Differentiation of stem cells to 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 progenitors, in which the differentiating 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

[0001] DIFFERENTIATION OF STEM CELLS TO PANCREATIC ENDOCRINE CELLS

[0002] TECHNICAL FIELD

[0003] The present invention relates to an in vitro method for obtaining pancreatic endocrine (PEC) cells involving steps wherein the cells are treated with an inhibitor of Vascular Endothelial Growth Factor (VEGF) receptor and / or Platelet-Derived Growth Factor (PDGF) receptor. The method provides islet-like cells, in particular beta-like cells.

[0004] BACKGROUND

[0005] Although insulin therapy is lifesaving, it can be difficult to obtain stable glycemia with exogenous insulin and poor control is associated with serious late state complications (Nathan, D.M., 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). Transplantation of pancreatic islets isolated from human donors to patients with typel diabetes have shown good results with some patients becoming completely insulin independent (Barton F.B. et al., 2012. Improvement in Outcomes of Clinical Islet Transplantation: 1999-2010. Diabetes Care, 35(7), pp.1436-1445). Despite such advances, one of the major challenges for islet transplantation is limited availability of donor islets. This donor material shortage can be overcome by generating functional insulin secreting cells in vitro by differentiation of human embryonic stem cells (hESCs). Protocols for the generation of functional insulin secreting cells in vitro from stem cells are continuously developing (Pagliuca F.W. et al., 2014. Generation of Functional Human Pancreatic p Cells In vitro. Cell, 159(2), pp.428-439; Rezania A etal. Reversal of diabetes with insulinproducing cells derived in vitro from human pluripotent stem cells. Nature Biotechnology, 32(11), pp.1121-1133); WO2012175633; WO2014033322; WO2015028614).

[0006] Beta cell (BC) transplantation potentially provides the ultimate cure for type I diabetes. However, the limited availability of donor beta cells constrains the use of this treatment as a clinical therapy. Pluripotent stem (PS) cells can proliferate infinitely and differentiate into many cell types. Thus, PS cells are a promising source for beta cells, but before PS cells can be used to treat diabetes, they need to be efficiently and reproducibly differentiated to pancreatic cells.

[0007] During vertebrate embryonic development, pluripotent stem cells give rise to the three germ layers: ectoderm, mesoderm and endoderm. Induction of definitive endoderm (DE) is the first step towards formation of endoderm derived tissues. Generation of pancreatic endoderm (PE) from DE cells is necessary for the generation of pancreatic endocrine progenitor (EP) cells and ultimately of insulin- producing beta cells. PE cells with the potential to become beta cells are characterized by co- expression of two important transcription factors, PDX1 and NKX6.1. Stepwise in vitro differentiation protocols have been established for generating pancreatic cells from PS cells. These protocols generally mimic the major events of pancreatic development, which includes several stages, such as formation of the DE which co-express SOX17 and FOXA2, primitive gut, posterior foregut, PE, EP, PEC (pancreatic endocrine cells). 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 beta cells is to generate PE that co-expresses PDX1 and NKX6.1 . Several 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) agonist in the induction of PE.

[0008] In summary, 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, and insulin producing cells derived from PS cells offer an attractive alternative to pancreatic islets. Key differentiation steps are differentiation to EP cells and to PEC cells to yield functional beta cells (BCs).

[0009] Current protocols for differentiating human pluripotent stem cells (hPSC) to EP cells and specific PEC cells are not efficient processes. Furthermore, with current protocols, a high degree of unwanted non-pancreatic endocrine cells, such as enterochromaffin cells, as well as non- endocrine cells, are formed during the differentiation. Thus, there is a need to increase the efficiency of current differentiation protocols.

[0010] SUMMARY

[0011] The present invention provides an in vitro method for producing pancreatic endocrine progenitors and / or pancreatic endocrine (PEC) cells comprising the steps of: i) differentiating a population of pancreatic endoderm (PE) cells into pancreatic endocrine progenitor (EP) cells, wherein the differentiating 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 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.

[0012] Notably, the present invention provides an in vitro method for producing pancreatic endocrine (PEC) cells comprising the steps of: 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 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.

[0013] In some embodiments the present invention provides an in vitro method for producing pancreatic endocrine (PEC) cells comprising the steps of: 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, or 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.

[0014] In some embodiments, the present invention provides an in vitro method for producing pancreatic endocrine (PEC) cells comprising the steps of: 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 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 Platelet-Derived Growth Factor (PDGF) receptor.

[0015] In embodiments, the in vitro method for producing a population of pancreatic endocrine progenitor (EP) cells comprises the step of: i) differentiating a population of pancreatic endoderm (PE) cells into pancreatic endocrine progenitors, by treating the PE cells with an inhibitor of Vascular Endothelial Growth Factor (VEGF) receptor and / or Platelet-Derived Growth Factor (PDGF) receptor. In embodiments, the in vitro method for producing a population of pancreatic endocrine (PEC) cells 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.

[0016] In embodiments, the 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 progenitors, by treating the PE cells with an inhibitor of Vascular Endothelial Growth Factor (VEGF) receptor and / or Platelet-Derived Growth Factor (PDGF) receptor, and ii) differentiating pancreatic endocrine progenitor (EP) cells into pancreatic endocrine (PEC) cells.

[0017] In embodiments, the 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 progenitors, 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.

[0018] A suitable inhibitor of Vascular Endothelial Growth Factor (VEGF) receptor and / or Platelet-Derived Growth Factor (PDGF) receptor for use in a method of the invention is 1 -(4-(3-amino-1 H-indazol-4- yl)phenyl)-3-(2-fluoro-5-methylphenyl)urea, also denoted linlfanib or ABT869.

[0019] The inventors have found that the use of an inhibitor of Vascular Endothelial Growth Factor (VEGF) receptor and / or Platelet-Derived Growth Factor (PDGF) receptor in step i) and / or step ii) above has the advantage of increasing the percentage of beta-like cells obtained, compared with a method wherein a Transforming Growth Factor beta (TGFb) receptor kinase inhibitor or TGFpRI kinase inhibitor or TGFP1R kinase inhibitor or TGFbRI inhibitor is used instead of the inhibitor of Vascular Endothelial Growth Factor (VEGF) receptor and / or Platelet-Derived Growth Factor (PDGF) receptor. Moreover, the percentage of pancreatic endocrine (PEC) cells is also increased. The concentration of inhibitor of Vascular Endothelial Growth Factor (VEGF) and / or Platelet- Derived Growth Factor (PDGF) receptor used in step ii) is in a range of from 1 nM to 12 μM such as from 5 nM to about 15 μM, 5 nM to about 12 μM, 10 nM to about 11 μM, from 25 nM to 10 μM, from 50 nM to 10 μM, from 100 nM to 10 μM, from 250 nM to 10 μM, from 500 nM to 10 μM, from 750 nM to 10 nM, from 1 μM to 10 μM, from 2 μM to 10 μM, from 2.5 μM to 10 μM, from 2.5 μM to 8 μM, from 2.5 μM to 7 μM, from 2.5 μM to 6 μM, from 2.5 μM to 5 μM, from 2.5 μM to 5.5 μM, from

[0020] 2.5 μM to 5 μM or in a range of from about 3 μM to about 4 μM, or in a range of from about 1 μM to about 12 μM, from about 1 μM to about 11 μM, from about 1 μM to about 10 μM, from about 1 μM to about 9 μM, from about 1 μM to about 8 μM, from about 1 μM to about 7 μM, from about 1 μM to about 6 μM, from about 1 μM to about 5 μM, from about 1 μM to about 4 μM, from about 1 μM to about 3 μM, from about 1 μM to about 2 μM, from about 2 μM to about 12 μM, from about 2 μM to about 11 μM, from about 2 μM to about 10 μM, from about 2 μM to about 9 μM, from about 2 μM to about 8 μM, from about 2 μM to about 7 μM, from about 2 μM to about 6 μM, from about 2 μM to about 5 μM, from about 2 μM to about 4 μM, from about 2 μM to about 3 μM, from about 3 μM to about 12 μM, from about 3 μM to about 11 μM, from about 3 μM to about 10 μM, from about 3 μM to about 9 μM, from about 3 μM to about 8 μM, from about 3 μM to about 7 μM, from about 3 μM to about 6 μM, from about 3 μM to about 5 μM, from about 3 μM to about 4 μM, from about 4 μM to about 12 μM, from about 4 μM to about 11 μM, from about 4 μM to about 10 μM, from about 4 μM to about 9 μM, from about 4 μM to about 8 μM, from about 4 μM to about 7 μM, from about 4 μM to about 6 μM, from about 4 μM to about 5 μM, from about 5 μM to about 12 μM, from about 5 μM to about 11 μM, from about 5 μM to about 10 μM, from about 5 μM to about 9 μM, from about 5 μM to about 8 μM, from about 5 μM to about 7 μM, from about 5 μM to about 6 μM, from about 6 μM to about 12 μM, from about 6 μM to about 11 μM, from about 6 μM to about 10 μM, from about 6 μM to about 9 μM, from about 6 μM to about 8 μM, from about 6 μM to about 7 μM, from about 7 μM to about 12 μM, from about 8 μM to about 11 μM, from about 9 μM to about 10 μM, from about 10 μM to about 12 μM, from about 10 μM to about 11 μM or from about 11 μM to about 12 μM, or in a concentration of 5nM, 50nM, 500nM, 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,

[0021] 11.5 μM, 12 μM or 15 μM.

