55. The pancreatic endoderm cells of embodiment 43, wherein the PE are further differentiated into beta cells having at least 40% C-PEP+ / NKX6.1+. 56. Pancreatic endoderm cells according to embodiment 43, in which the PE is derived selectively, more homogeneously and more efficiently from EP cells and also from beta cells. 57. Pancreatic endoderm cells according to embodiments 43 to 56, wherein the PE cells are derived in vitro or in vivo. 58. The pancreatic endoderm cells of embodiment 57, wherein the PE cells are derived in vitro. 59. The pancreatic endoderm cells of embodiment 57, wherein the PE cells are derived in vivo. 60. Pancreatic endoderm cells according to any one of embodiments 43 to 59 for further differentiation into beta cells, for use as a medicament. 61. Pancreatic endoderm cells according to any one of embodiments 43 to 59 for further differentiation into beta cells, for use as a medicament in the treatment of diabetes, by administering the stem cells or stem cell-derived tissue or organ to a subject, or by grafting the stem cells or stem cell-derived tissue or organ to a subject, or by transplanting the stem cells or stem cell-derived tissue or organ to a subject. 62. A culture medium containing a retinoic acid receptor (RAR) antagonist for generating pancreatic endoderm precursors from definitive endoderm cells derived from human pluripotent stem cells. 63. The culture medium of embodiment 62, further comprising a retinoic acid receptor (RAR) agonist for generating pancreatic endoderm cells, wherein the pancreatic endoderm cells are at least 70% PDX1+ / NKX6.1+ double positive. 64. The culture medium of embodiment 62, further comprising a BMP inhibitor. 65. The culture medium of embodiment 62, further comprising a lock inhibitor. 66. The culture medium of embodiment 63, further comprising FGF2. 67. The culture medium of embodiment 62, wherein the retinoic acid receptor (RAR) antagonist is AGN 193109. 68. The culture medium of embodiment 63, wherein the retinoic acid receptor (RAR) agonist is AM580. 69. The culture medium of embodiment 62, wherein the additional BMP inhibitor is LDN193189. 70. The culture medium of embodiment 62, wherein the Rock inhibitor is Tiger or Y27632. 71. Use of a retinoic acid receptor (RAR) antagonist at a concentration in the range of 0.5 to 100 μM to induce pancreatic endoderm cells from definitive endoderm cells derived from human pluripotent stem cells. 72. The use according to embodiment 71, wherein the retinoic acid receptor (RAR) antagonist is at a concentration ranging from 0.5 to 50 μM for inducing pancreatic endoderm cells from definitive endoderm cells derived from human pluripotent stem cells. 73. The use according to embodiment 71, wherein the retinoic acid receptor (RAR) antagonist is at a concentration ranging from 0.5 to 30 μM for inducing pancreatic endoderm cells from definitive endoderm cells derived from human pluripotent stem cells. 74. The use according to embodiment 71, wherein the retinoic acid receptor (RAR) antagonist is at a concentration ranging from 0.5 to 20 μM for inducing pancreatic endoderm cells from definitive endoderm cells derived from human pluripotent stem cells. 75. The use according to embodiment 71, wherein the retinoic acid receptor (RAR) antagonist concentration is in the range of 10 to 20 μM. 76. The use according to embodiment 71, wherein the retinoic acid receptor (RAR) antagonist concentration is about 10 μM. 77. The use according to embodiment 71, wherein the retinoic acid receptor (RAR) antagonist concentration is about 20 μM. 78. Use of the retinoic acid receptor (RAR) antagonist AGN193109 at concentrations ranging from 0.5 to 100 μM to induce pancreatic endoderm progenitor cells from definitive endoderm cells derived from human pluripotent stem cells. 79. The use according to embodiment 78, wherein the retinoic acid receptor (RAR) antagonist AGN193109 is at a concentration of 0.5 to 50 μM. 80. The use according to embodiment 78, wherein the retinoic acid receptor (RAR) antagonist AGN193109 is at a concentration of 0.5 to 30 μM. 81. The use according to embodiment 78, wherein the retinoic acid receptor (RAR) antagonist AGN193109 is at a concentration of 0.5 to 20 μM. 82. The use according to embodiment 78, wherein the retinoic acid receptor (RAR) antagonist AGN193109 is at a concentration of about 10 μM. 83. The use according to embodiment 78, wherein the retinoic acid receptor (RAR) antagonist AGN193109 is at a concentration of about 20 μM. 84. Use of the retinoic acid receptor (RAR) antagonist AGN193109 in combination with a BMP inhibitor, followed by AM580 in combination with an FGF2 and / or Rock inhibitor, to induce pancreatic endoderm cells from human pluripotent stem cells. 85. The use according to embodiment 84, wherein the BMP inhibitor is LDN193189. 86. The use according to embodiment 84, wherein the Rock inhibitor is Tiger or Y27632. 87. A synchronized pancreatic endoderm cell population derived from human pluripotent stem cells, exhibiting at least 70% pancreatic endoderm cells expressing PDX1+ / NKX6.1+ double positivity. 