Differentiation of stem cells into pancreatic endocrine cells

Abstract

An in vitro method for producing a population of pancreatic endocrine (PEC) cells comprising the steps of i) differentiating a population of pancreatic endoderm (PE) cells into pancreatic endocrine progenitor cells wherein the differentiation comprises treating the pancreatic endoderm (PE) cells with an inhibitor of vascular endothelial growth factor (VEGF) receptor and / or platelet-derived growth factor (PDGF) receptor, and ii) differentiating the pancreatic endocrine progenitor (EP) cells into pancreatic endocrine (PEC) cells by treating the pancreatic endocrine progenitor (EP) cells with an inhibitor of vascular endothelial growth factor (VEGF) receptor and / or platelet-derived growth factor (PDGF) receptor.

Description

Technical Field

[0001] The present invention relates to an in vitro method for obtaining pancreatic endocrine (PEC) cells, which comprises the step of treating the cells with an inhibitor of vascular endothelial growth factor (VEGF) receptor and / or platelet-derived growth factor (PDGF) receptor. The method provides pancreatic islet-like cells, in particular β-like cells. Background Art

[0002] Although insulin therapy can save lives, it can be difficult to achieve stable blood sugar with exogenous insulin, and poor control can lead to serious late complications (Nathan, DM, 2014. The diabetes control and complications trial / epidemiology of diabetes interventions and complications study at 30 years: overview. Diabetes care, 37 (1), pp. 9–16). Transplanting islets isolated from human donors into patients with type 1 diabetes has shown good results, with some patients becoming completely independent of insulin (Barton FB et al., 2012. Improvement in Outcomes of Clinical Islet Transplantation: 1999-2010. Diabetes Care, 35 (7), pp. 1436–1445). Despite these advances, one of the main challenges of islet transplantation is the limited availability of donor islets. This shortage of donor material can be overcome by generating functional insulin-secreting cells in vitro via differentiation of human embryonic stem cells (hESCs). Protocols for generating functional insulin-secreting cells from stem cells in vitro are under development (Pagliuca FW et al., 2014.

[0003] Generation of Functional Human PancreaticβCells In vitro. Cell, 159(2), pp.428–439; Rezania A et al., Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells. Nature Biotechnology, 32(11), pp.1121–1133); WO2012175633;

[0004] WO2014033322; WO2015028614).

[0005] β-cell (BC) transplantation has the potential to provide an eventual cure for type 1 diabetes. However, limited access to donor β-cells has limited the application of this treatment as a clinical therapy. Pluripotent stem (PS) cells can proliferate indefinitely and differentiate into a variety of cell types. Therefore, PS cells are a promising source of β-cells, but before PS cells can be used to treat diabetes, they need to be efficiently and reproducibly differentiated into pancreatic cells.

[0006] During vertebrate embryonic development, pluripotent stem cells generate three germ layers: ectoderm, mesoderm and endoderm. The induction of definitive endoderm (DE) is the first step in the formation of endoderm-derived tissues. The generation of pancreatic endoderm (PE) from DE cells is necessary for the generation of pancreatic endocrine progenitor (EP) cells and ultimately the generation of insulin-producing β cells. PE cells with the potential to become β cells are characterized by the co-expression of these two important transcription factors, PDX1 and NKX6.1.

[0007] Stepwise in vitro differentiation protocols for generating pancreatic cells from PS cells have been established. These protocols generally mimic the main events of pancreatic development, which include several stages, such as the formation of DE, archenteron, posterior foregut, PE, EP, PEC (pancreatic endocrine cells) that co-express SOX17 and FOXA2. To date, efficient DE differentiation of hESCs has been achieved by activin A treatment as well as treatment with Wnt pathway agonists. The next major step in generating pancreatic β cells is to generate PE that co-express PDX1 and NKX6.1. Several research groups have developed in vitro protocols that can differentiate PS cells into DE and PE, and almost all published protocols include a step of adding retinoic acid receptor (RAR) agonists in the induction of PE.

[0008] In summary, patients with type 1 diabetes can be treated by transplanting islets from human donors, and some patients achieve insulin independence. However, donor islets are scarce and of variable quality, and insulin-producing cells derived from PS cells offer an attractive islet alternative. The key differentiation step is differentiation into EP cells and PEC cells to generate functional β cells (BCs).

[0009] Current protocols for differentiating human pluripotent stem cells (hPSCs) into EP cells and specific PEC cells are not efficient processes. In addition, according to current protocols, a large number of unwanted non-pancreatic endocrine cells, such as enterochromaffin cells, as well as non-endocrine cells are formed during the differentiation process. Therefore, there is a need to improve the efficiency of current differentiation protocols. Summary of the invention

[0010] The present invention provides an in vitro method for producing pancreatic endocrine progenitor cells and / or pancreatic endocrine (PEC) cells, comprising the following steps:

[0011] i) differentiating a population of pancreatic endoderm (PE) cells into pancreatic endocrine progenitor (EP) cells, wherein the differentiation comprises treating the pancreatic endoderm (PE) cells with an inhibitor of vascular endothelial growth factor (VEGF) receptor and / or platelet-derived growth factor (PDGF) receptor, and / or

[0012] ii) treating the pancreatic endocrine progenitor (EP) cells with an inhibitor of vascular endothelial growth factor (VEGF) receptor and / or platelet-derived growth factor (PDGF) receptor;

[0013] cells, causing the pancreatic endocrine progenitor (EP) cells to differentiate into pancreatic endocrine progenitor (PEC) cells

[0014] cell.

[0015] In particular, the present invention provides an in vitro method for generating pancreatic endocrine (PEC) cells comprising the following steps:

[0016] i) differentiating a population of pancreatic endoderm (PE) cells into pancreatic endocrine progenitor (EP) cells by treating the pancreatic endoderm (PE) cells with an inhibitor of vascular endothelial growth factor (VEGF) receptor and / or platelet-derived growth factor (PDGF) receptor, and

[0017] ii) treating the pancreatic endocrine progenitor (EP) cells with an inhibitor of vascular endothelial growth factor (VEGF) receptor and / or platelet-derived growth factor (PDGF) receptor;

[0018] cells, causing the pancreatic endocrine progenitor (EP) cells to differentiate into pancreatic endocrine progenitor (PEC) cells

[0019] cell.

[0020] In some embodiments, the present invention provides an in vitro method for generating pancreatic endocrine (PEC) cells, comprising the steps of:

[0021] i) differentiating a population of pancreatic endoderm (PE) cells into pancreatic endocrine progenitor (EP) cells by treating the pancreatic endoderm (PE) cells with an inhibitor of the vascular endothelial growth factor (VEGF) receptor and / or the platelet-derived growth factor (PDGF) receptor, or

[0022] ii) treating the pancreatic endocrine progenitor (EP) cells with an inhibitor of vascular endothelial growth factor (VEGF) receptor and / or platelet-derived growth factor (PDGF) receptor;

[0023] cells, causing the pancreatic endocrine progenitor (EP) cells to differentiate into pancreatic endocrine progenitor (PEC) cells

[0024] cell.

[0025] In some embodiments, the present invention provides an in vitro method for generating pancreatic endocrine (PEC) cells, comprising the steps of:

[0026] i) differentiating a population of pancreatic endoderm (PE) cells into pancreatic endocrine progenitor (EP) cells by treating the pancreatic endoderm (PE) cells with inhibitors of vascular endothelial growth factor (VEGF) receptors and platelet-derived growth factor (PDGF) receptors, and

[0027] ii) differentiating the pancreatic endocrine progenitor (EP) cells into pancreatic endocrine (PEC) cells by treating the pancreatic endocrine progenitor (EP) cells with inhibitors of vascular endothelial growth factor (VEGF) receptor and platelet-derived growth factor (PDGF) receptor.

[0028] In embodiments, the in vitro method for generating a population of pancreatic endocrine progenitor (EP) cells comprises the following steps:

[0029] i) treating pancreatic endoderm (PE) cells with inhibitors of vascular endothelial growth factor (VEGF) receptor and / or platelet-derived growth factor (PDGF) receptor to induce PE

[0030] The cell population is differentiated into pancreatic endocrine progenitor cells.

[0031] In embodiments, the in vitro method for generating a pancreatic endocrine (PEC) cell population comprises the following steps:

[0032] ii) differentiating pancreatic endocrine progenitor (EP) cells into pancreatic endocrine (PEC) cells by treating the pancreatic endocrine progenitor (EP) cells with an inhibitor of vascular endothelial growth factor (VEGF) receptor and / or platelet-derived growth factor (PDGF) receptor.

[0033] In embodiments, the in vitro method for generating a pancreatic endocrine (PEC) cell population comprises the following steps:

[0034] i) treating pancreatic endoderm (PE) cells with inhibitors of vascular endothelial growth factor (VEGF) receptor and / or platelet-derived growth factor (PDGF) receptor to induce PE

[0035] Differentiation of cell populations into pancreatic endocrine progenitor cells, and

[0036] ii) Differentiating pancreatic endocrine progenitor (EP) cells into pancreatic endocrine (PEC) cells.

[0037] In embodiments, the in vitro method for generating a pancreatic endocrine (PEC) cell population comprises the following steps:

[0038] i) differentiating a population of pancreatic endoderm (PE) cells into pancreatic endocrine progenitor cells, and

[0039] ii) differentiating the pancreatic endocrine progenitor (EP) cells into pancreatic endocrine (PEC) cells by treating the pancreatic endocrine progenitor (EP) cells with an inhibitor of vascular endothelial growth factor (VEGF) receptor and / or platelet-derived growth factor (PDGF) receptor.

[0040] One inhibitor of vascular endothelial growth factor (VEGF) receptors and / or platelet-derived growth factor (PDGF) receptors suitable for use in the methods of the invention is 1-(4-(3-amino-1H-indazol-4-yl)phenyl)-3-(2-fluoro-5-methylphenyl)urea, also known as linifanib or ABT869.

[0041] The present inventors have found that, compared with the method of using a transforming growth factor β (TGFb) receptor kinase inhibitor or a TGFβR1 kinase inhibitor or a TGFβ1R kinase inhibitor or a TGFbR1 inhibitor instead of an inhibitor of a vascular endothelial growth factor (VEGF) receptor and / or a platelet-derived growth factor (PDGF) receptor, the use of an inhibitor of a vascular endothelial growth factor (VEGF) receptor and / or a platelet-derived growth factor (PDGF) receptor in the above step i) and / or step ii) has the advantage of increasing the percentage of β-like cells obtained. In addition, the percentage of pancreatic endocrine (PEC) cells is also increased.