[0022] The concentration of inhibitor of Vascular Endothelial Growth Factor (VEGF) and / or Platelet- Derived Growth Factor (PDGF) receptor used in step i) is in a range of from 1 nM to 12 μM such as from 5 nM to about 15 μM, 5 nM to about 12 μM,10 nM to about 11 μM, from 25 nM to 10 μM, from 50 nM to 10 μM, from 100 nM to 10 μM, from 250 nM to 10 μM, from 500 nM to 10 μM, from 750 nM to 10 nM, from 1 μM to 10 μM, from 2 μM to 10 μM, from 2.5 μM to 10 μM, from 2.5 μM to 8 μM, from 2.5 μM to 7 μM, from 2.5 μM to 6 μM, from 2.5 μM to 5 μM, from 2.5 μM to 5.5 μM, from

[0023] 2.5 μM to 5 μM or in a range of from about 3 μM to about 4 μM, , or in a range of from about 1 μM to about 12 μM, from about 1 μM to about 11 μM, from about 1 μM to about 10 μM, from about 1 μM to about 9 μM, from about 1 μM to about 8 μM, from about 1 μM to about 7 μM, from about 1 μM to about 6 μM, from about 1 μM to about 5 μM, from about 1 μM to about 4 μM, from about 1 μM to about 3 μM, from about 1 μM to about 2 μM, from about 2 μM to about 12 μM, from about 2 μM to about 11 μM, from about 2 μM to about 10 μM, from about 2 μM to about 9 μM, from about 2 μM to about 8 μM, from about 2 μM to about 7 μM, from about 2 μM to about 6 μM, from about 2 μM to about 5 μM, from about 2 μM to about 4 μM, from about 2 μM to about 3 μM, from about 3 μM to about 12 μM, from about 3 μM to about 11 μM, from about 3 μM to about 10 μM, from about

[0024] 3 μM to about 9 μM, from about 3 μM to about 8 μM, from about 3 μM to about 7 μM, from about 3 μM to about 6 μM, from about 3 μM to about 5 μM, from about 3 μM to about 4 μM, from about 4 μM to about 12 μM, from about 4 μM to about 11 μM, from about 4 μM to about 10 μM, from about

[0025] 4 μM to about 9 μM, from about 4 μM to about 8 μM, from about 4 μM to about 7 μM, from about 4 μM to about 6 μM, from about 4 μM to about 5 μM, from about 5 μM to about 12 μM, from about 5 μM to about 11 μM, from about 5 μM to about 10 μM, from about 5 μM to about 9 μM, from about 5 μM to about 8 μM, from about 5 μM to about 7 μM, from about 5 μM to about 6 μM, from about 6 μM to about 12 μM, from about 6 μM to about 11 μM, from about 6 μM to about 10 μM, from about 6 μM to about 9 μM, from about 6 μM to about 8 μM, from about 6 μM to about 7 μM, from about 7 μM to about 12 μM, from about 8 μM to about 11 μM, from about 9 μM to about 10 μM, from about 10 μM to about 12 μM, from about 10 μM to about 11 μM or from about 11 μM to about 12 μM, or in a concentration of 5nM, 50nM, 500nM, 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, or 15 μM.

[0026] In some embodiments, the pancreatic endocrine (PEC) cells obtained from step ii) include islet-like cells. Islet-like cells include alpha-like cells, beta-like cells, epsilon-like cells, delta-like cells and gamma-like cells.Steps i) and ii) of a method of the invention do not involve a TGFb receptor kinase inhibitor.

[0027] In some embodiments, step i) does not involve a TGFb receptor kinase inhibitor.

[0028] In some embodiments, step ii) does not involve a TGFb receptor kinase inhibitor.

[0029] As shown in FIG 1B, the population of pancreatic endocrine (PEC) cells obtained after step ii) comprises at least 20%, such as at least 25%, such as at least 28%, or at least 30% of beta-like cells, based on the total number of cells obtained after step ii), and the beta-like cells are double positive with respect to ISL1 and NKX6.1 (ISL1+NKX6.1+).

[0030] As mentioned above, the number of beta-like cells obtained in the population of pancreatic endocrine (PEC) cells obtained after step ii) is increased compared with a population of pancreatic endocrine (PEC) cells obtained by using a TGFb receptor kinase inhibitor instead of an inhibitor of VEGF and / or PDGF in step i) and / or in step ii), and wherein the beta-like cells are double positive with respect to ISL1 and NKX6.1 (ISL1+NKX6.1+). Thus, the population of pancreatic endocrine (PEC) cells obtained after step ii) contains at least 1% such as at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, or at least 20% more beta-like cells compared with a population of pancreatic endocrine (PEC) cells obtained by using a TGFb receptor kinase inhibitor instead of an inhibitor of VEGF and / or PDGF in step i) and / or in step ii), and wherein the beta-like cells are double positive with respect to ISL1 and NKX6.1 (ISL1+NKX6.1+). Moreover, more than 60% of the cells are ISL1+ compared with only about 40% of the cells are ISL1+ when a standard method is used, see e.g., FIG 3B.

[0031] The number of enterochromaffin (EC) cells produced by the method of the present invention is decreased, compared with an in vitro method for producing pancreatic endocrine (PEC) cells, wherein the pancreatic endocrine (PEC) cells are obtained by using a TGFb receptor kinase inhibitor instead of an inhibitor of VEGF and / or PDGF in step i) and / or in step ii). Thus, the population of pancreatic endocrine (PEC) cells obtained after step ii) contains at the most 35%, such as at the most 30%, or at the most 27% enterochromaffin cells, wherein the enterochromaffin cells are negative with respect to ISL1 and positive with respect to NKX6.1 (ISL1-NKX6.1+), see e.g., Fig. 3B.

[0032] The population of pancreatic endocrine (PEC) cells obtained after step ii) contains at least 1% 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 with a population of pancreatic endocrine (PEC) cells 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 with respect to ISL1 and positive with respect to NKX6.1 (ISL1-NKX6.1+), see e.g., Fig. 3B.

[0033] As can be seen in FIG 3E, the population of pancreatic endocrine (PEC) cells obtained after step ii) is also increased compared with a population of pancreatic endocrine (PEC) cells obtained by using a TGFb receptor kinase inhibitor instead of an inhibitor of VEGF and / or PDGF in step i) and / or step ii). Thus, the population of pancreatic endocrine (PEC) cells or cells obtained after step ii) contains at least 70% such as at least 75%, at least 78% or at least 80% pancreatic endocrine cells, wherein the endocrine cells are CHGA+.

[0034] The population of pancreatic endocrine (PEC) cells or cells obtained after step ii) contains at least 1% such as at least 2%, at least 3%, at least 4% or at least 5% more pancreatic endocrine (PEC) cells compared with a population of pancreatic endocrine (PEC) cells 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., Fig 3E. In a further aspect, a cell population or composition is disclosed according to the present invention for use as a medicament, such as in the treatment of diabetes type I. It is believed that transplantation of homogeneous populations of cells increases the safety and efficacy profile of cell therapy and decreases the risks of undesirable side-effects from undesirable cell types. A treatment with a cell population or composition according to the present invention provides a high percentage of beta-like cells, thereby making it suitable for the prevention, amelioration and / or treatment of a condition requiring the administration of such cells.

[0035] BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1

[0037] A) Schematic overview of differentiation of hPSC to pancreatic endocrine (PEC) cells through various cell stages namely DE, PE, EP and PEC. hPSC are first differentiated to definitive endoderm (DE), DE is differentiated to pancreatic endoderm (PE) and PE is differentiated to endocrine progenitor (EP) cells and finally, endocrine progenitor (EP) cells are differentiated towards pancreatic endocrine (PEC) cells.

[0038] B) hPSC were differentiated to PE and then treated with TGFpRI inhibitors [Standard (STD)] or Linifanib during the EP-PEC stage. Cells were analyzed at the PEC stage for expression of ISL1 and NKX6-1. Representative flow cytometry dot plots of three experiments.

[0039] C) Summary graph of percentage of cells within the individual quadrants (Q1 -Q4) of the flow cytometry dot plots shown in A). Graph shows mean ± standard deviation across three experiments.

[0040] Figure 2

[0041] A) hPSC were differentiated to PE and then treated during the EP stage with Linifanib added at concentrations ranging from 5nM to 15uM. After treatment during the EP stage all conditions received standard conditions towards PEC and cells were analyzed for expression of ISL1 and NKX6-1 .

[0042] B) hPSC differentiation carried out as described in A) but with expression of CHGA and VIM evaluated by flow cytometry.

[0043] C) Summary graph of percentage of cells within the ISL1+ / NKX6.1+ Q2 (hPSC-derived beta- like cells) of the flow cytometry dot plots shown in A.

[0044] D) Summary graph of percentage of cells within the CHGA+ Q1-Q2 (Endocrine cells) of the flow cytometry dot plots shown in B. Figure 3

[0045] A) Schematic overview of differentiation of hPSC to pancreatic endocrine cells (PEC). hPSC were differentiated to PE and then treated with TGFpIR inhibitors [Standard (STD)], without TGFpRI inhibitors (-TGFPR1 ) in two different treatment windows, without TGFpRI inhibitors but with Linifanib (Linifanib) during different treatment windows between the EP- PEC stage of the protocol (highlighted by bars).

[0046] B) Flow cytometry dot plots of cells differentiated as described A. Cells were analyzed for expression of ISL1 and NKX6-1.