88. The synchronized pancreatic endoderm cell population of embodiment 87, wherein the cell population further expresses PTF1a. 89. The synchronized pancreatic endoderm cell population of embodiment 87, wherein the cell population further expresses at least 30% DLK1. 90. The synchronized pancreatic endoderm cell population of embodiment 87, wherein the cell population further expresses 20-80% DLK1. 91. The synchronized pancreatic endoderm cell population of embodiment 87, wherein the cell population further expresses at least 30-80% DLK1. 92. The synchronized pancreatic endoderm cell population of embodiment 87, wherein the cell population expresses less than 1% NKX2.2. 93. The synchronized pancreatic endoderm cell population of embodiment 87, wherein expression of NGN3 and NeuroD is reduced. 94. The synchronized pancreatic endoderm cell population of embodiment 87, wherein expression of NGN3 and NeuroD is reduced by 2-fold. 95. The synchronized pancreatic endoderm cell population of embodiment 87, wherein the PEs are further differentiated into beta cells having at least 40% C-PEP+ / NKX6.1+. 96. A synchronized pancreatic endoderm cell population according to embodiments 87 to 95, wherein the PE is more selectively, more homogeneously and more efficiently induced into EP cells and more selectively into beta cells. 97. The synchronized pancreatic endoderm cell population of embodiments 87-95, wherein the PE cells are derived in vitro or in vivo. 98. The synchronized pancreatic endoderm cell population of embodiment 97, wherein the PE cells are derived in vitro. 99. The synchronized pancreatic endoderm cell population of embodiment 97, wherein the PE cells are derived in vivo. 100. A synchronized pancreatic endoderm cell population according to embodiments 87 to 99 for further differentiation into beta cells, for use as a medicament. 101. A synchronized pancreatic endoderm cell population according to embodiments 87 to 100 for further differentiation into beta cells, for use as a medicament in the treatment of diabetes by administering stem cells or stem cell-derived tissue or organs to a subject, or by grafting stem cells or stem cell-derived tissue or organs to a subject, or by transplanting stem cells or stem cell-derived tissue or organs to a subject. 102. Use of pancreatic endoderm cells according to embodiments 41 to 61 or synchronized cell populations according to embodiments 87 to 101 in the preparation of a medicament for stimulating or enhancing the formation and / or regeneration and / or repair of a tissue or organ in a subject. 103. a. A culture medium containing a retinoic acid receptor (RAR) antagonist (AGN 193109); b. A composition comprising definitive endoderm cells and / or pancreatic endoderm cells derived from human pluripotent stem cells. 104. A pharmaceutical composition comprising pancreatic endoderm cells for further differentiation of PE cells into beta cells or cell populations thereof according to any one of embodiments 41 to 61 or 87 to 101, and a pharmaceutically acceptable carrier. 105. A composition comprising pancreatic endoderm cells for further differentiation of PE cells into beta cells or cell populations thereof according to any one of embodiments 41 to 61 or 87 to 101, and a biocompatible scaffold or matrix. 106. A bioreactor comprising pancreatic endoderm cells obtained by the method according to embodiments 1 to 40. 107. A method for treating a patient with type 1 diabetes, comprising administering cells according to embodiments 41-61 or 87-101, cells obtained according to the methods described in embodiments 1-40, or cells according to embodiments 1-40 for further differentiation into beta cells. 108. A method of treating a patient with type 1 diabetes, comprising administering cells according to embodiments 41-61 or 87-101 for further differentiation into beta cells. 109. A method for preventing or treating diabetes, comprising administering, transplanting, or grafting to said subject an effective amount of pancreatic endoderm cells or a population of pancreatic endoderm cells of any one of embodiments 41-61 or 87-101 that have been further differentiated into beta cells, thereby preventing or treating diabetes in the subject. 110. A kit for regenerating and / or repairing and / or constructing tissue or organs, comprising: (i) a pancreatic endoderm cell or a pancreatic endoderm cell population according to any one of embodiments 41-61 or 87-101 for further differentiation into beta cells; (ii) a biocompatible scaffold or matrix; (iii) optionally, at least one growth factor or functional fragment thereof; (iv) optionally, an agent selected from the group consisting of a BMP inhibitor, a growth factor, and / or a Rock inhibitor, and combinations thereof; (iv) optionally, instructions for preparing, maintaining, and / or using the cells, including any cell culture or tissue or organ derived therefrom.