[0042] The concentration of the inhibitor of vascular endothelial growth factor (VEGF) and / or platelet derived growth factor (PDGF) receptor used in step ii) is in the range of 1 nM to 12 μM, such as 5 nM to about 15 μM, 5 nM to about 12 μM, 10 nM to about 11 μM, 25 nM to 10 μM, 50 nM to 10 μM, 100 nM to 10 μM, 250 nM to 10 μM, 500 nM to 10 μM, 750 nM to 10 nM, 1 μM to 10 μM, 2 μM to 10 μM, 2.5 μM to 10 μM, 2.5 μM to 8 μM, 2.5 μM to 7 μM, 2.5 μM to 6 μM, 2.5 μM to 5 ... The present invention relates to an aqueous solution of at least one embodiment of the present invention, or in the range of about 3 μM to about 4 μM, or in the range of about 1 μM to about 12 μM, about 1 μM to about 11 μM, about 1 μM to about 10 μM, about 1 μM to about 9 μM, about 1 μM to about 8 μM, about 1 μM to about 7 μM, about 1 μM to about 6 μM, about 1 μM to about 5 μM, about 1 μM to about 4 μM, about 1 μM to about 3 μM, about 1 μM to about 2 μM, about 2 μM to about 12 μM, about 2 μM to about 11 μM, about 2 μM to about 10 μM, about 2 μM to about 9 μM, about 2 μM to about 8 μM, about 2 μM to about 7 μM, about 2 μM to about 6 μM, about 2 μM to about 5 μM, about 2 μM to about 4 μM, about 1 μM to about 3 μM, about 1 μM to about 2 μM, about 2 μM to about 12 μM, about 3 μM to about 11 μM, about 2 μM to about 10 μM, about 2 μM to about 9 μM, about 2 μM to about 8 μM, about 2 μM to about 7 μM, about 2 μM to about μM to about 11 μM, about 3 μM to about 10 μM, about 3 μM to about 9 μM, about 3 μM to about 8 μM, about 3 μM to about 7 μM, about 3 μM to about 6 μM, about 3 μM to about 5 μM, about 3 μM to about 4 μM, about 4 μM to about 12 μM, about 4 μM to about 11 μM, about 4 μM to about 10 μM, about 4 μM to about 9 μM, about 4 μM to about 8 μM, about 4 μM to about 7 μM, about 4 μM to about 6 μM, about 4 μM to about 5 μM, about 5 μM to about 12 μM, about 5 μM to about 11 μM, about 5 μM to about 10 μM, about 5 μM to about 9 μM, about 5 μM to about 8 μM, about 5 μM to about 7 μM, about 5 μM to about 6 μM, about 6 μM to about 12 μM. In some embodiments, the present invention may be in the range of about 1 μM to about 11 μM, about 6 μM to about 10 μM, about 6 μM to about 9 μM, about 6 μM to about 8 μM, about 6 μM to about 7 μM, about 7 μM to about 12 μM, about 8 μM to about 11 μM, about 9 μM to about 10 μM, about 10 μM to about 12 μM, about 10 μM to about 11 μM, or about 11 μM to about 12 μM, or 5 nM, 50 nM, 500 nM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, 8.5 μM, 9 μM, 9.5 μM, 10 μM, 10.5 μM, 11 μM, 11.Concentrations of 5μM, 12μM or 15μM. .

[0043] The concentration of the inhibitor of vascular endothelial growth factor (VEGF) and / or platelet derived growth factor (PDGF) receptor used in step i) is in the range of 1 nM to 12 μM, such as 5 nM to about 15 μM, 5 nM to about 12 μM, 10 nM to about 11 μM, 25 nM to 10 μM, 50 nM to 10 μM, 100 nM to 10 μM, 250 nM to 10 μM, 500 nM to 10 μM, 750 nM to 10 nM, 1 μM to 10 μM, 2 μM to 10 μM, 2.5 μM to 10 μM, 2.5 μM to 8 μM, 2.5 μM to 7 μM, 2.5 μM to 6 μM, 2.5 μM to 5 ... , or in the range of about 3 μM to about 4 μM, or in the range of about 1 μM to about 12 μM, about 1 μM to about 11 μM, about 1 μM to about 10 μM, about 1 μM to about 9 μM, about 1 μM to about 8 μM, about 1 μM to about 7 μM, about 1 μM to about 6 μM, about 1 μM to about 5 μM, about 1 μM to about 4 μM, about 1 μM to about 3 μM, about 1 μM to about 2 μM, about 2 μM to about 12 μM, about 2 μM to about 11 μM, about 2 μM to about 10 μM, about 2 μM to about 9 μM, about 2 μM to about 8 μM, about 2 μM to about 7 μM, about 2 μM to about 6 μM, about 2 μM to about 5 μM, about 2 μM to about 4 μM, about 1 μM to about 3 μM, about 1 μM to about 2 μM, about 2 μM to about 12 μM, about 2 μM to about 11 μM, about 2 μM to about 10 μM, about 2 μM to about 9 μM, about 2 μM to about 8 μM, about 2 μM to about 7 μM, about 2 μM to about 6 μM M to about 11 μM, about 3 μM to about 10 μM, about 3 μM to about 9 μM, about 3 μM to about 8 μM, about 3 μM to about 7 μM, about 3 μM to about 6 μM, about 3 μM to about 5 μM, about 3 μM to about 4 μM, about 4 μM to about 12 μM, about 4 μM to about 11 μM, about 4 μM to about 10 μM, about 4 μM to about 9 μM, about 4 μM to about 8 μM, about 4 μM to about 7 μM, about 4 μM to about 6 μM, about 4 μM to about 5 μM, about 5 μM to about 12 μM, about 5 μM to about 11 μM, about 5 μM to about 10 μM, about 5 μM to about 9 μM, about 5 μM to about 8 μM, about 5 μM to about 7 μM, about 5 μM to about 6 μM, about 6 μM to about 12 μM. In some embodiments, the present invention may be in the range of 5 nM, 50 nM, 500 nM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, 8.5 μM, 9 μM, 9.5 μM, 10 μM, 10.5 μM, 11 μM, 11.5 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 21 μM, 22 μM, 23 μM, 24 μM, 25 μM, 26 μM, 27 μM, 28 μM, 29 μM, 30 μM, 31 μM, 32 μM, 33 μM, 34 μM, 35 μM, 36 μM, 37 μM, 38 μM, 39 μM, 40 μM, 41 μM, 42 μM, 43 μM, 44 μM, 45 μM, 46 μM, 47 μM, 48 μM, 49 μM, 50 μMConcentrations of 5μM, 12μM or 15μM. .

[0044] In some embodiments, the pancreatic endocrine (PEC) cells obtained from step ii) include islet-like cells. Islet-like cells include α-like cells, β-like cells, ε-like cells, δ-like cells and γ-like cells. Steps i) and ii) of the method of the present invention do not involve TGFb receptor kinase inhibitors.

[0045] In some embodiments, step i) does not involve a TGFb receptor kinase inhibitor.

[0046] In some embodiments, step ii) does not involve a TGFb receptor kinase inhibitor.

[0047] like Figure 1 As shown in B, the pancreatic endocrine (PEC) cell population obtained after step ii) contains at least 20%, such as at least 25%, such as at least 28%, or at least 30% β-like cells based on the total number of cells obtained after step ii), and the β-like cells are double positive for ISL1 and NKX6.1 (ISL1+NKX6.1+).

[0048] As described above, compared to the pancreatic endocrine (PEC) cell population obtained by replacing the inhibitor of VEGF and / or PDGF with a TGFb receptor kinase inhibitor in step i) and / or step ii), the number of β-like cells obtained in the pancreatic endocrine (PEC) cell population obtained after step ii) is increased, and wherein the β-like cells are double positive for ISL1 and NKX6.1 (ISL1+NKX6.1+). Therefore, compared to the pancreatic endocrine (PEC) cell population obtained by replacing the inhibitor of VEGF and / or PDGF with a TGFb receptor kinase inhibitor in step i) and / or step ii), the pancreatic endocrine (PEC) cell population obtained after step ii) contains at least 1% more, such as at least 2%, at least 3%, at least 4%, at least 5%, at least 10% or at least 20% more β-like cells, and wherein the β-like cells are double positive for ISL1 and NKX6.1 (ISL1+NKX6.1+). Furthermore, more than 60% of the cells were ISL1+, compared to only about 40% when using standard methods, see e.g. Figure 3 B.

[0049] Compared to an in vitro method for producing pancreatic endocrine (PEC) cells by using a TGFb receptor kinase inhibitor instead of an inhibitor of VEGF and / or PDGF in step i) and / or step ii), the number of enterochromaffin (EC) cells produced by the method of the present invention is reduced. Thus, the pancreatic endocrine (PEC) cell population obtained after step ii) contains up to 35%, such as up to 30% or up to 27% enterochromaffin cells, wherein the enterochromaffin cells are negative for ISL1 and positive for NKX6.1 (ISL1-NKX6.1+), see e.g. Figure 3 B.

[0050] The pancreatic endocrine (PEC) cell population obtained after step ii) contains at least 1% less, such as at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30% or at least 35% less enterochromaffin cells compared to a pancreatic endocrine (PEC) cell population obtained by using a TGFb receptor kinase inhibitor instead of an inhibitor of VEGF and / or PDGF in step i) and / or step ii), wherein the enterochromaffin cells are negative for ISL1 and positive for NKX6.1 (ISL1-NKX6.1+), see e.g. Figure 3 B.

[0051] from Figure 3 E, compared to the pancreatic endocrine (PEC) cell population obtained by using a TGFb receptor kinase inhibitor instead of an inhibitor of VEGF and / or PDGF in step i) and / or step ii), the pancreatic endocrine (PEC) cell population obtained after step ii) is also increased. Therefore, the pancreatic endocrine (PEC) cell population or cells obtained after step ii) contain at least 70%, such as at least 75%, at least 78% or at least 80% of pancreatic endocrine cells, wherein the endocrine cells are CHGA+.

[0052] The pancreatic endocrine (PEC) cell population or cells obtained after step ii) contain at least 1% more, such as at least 2%, at least 3%, at least 4% or at least 5% more pancreatic endocrine (PEC) cells compared to the pancreatic endocrine (PEC) cell population obtained by using a TGFb receptor kinase inhibitor instead of an inhibitor of VEGF and / or PDGF in step i) and / or step ii), wherein the pancreatic endocrine (PEC) cells are CHGA+, see e.g. Figure 3 E.

[0053] In a further aspect, a cell population or composition is disclosed according to the present invention for use as a medicament, e.g., for the treatment of type I diabetes. Transplantation of a homogenous cell population is believed to improve the safety and efficacy profile of cell therapy and reduce the risk of adverse side effects produced by unwanted cell types. Treatment with a cell population or composition according to the present invention provides a high percentage of β-like cells, making it suitable for preventing, ameliorating and / or treating conditions requiring administration of such cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1

[0055] A) Schematic overview of hPSC differentiation into pancreatic endocrine (PEC) cells through different cell stages (i.e., DE, PE, EP, and PEC). hPSCs first differentiate into definitive endoderm (DE), DE differentiate into pancreatic endoderm (PE), PE differentiate into endocrine progenitor (EP) cells, and finally, endocrine progenitor (EP) cells differentiate into pancreatic endocrine (PEC) cells.