[0047] C) Flow cytometry dot plots of cells differentiated as described A. Cells were analyzed for expression of CHGA and VIM.

[0048] D) Summary graph of percentage of cells within the ISL1+ / NKX6.1+ Q2 (hPSC-derived beta- like cells) of the flow cytometry dot plots shown in B.

[0049] E) Summary graph of percentage of cells within the CHGA+ Q1-Q2 (Endocrine cells) of the flow cytometry dot plots shown in C.

[0050] DETAILED DESCRIPTION

[0051] Methods are provided for obtaining pancreatic endocrine cells from pluripotent stem cells.

[0052] The pancreatic endocrine cells obtained by methods described herein are usable and / or intended for use in a method of providing pancreatic endocrine function to a mammal deficient in its production of at least one pancreatic hormone.

[0053] In some embodiments, the invention relates to a method of providing pancreatic endocrine function to a mammal deficient in its production of at least one pancreatic hormone, the method comprising the steps of implanting endocrine cells obtained by any of the methods described herein in an amount sufficient to produce a measurable amount of said at least one pancreatic hormone in said mammal.

[0054] Unless otherwise stated, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. The practice of the present invention employs, unless otherwise indicated, conventional methods of chemistry, biochemistry, biophysics, molecular biology, cell biology, genetics, immunology, and pharmacology, known to those skilled in the art.

[0055] It is noted that all headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way. The use of any and all examples, or exemplary language (e.g., “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.

[0056] As used herein, “a” or “an” or “the” can mean one or more than one. Unless otherwise indicated in the specification, terms presented in singular form also include the plural situation. As used herein, “and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or"). Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted.

[0057] General definitions hESC: human embryonic stem cells hiPSC: human induced pluripotent stem cells hPSC: human Pluripotent stem cells

[0058] DE: definitive endoderm

[0059] PE: pancreatic endoderm

[0060] EP: endocrine progenitor cells

[0061] PEC: pancreatic endocrine cells

[0062] BC: Beta cells, insulin-producing beta cells

[0063] Stem cells

[0064] By “stem cell” is to be understood an undifferentiated cell having proliferative capacity (particularly self-renewal competence) but maintaining differentiation potency. The term “stem cell” includes categories such as pluripotent stem cell, multipotent stem cell, and the like according to their differentiation potentiality.

[0065] As used herein, the term “pluripotent stem cell” (PSC) refers to a stem cell capable of being cultured in vitro and having a potency to differentiate into any cell lineage belonging to the three germ layers (ectoderm, mesoderm, endoderm) and / or extraembryonic tissue (pluripotency).

[0066] As used herein, the term “multipotent stem cell” means a stem cell having a potency to differentiate into plural types of tissues or cells, though not all kinds and is typically restricted to one germ layer. As used herein, the term “unipotent stem cell” means a stem cell having a potency to differentiate into only one particular tissue or cell.

[0067] A pluripotent stem cell can be induced from fertilized egg, clone embryo, germ stem cell, stem cell in a tissue, somatic cell and the like. Examples of the pluripotent stem cell (PSC) include embryonic stem cell (ESC), EG cell (embryonic germ cell), induced pluripotent stem cell (iPSC) and the like.

[0068] As used herein, the term “induced pluripotent stem cell” (also known as iPS cells or iPSCs) means a type of pluripotent stem cell that can be generated directly from adult cells. By the introduction of products of specific sets of pluripotency-associated genes, non-pluripotent cells can be converted into pluripotent stem cells. Pluripotent embryonic stem cells may also be derived from parthenotes as described in e.g., WO 2003 / 046141 , the contents of which are incorporated by reference in their entirety. Additionally, embryonic stem cells can be produced from a single blastomere or by culturing an inner cell mass obtained without the destruction of the embryo. Embryonic stem cells are available from given organizations and are also commercially available. Preferably, in some embodiment, methods and products described herein are based on hPSCs, i.e., stem cells derived from either induced pluripotent stem cells or embryonic stem cells, including parthenotes.

[0069] Definite endoderm (DE)

[0070] As used herein, the term “definitive endoderm”, “definitive endoderm cells”, or “DE” refers to cells characterized by expression of the marker SOX17. Optionally, further markers of DE are one or more of the following FOXA2 and CXCR4. Definitive endoderm cells are important for development of e.g., pancreatic cells.

[0071] "SOX17" (SRY-box 17) as used herein is a member of the SOX (SRY-related HMGbox) family of transcription factors involved in the regulation of embryonic development and in the determination of the cell fate.

[0072] "FOXA2" (forkhead box A2) as used herein is a member of the forkhead class of DNA-binding proteins

[0073] "CXCR4" (C-X-C motif chemokine receptor 4) as used herein is a CXC chemokine receptor specific for stromal cell-derived factor-1 .

[0074] Non-limiting examples of DE inducing protocols is the conventional D'Amour protocol (Nature Biotechnology 2006, 2008) and the protocol described in WO2012 / 175633 (which is incorporated herein by reference in its entirety). Pancreatic endoderm (PE)

[0075] As used herein, the term “pancreatic endoderm”, “pancreatic endoderm cells”, “pancreatic progenitors" or “PE” refers to cells characterized by expressing the markers PDX1 and NKX6.1 . In some embodiments, at least 5% of the cells are NKX6.1+ / PDX1+ double positive. Optionally, further markers of PE are one or more of SOX9, and PTF1 A.

[0076] "PDX1" as used herein, refers to a homeodomain transcription factor implicated in pancreas development.

[0077] "NKX6.1" as used herein is a member of the NKX transcription factor family.

[0078] “SOX9” (SRY-Box Transcription Factor 9) as used herein is a transcription factor that plays a critical role during embryonic development and cell lineage allocation.

[0079] "PTF1A" as used herein is a protein that is a component of the pancreas transcription factor 1 complex (PTF1) and is known to have a role in mammalian pancreatic development.

[0080] "CPA1" as used herein is a member of the carboxypeptidase A family of zinc metalloproteases. This enzyme is produced in the pancreas.

[0081] Non-limiting examples of PE inducing protocols is described in WO2014 / 033322, which is incorporated herein by reference in its entirety.

[0082] Pancreatic endocrine progenitor (EP) cells

[0083] As used herein, the term “pancreatic endocrine progenitors” or “endocrine progenitor cells” or “EP” refers to cells characterized by expressing NEUROG3, and optionally one or more of, NeuroD and NKX2.2, hallmarks for EP cells committed to an endocrine cell fate.

[0084] "NEUROG3" as used herein, is a member of the neurogenin family of basic loop- helix-loop transcription factors.

[0085] "NKX2.2" and "NKX6.1" as used herein are members of the NKX transcription factor family.

[0086] "NeuroD" as used herein is a member of the NeuroD family of basic helix-loop-helix (bHLH) transcription factors.

[0087] A protocol for generating pancreatic endocrine progenitor cells is described in WO2015 / 028614 which is incorporated herein by reference in its entirety. Pancreatic endocrine cells (PEC)

[0088] As used herein, the term “pancreatic endocrine cells” or “PEC” refers to cells expressing CHGA and ISL1.

[0089] The pancreatic endocrine (PEC) cells obtained with the method of the present invention include islet-like cells. Islet-like cells include alpha-like cells, beta-like cells, epsilon-like cells, delta-like cells and gamma-like cells.

[0090] As used herein, the term “islet-like cells” refers to islet cells obtained in vitro after culturing of stem cells. Islet-like cells include beta cells, alpha cells, delta cells, gamma cells.

[0091] As used herein, the term “alpha cells” refer to cells expressing GCG, and optionally one of more of ISL1 and ARX. In pancreas, the alpha cells produce the hormone glucagon.

[0092] As used herein, the term “beta-cells” or “beta-like cells” refers to cells expressing INS, and optionally one or more of PDX1 , ISL1 and NKX6.1 . In pancreas, the beta cells produce the hormone insulin and amylin.

[0093] As used herein, the term “delta cells” refer to cells expressing SST, and optionally one or more of ISL1 and HHEX. In pancreas, the delta cells secrete the peptide hormone somatostatin.

[0094] As used herein, the term “epsilon cells” refer to cells expressing GHRL, and optionally one or more of ISL1 , ARX and ETV1. In the pancreas, epsilon cells produce the hormone ghrelin.

[0095] As used herein, the term “gamma cells” is in the current context used interchangeably with “Pancreatic polypeptide cells”, “PP cells”, “y-cells”, or “F cells” and refers to endocrine cells expressing PPY, and optionally one or more of ISL1 and PAX6. In the pancreas, they help synthesize and regulate the release of pancreatic polypeptide (PP).

[0096] As used herein, the term “enterochromaffin cells” is used interchangeably with “EC cells” and “Kulchitsky cells” and refers to endocrine cells expressing TPH1 , and optionally one or more of LMX1 A and FEV. The enterochromaffin cells are a type of enteroendocrine cells and neuroendocrine cells. In humans, they are located in the epithelial layer of the entire gastrointestinal tract. EC cells modulate neuron signalling in the enteric nervous system (ENS) via the secretion of the neurotransmitter serotonin and other peptides.

[0097] Expression of markers

[0098] As used herein, the term "expression level" refers to the degree of gene expression and / or gene product activity in a cell. Expression level can be determined in arbitrary absolute units or normalized units (relative to known expression levels of a control reference). As used herein, the term "marker" refers to a naturally occurring identifiable expression made by a cell which can be correlated with certain properties of the cell and serves to identify, predict or characterise a cell or cell population. A marker may be referred to by gene. A marker may be in the form of mRNA or protein for e.g., protein on the cell surface.

