WO2024008810A1 - Différenciation de cellules souches en cellules endocrines pancréatiques - Google Patents

Différenciation de cellules souches en cellules endocrines pancréatiques Download PDF

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WO2024008810A1
WO2024008810A1 PCT/EP2023/068586 EP2023068586W WO2024008810A1 WO 2024008810 A1 WO2024008810 A1 WO 2024008810A1 EP 2023068586 W EP2023068586 W EP 2023068586W WO 2024008810 A1 WO2024008810 A1 WO 2024008810A1
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cells
pancreatic endocrine
pancreatic
receptor
growth factor
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Christian Le Fèvre HONORÉ
Djordje DJORDJEVIC
Hanni WILLENBROCK
Thomas Henri KLEIBER
Jeannette Schlichting KIRKEGAARD
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Novo Nordisk A/S
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    • C12N2506/22—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from pancreatic cells

Definitions

  • 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.
  • VEGF Vascular Endothelial Growth Factor
  • PDGF Platelet-Derived Growth Factor
  • 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).
  • islet transplantation is limited availability of donor islets.
  • Beta cell (BC) transplantation potentially provides the ultimate cure for type I diabetes.
  • Pluripotent stem (PS) cells can proliferate infinitely and differentiate into many cell types.
  • 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.
  • 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.
  • pancreatic 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).
  • 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.
  • PE pancreatic endoderm
  • EP pancreatic endocrine progenit
  • 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.
  • PE pancreatic endoderm
  • EP pancreatic endocrine progenitor
  • VEGF Vascular Endothelial Growth Factor
  • PDGF Platelet
  • 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.
  • PE pancreatic endoderm
  • EP pancreatic endocrine progenitor
  • VEGF Vascular Endothelial Growth Factor
  • PDGF Platelet
  • 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.
  • PE pancreatic endoderm
  • EP pancreatic endocrine progenitor
  • VEGF Vascular Endothelial Growth Factor
  • PDGF Platelet-Derive
  • 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.
  • VEGF Vascular Endothelial Growth Factor
  • PDGF Platelet-Derived Growth Factor
  • 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.
  • VEGF Vascular Endothelial Growth Factor
  • PDGF Platelet-Derived Growth Factor
  • 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.
  • PE pancreatic endoderm
  • PDGF Platelet-Derived Growth Factor
  • 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.
  • PE pancreatic endoderm
  • EP pancreatic endocrine progenitor
  • VEGF Vascular Endothelial Growth Factor
  • PDGF Platelet-Derived Growth Factor
  • 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.
  • VEGF Vascular Endothelial Growth Factor
  • PDGF Platelet-Derived Growth Factor
  • 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 ⁇ M such as from 5 nM to about 15 ⁇ M, 5 nM to about 12 ⁇ M, 10 nM to about 11 ⁇ M, from 25 nM to 10 ⁇ M, from 50 nM to 10 ⁇ M, from 100 nM to 10 ⁇ M, from 250 nM to 10 ⁇ M, from 500 nM to 10 ⁇ M, from 750 nM to 10 nM, from 1 ⁇ M to 10 ⁇ M, from 2 ⁇ M to 10 ⁇ M, from 2.5 ⁇ M to 10 ⁇ M, from 2.5 ⁇ M to 8 ⁇ M, from 2.5 ⁇ M to 7 ⁇ M, from 2.5 ⁇ M to 6 ⁇ M, from 2.5 ⁇ M
  • 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 ⁇ M such as from 5 nM to about 15 ⁇ M, 5 nM to about 12 ⁇ M,10 nM to about 11 ⁇ M, from 25 nM to 10 ⁇ M, from 50 nM to 10 ⁇ M, from 100 nM to 10 ⁇ M, from 250 nM to 10 ⁇ M, from 500 nM to 10 ⁇ M, from 750 nM to 10 nM, from 1 ⁇ M to 10 ⁇ M, from 2 ⁇ M to 10 ⁇ M, from 2.5 ⁇ M to 10 ⁇ M, from 2.5 ⁇ M to 8 ⁇ M, from 2.5 ⁇ M to 7 ⁇ M, from 2.5 ⁇ M to 6 ⁇ M, from 2.5 ⁇ M to 5 ⁇ M, from 2.5 ⁇ M to 5.5 ⁇ M, from
  • 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.
  • step i) does not involve a TGFb receptor kinase inhibitor.
  • step ii) does not involve a TGFb receptor kinase inhibitor.
  • 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+).
  • 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+).
  • 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+).
  • 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.
  • 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).
  • 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.
  • 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.
  • 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).
  • 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+.
  • 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.
  • 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.
  • 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.
  • hPSC pancreatic endocrine 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.
  • DE definitive endoderm
  • PE pancreatic endoderm
  • EP endocrine progenitor
  • EP endocrine progenitor
  • hPSC were differentiated to PE and then treated with TGFpRI inhibitors [Standard (STD)] or Linifanib during the EP-PEC stage.
  • TGFpRI inhibitors Standard (STD)
  • 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.
  • 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 .
  • hPSC pancreatic endocrine cells