[0056] B) hPSCs were differentiated into PE and then treated with TGFβR1 inhibitor [STD] or Linifanib during the EP-PEC stage. Cells were analyzed for ISL1 and NKX6-1 expression at the PEC stage. Representative flow cytometry dot plots of three experiments.

[0057] C) Summary of the percentage of cells in each quadrant (Q1-Q4) of the flow cytometry dot plots shown in B). The mean ± SD of three experiments is shown.

[0058] Figure 2

[0059] A) hPSCs were differentiated into PE and then treated with linifanib added at concentrations ranging from 5 nM to 15 uM during the EP stage. After treatment during the EP stage, all conditions were subjected to standard conditions for differentiation into PECs and cells were analyzed for ISL1 and NKX6-1 expression.

[0060] B) hPSC differentiation was performed as described in A), but the expression of CHGA and VIM was assessed by flow cytometry.

[0061] C) Summary graph of the percentage of ISL1+ / NKX6.1+ cells in Q2 (hPSC-derived β-like cells) from the flow cytometry dot plots shown in A.

[0062] D) Summary graph of the percentage of CHGA+ cells (endocrine cells) within Q1-Q2 from the flow cytometry dot plots shown in B.

[0063] Figure 3

[0064] A) Schematic overview of hPSC differentiation into pancreatic endocrine cells (PECs). hPSCs were differentiated into PEs and then treated with a TGFβ1R inhibitor [standard (STD)], without a TGFβR1 inhibitor during two different treatment windows (-TGFβR1), and without a TGFβR1 inhibitor but with linivasib (linivasib) during different treatment windows between the EP-PEC phase of the protocol (highlighted by lines).

[0065] B) Flow cytometry dot plots of cells differentiated as described in A. Cells were analyzed for ISL1 and NKX6-1 expression.

[0066] C) Flow cytometry dot plots of cells differentiated as described in A. Cells were analyzed for CHGA and VIM expression.

[0067] D) Summary graph of the percentage of ISL1+ / NKX6.1+ Q2 inner cells (hPSC-derived β-like cells) from the flow cytometry dot plots shown in B.

[0068] E) Summary graph of the percentage of cells (endocrine cells) within CHGA+Q1-Q2 of the flow cytometry dot plots shown in C. DETAILED DESCRIPTION

[0069] A method for obtaining pancreatic endocrine cells from pluripotent stem cells is provided.

[0070] The pancreatic endocrine cells obtained by the methods described herein may be used and / or intended for use in methods of providing pancreatic endocrine function to a mammal that has deficient production of at least one pancreatic hormone.

[0071] In some embodiments, the present invention relates to a method of providing pancreatic endocrine function to a mammal having deficient production of at least one pancreatic hormone, the method comprising the step of implanting endocrine cells obtained by any of the methods described herein in an amount sufficient to produce a measurable amount of the at least one pancreatic hormone in the mammal.

[0072] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which the invention belongs. Unless otherwise specified, conventional methods of chemistry, biochemistry, biophysics, molecular biology, cell biology, genetics, immunology and pharmacology known to those skilled in the art are used in the practice of the present invention.

[0073] It should be noted that all headings and subheadings used herein are for convenience only and should not be construed as limiting the invention in any way.

[0074] The use of any and all examples, or exemplary language (eg, "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0075] As used herein, "a" or "an" or "the" may mean one (kind) or more than one (kind). Unless otherwise stated in this specification, terms presented in the singular also include plural cases. As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the relevant listed items, and when interpreted in an alternative manner ("or"), refers to the lack of a combination. In addition, the present invention also contemplates that in some embodiments of the present invention, any feature or combination of features set forth herein may be excluded or omitted.

[0076] General Definition

[0077] hESC: human embryonic stem cell

[0078] hiPSC: human induced pluripotent stem cells

[0079] hPSC: human pluripotent stem cells

[0080] DE: Definitive endoderm

[0081] PE: pancreatic endoderm

[0082] EP: Endocrine progenitor cells

[0083] PEC: pancreatic endocrine cells

[0084] BC: beta cells, insulin-producing beta cells

[0085] Stem Cells

[0086] "Stem cells" should be understood as undifferentiated cells that have proliferation capacity (especially self-renewal capacity) but retain differentiation potential. According to their differentiation potential, the term "stem cell" includes categories such as pluripotent stem cells, multipotent stem cells, etc.

[0087] As used herein, the term "pluripotent stem cell" (PSC) refers to a stem cell that can be cultured in vitro and has the potential (pluripotency) to differentiate into any cell lineage belonging to the three germ layers (ectoderm, mesoderm, endoderm) and / or extraembryonic tissues.

[0088] As used herein, the term "multipotent stem cell" means a stem cell that has the potential to differentiate into multiple types of tissues or cells (but not all kinds), and is usually restricted to one germ layer.

[0089] As used herein, the term "unipotent stem cell" refers to a stem cell that has the potential to differentiate into only one specific tissue or cell.

[0090] Pluripotent stem cells can be induced from fertilized eggs, cloned embryos, germ stem cells, stem cells in tissues, somatic cells, etc. Examples of pluripotent stem cells (PSCs) include embryonic stem cells (ESCs), EG cells (embryonic germ cells), induced pluripotent stem cells (iPSCs), and the like.

[0091] As used herein, the term "induced pluripotent stem cell" (also referred to as iPS cell or iPSC) means a type of pluripotent stem cell that can be generated directly from adult cells. By introducing the product of a specific group of pluripotency-related genes, non-pluripotent cells can be converted into pluripotent stem cells. Pluripotent embryonic stem cells can also be derived from parthenogenetic bodies (parthenotes), as described in, for example, WO 2003 / 046141, the contents of which are incorporated herein by reference in their entirety. In addition, embryonic stem cells can be produced from a single blastomere or produced by culturing an inner cell mass obtained without destroying the embryo. Embryonic stem cells can be obtained from a given organizational structure or can be commercially available. Preferably, in some embodiments, the methods and products described herein are based on hPSC, i.e., stem cells derived from induced pluripotent stem cells or embryonic stem cells (including parthenogenetic bodies).

[0092] Definitive endoderm (DE)

[0093] As used herein, the term "definitive endoderm", "definitive endoderm cells" or "DE" refers to cells characterized by the expression of the marker SOX17. Optionally, other markers of DE are one or more of the following: FOXA2 and CXCR4. Definitive endoderm cells are essential for the development of, for example, pancreatic cells.

[0094] As used herein, "SOX17" (SRY-box 17) is a member of the SOX (SRY-related HMG box) transcription factor family that is involved in the regulation of embryonic development and cell fate determination.

[0095] "FOXA2" (forkhead box A2) as used herein is a member of the forkhead class of DNA binding proteins.

[0096] As used herein, "CXCR4" (CXC motif chemokine receptor 4) is a CXC chemokine receptor specific for stromal cell-derived factor-1.

[0097] Non-limiting examples of DE induction protocols are the conventional D'Amour protocol (Nature Biotechnology 2006, 2008) and the protocol described in WO2012 / 175633 (incorporated herein by reference in its entirety).

[0098] Pancreatic endoderm (PE)

[0099] As used herein, the term "pancreatic endoderm", "pancreatic endoderm cells", "pancreatic progenitor cells" or "PE" refers to cells characterized by the expression of markers PDX1 and NKX6.1. In some embodiments, at least 5% of the cells are NKX6.1+ / PDX1+ double positive. Optionally, the other markers of PE are one or more of SOX9 and PTF1A.

[0100] "PDX1" as used herein refers to a homeodomain transcription factor involved in pancreatic development.

[0101] "NKX6.1" as used herein is a member of the NKX transcription factor family.

[0102] As used herein, "SOX9" (SRY-box transcription factor 9) is a transcription factor that plays a key role during embryonic development and cell lineage assignment.

[0103] "PTF1A" as used herein is a protein that is a component of the pancreatic transcription factor 1 complex (PTF1) and is known to play a role in mammalian pancreas development.

[0104] "CPAl" as used herein is a member of the carboxypeptidase A family of zinc metalloproteinases. This enzyme is produced in the pancreas.

[0105] Non-limiting examples of PE induction protocols are described in WO2014 / 033322, which is incorporated herein by reference in its entirety.

[0106] Pancreatic endocrine progenitor (EP) cells

[0107] As used herein, the term "pancreatic endocrine progenitor cell" or "endocrine progenitor cell" or "EP" refers to a cell characterized by expression of NEUROG3 and optionally one or more of NeuroD and NKX2.2, which are hallmarks of EP cells committed to an endocrine cell fate.

[0108] "NEUROG3" as used herein is a member of the neurogenin family of basic loop-helix-loop transcription factors.

[0109] As used herein, "NKX2.2" and "NKX6.1" are members of the NKX family of transcription factors.

[0110] "NeuroD" as used herein is a member of the NeuroD family of basic helix-loop-helix (bHLH) transcription factors.

[0111] Protocols for generating pancreatic endocrine progenitor cells are described in WO2015 / 028614, which is incorporated herein by reference in its entirety.

[0112] Pancreatic endocrine cells (PEC)

[0113] As used herein, the term "pancreatic endocrine cell" or "PEC" refers to a cell that expresses CHGA and ISL1.

[0114] The pancreatic endocrine (PEC) cells obtained by the method of the present invention include islet-like cells, which include α-like cells, β-like cells, ε-like cells, δ-like cells and γ-like cells.

[0115] As used herein, the term "islet-like cells" refers to islet cells obtained after culturing stem cells in vitro. Islet-like cells include β cells, α cells, δ cells, and γ cells.

[0116] As used herein, the term "alpha cell" refers to a cell that expresses GCG and optionally one or more of ISL1 and ARX. In the pancreas, alpha cells produce the hormone glucagon.

[0117] As used herein, the term "β cell" or "β-like cell" refers to a cell that expresses INS and optionally expresses one or more of PDX1, ISL1, and NKX6.1. In the pancreas, β cells produce the hormones insulin and amylin.

[0118] As used herein, the term "delta cell" refers to a cell that expresses SST and optionally one or more of ISL1 and HHEX. In the pancreas, delta cells secrete the peptide hormone somatostatin.

[0119] As used herein, the term "epsilon cell" refers to a cell that expresses GHRL and optionally one or more of ISL1, ARX, and ETV1. In the pancreas, epsilon cells produce the hormone ghrelin.

[0120] As used herein, the term "γ cell" is used interchangeably with "pancreatic polypeptide cell", "PP cell", "gamma cell" or "F cell" in the present context and refers to an endocrine cell that expresses PPY and optionally one or more of ISL1 and PAX6. In the pancreas, they help synthesize and regulate the release of pancreatic polypeptide (PP).