[0099] As used herein, the term "expression" in reference to a marker refers to the presence or lack of presence in the cell of a molecule, which can be detected. In an embodiment, the expressed molecule is mRNA or a protein. The expression of the marker may be detected at any suitable level, such as at mRNA or protein level. A person skilled in the art will readily appreciate that a cell can be defined by the positive or negative expression of a marker, i e. the properties and state of a cell may equally be correlated based on the expression of a certain marker as well as the lack thereof. When referring to specific markers the presence or lack of expression may be denoted with + (plus) or - (minus) signs, respectively.

[0100] Method steps

[0101] As used herein, the term “step” in relation to methods as described herein is to be understood as a stage, where something is undertaken and / or an action is performed. It will be understood by one of ordinary skill in the art when the steps to be performed and / or the steps undertaking are concurrent and / or successive and / or continuous.

[0102] Throughout this application the terms “method” and “protocol”, when referring to processes for differentiating cells, may be used interchangeably.

[0103] As used herein, the term “day” and similarly day in vitro (DIV), in reference to the protocols, refers to a specific time for carrying out certain steps during the differentiation procedure.

[0104] In general, and unless otherwise stated, “day 0” refers to the initiation of the protocol, this be by for example but not limited to plating the cells or transferring the cells to an incubator or contacting the cells in their current cell culture medium with a compound prior to transfer of the cells. Typically, the initiation of the protocol will be by transferring the cells, such as e.g. undifferentiated stem cells, definitive endoderm cells, pancreatic endoderm cell, 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 incubating, and / or with the first contacting of the cells with a compound or compounds that affects the undifferentiated stem cells in such a way that a differentiation process is initiated.

[0105] When referring to “day X”, such as day 1 , day 2 etc., it is relative to the initiation of the protocol at day 0. One of ordinary skill in the art will recognize that unless otherwise specified the exact time of the day for carrying out the step may vary. Accordingly, “day X” is meant to encompass a time span such as of + / -10 hours, + / -8 hours, + / -6 hours, + / -4 hours, + / -2 hours, or + / -1 hours.

[0106] As used herein, the phrase “from at about day X to at about day Y” refers to a day at which an event starts from. The phrase provides an interval of days on which the event may start from. For example, if “cells are contacted with a differentiating factor from at about day 3 to at about day 5” then this is to be construed as encompassing all the options: “the cells are contacted with a differentiating factor from about day 3”, “the cells are contacted with a differentiating factor from about day 4”, and “the cells are contacted with a differentiating factor from about day 5”. Accordingly, this phrase should not be construed as the event only occurring in the interval from day 3 to day 5. This applies mutatis mutandis to the phrase “to at about day X to at about day Y”.

[0107] Differentiation

[0108] As used herein "differentiate" or "differentiation" refers to a process where 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 are able to proliferate and express characteristics markers, like PDX1 , NKX6.1 and PTF1a. Mature or differentiated pancreatic cells do not proliferate and do secrete high levels of pancreatic endocrine hormones or digestive enzymes. E.g., fully differentiated beta cells secrete insulin at high levels in response to glucose. Changes in cell interaction and maturation occur as cells lose markers of undifferentiated cells or gain markers of differentiated cells. Loss or gain of a single marker can indicate that a cell has "matured or fully differentiated". The term "differentiation factor" refers to a compound added to pancreatic cells to enhance their differentiation to mature endocrine cells also containing insulin producing beta cells. 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 differentiation of the cells comprises culturing the cells in a medium comprising one or more differentiation factors.

[0109] 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 , indolactam V, and retinoic acid.

[0110] In aspects of the invention, differentiation of the cells comprises culturing the cells in a medium comprising one or more differentiation factors. In a preferred embodiment, the method is carried out in vitro. By the term “in vitro” is meant that the cells are provided and maintained outside of the human or animal body. In an embodiment, the cells are non-native. By the term “non-native” is meant that the cells although derived from pluripotent stem cells, which may have human origin, is an artificial construct, that does not exist in nature. As used herein, the term "artificial” may comprise material naturally occurring in nature but modified to a construct not naturally occurring. This includes human stem cells, which are differentiated into non-naturally occurring cells mimicking the cells of the human body.

[0111] Known protocols for the individual differentiation steps from hPSC to pancreatic endocrine cells

[0112] In the following, references are given to already known protocols for the individual differentiation steps from hPSC to pancreatic endocrine cells. The protocols, as well as the references given in the paragraph “Background of the invention”, can be used for providing definitive endoderm cells, for providing pancreatic endoderm (PE) cells, or for providing pancreatic endocrine progenitors. hPSC are differentiated towards pancreatic endocrine (PEC) cells in a stepwise manner through distinct stages. These stages include definitive endoderm (DE), pancreatic endoderm (PE), endocrine progenitor (EP) cells (EP) and finally to pancreatic islet cells (also denoted PEC) (Madsen et al. - Nat Biotechnol. - 2006 Dec, 24(12): 1481-3).

[0113] DE is commonly derived by treating hPSC with transforming growth factor [3 and WNT / p-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 is further specified into PDX1+ NKX6.1+ PE population in vitro. Fibroblast growth factor, retinoic acid, sonic hedgehog, epidermal growth factor and bone morphogenic protein signalling pathways have all been implicated in pancreas development and manipulation of these pathways at distinct stages of the differentiation promote highly enriched populations of PE (: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 Aug;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). Additional pathways including protein kinase C, Nicotinamide, WNT, Rho associated kinase and TGFp have also been identified to participate in the specification of hPSC towards 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). Pancreatic endocrine specification from PE is dependent on the expression of the transcription factor NEUROG3 (McGrath et al. - Diabetes - 2015 Jul;64(7):2497-505, Zhang et al. - Dev. Cell - 2019 Aug 5;50(3):367-380.e7_). Several approaches have been explored to induce EP as well as PEC from PE. Culturing PE on air-liquid interface culture resulted in upregulation of NEUROG3 transcript, as well as the pancreatic hormones insulin (INS) and glucagon (GCG), compared with cells cultured in planar culture Rezania et al. - Nat Biotechnol. - 2014 Nov;32(11):1121-33). Expression of a NEUROG3 transgene in PE was shown to induce endocrine differentiation (Zhu et al. - Cell Stem Cell. - 2016 Jun 2;18(6):755-768). Modulation of the actin cytoskeleton as well as dispersion of PE to single cells followed by reaggregation cells to clusters can induce NEUR0G3 expression and differentiation to 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 TGFp signalling and Notch signalling progressed PE to a pancreatic endocrine phenotype (Rezania et al. - Diabetes. - 2011 Jan;60(1):239-47, Nostro et al. - Development. - 2011 Mar;138(5):861-71, Pagliuca et al. - Cell. - 2014 Oct 9;159(2):428-39, Rezania et al. - Nat Biotechnol. - 2014 Nov;32(11):1121-33, Rezania et al. - Differentiation of human embryonic stem cells - 2015 Jun 23). However, permitting TGFp signalling appears to be required for differentiation to more mature beta-like cells (Velazco-Cruz et al. - Stem Cell Reports. - 2019 Feb 12;12(2):351- 365).

[0114] Several other signalling pathways has been shown to promote EP induction and further differentiation to hPSC-endocrine cells. Bromodomain and extraterminal (BET) protein inhibition with l-BET 151 or JQ1 enhanced the number of NEUROG3 endocrine progenitor (EP) cells (Huijbregts et al. - Diabetes. - 2019 Apr;68(4):761 -773). Sodium cromoglicate (SCG), was identified in a small molecule screen and induced pancreatic endocrine differentiation in PE through the inhibition of bone morphogenetic protein 4 signalling pathway (Kondo et al. - Diabetologia. - 2017 Aug;60(8):1454-1466.). Bone morphogenetic protein has been implicated 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 is commonly used in differentiation of hPSC to EP and PEC. Treatment of PE with the WNT-tankyrase inhibitor IWR1-endo increased the expression of endocrine markers and downregulated progenitor markers demonstrating that small- molecule WNT inhibitors increases the endocrine induction (Sharon et al. - Cell Rep. - 2019 May 21; 27(8): 2281-2291 .e5.).

[0115] Finally, specific pancreatic endocrine cell types resembling their in vivo counterparts have been derived and characterized in detail. Glucagon expressing alpha-like cells derived from hPSC display molecular and functional characteristics of bona fide pancreatic alpha cells (Rezania et al. - Diabetes - 2011 Jan;60(1):239-47, Peterson et al. - Nat Commun. - 2020 May 7;11(1):2241). Differentiation protocols for maturing hPSC-derived beta-like cells that are capable of secreting insulin in response to elevated glucose concentrations have recently been reported (Rezania et al.

[0116] - 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.

[0117] - Nat Cell Biol. - 2019 Feb;21(2):263-274).

[0118] Single cell gene expression analysis has delineated the differentiation path of hPSC towards pancreatic endocrine (PEC) cells including beta-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 the hPSC-derived beta-like cells both in vitro and in vivo have revealed many similarities to bona fide pancreatic beta cells (Velazco-Cruz et al. - Stem Cell Reports. - 2019 Feb 12;12(2):351-365, Augsornworawat et al. - Cell Rep. - 2020 Aug 25;32(8):108067, Balboa et al. - Functional, metabolic and transcriptional maturation of stem cell derived beta cells - 2021.03.31.437748v1). Interestingly, formation of non-endocrine cells as well as non-pancreas enterochromaffin cells has recently been reported for protocols aiming and differentiation hPSC towards the pancreatic endocrine lineage (Petersen et al. - Stem Cell Reports.