  • PEC pancreatic endocrine cells
  • Methods are provided for obtaining pancreatic endocrine cells from pluripotent stem cells.
  • 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.
  • 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.
  • hESC human embryonic stem cells
  • hiPSC human induced pluripotent stem cells
  • hPSC human Pluripotent stem cells
  • PE pancreatic endoderm
  • PEC pancreatic endocrine cells
  • BC Beta cells, insulin-producing beta cells
  • stem cell is to be understood an undifferentiated cell having proliferative capacity (particularly self-renewal competence) but maintaining differentiation potency.
  • stem cell includes categories such as pluripotent stem cell, multipotent stem cell, and the like according to their differentiation potentiality.
  • pluripotency 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).
  • 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.
  • unipotent stem cell means a stem cell having a potency to differentiate into only one particular tissue or cell.
  • 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.
  • induced pluripotent stem cell means a type of pluripotent stem cell that can be generated directly from adult cells.
  • 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.
  • 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.
  • the term “definitive endoderm”, “definitive endoderm cells”, or “DE” refers to cells characterized by expression of the marker SOX17.
  • 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.
  • SOX17 SRY-box 17
  • SOX SRY-related HMGbox
  • FOXA2 forkhead box A2
  • forkhead box A2 is a member of the forkhead class of DNA-binding proteins
  • CXCR4 C-X-C motif chemokine receptor 4
  • CXC chemokine receptor 4 is a CXC chemokine receptor specific for stromal cell-derived factor-1 .
  • 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) Pancreatic endoderm
  • pancreatic endoderm 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.
  • PDX1 refers to a homeodomain transcription factor implicated in pancreas development.
  • NKX6.1 as used herein is a member of the NKX transcription factor family.
  • 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.
  • PTF1A 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.
  • CUA1 as used herein is a member of the carboxypeptidase A family of zinc metalloproteases. This enzyme is produced in the pancreas.
  • Non-limiting examples of PE inducing protocols is described in WO2014/033322, which is incorporated herein by reference in its entirety.
  • 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.
  • NEUROG3 as used herein, is a member of the neurogenin family of basic loop- helix-loop transcription factors.
  • NKX2.2 and “NKX6.1” as used herein are members of the NKX transcription factor family.
  • NeuroD as used herein is a member of the NeuroD family of basic helix-loop-helix (bHLH) transcription factors.
  • pancreatic endocrine progenitor cells 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
  • pancreatic endocrine cells or “PEC” refers to cells expressing CHGA and ISL1.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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).
  • 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.
  • 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.
  • 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 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.
  • the presence or lack of expression may be denoted with + (plus) or - (minus) signs, respectively.
  • 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.
  • 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.
  • 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.
  • 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.
  • day X When referring to “day X”, such as day 1 , day 2 etc., it is relative to the initiation of the protocol at day 0.
  • 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.
  • 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”.
  • differentiate 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.
  • 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.
  • 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.
  • 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.
  • differentiation of the cells comprises culturing the cells in a medium comprising one or more differentiation factors.
  • 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.
  • differentiation of the cells comprises culturing the cells in a medium comprising one or more differentiation factors.
  • the method is carried out in vitro.
  • in vitro is meant that the cells are provided and maintained outside of the human or animal body.
  • the cells are non-native.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.).
  • 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.
  • 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.
  • 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.
  • Vascular Endothelial Growth Factor receptors VEGFR
  • PDGFR Platelet-derived Growth Factor receptors
  • 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.
  • VEGF Vascular Endothelial Growth Factor
  • PDGF Platelet-Derived Growth Factor
  • 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.
  • PE pancreatic endoderm
  • pancreatic endoderm cells PE