[0121] As used herein, the term "enterochromaffin cell" is used interchangeably with "EC cell" and "Kulchitsky cell" and refers to an endocrine cell that expresses TPH1 and optionally expresses one or more of LMX1A and FEV. Enterochromaffin cells are a type of enteroendocrine cell and neuroendocrine cell. In humans, they are located in the epithelial layer throughout the gastrointestinal tract. EC cells regulate neuronal signaling in the enteric nervous system (ENS) by secreting the neurotransmitter serotonin and other peptides.

[0122] Expression of markers

[0123] As used herein, the term "expression level" refers to the degree of gene expression and / or gene product activity in a cell. Expression levels can be determined using any absolute unit or normalized unit (relative to a known expression level of a control reference).

[0124] As used herein, the term "marker" refers to a naturally occurring identifiable expression by a cell that can be associated with certain properties of the cell and used to identify, predict or characterize a cell or cell population. A marker can be referred to as a gene. A marker can be in the form of mRNA or protein, such as a protein on the surface of a cell.

[0125] As used herein, the term "expression" associated with a marker refers to the presence or absence of a detectable molecule in a cell. In an embodiment, the expressed molecule is an mRNA or a protein. The expression of a marker can be detected at any appropriate level, for example, at the mRNA or protein level. It will be readily understood by those skilled in the art that cells can be defined by the positive or negative expression of a marker, i.e., the nature and state of a cell can be similarly associated based on the expression of a certain marker and its lack. When referring to a specific marker, the presence or absence of expression can be represented by + (plus sign) or - (minus sign) symbols, respectively.

[0126] Method steps

[0127] As used herein, the term "step" in connection with the methods described herein should be understood as a stage of embarking on something and / or performing an action. One of ordinary skill in the art will understand when the steps to be performed and / or the steps performed are simultaneous and / or sequential and / or continuous.

[0128] Throughout this application, the terms "method" and "protocol" are used interchangeably when referring to a cell differentiation process.

[0129] As used herein, the term "day" and the similar term day in vitro (DIV) in relation to a protocol refer to the specific time during which certain steps are performed during a differentiation procedure.

[0130] In general, unless otherwise indicated, "Day 0" refers to the start of a protocol, which is performed by, for example, but not limited to, plating or transferring cells to an incubator, or contacting cells with a compound in their current cell culture medium prior to transferring cells. Typically, a protocol is initiated by transferring cells, such as undifferentiated stem cells, definitive endoderm cells, pancreatic endoderm cells, pancreatic endocrine progenitor (EP) cells, or pancreatic endocrine (PEC) cells, to a different cell culture medium and / or container, such as, but not limited to, by plating or incubation, and / or first contacting cells with one or more compounds that affect undifferentiated stem cells in a manner that initiates the differentiation process.

[0131] When reference is made to "Day X," such as Day 1, Day 2, etc., this is relative to the start of the protocol on Day 0. One of ordinary skill in the art will recognize that the exact time of day that the step is performed may vary unless otherwise specified. Thus, "Day X" is intended to encompass time spans such as + / -10 hours, + / -8 hours, + / -6 hours, + / -4 hours, + / -2 hours, or + / -1 hour.

[0132] As used herein, the phrase "from about day X to about day Y" refers to the day when an event begins. The phrase provides an interval of days in which an event may begin. For example, if "cells are contacted with a differentiation factor from about day 3 to about day 5", this should be interpreted as covering all of the following options: "cells are contacted with a differentiation factor from about day 3", "cells are contacted with a differentiation factor from about day 4", and "cells are contacted with a differentiation factor from about day 5". Therefore, the phrase should not be interpreted as an event that occurs only within the interval of day 3 to day 5. This also applies to the phrase "to about day X to about day Y" with necessary adjustments.

[0133] Differentiation

[0134] As used herein, "differentiation" refers to the process by which cells progress from an undifferentiated state to a differentiated state, from an immature state to a more mature 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 can secrete high levels of pancreatic endocrine hormones or digestive enzymes. For example, fully differentiated β cells secrete insulin at high levels in response to glucose. When cells lose markers of undifferentiated cells or gain markers of differentiated cells, changes in cell interactions and cell maturation occur. The loss or gain of a single marker can indicate that the cell has "matured or fully differentiated". The term "differentiation factor" refers to a compound added to pancreatic cells to enhance their differentiation into mature endocrine cells (also including β cells that produce insulin). 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. In some aspects, differentiating the cells comprises culturing the cells in a medium comprising one or more differentiation factors.

[0135] 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, glucagon-like peptide 1, indole lactam V, and retinoic acid.

[0136] In aspects of the invention, differentiating the cells comprises culturing the cells in a medium comprising one or more differentiation factors.

[0137] In preferred embodiments, the method is performed in vitro. The term "in vitro" refers to cells provided and maintained outside the human or animal body. In embodiments, the cells are non-natural. The term "non-natural" refers to cells that, although derived from pluripotent stem cells that may have human origin, are artificial constructs that do not exist in nature. As used herein, the term "artificial" may include materials that occur naturally in nature but are modified to be non-naturally occurring constructs. This includes human stem cells that differentiate into non-naturally occurring cells that mimic human cells.

[0138] Known protocols for the individual differentiation steps from hPSCs to pancreatic endocrine cells

[0139] Below, will provide about the reference of known scheme of the separate differentiation step of hPSC to pancreatic endocrine cell.These schemes, and the reference provided in " background technology " paragraph, can be used for providing definitive endoderm cell, providing pancreatic endoderm (PE) cell or providing pancreatic endocrine progenitor cell.

[0140] hPSCs differentiate into pancreatic endocrine (PEC) cells through different stages, including definitive endoderm (DE), pancreatic endoderm (PE), endocrine progenitor (EP) cells (EP), and finally pancreatic islet cells (also referred to as PEC) (Madsen et al. - Nat Biotechnol. - 2006 Dec, 24(12): 1481-3).

[0141] DE are usually derived by treating hPSCs with transforming growth factor β and WNT / β-catenin agonists (D'Amour et al. - Nat Biotechnol. - 2005 Dec; 23(12): 1534-41, Rezania et al. - Diabetes - 2011 Jan; 60(1): 239-47, Kubo et al. - Development - 2004 Apr; 131(7): 1651-62, Rezania et al. - Nat Biotechnol. - 2014 Nov; 32(11): 1121-33, Funa et al. - Cell Stem Cell. - 2015 Jun 4; 16(6): 639-52). DE are further specialized into the PDX1+NKX6.1+PE population in vitro. Fibroblast growth factor, retinoic acid, sonic hedgehog, epidermal growth factor, and bone morphogenetic protein signaling pathways have all been implicated in pancreatic development, and manipulation of these pathways at different stages of differentiation promotes a highly enriched PE population (D'Amour et al. - Nat Biotechnol. - 2006 Nov;24(11):1392-401, Kroon et al. - Nat Biotechnol. - 2008 Apr;26(4):443-52, Nostro et al. - Development - 2011 Mar;138(5):861-71, Rezania et al. - Diabetes - 2012 Apr;26(4):443-52).

[0142] May;61(8):2016-29, Mfopou et al. - Gastroenterology - 2010 Jun;138(7):2233-45, Ameri et al. - Stam cells - 2010 Jan;28(1):45-56, Russ et al. - EMBO J. - 2015 Jul 2;34(13):1759-72). Other pathways including protein kinase C, nicotinamide, WNT, Rho-associated kinase, and TGFβ have also been identified as being involved in hPSC specification to the pancreatic lineage (Chen et al. - Nat Chem Biol. - 2009 Apr;5(4):258-65, Rezania et al. - Diabetes - 2012 Aug;61(8):2016-29, Rezania et al. - Stem Cell - 2013 Nov;31(11):2432-42, Nostro et al. - Stem Cell Reports - 2015 Apr 14;4(4):591-604, Sharon et al. - Cell Rep. - 2019 May 21;27(8):2281-2291.e5, Toyoda et al. - Stem Cell Rep. - 2017 Aug 8;9(2):419-428).

[0143] Pancreatic endocrine specialization from PE depends on the expression of the transcription factor NEUROG3 (McGrath et al. - Diabetes - July 2015; 64(7): 2497-505, Zhang et al. - Dev. Cell - August 5, 2019; 50(3): 367-380.e7). Several methods for inducing EP and PEC from PE have been explored. Compared with cells cultured in planar culture, culturing PE in air-liquid interface culture leads to upregulation of NEUROG3 transcripts and pancreatic hormones insulin (INS) and glucagon (GCG) (Rezania et al. - Nat Biotechnol. - November 2014; 32(11): 1121-33). Studies have shown that expression of NEUROG3 transgenes in PE induces endocrine differentiation (Zhu et al. - Cell Stem Cell. - June 2, 2016; 18(6): 755-768). Regulation of the actin cytoskeleton and the dispersion of PE into single cells followed by cell reaggregation into clusters can induce NEUROG3 expression and differentiation into EP and hPSC endocrine cells (Mamidi-Nature.-2018 Dec;564(7734):114-118, Hogrebe et al.-Nat Biotechnol.-2020 Apr;38(4):460-470). Inhibition of TGFβ signaling and Notch signaling allows PE to progress to a pancreatic endocrine phenotype (Rezania et al. - Diabetes. - January 2011; 60(1): 239-47, Nostro et al. - Development. - March 2011; 138(5): 861-71, Pagliuca et al. - Cell. - October 9, 2014; 159(2): 428-39, Rezania et al. - Nat Biotechnol. - November 2014; 32(11): 1121-33, Rezania et al. - Differentiation of human embryonic stem cells - June 23, 2015). However, allowing TGFβ signaling appears to be required for differentiation into more mature β-like cells (Velazco-Cruz et al. - Stem Cell Reports. - February 12, 2019; 12(2): 351-365).

[0144] Several other signaling pathways have been shown to promote EP induction and further differentiation into hPSC endocrine cells. Inhibition of bromodomain and extra-terminal (BET) proteins with I-BET151 or JQ1 increased the number of NEUROG3 endocrine progenitor (EP) cells (Huijbregts et al. - Diabetes. - 2019 Apr; 68(4): 761-773). Cromolyn sodium (SCG) was identified in a small molecule screen that induces pancreatic endocrine differentiation in PE by inhibiting the bone morphogenetic protein 4 signaling pathway (Kondo et al. - Diabetologia. - 2017 Aug; 60(8): 1454-1466). Bone morphogenetic proteins are involved in endocrine induction (Nostro et al. - Development. - 2011 Mar; 138(5):861-71, Sharon et al. - Cell Rep. - 2019 May 21; 27(8):2281-2291.e5), and inhibitors of this pathway are commonly used in the differentiation of hPSCs to EPs and PECs. Treatment of PEs with the WNT-tankyrase inhibitor IWR1-endo increased the expression of endocrine markers and downregulated progenitor markers, demonstrating that small molecule WNT inhibitors can increase endocrine induction (Sharon et al. - Cell Rep. - 2019 May 21; 27(8):2281–2291.e5).