[0119] - 2017 Oct 10;9(4):1246-1261 , Veres et al. - Nature. - 2019 May;569(7756):368-373).

[0120] Hereinafter, the methods according to the present invention are described in more detail by non- limiting embodiments and examples.

[0121] An in vitro method for obtaining pancreatic endocrine cells

[0122] Described herein are in vitro methods for obtaining pancreatic endocrine cells from pluripotent stem cells which are usable and / or intended for use in a method of providing pancreatic endocrine function to a mammal deficient in its production of at least one pancreatic hormone.

[0123] The present invention identifies a small molecule inhibitor (Linifanib) of Vascular Endothelial Growth Factor receptors (VEGFR) and / or Platelet-derived Growth Factor receptors (PDGFR) that promotes differentiation of pancreatic endoderm to pancreatic endocrine progenitor (EP) cells and pancreatic endocrine cells, including beta-like cells.

[0124] The present invention provides 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 progenitors, wherein the differentiating 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 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.

[0125] In some embodiments, step i) is performed 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.

[0126] Differentiation of pancreatic endoderm cells (PE) into pancreatic endocrine progenitor (EP) cells (EP or PEP) - step I)

[0127] In some embodiments, the pancreatic endoderm (PE) cells for use in step I) of the methods described herein are cells having the markers PDX1 and NKX6.

[0128] In some embodiments, the step I) of culturing pancreatic endoderm (PE) cells into pancreatic endocrine progenitors, the pancreatic endodermal cells are treated with one or more compounds, selected from the group consisting of thyroid hormones, epidermal growth factor (EGF) agonists, staurosporine, NOTCH pathway inhibitors, BMP pathway inhibitors, EZH2 histone methyltransferase inhibitors, JNK pathway inhibitors and TGFbRI inhibitors.

[0129] In some embodiments, suitable examples of thyroid hormones are T3 (Cas No, 6893-02-3), or GC1 (Cas No. 211110-63-3). In embodiments the thyroid hormone is T3.

[0130] For use in a method of the invention, suitable examples of EGF agonists (EGF pathway activators) are Betacellulin (gene name BTC), Epidermal Growth Factor (gene name EGF), Amphiregulin

[0131] SUBSTITUTE SHEET (RULE 26) cells with an inhibitor of Vascular Endothelial Growth Factor (VEGF) receptor and / or Platelet-Derived Growth Factor (PDGF) receptor, and / or 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.

[0132] In some embodiments, step i) is performed 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.

[0133] Differentiation of pancreatic endoderm cells (PE) into pancreatic endocrine progenitor (EP) cells (EP or PEP) - step i)

[0134] In some embodiments, the pancreatic endoderm (PE) cells for use in step i) of the methods described herein are cells having the markers PDX1 and NKX6.

[0135] In some embodiments, the step i) of culturing pancreatic endoderm (PE) cells into pancreatic endocrine progenitors, the pancreatic endodermal cells are treated with one or more compounds, selected from the group consisting of thyroid hormones, epidermal growth factor (EGF) agonists, staurosporine, NOTCH pathway inhibitors, BMP pathway inhibitors, EZH2 histone methyltransferase inhibitors, JNK pathway inhibitors and TGFbRI inhibitors.

[0136] In some embodiments, suitable examples of thyroid hormones are T3 (Cas No, 6893-02-3), or GC1 (Cas No. 211110-63-3). In embodiments the thyroid hormone is T3.

[0137] For use in a method of the invention, suitable examples of EGF agonists (EGF pathway activators) are Betaceliulin (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 embodiments the EGF pathway activator is Betacellulin.

[0138] 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.

[0139] In some embodiments, suitable examples of BMP pathway inhibitors include LDN 193189 dihydrochloride (Cas No. 1435934-00-1), DMH-1 (Cas No. 1206711-16-1), Dorsomorphin dihydrochloride (Cas No. 1219168-18-9) and Noggin. In embodiments the BMP pathway inhibitor is LDN.

[0140] In some embodiments, suitable examples of EZH2 histone methyltransferase inhibitors are 3- Deazaneplanocin A hydrochloride (DZNep) (Cas No. 120964-45-6), GSK 126 (Cas No. 1346574- 57-9), EPZ005687 (Cas No. 1396772-26-1). In embodiments the EZH2 histone methyltransferase inhibitor is DZNep.

[0141] In some embodiments, suitable examples of JNK pathway inhibitors are TCS JNK 60 / JNK Inhibitor VIII (Cas, No. 894804-07-0), SP 600125 (Cas No. 129-56-6), TCS JNK 5a (Cas No. 312917-14-9), and JNK-IN-8 (Cas No. 1410880-22-6). In embodiments, the JNK pathway inhibitor is JNK Inhibitor VIII.

[0142] In some embodiments, Staurosporine is a broad-spectrum protein kinase inhibitor (Cas No. 62996- 74-1). Other broad-spectrum protein kinase inhibitors for use in a method of the invention include Apigenin, H-7 dihydrochloride, 5-lodotubercidin, K 252a, PKC 412, and Ro 31-8220 mesylate. In embodiments, the broad-spectrum protein kinase inhibitor is Staurosporine.

[0143] In some embodiments, suitable examples of TGFb receptor kinase inhibitor include RepSox (ALK5i II) (Cas No. 446859-33-2), SB431542 (Cas No. 301836-41-9), LY 364947 (Cas No. 396129-53-6), and A 83-01 (Cas No. 909910-43-6). In some embodiments, the TGFb receptor kinase inhibitors are RepSox and SB431542.

[0144] The differentiation in step i) may also include other differentiation factors such as, e.g., Rho kinase (ROCK inhibitor) such as, e.g., Tiger, Chroman-1 (Cas. No. 1273579-40-0) and Thiazovivin (Cas No. 1226056-71-8); Heparin; and Forskolin or NKH 477.

[0145] In step i), the pancreatic endoderm (PE) cells may also be treated with an inhibitor of Vascular Endothelial Growth Factor (VEGF) receptor and / or 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 an embodiment the cells are treated with such an inhibitor. The concentration of inhibitor of Vascular Endothelial Growth Factor (VEGF) and / or Platelet- Derived Growth Factor (PDGF) receptors used in step i) is in a range of from 5 nM to about 15 μM, such as from 5 nM to about 12 μM, 1 nM to 12 μM, from 10 nM to about 11 μM, from 25 nM to 10 μM, from 50 nM to 10 μM, from 100 nM to 10 μM, from 250 nM to 10 μM, from 500 nM to 10 μM, from 750 nM to 10 nM, from 1 μM to 10 μM, from 2 μM to 10 μM, from 2.5 μM to 10 μM, from 2.5 μM to 8 μM, from 2.5 μM to 7 μM, from 2.5 μM to 6 μM, from 2.5 μM to 5 μM, from 2.5 μM to 5.5 μM, from 2.5 μM to 5 μM or in a range of from about 3 μM to about 4 μM, or in a range of from about 1 μM to about 12 μM, from about 1 μM to about 11 μM, from about 1 μM to about 10 μM, from about 1 μM to about 9 μM, from about 1 μM to about 8 μM, from about 1 μM to about 7 μM, from about 1 μM to about 6 μM, from about 1 μM to about 5 μM, from about 1 μM to about 4 μM, from about 1 μM to about 3 μM, from about 1 μM to about 2 μM, from about 2 μM to about 12 μM, from about 2 μM to about 11 μM, from about 2 μM to about 10 μM, from about 2 μM to about 9 μM, from about 2 μM to about 8 μM, from about 2 μM to about 7 μM, from about 2 μM to about 6 μM, from about 2 μM to about 5 μM, from about 2 μM to about 4 μM, from about 2 μM to about 3 μM, from about 3 μM to about 12 μM, from about 3 μM to about 11 μM, from about 3 μM to about 10 μM, from about 3 μM to about 9 μM, from about 3 μM to about 8 μM, from about 3 μM to about 7 μM, from about 3 μM to about 6 μM, from about 3 μM to about 5 μM, from about 3 μM to about 4 μM, from about 4 μM to about 12 μM, from about 4 μM to about 11 μM, from about 4 μM to about

[0146] 10 μM, from about 4 μM to about 9 μM, from about 4 μM to about 8 μM, from about 4 μM to about 7 μM, from about 4 μM to about 6 μM, from about 4 μM to about 5 μM, from about 5 μM to about 12 μM, from about 5 μM to about 11 μM, from about 5 μM to about 10 μM, from about 5 μM to about 9 μM, from about 5 μM to about 8 μM, from about 5 μM to about 7 μM, from about 5 μM to about 6 μM, from about 6 μM to about 12 μM, from about 6 μM to about 11 μM, from about 6 μM to about 10 μM, from about 6 μM to about 9 μM, from about 6 μM to about 8 μM, from about 6 μM to about 7 μM, from about 7 μM to about 12 μM, from about 8 μM to about 11 μM, from about 9 μM to about 10 μM, from about 10 μM to about 12 μM, from about 10 μM to about 11 μM or from about

[0147] 11 μM to about 12 μM, or in a concentration of 5nM, 50nM, 500nM, 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 or 15 μM.