  • pancreatic endocrine progenitor cells EP or PEP
  • the pancreatic endoderm (PE) cells for use in step I) of the methods described herein are cells having the markers PDX1 and NKX6.
  • the step I) of culturing pancreatic endoderm (PE) cells into pancreatic endocrine progenitors 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.
  • EGF epidermal growth factor
  • 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.
  • EGF agonists include Betacellulin (gene name BTC), Epidermal Growth Factor (gene name EGF), Amphiregulin
  • 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.
  • VEGF Vascular Endothelial Growth Factor
  • PDGF Platelet-Derived Growth Factor
  • 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.
  • PE pancreatic endoderm
  • pancreatic endoderm cells PE
  • pancreatic endocrine progenitor cells EP or PEP
  • the pancreatic endoderm (PE) cells for use in step i) of the methods described herein are cells having the markers PDX1 and NKX6.
  • 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.
  • EGF epidermal growth factor
  • 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.
  • 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).
  • EGF pathway activator is Betacellulin.
  • 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).
  • the NOTCH pathway inhibitor is XX.
  • 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.
  • the BMP pathway inhibitor is LDN.
  • 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).
  • DZNep 3- Deazaneplanocin A hydrochloride
  • GSK 126 Cas No. 1346574- 57-9
  • EPZ005687 Cas No. 1396772-26-1
  • the EZH2 histone methyltransferase inhibitor is DZNep.
  • 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).
  • the JNK pathway inhibitor is JNK Inhibitor VIII.
  • 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.
  • the broad-spectrum protein kinase inhibitor is Staurosporine.
  • TGFb receptor kinase inhibitor examples 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).
  • the TGFb receptor kinase inhibitors are RepSox and SB431542.
  • 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.
  • Rho kinase e.g., Tiger, Chroman-1 (Cas. No. 1273579-40-0) and Thiazovivin (Cas No. 1226056-71-8); Heparin; and Forskolin or NKH 477.
  • 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.
  • VEGF Vascular Endothelial Growth Factor
  • PDGF Platelet-Derived Growth Factor
  • 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 ⁇ M, such as from 5 nM to about 12 ⁇ M, 1 nM to 12 ⁇ M, from 10 nM to about 11 ⁇ M, from 25 nM to 10 ⁇ M, from 50 nM to 10 ⁇ M, from 100 nM to 10 ⁇ M, from 250 nM to 10 ⁇ M, from 500 nM to 10 ⁇ M, from 750 nM to 10 nM, from 1 ⁇ M to 10 ⁇ M, from 2 ⁇ M to 10 ⁇ M, from 2.5 ⁇ M to 10 ⁇ M, from 2.5 ⁇ M to 8 ⁇ M, from 2.5 ⁇ M to 7 ⁇ M, from 2.5 ⁇ M to 6 ⁇ M, from 2.5 ⁇ M to 5 ⁇ M, from 2.5 ⁇ M to 5.5 ⁇ M, from
  • 11 ⁇ M to about 12 ⁇ M or in a concentration of 5nM, 50nM, 500nM, 1 ⁇ M, 1.5 ⁇ M, 2 ⁇ M, 2.5 ⁇ M, 3 ⁇ M, 3.5 ⁇ M, 4 ⁇ M, 4.5 ⁇ M, 5 ⁇ M, 5.5 ⁇ M, 6 ⁇ M, 6.5 ⁇ M, 7 ⁇ M, 7.5 ⁇ M, 8 ⁇ M, 8.5 ⁇ M, 9 ⁇ M, 9.5 ⁇ M, 10 ⁇ M, 10.5 ⁇ M, 11 ⁇ M, 11.5 ⁇ M, 12 ⁇ M or 15 ⁇ M.
  • step i) 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.
  • 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).
  • 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.
  • pancreatic endocrine progenitor obtained in step i) typically have the markers NEUROG3, NKX2.2 and NEUROD1.
  • differentiation is carried out in a suitable culture medium such as MCDB131 (basal medium), RPMI, DMEM, DMEM/F12, CMRL, MEM and the like.
  • 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.
  • HSA human serum albumin
  • 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.
  • the pancreatic endocrine progenitor (EP) cells obtained comprises at least one of the markers NEUROG3, NKX2.2, and NEUROD1.
  • pancreatic endocrine progenitor into pancreatic endocrine (PEC) cells step ii)
  • 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.
  • pancreatic endocrine progenitor (EP) cells may also be used as starting material for step ii) when obtained using already published methods.
  • 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.
  • step ii) pancreatic endoderm (PE) cells to pancreatic endocrine progenitor (EP) cells - step i)
  • 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.
  • Rho kinase e.g., Tiger, Chroman-1 (Cas. No. 1273579-40-0) and Thiazovivin (Cas No. 1226056-71-8
  • Heparin and Forskolin or NKH 477.
  • 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.
  • VEGF Vascular Endothelial Growth Factor
  • PDGF Platelet-Derived Growth Factor
  • 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.
  • 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 ⁇ M, such as from 5 nM to about 12 ⁇ M, 1 nM to 12 ⁇ M.from 10 nM to about 11 ⁇ M, from 25 nM to 10 ⁇ M, from 50 nM to 10 ⁇ M, from 100 nM to 10 ⁇ M, from 250 nM to 10 ⁇ M, from 500 nM to 10 ⁇ M, from 750 nM to 10 nM, from 1 ⁇ M to 10 ⁇ M, from 2 ⁇ M to 10 ⁇ M, from 2.5 ⁇ M to
  • step ii) 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.
  • 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).
  • 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.
  • MCDB131 basic medium
  • 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.
  • HSA human serum albumin
  • pancreatic endocrine progenitor (EP) cells to pancreatic endocrine (PEC) cells