[0145] Finally, a specific pancreatic endocrine cell type similar to its in vivo counterpart was derived and characterized in detail. Glucagon-expressing α-like cells derived from hPSCs exhibit molecular and functional properties of authentic pancreatic α-cells (Rezania et al. - Diabetes -

[0146] 2011 Jan;60(1):239-47, Peterson et al. - Nat Commun. - 2020 May 7;11(1):2241). Differentiation protocols for maturing hPSC-derived β-like cells capable of secreting insulin in response to elevated glucose concentrations have recently been reported (Rezania et al. - Nat Biotechnol. - 2014 Nov; 32(11): 1121-33, Pagliuca et al. - Cell. - 2014 Oct 9; 159(2): 428-39, Velazco-Cruz et al. - Stem Cell Reports. - 2019 Feb 12; 12(2): 351-365, Hogrebe et al. - Nat Biotechnol. - 2020 Apr; 38(4): 460-470, Liu et al. - Nat Commun. - 2021 Jun 7; 12(1): 3330, Nair et al. - Nat Cell Biol. - 2019 Feb; 21(2): 263-274).

[0147] Single-cell gene expression analysis has outlined the differentiation pathway of hPSCs into pancreatic endocrine (PEC) cells, including β-like cells (Petersen et al. - Stem Cell Reports. - 2017 Oct 10;9(4):1246-1261, Ramond et al. - Development. - 2018 Aug 15;145(16):dev165480, Docherty et al. - Diabetes. - 2021 Nov;70(11):2554-2567, Veres et al. - Nature. - 2019 May;569(7756):368-373), and detailed characterization of hPSC-derived β-like cells in vitro and in vivo has revealed many similarities with authentic pancreatic β cells (Velazco-Cruz et al. - Stem Cell Reports.-February 12, 2019; 12(2):351-365, Augsornworawat et al.-Cell Rep.-August 25, 2020; 32(8):108067, Balboa et al.-Functional, metabolic and transcriptional maturation of stem cell derived beta cells-2021.03.31.437748v1). Interestingly, the formation of non-endocrine cells and non-pancreatic enterochromaffin cells has recently been reported for protocols for hPSC differentiation to the pancreatic endocrine lineage (Petersen et al.-Stem Cell Reports.-October 10, 2017; 9(4):1246-1261, Veres et al.-Nature.-May 2019; 569(7756):368-373).

[0149] In the following, the method of the present invention is described in more detail by means of non-limiting embodiments and examples.

[0150] In vitro method for obtaining pancreatic endocrine cells

[0151] Described herein are in vitro methods of obtaining pancreatic endocrine cells from pluripotent stem cells, which pancreatic endocrine cells can be used and / or are intended for use in methods of providing pancreatic endocrine function to a mammal that has deficient production of at least one pancreatic hormone.

[0152] The present invention identifies a small molecule inhibitor of vascular endothelial growth factor receptor (VEGFR) and / or platelet-derived growth factor receptor (PDGFR) (linifanib) that promotes the differentiation of pancreatic endoderm into pancreatic endocrine progenitor (EP) cells and pancreatic endocrine cells including β-like cells.

[0153] The present invention provides an in vitro method for generating a pancreatic endocrine (PEC) cell population, comprising the following steps:

[0154] i) differentiating a population of pancreatic endoderm (PE) cells into pancreatic endocrine progenitor cells, wherein

[0155] The differentiation comprises treating the pancreatic endoderm (PE) with an inhibitor of a vascular endothelial growth factor (VEGF) receptor and / or a platelet-derived growth factor (PDGF) receptor.

[0156] cells, and / or

[0157] ii) treating the pancreatic endocrine progenitor (EP) cells with an inhibitor of vascular endothelial growth factor (VEGF) receptor and / or platelet-derived growth factor (PDGF) receptor;

[0158] cells, causing the pancreatic endocrine progenitor (EP) cells to differentiate into pancreatic endocrine progenitor (PEC) cells

[0159] cell.

[0160] In some embodiments, step i) is performed by treating pancreatic endoderm (PE) cells with an inhibitor of vascular endothelial growth factor (VEGF) receptor and / or platelet-derived growth factor (PDGF) receptor.

[0161] Differentiation of pancreatic endoderm (PE) cells into pancreatic endocrine progenitor (EP) cells (EP or PEP) - Step i)

[0162] In some embodiments, the pancreatic endoderm (PE) cells used in step i) of the methods described herein are cells having the markers PDX1 and NKX6.

[0163]

[0164] In some embodiments, in step i) of culturing pancreatic endoderm (PE) cells into pancreatic endocrine progenitor cells, the pancreatic endoderm cells are treated with one or more compounds selected from thyroid hormones, epidermal growth factor (EGF) agonists, staurosporine, NOTCH pathway inhibitors, BMP pathway inhibitors, EZH2 histone methyltransferase inhibitors, JNK pathway inhibitors and TGFbR1 inhibitors.

[0165] In some embodiments, suitable examples of thyroid hormones are T3 (Cas number 6893-02-3) or GC1 (Cas number 211110-63-3). In embodiments, the thyroid hormone is T3.

[0166] For use in the methods of the invention, suitable examples of EGF agonists (EGF pathway activators) are β-cellulin (gene name BTC), epidermal growth factor (gene name EGF), amphiregulin (gene name AREG), transforming growth factor alpha (gene name TGFA) and neuregulin 1 (gene name NRG1). In an embodiment, the EGF pathway activator is β-cellulin.

[0167] In some embodiments, suitable examples of NOTCH pathway inhibitors are DBZ (XX) (Gas No. 209984-56-5), DAPT (Cas No. 208255-80-5), Compound E (Cas No. 209986-17-4) and L-685,485 (Cas No. 292632-98-5). In embodiments, the NOTCH pathway inhibitor is XX.

[0168] In some embodiments, suitable examples of BMP pathway inhibitors include LDN 193189 dihydrochloride (Cas number 1435934-00-1), DMH-1 (Cas number 1206711-16-1), Dorsomorphin dihydrochloride (Cas number 1219168-18-9) and Noggin. In embodiments, the BMP pathway inhibitor is LDN.

[0169] In some embodiments, suitable examples of EZH2 histone methyltransferase inhibitors are 3-deazaadenine A hydrochloride (DZNep) (Cas number 120964-45-6), GSK 126 (Cas number 1346574-57-9), EPZ005687 (Cas number 1396772-26-1). In embodiments, the EZH2 histone methyltransferase inhibitor is DZNep.

[0170] In some embodiments, suitable examples of JNK pathway inhibitors are TCS JNK 6o / JNK inhibitor VIII (Cas number 894804-07-0), SP 600125 (Cas number 129-56-6), TCS JNK 5a (Cas number 312917-14-9) and JNK-IN-8 (Cas number 1410880-22-6). In embodiments, the JNK pathway inhibitor is JNK inhibitor VIII.

[0171] In some embodiments, staurosporine is a broad-spectrum protein kinase inhibitor (Cas number 62996-74-1). Other broad-spectrum protein kinase inhibitors for use in the methods of the invention include apigenin, H-7 dihydrochloride, 5-iodotuberculocidin, K 252a, PKC 412, and Ro31-8220 mesylate. In embodiments, the broad-spectrum protein kinase inhibitor is staurosporine.

[0172] In some embodiments, suitable examples of TGFb receptor kinase inhibitors include RepSox (ALK5i II) (Cas number 446859-33-2), SB431542 (Cas number 301836-41-9), LY 364947 (Cas number 396129-53-6) and A 83-01 (Cas number 909910-43-6). In some embodiments, the TGFb receptor kinase inhibitor is RepSox and SB431542.

[0173] The differentiation in step i) may also include other differentiation factors, such as Rho kinase (ROCK inhibitor), such as Tiger, Chroman-1 (Cas No. 1273579-40-0) and Thiazovivin (Cas No. 1226056-71-8); Heparin; and Forskolin or NKH 477.

[0174] In step i), pancreatic endoderm (PE) cells may also be treated with inhibitors of vascular endothelial growth factor (VEGF) receptors and / or platelet-derived growth factor (PDGF) receptors. Linifanib is an example of such a substance. Linifanib is 1-(4-(3-amino-1H-indazol-4-yl)phenyl)-3-(2-fluoro-5-methylphenyl)urea. In an embodiment, cells are treated with such inhibitors.

[0175] The concentration of the inhibitor of vascular endothelial growth factor (VEGF) and / or platelet derived growth factor (PDGF) receptor used in step i) is in the range of 5 nM to about 15 μM, such as 5 nM to about 12 μM, 1 nM to 12 μM, 10 nM to about 11 μM, 25 nM to 10 μM, 50 nM to 10 μM, 100 nM to 10 μM, 250 nM to 10 μM, 500 nM to 10 μM, 750 nM to 10 nM, 1 μM to 10 μM, 2 μM to 10 μM, 2.5 μM to 10 μM, 2.5 μM to 8 μM, 2.5 μM to 7 μM, 2.5 μM to 6 μM, 2.5 μM to 5 ... , or in the range of about 3 μM to about 4 μM, or in the range of about 1 μM to about 12 μM, about 1 μM to about 11 μM, about 1 μM to about 10 μM, about 1 μM to about 9 μM, about 1 μM to about 8 μM, about 1 μM to about 7 μM, about 1 μM to about 6 μM, about 1 μM to about 5 μM, about 1 μM to about 4 μM, about 1 μM to about 3 μM, about 1 μM to about 2 μM, about 2 μM to about 12 μM, about 2 μM to about 11 μM, about 2 μM to about 10 μM, about 2 μM to about 9 μM, about 2 μM to about 8 μM, about 2 μM to about 7 μM, about 2 μM to about 6 μM, about 2 μM to about 5 μM, about 2 μM to about 4 μM, about 1 μM to about 3 μM, about 1 μM to about 2 μM, about 2 μM to about 12 μM, about 2 μM to about 11 μM, about 2 μM to about 10 μM, about 2 μM to about 9 μM, about 2 μM to about 8 μM, about 2 μM to about 7 μM, about 2 μM to about 6 μM M to about 11 μM, about 3 μM to about 10 μM, about 3 μM to about 9 μM, about 3 μM to about 8 μM, about 3 μM to about 7 μM, about 3 μM to about 6 μM, about 3 μM to about 5 μM, about 3 μM to about 4 μM, about 4 μM to about 12 μM, about 4 μM to about 11 μM, about 4 μM to about 10 μM, about 4 μM to about 9 μM, about 4 μM to about 8 μM, about 4 μM to about 7 μM, about 4 μM to about 6 μM, about 4 μM to about 5 μM, about 5 μM to about 12 μM, about 5 μM to about 11 μM, about 5 μM to about 10 μM, about 5 μM to about 9 μM, about 5 μM to about 8 μM, about 5 μM to about 7 μM, about 5 μM to about 6 μM, about 6 μM to about 12 μM. In some embodiments, the present invention may be in the range of 5 nM, 50 nM, 500 nM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, 8.5 μM, 9 μM, 9.5 μM, 10 μM, 10.5 μM, 11 μM, 11.5 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 21 μM, 22 μM, 23 μM, 24 μM, 25 μM, 26 μM, 27 μM, 28 μM, 29 μM, 30 μM, 31 μM, 32 μM, 33 μM, 34 μM, 35 μM, 36 μM, 37 μM, 38 μM, 39 μM, 40 μM, 41 μM, 42 μM, 43 μM, 44 μM, 45 μM, 46 μM, 47 μM, 48 μM, 49 μM, 50 μMConcentrations of 5μM, 12μM or 15μM. .