[0148] When the pancreatic endoderm (PE) cells in step i) are treated with an inhibitor of Vascular Endothelial Growth Factor (VEGF) receptor and / or Platelet-Derived Growth Factor (PDGF) receptor, then step i) does not involve a TGFb receptor kinase inhibitor. No other changes to the protocol are made, allowing for a direct comparison to the protocol using TGFb receptor kinase inhibitors. Examples of TGFbR inhibitors include RepSox (ALK5i II) (Gas No. 446859-33-2), SB431542 (Cas No. 301836-41-9), LY 364947 (Gas No. 396129-53-6), and A 83-01 (Gas No. 909910-43-6). Such a population of pancreatic endoderm (PE) cells may be used as starting material in step i) of the method of the invention, but in a separate aspect of the invention, pancreatic endoderm (PE) cells may also be used as starting material for step i) when obtained using already published methods.

[0149] The pancreatic endocrine progenitor obtained in step i) typically have the markers NEUROG3, NKX2.2 and NEUROD1.

[0150] In some embodiments, differentiation is carried out in a suitable culture medium such as MCDB131 (basal medium), RPMI, DMEM, DMEM / F12, CMRL, MEM and the like.

[0151] The medium may be supplemented with e.g., human serum albumin (HSA), antibiotics such as penicillin and / or streptomycin, glucose, sodium hydrogen carbonate, ITSX, glutamax, ascorbic acid and zinc sulfate.

[0152] In some embodiments, the differentiation of the pancreatic endoderm to pancreatic endocrine progenitor (EP) cells is carried out over a time period of from about 1 to about 15 days or from about 1 to about 12 days, from about 1 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. In general, about 4 days.

[0153] The pancreatic endocrine progenitor (EP) cells obtained comprises at least one of the markers NEUROG3, NKX2.2, and NEUROD1.

[0154] Differentiation of pancreatic endocrine progenitor into pancreatic endocrine (PEC) cells - step ii)

[0155] In some embodiments, the population obtained from step i) comprises pancreatic endocrine progenitor (EP) cells that express at least one of the markers NEUROG3, NKX2.2, and NEUROD1. Such a population obtained from step i) may be used as starting material in step ii) of the method of the invention, but in a separate aspect of the invention, pancreatic endocrine progenitor (EP) cells may also be used as starting material for step ii) when obtained using already published methods.

[0156] 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 hormones, staurosporine, BMP pathway inhibitors, and EZH2 histone methyltransferase inhibitors.

[0157] Examples of thyroid hormones, staurosporine, BMP pathway inhibitors, EZH histone methyltransferase inhibitors and TGFb receptor kinase inhibitors are given in paragraph “Differentiation of pancreatic endoderm (PE) cells to pancreatic endocrine progenitor (EP) cells - step i)” above and they are also suitable for use in step ii) of the method of the invention. The differentiation in step ii) may also include other differentiation factors such as, e.g., Rho kinase (ROCK inhibitor) such as, e.g., Tiger, Chroman-1 (Cas. No. 1273579-40-0) and Thiazovivin (Cas No. 1226056-71-8); Heparin; and Forskolin or NKH 477.

[0158] In some embodiments, the pancreatic endocrine progenitor in step ii) are treated with an inhibitor of Vascular Endothelial Growth Factor (VEGF) receptor and / or Platelet-Derived Growth Factor (PDGF) receptor. Linifanib is an example of such a substance. Linifanib is 1-(4-(3-amino-1 H- indazol-4-yl)phenyl)-3-(2-fluoro-5-methylphenyl)urea. In an embodiment the cells are treated with such an inhibitor.

[0159] In some embodiments, the concentration of inhibitor of Vascular Endothelial Growth Factor (VEGF) and / or Platelet-Derived Growth Factor (PDGF) receptors used in step ii) is in a range of from 5 nM to about 15 μM, such as from 5 nM to about 12 μM, 1 nM to 12 μM.from 10 nM to about 11 μM, from 25 nM to 10 μM, from 50 nM to 10 μM, from 100 nM to 10 μM, from 250 nM to 10 μM, from 500 nM to 10 μM, from 750 nM to 10 nM, from 1 μM to 10 μM, from 2 μM to 10 μM, from 2.5 μM to

[0160] 10 μM, from 2.5 μM to 8 μM, from 2.5 μM to 7 μM, from 2.5 μM to 6 μM, from 2.5 μM to 5 μM, from 2.5 μM to 5.5 μM, from 2.5 μM to 5 μM or in a range of from about 3 μM to about 4 μM, or in a range of from about 1 μM to about 12 μM, from about 1 μM to about 11 μM, from about 1 μM to about 10 μM, from about 1 μM to about 9 μM, from about 1 μM to about 8 μM, from about 1 μM to about 7 μM, from about 1 μM to about 6 μM, from about 1 μM to about 5 μM, from about 1 μM to about 4 μM, from about 1 μM to about 3 μM, from about 1 μM to about 2 μM, from about 2 μM to about 12 μM, from about 2 μM to about 11 μM, from about 2 μM to about 10 μM, from about 2 μM to about 9 μM, from about 2 μM to about 8 μM, from about 2 μM to about 7 μM, from about 2 μM to about 6 μM, from about 2 μM to about 5 μM, from about 2 μM to about 4 μM, from about 2 μM to about 3 μM, from about 3 μM to about 12 μM, from about 3 μM to about 11 μM, from about 3 μM to about 10 μM, from about 3 μM to about 9 μM, from about 3 μM to about 8 μM, from about 3 μM to about 7 μM, from about 3 μM to about 6 μM, from about 3 μM to about 5 μM, from about 3 μM to about 4 μM, from about 4 μM to about 12 μM, from about 4 μM to about 11 μM, from about 4 μM to about 10 μM, from about 4 μM to about 9 μM, from about 4 μM to about 8 μM, from about 4 μM to about 7 μM, from about 4 μM to about 6 μM, from about 4 μM to about 5 μM, from about 5 μM to about 12 μM, from about 5 μM to about 11 μM, from about 5 μM to about 10 μM, from about 5 μM to about 9 μM, from about 5 μM to about 8 μM, from about 5 μM to about 7 μM, from about 5 μM to about 6 μM, from about 6 μM to about 12 μM, from about 6 μM to about 11 μM, from about 6 μM to about 10 μM, from about 6 μM to about 9 μM, from about 6 μM to about 8 μM, from about 6 μM to about 7 μM, from about 7 μM to about 12 μM, from about 8 μM to about 11 μM, from about 9 μM to about 10 μM, from about 10 μM to about 12 μM, from about 10 μM to about 11 μM or from about

[0161] 11 μM to about 12 μM, or in a concentration of 5nM, 50nM, 500nM, 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, 10.5 μM, 11 μM, 11.5 μM, 12 μM or 15 μM.

[0162] When the pancreatic endocrine cells (EP) cells in step ii) are treated with an inhibitor of Vascular Endothelial Growth Factor (VEGF) receptor and / or Platelet-Derived Growth Factor (PGDF) receptor, then step ii) does not involve a TGFb receptor kinase inhibitor. No other changes to the protocol are made, allowing for a direct comparison to the protocol using TGFb receptor kinase inhibitors. Examples of TGFbR inhibitors include RepSox (ALK5i II) (Gas No. 446859-33-2), SB431542 (Cas No. 301836-41-9), LY 364947 (Gas No. 396129-53-6), and A 83-01 (Gas No. 909910-43-6).

[0163] The differentiation is typically carried out in a suitable culture medium such as MCDB131 (basal medium) or in one of the culture media mentioned above or their equivalents.

[0164] The medium may be supplemented with e.g., human serum albumin (HSA), antibiotics such as penicillin and / or streptomycin, glucose, sodium hydrogen carbonate, ITSX, glutamax, ascorbic acid and zinc sulfate.

[0165] In general, the differentiation of the pancreatic endocrine progenitor (EP) cells to pancreatic endocrine (PEC) cells is carried out over a time period of from about 2 to about 30 days or from about 2 to 25 days, from about 2 to 20 days, from about 3 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 normally added to the culture medium when the culture medium is changed. The culture medium is typically changed after 1 or 2 days of culturing.

[0166] 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 the initiation of step i) as described herein.

[0167] When the population of pancreatic endocrine (PEC) cells contains beta-cells, the culturing may be continued. The culture medium may be the same as above, but in general zinc sulfate is excluded. Moreover, the differentiation factors mentioned above are not included, but the inhibitor of Vascular Endothelial Growth Factor (VEGF) receptor and / or Platelet-Derived Growth Factor (PDGF) receptor may be included for further culturing in at least 2 days such as 2 days, 3 days, or 4 days.

[0168] If the population of endocrine cells obtained contains pancreatic endocrine cell aggregates the aggregates may be dissociated into single cells.

[0169] The pancreatic endocrine (PEC) cells may be further treated with a cryopreservation medium and lowering temperature to obtain cryopreserved single cells. A suitable method for cryopreservation of pancreatic endocrine (PEC) cells is described in WO 2019 / 048690 to which reference is made and which is incorporated by reference in its entirety.

[0170] The cryopreservation may be performed after culturing the cells after 1 day or longer such after culturing from about 1 to about 30 days.

[0171] In some embodiments, pancreatic endocrine (PEC) cells obtained include islet-like cells such as beta cells (with marker INS, PDX1 and / or NKX6.1 ), alpha-cells (with markers GCG and / or ARX), delta cells (with markers SST and / or HHEX).