[0209] material and method List of Abbreviations +ve:positive BC: Beta cells (e.g., BC7: day 7 of beta cell differentiation) bFGF: basic fibroblast growth factor (FGF) (also known as FGF2) Cyc: Cyclopamine db: double positive DE: Definitive endoderm EP: Endocrine precursor cells hBS: human blastocyst-derived stem cells hBSC; human blastocyst-derived stem cells hES: human embryonic stem cells hESC: human embryonic stem cells hiPSC: human induced pluripotent stem cells hPSC: human pluripotent stem cells KOSR: Knockout Serum Replacement NKX6.1: NK6 homeobox 1 PDX1: pancreatic and duodenal homeobox 1 PE: pancreatic endoderm (e.g., PE10: day 10 of pancreatic endoderm differentiation) PEST: Penicillin Streptomycin PS: Pluripotent stem Rocki: Rho kinase inhibitor III RT: room temperature

[0210] General Preparation Method Pluripotent stem cell culture Human embryonic stem (hES) cell line SA121 (Cellectis) is grown in DEF-CS culture medium (Takara BIO Europe) in fibronectin (Sigma)-coated culture flasks (Corning) with 30 ng / mL bFGF (Peprotech) and 10 ng / mL Noggin (Peprotech). Cells are passaged in 10 μM Rockland-Aldrich inhibitor Y-27632 (Sigma #Y0503) and seeded at a density of 0.5–1 μg / mL in shake flasks or bioreactors (DASBOX, DASGIP, Eppendorf) to allow single cells to form clusters under constant agitation.

[0211] Differentiation of pluripotent stem cells into definitive endoderm (DE) After 1-3 days in DEF-CS medium with daily medium changes, clusters were differentiated into DE cells as described in WO2012 / 175633 (incorporated herein by reference in its entirety). Cell clusters were washed once in RPMI 1640 (Gibco #61870) and treated with 2-7 μM CHIR99021 (Axon #1386) in RPMI 1640. After 24 hours, cells were washed with RPMI 1640 and treated with 25-100 ng / mL activin A (Peprotech #120-14E) and 2% B27 (Invitrogen #17504-044) in RPMI 1640 for 3 days, with medium changes every 24-48 hours. During differentiation into the DE, cells are maintained at 37°C and 5% CO in a humidified shaking incubator (Infors) or in a bioreactor system (DASBOX, DASGIP, Eppendorf). At the end of differentiation into the DE, 95% of the population expresses SOX17 and less than 1% expresses the pluripotency marker OCT4.

[0212] [Example 1] [Definitive endoderm differentiation to pancreatic endoderm is improved by adding AGN193109 at the LDN stage] In a standard protocol for differentiation into pancreatic endoderm, as described in WO2014 / 033322 (incorporated herein by reference in its entirety), clusters to be differentiated into the DE were washed once in RPMI1640 (Gibco #61870) and differentiated for 4 days in the presence of LDN193189 (Stemgent 04-0074) and Rock-I (Sigma Y27632-Y0503) in RPMI1640 with 12% KOSR (Gibco 10828-028), then subjected to medium containing AM580 (ENZO / Biomol GR104-0025), bFGF (#100-18B, Peprotech), JnkiII (Calbiochem 420119), and Rock-I for 6–9 days. The medium was changed daily or every other day. At the end of PE differentiation, clusters were analyzed by flow cytometry or gene expression analysis (Nanostring). 10 μM AGN193109 (Tocris 5758) was added to the LDN stage of PE differentiation, but the rest of the protocol was unchanged.