[0176] When the pancreatic endoderm (PE) cells in step i) are treated with inhibitors of vascular endothelial growth factor (VEGF) receptors and / or platelet-derived growth factor (PDGF) receptors, step i) does not involve a TGFb receptor kinase inhibitor. No other changes were made to the protocol, allowing direct comparison with the protocol using a TGFb receptor kinase inhibitor. Examples of TGFbR inhibitors include RepSox (ALK5i II) (Cas number 446859-33-2), SB431542 (Cas number 301836-41-9), LY364947 (Cas number 396129-53-6) and A 83-01 (Cas number 909910-43-6).

[0177] Such a pancreatic endoderm (PE) cell population can be used as the starting material in step i) of the method of the present invention, but in a separate aspect of the present invention, when the pancreatic endoderm (PE) cells are obtained using a disclosed method, they can also be used as the starting material of step i).

[0178] The pancreatic endocrine progenitor cells obtained in step i) typically have the markers NEUROG3, NKX2.2 and NEUROD1.

[0179] In some embodiments, differentiation is performed in a suitable culture medium such as MCDB131 (basal medium), RPMI, DMEM, DMEM / F12, CMRL, MEM, etc.

[0180] The culture medium may be supplemented with, for example, human serum albumin (HSA), antibiotics such as penicillin and / or streptomycin, glucose, sodium bicarbonate, ITSX, glutamax, ascorbic acid and zinc sulfate.

[0181] In some embodiments, the differentiation of pancreatic endoderm to pancreatic endocrine progenitor (EP) cells is carried out for a period of about 1 day to about 15 days, about 1 day to about 12 days, about 1 day to 8 days, such as about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days or about 8 days. Typically, about 4 days.

[0182] The obtained pancreatic endocrine progenitor (EP) cells contain at least one of the markers NEUROG3, NKX2.2 and NEUROD1.

[0183] Differentiation of pancreatic endocrine progenitor cells into pancreatic endocrine (PEC) cells - step ii)

[0184] In some embodiments, the population obtained from step i) comprises pancreatic endocrine progenitor (EP) cells expressing at least one of the markers NEUROG3, NKX2.2 and NEUROD1. Such a population obtained from step i) can be used as a starting material in step ii) of the method of the present invention, but in a separate aspect of the present invention, when the pancreatic endocrine progenitor (EP) cells are obtained using a disclosed method, they can also be used as a starting material for step ii).

[0185] In some embodiments, in step ii), the pancreatic endocrine progenitor (EP) cells are treated with one or more compounds selected from the group consisting of thyroid hormone, staurosporine, a BMP pathway inhibitor, and an EZH2 histone methyltransferase inhibitor.

[0186] Examples of thyroid hormones, staurosporine, BMP pathway inhibitors, EZH histone methyltransferase inhibitors and TGFb receptor kinase inhibitors are given in the above paragraph "Differentiation of pancreatic endoderm (PE) cells into pancreatic endocrine progenitor (EP) cells - step i)", and they are also suitable for step ii) of the method of the present invention.

[0187] The differentiation in step ii) may also include other differentiation factors, such as Rho kinase (ROCK inhibitor), such as Tiger, Chroman-1 (Cas No. 1273579-40-0) and Thiazovivin (Cas No. 1226056-71-8); Heparin; and Forskolin or NKH 477.

[0188] In some embodiments, the pancreatic endocrine progenitor cells in step ii) are treated with an inhibitor of the vascular endothelial growth factor (VEGF) receptor and / or the platelet-derived growth factor (PDGF) receptor. Linifanib is an example of such a substance. Linifanib is 1-(4-(3-amino-1H-indazol-4-yl)phenyl)-3-(2-fluoro-5-methylphenyl)urea. In embodiments, the cells are treated with such an inhibitor.

[0189] In some embodiments, the concentration of the inhibitor of vascular endothelial growth factor (VEGF) and / or platelet-derived growth factor (PDGF) receptor used in step ii) is between 5 nM and about 15 μM, such as 5 nM to about 12 μM, 1 nM to 12 μM, 10 nM to about 11 μM, 25 nM to 10 μM, 50 nM to 10 μM, 100 nM to 10 μM, 250 nM to 10 μM, 500 nM to 10 μM, 750 nM to 10 nM, 1 μM to 10 μM, 2 μM to 10 μM, 2.5 μM to 10 μM, 2.5 μM to 8 μM, 2.5 μM to 7 μM, 2.5 μM to 5 μM, 2.5 μM to 5 μM, 2.5 μM to 6 ... In the range of about 5 μM to 5 μM, or in the range of about 3 μM to about 4 μM, or in the range of about 1 μM to about 12 μM, about 1 μM to about 11 μM, about 1 μM to about 10 μM, about 1 μM to about 9 μM, about 1 μM to about 8 μM, about 1 μM to about 7 μM, about 1 μM to about 6 μM, about 1 μM to about 5 μM, about 1 μM to about 4 μM, about 1 μM to about 3 μM, about 1 μM to about 2 μM, about 2 μM to about 12 μM, about 2 μM to about 11 μM, about 2 μM to about 10 μM, about 2 μM to about 9 μM, about 2 μM to about 8 μM, about 2 μM to about 7 μM, about 2 μM to about 6 μM, about 2 μM to about 5 μM, about 2 μM to about 4 μM, about 2 μM to about 3 μM, about 3 μM to about 12μM, about 3μM to about 11μM, about 3μM to about 10μM, about 3μM to about 9μM, about 3μM to about 8μM, about 3μM to about 7μM, about 3μM to about 6μM, about 3μM to about 5μM, about 3μM to about 4μM, about 4μM to about 12μM, about 4μM to about 11μM, about 4μM to about 10μM, about 4μM to about 9μM, about 4μM to about 8μM, about 4μM to about 7μM, about 4μM to about 6μM, about 4μM to about 5μM, about 5μM to about 12μM, about 5μM to about 11μM, about 5μM to about 10μM, about 5μM to about 9μM, about 5μM to about 8μM, about 5μM to about 7μM, about 5μM to about 6μM, In some embodiments, the present invention relates to an agent that is in the range of 6 μM to about 12 μM, about 6 μM to about 11 μM, about 6 μM to about 10 μM, about 6 μM to about 9 μM, about 6 μM to about 8 μM, about 6 μM to about 7 μM, about 7 μM to about 12 μM, about 8 μM to about 11 μM, about 9 μM to about 10 μM, about 10 μM to about 12 μM, about 10 μM to about 11 μM, or about 11 μM to about 12 μM, or is 5 nM, 50 nM, 500 nM, 1 μM, 1 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, 8.5 μM, 9 μM, 9.5 μM, 10 μM, 10.Concentrations of 5μM, 11μM, 11.5μM, 12μM or 15μM. .

[0190] When the pancreatic endocrine progenitor cells (EP) in step ii) are treated with inhibitors of vascular endothelial growth factor (VEGF) receptors and / or platelet-derived growth factor (PGDF) receptors, step ii) does not involve a TGFb receptor kinase inhibitor. No other changes were made to the protocol, allowing direct comparison with the protocol using a TGFb receptor kinase inhibitor. Examples of TGFbR inhibitors include RepSox (ALK5i II) (Cas number 446859-33-2), SB431542 (Cas number 301836-41-9), LY 364947 (Cas number 396129-53-6) and A 83-01 (Cas number 909910-43-6).

[0191] Differentiation is typically performed in a suitable culture medium such as MCDB131 (basal medium) or one of the above-mentioned culture media or their equivalents.

[0192] The culture medium may be supplemented with, for example, human serum albumin (HSA), antibiotics such as penicillin and / or streptomycin, glucose, sodium bicarbonate, ITSX, glutamax, ascorbic acid and zinc sulfate.

[0193] Typically, the differentiation of pancreatic endocrine progenitor (EP) cells to pancreatic endocrine (PEC) cells is carried out for a period of about 2 days to about 30 days, or about 2 days to 25 days, about 2 days to 20 days, about 3 days to about 12 days, such as about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 15 days, about 20 days, about 25 days or about 30 days. Linifanib is usually added to the culture medium when the culture medium is replaced. The culture medium is usually replaced after 1 or 2 days of cultivation.

[0194] In some embodiments, the inhibitor of vascular endothelial growth factor (VEGF) receptor and / or platelet derived growth factor (PDGF) receptor is administered about 2 days, about 3 days, about 4 days, about 5 days, or about 6 days after initiation of step i) as described herein.

[0195] When the pancreatic endocrine (PEC) cell colony contains β-cells, the culture can be continued. The culture medium can be the same as above, but usually does not contain zinc sulfate. In addition, it does not contain the above-mentioned differentiation factors, but can contain inhibitors of vascular endothelial growth factor (VEGF) receptors and / or platelet-derived growth factor (PDGF) receptors for further culture for at least 2 days, such as 2 days, 3 days or 4 days.

[0196] If the obtained endocrine cell population contains pancreatic endocrine cell aggregates, the aggregates can be dissociated into single cells.

[0197] Pancreatic endocrine (PEC) cells can be further treated with a cryopreservation medium and a reduced temperature to obtain cryopreserved single cells. Methods suitable for cryopreserving pancreatic endocrine (PEC) cells are described in WO 2019 / 048690, which is referenced herein and incorporated herein by reference in its entirety.

[0198] Cryopreservation can be performed after culturing the cells for 1 day or longer, for example, after culturing for about 1 day to about 30 days.

[0199] In some embodiments, the obtained pancreatic endocrine (PEC) cells include islet-like cells, such as β cells (having markers INS, PDX1 and / or NKX6.1), α cells (having markers GCG and / or ARX), δ cells (having markers SST and / or HHEX).

[0200] In some embodiments, the pancreatic endocrine (PEC) cell population obtained after step ii) comprises at least 20%, such as at least 25%, such as at least 28% or at least 30% β-like cells based on the total number of cells obtained after step ii), and wherein the β-like cells are double positive for ISL1 and NKX6.1 (ISL1+NKX6.1+), see e.g. Figure 1 B.