[0172] In some embodiments, the population of pancreatic endocrine (PEC) cells obtained after step ii) comprises at least 20% such as at least 25% such as at least 28% or at least 30% of beta-like cells based on the total number of cells obtained after step ii), and wherein the beta-like cells are double positive with respect to ISL1 and NKX6.1 (ISL1+NKX6.1+), see e.g., Fig 1B.

[0173] Thus, the population of pancreatic endocrine (PEC) cells obtained after step ii) may contain at least 1% 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 beta-like cells compared with a population of pancreatic endocrine (PEC) cells obtained by using a TGFb receptor kinase inhibitor instead of an inhibitor of VEGF and / or PDGF in step i) and / or in step ii), and wherein the beta-like cells are double positive with respect to ISL1 and NKX6.1 (ISL1+NKX6.1+), see e.g., FIG 3B.

[0174] In embodiments, the population of pancreatic endocrine (PEC) cells obtained after step ii) contains at the most 35% such as at the most 30% or at the most 27% enterochromaffin cells, and wherein the enterochromaffin cells are negative with respect to ISL1 and positive with respect to NKX6.1 (ISL1-NKX6.1+), see e.g., Fig. 3B

[0175] Thus, the population of pancreatic endocrine (PEC) cells obtained after step ii) may contain at least 1% 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 with a population of pancreatic endocrine (PEC) cells 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 with respect to ISL1 and positive with respect to NKX6.1 (ISL1-NKX6.1+), see e.g., Fig. 3B.

[0176] In embodiments, the population of pancreatic endocrine (PEC) cells obtained after step ii) contains at least 78% such as at least 80% pancreatic endocrine cells, and wherein the endocrine cells are CHGA+, see e.g., Fig 3E.

[0177] Thus, the population of pancreatic endocrine (PEC) cells obtained after step ii) may contain at least 1% such as at least 2%, at least 3%, at least 4% or at least 5% more pancreatic endocrine (PEC) cells compared with a population of pancreatic endocrine (PEC) cells 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., Fig 3E.

[0178] Differentiation of stem cells into pancreatic endocrine cells

[0179] In some embodiments, hPSCs, are obtained from any suitable source as referred to in the above. In some embodiments, the methods described herein include culturing hPSCs. By the term “culturing” is meant that the hPSCs are cultured in a cell culture medium, which is suitable for viability in their current state of development. In some embodiments, culturing the stem cells involves transferring the stem cells into a different environment, such as by seeding onto a new substrate or suspending in an incubator. One of ordinary skill in the art will readily recognize that stem cells are fragile to such a transfer and the procedure requires diligence and that maintaining the stem cells in the origin cell culture medium may facilitate a more sustainable transfer of the cells before replacing the cell culture medium with another cell culture medium more suitable for the differentiation process.

[0180] In some embodiments, the methods described herein relate to an in vitro method for producing pancreatic endocrine (PEC) cells from human pluripotent stem cells comprising the steps of i) differentiating hPSC cells into definitive endoderm cells, ii) differentiating definitive endoderm cells into pancreatic endoderm cells, iii) differentiating pancreatic endoderm (PE) cells into pancreatic endocrine progenitor cells, and iv) differentiating said pancreatic endocrine progenitor (EP) cells to pancreatic endocrine (PEC) cells (islet-like cells including beta-cells), wherein step iii) and / or step iv) are performed using an inhibitor of Vascular Endothelial Growth Factor (VEGF) and / or Platelet-Derived Growth Factor (PDGF) receptors.

[0181] Step iii) is described as step i) under the heading “Differentiation of pancreatic endoderm cells (PE) into pancreatic endocrine progenitors (EP or PEP) - step i)” to which reference is made, and step iv) is described as step ii) under the heading “Differentiation of pancreatic endocrine progenitor into pancreatic endocrine (PEC) cells - step ii)” to which reference is made.

[0182] The steps of differentiating hPSC cells into definitive endoderm cells, and of differentiating definitive endoderm cells into pancreatic endoderm (PE) cells can follow standard protocol, such as, e.g.,

[0183] D'Amour et al. - Nat Biotechnol. - 2005 Dec;23(12): 1534-41. - Funa et al. - Cell Stem Cell. - 2015 Jun 4;16(6):639-52

[0184] ■ Teo et al. - Stem Cell Reports. - 2014 Jun 12;3(1 ):5-14

[0185] * Kubo et al. - Development. - 2004 Apr;131(7):1651-62

[0186] - Rezania et al. - Nat Biotechnol. - 2014 Nov;32(11):1121-33

[0187] ■ Rezania et al. - Diabetes. - 2011 Jan;60(1 ):239-47

[0188] - Kroon et al. - Nat Biotechnol. - 2008 Apr;26(4):443-52.

[0189] * Ameri et al. - Stem Cells. - 2010 Jan;28(1 ):45-56

[0190] * Nostro et al. - Development. - 2011 Mar;138(5):861-71 .

[0191] * Rezania et al. - Diabetes. - 2012 Aug;61(8):2016-29

[0192] ■ Nostro et al. - Stem Cell Reports. - 2015 Apr 14;4(4):591 -604

[0193] - Russ et al. - EMBO J - 2015 Jul 2;34(13):1759-72.

[0194] Other aspects of the invention

[0195] Compositions comprising the pancreatic endocrine cells (PEC) obtained by any of the methods of the invention

[0196] In a further aspect, it is described herein a medicament comprising pancreatic endocrine cells (PEC) obtained by any of the methods of the invention according to the present description. In particular, the PEC obtained by the method described herein have i) more pancreatic islet cells (ISL1+), more beta-like cells (ISL1+ / NKX6.1+) and less EC cells (ISL1- / NKX6.1+) compared with a standard method using TGFbRi.

[0197] In a preferred embodiment, the medicament described herein comprises enriched or homogenous, thawed and re-aggregated cryopreserved pancreatic endocrine cells (PEC), obtained by any of the methods of the present invention.

[0198] Medical use of pancreatic endocrine (PEC) cells obtained by any of the methods of the invention

[0199] In some embodiments, the invention relates to a method of providing pancreatic endocrine function to a mammal deficient in its production of at least one pancreatic hormone, the method comprising the steps of implanting pancreatic endocrine cells obtained by any of the methods of the invention in an amount sufficient to produce a measurable amount of said at least one pancreatic hormone in said mammal.

[0200] In the current context, the term “mammal” includes human and veterinary subjects.

[0201] In the current context, the term “mammal” relates to e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig or rodent. The term does not denote a particular age or sex. Pancreatic Islet cell transplantation can e.g., be used to restore insulin production and glycemic control to the diabetic mammal.

[0202] Methods of treating diabetes (type 1 or type 2) are also provided herein. For example, provided herein is a method of treating type-1 diabetes in a mammal. In some embodiments, the method includes the steps of selecting a mammal with type-1 diabetes and administering to the mammal pancreatic endocrine cells obtained by any of the methods of the invention. In other embodiments, methods include preventing type 1 diabetes in a mammal at risk for developing type 1 diabetes by administering to the mammal endocrine cells obtained by any of the methods of the invention.

[0203] One of skill in the art identifies and selects mammals with type 1 diabetes and mammals at risk for developing type 1 diabetes using any methods of diagnosis and identification. For example, diagnosis is based on an elevated blood glucose level after fasting or on a glucose tolerance test. Furthermore, diagnosis of type 1 diabetes includes various physical symptoms and characteristics.

[0204] Identification of a mammal at risk for developing type 1 diabetes is also within an artisan's skills. For example, a mammal at risk for type 1 diabetes is an individual with a genetic predisposition or an individual with a surgically excised pancreas or portion thereof. A mammal with a surgically removed pancreas includes a mammal with chronic pancreatitis or a mammal with an injury necessitating surgical removal of the pancreas.

[0205] Mammals with insulin dependent type 2 diabetes or at risk for developing type 2 diabetes similarly benefit from the administration of pancreatic endocrine cells obtained by any of the methods of the invention. Thus, provided herein is a method that includes the steps of selecting a mammal with, or at risk of developing, type-2 diabetes and administering to the mammal pancreatic endocrine cells obtained by any of the methods of the invention in an amount sufficient. Diagnosis is usually based on fasting glucose levels, on a glucose tolerance test, or on the level of blood insulin.

[0206] The pancreatic endocrine cells obtained by any of the methods of the invention in an amount sufficient herein are administered in a number of ways. Transplant compositions are frequently administered intrahepatically, for example by percutaneous direct puncture of the liver. The right or the left branch of the portal vein can be chosen for cannulation and the puncture site is chosen accordingly by the interventional radiologist. In some embodiments, several transplants are performed. One of skill in the art, however, readily determines the concentration of cells to be include in the transplant composition and recognizes the need for a second or subsequent transplant based on such clinical signs as hyperglycemia and the like.

[0207] The methods taught herein for preparing a population of islet cells for transplantation are also combined with treatment. Thus, for example, provided herein is a method of treating diabetes in a mammal that includes the steps of preparing an insulin secreting cell population (e.g., a pancreatic islet cell population) for transplantation according to any one of the in vitro method described above and transplanting the cell population to the mammal to be treated (i.e., to the transplant recipient).

[0208] EXAMPLES

[0209] Example 1

[0210] Pancreatic endocrine progenitors (EP) generated in vitro according to the present invention are obtained through the following steps.