[0213] Addition of 10 μM AGN 193109 during the last 2 days of the LDN stage (PE2-3) significantly increased the percentage of Pdx1 / Nkx6.1 double-positive pancreatic endoderm at day 10 of PE differentiation (PE10) (Figure 1A, B). With AGN193109, at least 70% Pdx1 / Nkx6.1 double-positive cells were observed, whereas in standard conditions, this was approximately 40% and less than 10% Pdx1 / Nkx6.1 double-negative cells.

[0214] Furthermore, it appears that the addition of AGN193109 as described can rescue otherwise suboptimal PE differentiation, as an increase in Pdx1 / Nkx6.1 double-positive cells from 20% to 75% was observed with AGN193109 treatment ( Figure 1C ).

[0215] Furthermore, addition of AGN 193109 also increased other markers of pancreatic endoderm, such as PTF1A, CPA1, and DLK1 (Figure 2A). Instead, when RAR signaling was activated with AM580 on PE2-3, a significant downregulation of Nkx6.1, PTF1A, and DLK1 was observed, indicating that the effects of AGN193109 are mediated specifically by the inhibition of RAR signaling (Figure 2A).

[0216] Furthermore, flow cytometry confirmed that DLK1 was expressed in a higher proportion of pancreatic endoderm cells when AGN 193103 was added to PE2-3 (31% vs. 13%). DLK1 was identified as a marker of pancreatic endoderm, primarily in cells expressing high levels of Nkx6.1 (Figure 2B).

[0217] Furthermore, differentiation is more synchronized with a lower degree of immature endocrine differentiation at the PE stage, characterized by lower expression of Ngn3 and NeuroD1 compared to conditions without AGN193109 addition (Figure 3A). Again, activation of RAR signaling by AM580 appears to have the opposite effect, resulting in a significant increase in Ngn3 and NeuroD1 already at PE10 (Figure 3A).

[0218] The percentage of Nkx2.2-positive cells, representing cells committed to an endocrine fate, was reduced to less than 1% with AGN193109 treatment compared to 2.9% in controls at the PE stage (Fig. 3B).

[0219] Furthermore, flow cytometry demonstrated that the effect of AGN193109, as evidenced by gene expression analysis, is likely a specific effect of retinoic acid receptor (RAR) inhibition. Addition of the RAR agonist AM580 instead produced the opposite phenotype: a significant reduction in the proportion of PDX1 / NKX6.1 double-positive pancreatic endoderm in PE10 cells (Figure 4A). Treatment with AGN193109 increased the number of PDX1 / NKX6.1 double-positive cells from 52% to 76.9%, while treatment with AM580 reduced the number to approximately 14%.

[0220] The positive effects of AGN193109 could be reproduced in a 1 L bioreactor (DASgip, Eppendorf). The data demonstrate that the modifications to the AGN193109 protocol are compatible with the bioreactor format. Although experiments were not performed in parallel, the results suggest that AGN193109 in the bioreactor format offers advantages in PE induction compared to the standard protocol (Figure 8A). Using the standard protocol, we obtained 58.4% Pdx1 / Nkx6.1 double positivity, whereas with AGN193109 treatment, the figure was 82.2.

[0221] The addition of AGN193109 could be extended from 2 to 4 days at the LDN stage without any adverse effect on PE induction, as measured by Pdx1 / Nkx6.1 double positivity (Fig. 6A).

[0222] Quite surprisingly, the length of the LDN stage could be reduced to only 2 days in the presence of AGN 193109, thus shortening the length of the protocol for induction of PE by 2 days (Figure 6B).

[0223] The effect of AGN193109 is dose-responsive and is demonstrated in the range of 0.5 µM to 20 µM. An increase in the number of Pdx1 / Nkx6.1 double-positive cells and a decrease in Pdx1-only positive cells are observed without increasing the concentration (Figure 5A). No further effect is observed by increasing the concentration from 10 µM to 20 µM (Figure 5B).