[0201] Thus, the pancreatic endocrine (PEC) cell population obtained after step ii) may contain at least 1% more, such as at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 25%, at least 30% more β-like cells compared to the pancreatic endocrine (PEC) cell population obtained by using a TGFb receptor kinase inhibitor instead of an inhibitor of VEGF and / or PDGF in step i) and / or step ii), and wherein the β-like cells are double positive for ISL1 and NKX6.1 (ISL1+NKX6.1+), see e.g. Figure 3 B.

[0202] In an embodiment, the pancreatic endocrine (PEC) cell population obtained after step ii) contains at most 35%, such as at most 30% or at most 27% enterochromaffin cells, and wherein the enterochromaffin cells are negative for ISL1 and positive for NKX6.1 (ISL1-NKX6.1+), see e.g. Figure 3 B.

[0203] Thus, the pancreatic endocrine (PEC) cell population obtained after step ii) may contain at least 1% less, such as at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30% or at least 35% less enterochromaffin cells compared to a pancreatic endocrine (PEC) cell population obtained by using a TGFb receptor kinase inhibitor instead of an inhibitor of VEGF and / or PDGF in step i) and step ii), and wherein the enterochromaffin cells are negative for ISL1 and positive for NKX6.1 (ISL1-NKX6.1+), see e.g. Figure 3 B.

[0204] In an embodiment, the pancreatic endocrine (PEC) cell population obtained after step ii) contains at least 78%, such as at least 80%, of pancreatic endocrine cells, and wherein said endocrine cells are CHGA+, see e.g. Figure 3 E.

[0205] Thus, the pancreatic endocrine (PEC) cell population obtained after step ii) may contain at least 1% more, such as at least 2%, at least 3%, at least 4% or at least 5% more pancreatic endocrine (PEC) cells compared to a pancreatic endocrine (PEC) cell population obtained by using a TGFb receptor kinase inhibitor instead of an inhibitor of VEGF and / or PDGF in step i) and step ii), and wherein the pancreatic endocrine (PEC) cells are CHGA+, see e.g. Figure 3 E.

[0206] Differentiation of stem cells into pancreatic endocrine cells

[0207] In some embodiments, hPSC is obtained from any suitable source mentioned above. In some embodiments, the methods described herein include culturing hPSC. The term "culture" refers to culturing hPSC in a cell culture medium suitable for the viability of its current developmental state. In some embodiments, culturing stem cells involves transferring stem cells to a different environment, such as by inoculating them on a new substrate or suspending them in a culture vessel. Those of ordinary skill in the art will readily recognize that stem cells are fragile to such transfers, so the procedure requires caution, and maintaining stem cells in the original cell culture medium before replacing the cell culture medium with another cell culture medium that is more suitable for the differentiation process can promote more sustainable cell transfer.

[0208] In some embodiments, the methods described herein relate to an in vitro method of generating pancreatic endocrine (PEC) cells from human pluripotent stem cells, comprising the steps of:

[0209] i) differentiating hPSC cells into definitive endoderm cells,

[0210] ii) differentiating definitive endoderm cells into pancreatic endoderm cells,

[0211] iii) differentiating pancreatic endoderm (PE) cells into pancreatic endocrine progenitor cells, and

[0212] iv) differentiating the pancreatic endocrine progenitor (EP) cells into pancreatic endocrine (PEC) cells (islet-like cells, including β-cells),

[0213] Wherein step iii) and / or step iv) is performed using an inhibitor of vascular endothelial growth factor (VEGF) and / or platelet-derived growth factor (PDGF) receptor.

[0214] Step iii) is described as step i) under the referenced title “Differentiation of pancreatic endoderm cells (PE) into pancreatic endocrine progenitor cells (EP or PEP) - step i)”, while step iv) is described as step ii) under the referenced title “Differentiation of pancreatic endocrine progenitor cells into pancreatic endocrine (PEC) cells - step ii)”.

[0215] The steps of differentiating hPSC cells into definitive endoderm cells and differentiating definitive endoderm cells into pancreatic endoderm (PE) cells can follow standard protocols, e.g.,

[0216] ■ D'Amour et al. - Nat Biotechnol. - 2005 Dec;23(12):1534-41.

[0217] ■Funa et al. - Cell Stem Cell. - 2015 Jun 4;16(6):639-52

[0218] ■Teo et al. - Stem Cell Reports. - 2014 Jun 12;3(1):5-14

[0219] ■ Kubo et al. - Development. - 2004 April; 131(7): 1651-62

[0220] ■ Rezania et al. - Nat Biotechnol. - 2014 Nov;32(11):1121-33

[0221] ■ Rezania et al. - Diabetes. - 2011 Jan; 60(1): 239-47

[0222] ■ Kroon et al. - Nat Biotechnol. - 2008 Apr;26(4):443-52.

[0223] ■Ameri et al. - Stem Cells. - 2010 Jan; 28(1): 45-56

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[0227] ■Russ et al. - EMBO J - 2015 Jul 2;34(13):1759-72.

[0228] Other aspects of the invention

[0229] Compositions comprising pancreatic endocrine cells (PEC) obtained by any method of the present invention

[0230] In a further aspect, described herein is a medicament comprising pancreatic endocrine cells (PECs) obtained by any method of the present invention according to the present specification. In particular, compared with the standard method using TGFbRi, the PECs obtained by the method described herein have i) more islet cells (ISL1+), more β-like cells (ISL1+ / NKX6.1+) and fewer EC cells (ISL1- / NKX6.1+).

[0231] In a preferred embodiment, the medicament described herein comprises enriched or homogenized, thawed and reaggregated cryopreserved pancreatic endocrine cells (PECs) obtained by any of the methods of the present invention.

[0232] Medical use of pancreatic endocrine (PEC) cells obtained by any method of the present invention

[0233] In some embodiments, the present invention relates to a method of providing pancreatic endocrine function to a mammal having deficient production of at least one pancreatic hormone, the method comprising the step of implanting pancreatic endocrine cells obtained by any method of the present invention in an amount sufficient to produce a measurable amount of the at least one pancreatic hormone in the mammal.

[0234] In the present context, the term "mammal" includes both human and veterinary subjects.

[0235] In the present context, the term "mammal" relates to, for example, humans, horses, pigs, rabbits, dogs, sheep, goats, non-human primates, cows, cats, guinea pigs or rodents. The term does not denote a particular age or sex.

[0236] For example, pancreatic islet cell transplantation can be used to restore insulin production and blood sugar control in diabetic mammals.

[0237] Also provided herein is a method for treating diabetes (type 1 or type 2). For example, provided herein is a method for treating type 1 diabetes in a mammal. In some embodiments, the method includes selecting a mammal suffering from type 1 diabetes and administering to the mammal a step of pancreatic endocrine cells obtained by any method of the present invention. In other embodiments, the method includes preventing type 1 diabetes in the mammal by administering to a mammal at risk of suffering from type 1 diabetes an endocrine cell obtained by any method of the present invention.

[0238] Those skilled in the art use any diagnostic and identification methods to identify and select mammals suffering from type 1 diabetes and mammals at risk of suffering from type 1 diabetes. For example, diagnosis is based on elevated blood sugar levels after fasting or based on a glucose tolerance test. In addition, the diagnosis of type 1 diabetes includes various physical symptoms and characteristics.

[0239] Identification of mammals at risk for type 1 diabetes is also within the skill of the skilled artisan. For example, a mammal at risk for type 1 diabetes is an individual with a genetic predisposition or an individual who has had the pancreas or a portion thereof surgically removed. Mammals that have had the pancreas surgically removed include mammals with chronic pancreatitis or mammals that require surgical removal of the pancreas due to injury.

[0240] Mammals suffering from insulin-dependent type 2 diabetes or at risk of type 2 diabetes similarly benefit from administering pancreatic endocrine cells obtained by any of the methods of the present invention. Therefore, provided herein is a method comprising the steps of selecting a mammal suffering from type 2 diabetes or at risk of type 2 diabetes, and administering to the mammal a sufficient amount of pancreatic endocrine cells obtained by any of the methods of the present invention. Diagnosis is typically based on fasting blood glucose levels, based on a glucose tolerance test, or based on blood insulin levels.

[0241] A sufficient amount of pancreatic endocrine cells obtained by any method of the present invention are administered in various ways herein. Transplant compositions are often administered intrahepatically, for example, by direct percutaneous puncture of the liver. The right or left branch of the portal vein can be selected for cannulation, and the puncture site is selected accordingly by an interventional radiologist. In some embodiments, several transplants are performed. However, those skilled in the art can easily determine the concentration of cells to be included in the transplant composition, and recognize whether a second or subsequent transplant is needed based on clinical signs such as hyperglycemia.

[0242] The method for preparing the islet cell colony for transplantation taught herein is also combined with treatment. Therefore, for example, a method for treating diabetes in a mammal is provided herein, comprising the following steps: preparing an insulin-secreting cell colony (e.g., islet cell colony) for transplantation according to any in vitro method described above, and transplanting the cell colony into a mammal to be treated (i.e., a transplant recipient).

[0243] Example

[0244] Example 1

[0245] The pancreatic endocrine progenitor cells (EP) produced in vitro according to the present invention are obtained by the following steps.

[0246] The pancreatic endoderm cell aggregates generated from hPSCs were cultured in a suitable suspension culture format. The aggregates were washed by settling the cell aggregates and removing excess culture medium. Washing medium (MCDB 131 culture medium, Gibco, catalog number 10372019) was added to the cell aggregates and then removed.

[0247] Differentiation into EPs was performed in MCDB131 medium supplemented with Glutamax (Gibco, catalog number 35050038), 0.05% human serum albumin (Origin, catalog number ART-3003), 20 mM glucose (Sigma-Aldrich, catalog number G8769), 14.64 mM NaHCO3 (Gibco, catalog number 25080094), ITS-X (Gibco, catalog number 51500056), 0.25 mM ascorbic acid (FisherScientific, catalog number 0937-07) and 10 uM ZnSO4 (Merck, catalog number 1088811000). The medium was further supplemented with the following compounds: 2uM XX (Tocris, catalog no. 4489), 1uM T3 (Tocris, catalog no. 6666), 5uM Tiger (Tocris, catalog no. 1254), 100nM LDN-193189 (Tocris, catalog no. 6053), 20ng / ml β-cellulin (R&D systems, catalog no. 261-CE-250), 10ug / ml heparin (Merck, catalog no. H3393), 3.3nM staurosporine (Tocris, catalog no. 1258), 0.1uM DZNep (Tocris, catalog no. 4703), 10uM Forskolin (Tocris, catalog no. 1099), 5uM JNKi VIII (Tocris, catalog no. 3222), 10uM RepSox (Tocris, catalog no. 3742) and 6uM SB431542 (Tocris, catalog number 1614). This regimen is referred to as the standard (STD) regimen. When evaluating the effects of using VEGFR / PDGFR inhibitors, RepSox and SB431542 were removed from the above regimen and 4uM linifanib (ABT-869, SelleckChem, catalog number S1003) was added instead.

[0248] The culture medium was replenished every 48 hours, and differentiation from pancreatic endoderm to endocrine progenitor cells was performed for four days.