  • 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.
  • 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.
  • VEGF Vascular Endothelial Growth Factor
  • PDGF Platelet-Derived Growth Factor
  • 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.
  • 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.
  • VEGF Vascular Endothelial Growth Factor
  • PDGF Platelet-Derived Growth Factor
  • the aggregates may be dissociated into single cells.
  • 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.
  • the cryopreservation may be performed after culturing the cells after 1 day or longer such after culturing from about 1 to about 30 days.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • hPSCs are obtained from any suitable source as referred to in the above.
  • the methods described herein include culturing hPSCs.
  • culturing is meant that the hPSCs are cultured in a cell culture medium, which is suitable for viability in their current state of development.
  • 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.
  • 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.
  • 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.
  • VEGF Vascular Endothelial Growth Factor
  • PDGF Platelet-Derived Growth Factor
  • 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
  • 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.
  • 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.,
  • compositions comprising the pancreatic endocrine cells (PEC) obtained by any of the methods of the invention
  • a medicament comprising pancreatic endocrine cells (PEC) obtained by any of the methods of the invention according to the present description.
  • PEC pancreatic endocrine cells
  • 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.
  • 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.
  • PEC pancreatic endocrine cells
  • pancreatic endocrine (PEC) cells obtained by any of the methods of the invention
  • 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.
  • the term “mammal” includes human and veterinary subjects.
  • 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.
  • Methods of treating diabetes are also provided herein.
  • a method of treating type-1 diabetes in a mammal 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.
  • 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.
  • diagnosis is based on an elevated blood glucose level after fasting or on a glucose tolerance test.
  • diagnosis of type 1 diabetes includes various physical symptoms and characteristics.
  • a mammal at risk for developing 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.
  • 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.
  • 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.
  • 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.
  • several transplants are performed.
  • One of skill in the art 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.
  • 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).
  • an insulin secreting cell population e.g., a pancreatic islet cell population
  • Pancreatic endocrine progenitors (EP) generated in vitro according to the present invention are obtained through the following steps.
  • 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.
  • Wash medium MCDB 131 medium, Gibco, cat. no. 10372019
  • Pancreatic endocrine cells (PEC) generated in vitro according to the present invention are obtained through the following steps.
  • 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.
  • ITS-X Gabco, cat. no. 51500056
  • 0.25mM Ascorbic acid Fisher Scientific, cat. no. 0937-07
  • 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.
  • 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.
  • 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.
  • Glutamax Gibco, cat. no. 35050038
  • human serum albumin Olin, 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
  • 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.
  • the cells obtained are re-suspended in cryopreservation media and preserved by a sequential lowering of temperature to below -80°C.
  • 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 ⁇ M Rocki (Sigma#Y27632-Y0503).
  • 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.

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Abstract

Procédé in vitro de production d'une population de cellules endocrines pancréatiques (PEC) comprenant les étapes suivantes : i) différenciation d'une population de cellules de l'endoderme pancréatique (PE) en progéniteurs endocriniens pancréatiques, la différenciation consistant à traiter les cellules de l'endoderme pancréatique (PE) avec un inhibiteur du récepteur du facteur de croissance de l'endothélium vasculaire (VEGF) et/ou du récepteur du facteur de croissance dérivé des plaquettes (PDGF), et ii) différenciation des cellules progénitrices endocrines pancréatiques (PE) en cellules endocrines pancréatiques (PEC) en traitant les cellules progénitrices endocrines pancréatiques (PE) avec un inhibiteur du récepteur du facteur de croissance endothélial vasculaire (VEGF) et/ou du récepteur du facteur de croissance dérivé des plaquettes (PDGF).
PCT/EP2023/068586 2022-07-06 2023-07-05 Différenciation de cellules souches en cellules endocrines pancréatiques WO2024008810A1 (fr)

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