[0211] Pancreatic endoderm cell aggregates generated from hPSC are cultured in a suitable suspension culture format. The aggregates are washed by sedimentation of the cell aggregates and removing excess culture medium. Wash medium (MCDB 131 medium, Gibco, cat. no. 10372019) is added to the cell aggregates and subsequently removed.

[0212] Differentiation to EP is performed in MCDB131 medium supplemented with Glutamax (Gibco, cat. no. 35050038), 0.05% human serum albumin (Origin, cat. no. ART-3003), 20mM glucose (Sigma- Aldrich, cat. no. G8769), 14.64mM NaHCO3 (Gibco, cat. no. 25080094), ITS-X (Gibco, cat. no. 51500056), 0.25mM Ascorbic acid (Fisher Scientific, cat. no. 0937-07) and 10uM ZnSO4 (Merck, cat. no. 1088811000). The following compounds are further supplemented to the medium: 2uM XX (Tocris, cat. no. 4489), 1uM T3 (Tocris, cat. no. 6666), 5uM Tiger (Tocris, cat. no. 1254), 100nM LDN-193189 (Tocris, cat. no. 6053), 20ng / ml Betacellulin (R&D systems, cat. no. 261-CE-250), 10ug / ml Heparin (Merck, cat. no. H3393), 3.3nM Staurosporine (Tocris, cat. no. 1258), 0.1 uM DZNep (Tocris, cat. no. 4703), 10uM Forskolin (Tocris, cat. no. 1099), 5uM JNKi VIII (Tocris, cat. no. 3222), 10uM RepSox (Tocris, cat. no. 3742) and 6uM SB431542 (Tocris, cat. no. 1614). This protocol is referred to as the standard (STD) protocol. When evaluating the effect of using VEGFR / PDGFR inhibitors, RepSox and SB431542 are excluded from the above protocol and 4uM Linifanib (ABT-869, SelleckChem, cat. No. S1003) is added instead.

[0213] Medium is replenished every 48h and the differentiation from pancreatic endoderm to endocrine progenitors is carried out over four days.

[0214] Example 2

[0215] Pancreatic endocrine cells (PEC) generated in vitro according to the present invention are obtained through the following steps.

[0216] Pancreatic endocrine progenitor cell aggregates generated from pancreatic endoderm are cultured in a suitable suspension culture format. The aggregates are washed by sedimentation of the cell aggregates and removing excess culture medium. Wash medium (MCDB 131 medium, Gibco, cat. no. 10372019) is added to the cell aggregates and subsequently removed. Differentiation to PEC is performed in MCDB131 medium supplemented with Glutamax (Gibco, cat. no. 35050038), 0.05% human serum albumin (Origin, cat. no. ART-3003), 20mM glucose (Sigma- Aldrich, cat. no. G8769), 14.64mM NaHCO3 (Gibco, cat. no. 25080094), ITS-X (Gibco, cat. no. 51500056), 0.25mM Ascorbic acid (Fisher Scientific, cat. no. 0937-07) and 10uM ZnSO4 (Merck, cat. no. 1088811000). The following compounds are further supplemented to the medium: 1uM XX (Tocris, cat. no. 4489), 1uM T3 (Tocris, cat. no. 6666), 5uM Tiger (Tocris, cat. no. 1254), 100nM LDN-193189 (Tocris, cat. no. 6053), 10ug / ml Heparin (Merck, cat. no. H3393), 3.3nM Staurosporine (Tocris, cat. no. 1258), 0.1uM DZNep (Tocris, cat. no. 4703), and 10uM RepSox (Tocris, cat. no. 3742). This protocol is referred to as the standard (STD) protocol. When evaluating the effect of using VEGFR / PDGFR inhibitors, RepSox is excluded from the above protocol and 4uM Linifanib (ABT-869, SelleckChem, cat. No. S1003) is added instead.

[0217] Medium is replenished every 48h and the differentiation from endocrine progenitors to pancreatic endocrine cells is carried out over three days. At this stage, cells can be cryopreserved or further differentiated using the following medium compositions.

[0218] Two days of differentiation in MCDB131 medium supplemented with Glutamax (Gibco, cat. no. 35050038), 0.05% human serum albumin (Origin, cat. no. ART-3003), 2.5mM glucose (Sigma- Aldrich, cat. no. G8769), 14.64mM NaHCO3 (Gibco, cat. no. 25080094), ITS-X (Gibco, cat. no. 51500056), 0.25mM Ascorbic acid (Fisher Scientific, cat. no. 0937-07) and 10uM ZnSO4 (Merck, cat. no. 1088811000). The following compounds are further supplemented to the medium: 1 uM XX (Tocris, cat. no. 4489), 1uM T3 (Tocris, cat. no. 6666), 5uM Tiger (Tocris, cat. no. 1254), 10ug / ml Heparin (Merck, cat. no. H3393), 0.1uM DZNep (Tocris, cat. no. 4703), and further supplemented with 10uM RepSox (Tocris, cat. no. 3742) (STD) or 4uM Linifanib (ABT-869, SelleckChem, cat. No. S1003).

[0219] Two days of differentiation in MCDB131 medium supplemented with Glutamax (Gibco, cat. no. 35050038), 0.05% human serum albumin (Origin, cat. no. ART-3003), 2.5mM glucose (Sigma- Aldrich, cat. no. G8769), 14.64mM NaHCO3 (Gibco, cat. no. 25080094), ITS-X (Gibco, cat. no. 51500056), 0.25mM Ascorbic acid (Fisher Scientific, cat. no. 0937-07) and further supplemented with 10uM RepSox (Tocris, cat. no. 3742) (STD) or 4uM Linifanib (ABT-869, SelleckChem, cat. No. S1003).

[0220] Pancreatic endocrine cells can subsequently be maintained for days to weeks in MCDB131 medium supplemented with Glutamax (Gibco, cat. no. 35050038), 0.05% human serum albumin (Origin, cat. no. ART-3003), 2.5mM glucose (Sigma-Aldrich, cat. no. G8769), 14.64mM NaHCO3 (Gibco, cat. no. 25080094), ITS-X (Gibco, cat. no. 51500056) and 0.25mM Ascorbic acid (Fisher Scientific, cat. 0937-07) with medium being replenished every 48h. Example 3 - Cryopreservation

[0221] Pancreatic endocrine cell aggregates that have been obtained in vitro according to the present invention are subjected to cryopreservation as described in WO 2019 / 048690. In short, the cells obtained are re-suspended in cryopreservation media and preserved by a sequential lowering of temperature to below -80°C.

[0222] Thawing crvopreserved single cells

[0223] To bring the cells back in culture, the cells are quickly brought 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 are re-suspended in stage specific medium supplemented with 50 pg / mL DNasel (Sigma#11284932001) and 10 μM Rocki (Sigma#Y27632-Y0503).

[0224] Re-aggregating the cells obtained after thawing

[0225] The cells obtained after thawing may be re-aggregated in Erlenmeyer flasks in a reduced volume with a density of 0.5-2 mio viable cells / ml. Re-aggregation is performed at 37°C with horizontal shaking at 70rpm for two days and is followed by a media change. After cryopreservation the cells with viability in a range of from 60% to 90% are recovered. Upon re-aggregation of cells the glucose responsive insulin secreting phenotype is improved.

Claims

CLAIMS1 . 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 progenitors, wherein the differentiating 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.

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

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

4. A method according to any one of claims 1-3, 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 inhibitor of Vascular Endothelial Growth Factor (VEGF) and / or Platelet-Derived Growth Factor (PDGF) receptors used in step ii) is in a range of from 5 nM to about 15 μM, such as from 5 nM to about 12 μM, 1 μM to 10 μM, from about 1 μM to about 5 μM, or from about 3 μM to about 4 μM.

6. A method according to any one of claims 2-5, wherein the concentration of inhibitor of Vascular Endothelial Growth Factor (VEGF) receptor and / or Platelet-Derived Growth Factor (PDGF) receptor used in step I) is in a range of from 5 nM to about 15 μM, such as from 5 nM to about 12 μM, 1 μM to about 10 μM from about 1 μM to about 5 μM or from about 3 μM to about 4 μM, (Fig. 2C, 2D).

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

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

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

10. A 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% pancreatic endocrine cells, and wherein the endocrine cells are CHGA+.11 . A method according to any one of the preceding claims, wherein the population of pancreatic endocrine (PEC) cells obtained after step ii) contains at least 1% such as at least 2%, at least 3%, at least 4% or at least 5% more pancreatic endocrine (PEC) cells compared with a population of pancreatic endocrine (PEC) cells 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 step of i) differentiating a population of pancreatic endoderm (PE) cells into pancreatic endocrine progenitors, by treating the PE cells with an inhibitor of Vascular Endothelial Growth Factor (VEGF) receptor and / or Platelet-Derived Growth Factor (PDGF) receptor.

13. An in vitro method for producing a population of pancreatic endocrine (PEC) cells 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 producing a population of pancreatic endocrine (PEC) cells comprising the steps of i) differentiating a population of pancreatic endoderm (PE) cells into pancreatic endocrine progenitors, by treating the PE cells with an inhibitor of Vascular Endothelial Growth Factor (VEGF) receptor and / or Platelet-Derived Growth Factor (PDGF) receptor, andii) differentiating pancreatic endocrine progenitor (EP) cells into pancreatic endocrine (PEC) cells.

15. 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 progenitors, 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.

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