[0249] Example 2

[0250] The pancreatic endocrine cells (PEC) produced in vitro according to the present invention are obtained by the following steps.

[0251] The pancreatic endocrine progenitor cell aggregates generated from pancreatic endoderm are cultured in a suitable suspension culture format. Aggregates are washed by settling cell aggregates and removing excess culture medium. Washing medium (MCDB 131 culture medium, Gibco, catalog number 10372019) is added to the cell aggregates and then removed.

[0252] Differentiation into PECs was performed in MCDB131 medium supplemented with Glutamax (Gibco, catalog number 35050038), 0.05% human serum albumin (Origin, catalog number ART-3003), 20 mM glucose (Sigma-Aldrich, catalog number G8769), 14.64 mM NaHCO3 (Gibco, catalog number 25080094), ITS-X (Gibco, catalog number 51500056), 0.25 mM ascorbic acid (FisherScientific, catalog number 0937-07) and 10 uM ZnSO4 (Merck, catalog number 1088811000). The following compounds were further supplemented to the culture medium: 1uM XX (Tocris, catalog number 4489), 1uM T3 (Tocris, catalog number 6666), 5uM Tiger (Tocris, catalog number 1254), 100nM LDN-193189 (Tocris, catalog number 6053), 10ug / ml heparin (Merck, catalog number H3393), 3.3nM staurosporine (Tocris, catalog number 1258), 0.1uM DZNep (Tocris, catalog number 4703) and 10uM RepSox (Tocris, catalog number 3742). This regimen is referred to as the standard (STD) regimen. When evaluating the effect of using VEGFR / PDGFR inhibitors, RepSox was removed from the above regimen and replaced by adding 4uM linifanib (ABT-869, SelleckChem, catalog number S1003).

[0253] The medium was replenished every 48 hours and differentiation from endocrine progenitor cells to pancreatic endocrine cells was performed for three days. At this stage, the cells could be cryopreserved or further differentiated using the following medium composition.

[0254] Differentiation was performed for two days in MCDB131 medium supplemented with Glutamax (Gibco, catalog number 35050038), 0.05% human serum albumin (Origin, catalog number ART-3003), 2.5 mM glucose (Sigma-Aldrich, catalog number G8769), 14.64 mM NaHCO3 (Gibco, catalog number 25080094), ITS-X (Gibco, catalog number 51500056), 0.25 mM ascorbic acid (Fisher Scientific, catalog number 0937-07) and 10 uM ZnSO4 (Merck, catalog number 1088811000). The culture medium was further supplemented with the following compounds: 1uM XX (Tocris, catalog no. 4489), 1uM T3 (Tocris, catalog no. 6666), 5uM Tiger (Tocris, catalog no. 1254), 10ug / ml heparin (Merck, catalog no. H3393), 0.1uM DZNep (Tocris, catalog no. 4703), and further supplemented with 10uM RepSox (Tocris, catalog no. 3742) (STD) or 4uM Linifanib (ABT-869, SelleckChem, catalog no. S1003).

[0255] Differentiation was performed for two days in MCDB131 medium supplemented with Glutamax (Gibco, catalog number 35050038), 0.05% human serum albumin (Origin, catalog number ART-3003), 2.5 mM glucose (Sigma-Aldrich, catalog number G8769), 14.64 mM NaHCO3 (Gibco, catalog number 25080094), ITS-X (Gibco, catalog number 51500056), 0.25 mM ascorbic acid (Fisher Scientific, catalog number 0937-07) and further supplemented with 10 uM RepSox (Tocris, catalog number 3742) (STD) or 4 uM linifanib (ABT-869, SelleckChem, catalog number S1003).

[0256] Subsequently, pancreatic endocrine cells can be maintained for several days to weeks in MCDB131 medium supplemented with Glutamax (Gibco, catalog number 35050038), 0.05% human serum albumin (Origin, catalog number ART-3003), 2.5 mM glucose (Sigma-Aldrich, catalog number G8769), 14.64 mM NaHCO3 (Gibco, catalog number 25080094), ITS-X (Gibco, catalog number 51500056) and 0.25 mM ascorbic acid (Fisher Scientific, catalog number 0937-07), with the medium replenished every 48 hours.

[0257] Example 3 - Cryopreservation

[0258] The pancreatic endocrine cell aggregates obtained in vitro according to the present invention are cryopreserved as described in WO 2019 / 048690. In brief, the obtained cells are resuspended in a cryopreservation medium and stored by continuously lowering the temperature to below -80°C.

[0259] Thawing cryopreserved single cells

[0260] To recover the cells, the cells were quickly warmed to 37°C and washed once in pre-warmed RPMI1640 medium (Gibco #61870-044) supplemented with 12% KOSR (Gibco #10828-0280). After counting, the cells were resuspended in stage-specific medium supplemented with 50 μg / mL DNaseI (Sigma #11284932001) and 10 μM Rocki (Sigma #Y27632-Y0503).

[0261] Reaggregate cells obtained after thawing

[0262] The cells obtained after thawing can be reaggregated in a conical flask at a density of 0.5-2 mio viable cells / ml in a reduced volume. Reaggregation is performed at 37°C and 70 rpm horizontal shaking for two days, followed by a culture medium change.

[0263] After cryopreservation, cells with viability ranging from 60% to 90% recovered. After cell reaggregation, the glucose-responsive insulin secretion phenotype was improved.

Claims

1. An in vitro method for generating a pancreatic endocrine (PEC) cell population, comprising the following steps: i) differentiating a population of pancreatic endoderm (PE) cells into pancreatic endocrine progenitor cells, wherein The differentiation includes the use of vascular endothelial growth factor (VEGF) receptors and / or platelet derivatives The pancreatic endoderm (PE) is treated with an inhibitor of the PDGF receptor cells, and ii) treating the pancreatic endocrine progenitor (EP) cells with an inhibitor of vascular endothelial growth factor (VEGF) receptor and / or platelet-derived growth factor (PDGF) receptor; cells, causing the pancreatic endocrine progenitor (EP) cells to differentiate into pancreatic endocrine progenitor (PEC) cells cell.

2. The method of claim 1, wherein the inhibitor of VEGF receptor and / or PDGF receptor is 1-(4-(3-amino-1H-indazol-4-yl)phenyl)-3-(2-fluoro-5-methylphenyl)urea.

3. The method according to any one of claims 1-2, wherein step i) does not involve a TGFb receptor kinase inhibitor.

4. The method according to any one of claims 1 to 3, wherein step ii) does not involve a TGFb receptor kinase inhibitor.

5. A method according to any one of the preceding claims, wherein the concentration of the inhibitor of vascular endothelial growth factor (VEGF) and / or platelet-derived growth factor (PDGF) receptor used in step ii) is in the range of 5 nM to about 15 μM, such as 5 nM to about 12 μM, 1 μM to 10 μM, about 1 μM to about 5 μM or about 3 μM to about 4 μM.

6. The method according to any one of claims 2-5, wherein the concentration of the inhibitor of vascular endothelial growth factor (VEGF) receptor and / or platelet-derived growth factor (PDGF) receptor used in step i) is in the range of 5 nM to about 15 μM, such as 5 nM to about 12 μM, 1 μM to about 10 μM, about 1 μM to about 5 μM or about 3 μM to about 4 μM (Figures 2C, 2D).

7. The method according to any one of the preceding claims, wherein the pancreatic endocrine (PEC) cell population obtained after step ii) comprises at least 20%, such as at least 25%, such as at least 28%, or at least 30% β-like cells based on the total number of cells obtained after step ii), and wherein the β-like cells are doubly positive for ISL1 and NKX6.1 (ISL1+NKX6.1+).

8. The method according to any of the preceding claims, wherein the pancreatic endocrine (PEC) cell population obtained after step ii) contains at least 1% more, such as at least 2%, at least 3%, at least 4%, at least 5%, at least 10% or at least 20% more β-like cells compared to the pancreatic endocrine (PEC) cell population obtained by using a TGFb receptor kinase inhibitor instead of an inhibitor of VEGF and / or PGDF in step i) and step ii), and wherein the β-like cells are double positive for ISL1 and NKX6.1 (ISL1+NKX6.1+).

9. The method according to any one of the preceding claims, wherein the pancreatic endocrine (PEC) cell population obtained after step ii) contains at most 35%, such as at most 30% or at most 27% enterochromaffin cells, and wherein the enterochromaffin cells are negative for ISL1 and positive for NKX6.1 (ISL1-NKX6.1+).

10. The method according to any one of the preceding claims, wherein the cells obtained after step ii) contain at least 78%, such as at least 80%, of pancreatic endocrine cells, and wherein the endocrine cells are CHGA+.

11. A method according to any of the preceding claims, wherein the pancreatic endocrine (PEC) cell population obtained after step ii) contains at least 1% more, such as at least 2%, at least 3%, at least 4% or at least 5% more pancreatic endocrine (PEC) cells compared to a pancreatic endocrine (PEC) cell population obtained by using a TGFb receptor kinase inhibitor instead of an inhibitor of VEGF and / or PDGF in step i) and step ii), and wherein the pancreatic endocrine (PEC) cells are CHGA+.

12. An in vitro method for producing a population of pancreatic endocrine progenitor (EP) cells, comprising the steps of: i) Differentiating a population of pancreatic endoderm (PE) cells into pancreatic endocrine progenitor cells by treating the PE cells with inhibitors of the vascular endothelial growth factor (VEGF) receptor and / or the platelet-derived growth factor (PDGF) receptor.

13. An in vitro method for generating a pancreatic endocrine (PEC) cell population, comprising the steps of: ii) differentiating pancreatic endocrine progenitor (EP) cells into pancreatic endocrine (PEC) cells by treating the pancreatic endocrine progenitor (EP) cells with an inhibitor of vascular endothelial growth factor (VEGF) receptor and / or platelet-derived growth factor (PDGF) receptor.

14. An in vitro method for generating a pancreatic endocrine (PEC) cell population, comprising the steps of: i) differentiating a population of pancreatic endoderm (PE) cells into pancreatic endocrine progenitor cells by treating the PE cells with an inhibitor of the vascular endothelial growth factor (VEGF) receptor and / or the platelet-derived growth factor (PDGF) receptor, and ii) Differentiating pancreatic endocrine progenitor (EP) cells into pancreatic endocrine (PEC) cells.

15. An in vitro method for generating a pancreatic endocrine (PEC) cell population, comprising the steps of: i) differentiating a population of pancreatic endoderm (PE) cells into pancreatic endocrine progenitor cells, and ii) treating the pancreatic endocrine progenitor (EP) cells with an inhibitor of vascular endothelial growth factor (VEGF) receptor and / or platelet-derived growth factor (PDGF) receptor; cells, causing the pancreatic endocrine progenitor (EP) cells to differentiate into pancreatic endocrine progenitor (PEC) cells cell.

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