[0224] Example 2 [Improvement of differentiation into Cpep / Nkx6.1 double-positive beta-like cells after addition of AGN193109 during the LDN stage] Human ES cell- or human iPS cell-derived PE clusters were washed and further differentiated into endocrine precursor cells (EP) and beta cell-like cells (BC) using protocols for endocrine and beta cell induction as described in WO2015 / 028614, WO2017 / 144695, and WO / 2019 / 048690 (all of which are incorporated herein by reference in their entireties).

[0225] At the end of BC differentiation, clusters were sampled, dissociated into single cells with TrypLE-Select (Gibco 12563-011), fixed in 10% formalin, stained for Cpep / Nkx6.1 and glucagon, and analyzed by flow cytometry.

[0226] After including AGN193109 in the LDN stage of the PE protocol, a clear increase in the percentage of Cpep / Nkx6.1 double-positive beta-like cells was observed, indicating improved PE quality when AGN193109 was used. Cpep / Nkx6.1 double positivity increased from approximately 35% to approximately 50% with the addition of AGN193109 (Figure 7A,B). This increase was not accompanied by an increase in glucagon-positive cells, indicating a specific effect of AGN193109 on the ability of PE to form beta-like cells in later differentiation (Figure 7B).

[0227] From a cell therapy perspective, a stable phenotype of the therapeutic cell type is a major advantage, as it allows for the transportation and storage of the cell product prior to transplantation into the patient. Addition of AGN193109 during PE induction was observed to improve the stability of the beta cell phenotype over long-term in vitro culture. In experiments with a nearly identical phenotype on day 6 of BC differentiation, a dramatic decrease was observed one week later on day 13 of BC for the standard protocol. From day 6 to day 13 of BC, the standard protocol reduced Cpep / Nkx6.1 double positivity from 53% to 27%, while the AGN193109 protocol had only a slight decrease in double positivity, from 59% to 57% (Figure 9A).

[0228] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those skilled in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes that fall within the true spirit of the invention.

Claims

1. A method for inducing pancreatic endoderm cells from definitive endoderm derived from human pluripotent stem cells, comprising the steps of culturing the definitive endoderm in a culture medium containing a retinoic acid receptor (RAR) antagonist to obtain pancreatic endoderm precursors, and then culturing the pancreatic endoderm precursors in a cell culture medium containing an RAR agonist, thereby inducing pancreatic endoderm, wherein the pancreatic endoderm cells are PDX1+ / NKX6.1+ double positive.

2. 2. The method of claim 1, wherein the retinoic acid receptor (RAR) antagonist is selected from the group consisting of AGN 193109, AGN 194431, AGN 194301, SR 11335, BMS 453, BMS 195614, LE 135, LG 100815, MM11253, CD 2665, and ER 50891.

3. 3. The method of claim 2, wherein the retinoic acid receptor (RAR) antagonist is AGN 193109.

4. 4. The method of any one of claims 1 to 3, wherein the concentration of the retinoic acid receptor (RAR) antagonist is 0.5 to 100 μM, 1 to 50 μM, 1 to 300 μM, 1 to 250 μM, 1 to 20 μM, 3 to 17 μM, 5 to 15 μM, or 7 to 12 μM.

5. A method according to any one of claims 1 to 4, wherein the step of culturing the definitive endoderm in a culture medium containing an RAR antagonist to obtain pancreatic endoderm precursors has a duration of 1 hour to 6 days or 1 to 4 days.

6. 6. The method according to any one of claims 1 to 5, wherein the steps defined in claim 5 are reduced to two days.

7. A synchronized pancreatic endoderm cell population derived from human pluripotent stem cells, wherein at least 70% of the pancreatic endoderm cells are PDX1+ / NKX6.1+ double positive, the cell population further expresses PTF1a and at least 30% DLK1, and the expression of NGN3 and NeuroD is reduced by at least 2-fold compared to a pancreatic endoderm cell population obtained without a retinoic acid receptor (RAR) antagonist.

8. The synchronized cell population of claim 7, wherein the cell population expresses less than 1% NKX2.

2.

9. 9. The synchronized cell population of claim 7 or 8 for further differentiation into beta cells for use as a medicament.

10. 9. The synchronized cell population of claim 7 or 8 for further differentiation into beta cells, for use as a medicament in the treatment of diabetes by administering the stem cells or stem cell-derived tissue or organ to a subject, or by grafting the stem cells or stem cell-derived tissue or organ to a subject, or by transplanting the stem cells or stem cell-derived tissue or organ to a subject.

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