Generation of pancreatic endoderm from pluripotent stem cells using small molecules

• US 10,221,392 B2
• Filed: 07/21/2016
• Issued: 03/05/2019
• Est. Priority Date: 09/03/2012
• Status: Active Grant

• Current Assignee: Takara Bio Europe SAS
• Original Assignee: Novo Nordisk A/S, Takara Bio Europe AB
• Inventors: Ekberg, Jenny, Hansson, Mattias, Doehn, Ulrik, Hess, Katja, Funa, Nina
• Primary Examiner(s): Schultz, James D
• Application Number:
  US15/215,807
• Publication Number:
  US 20160326495A1
• Time in Patent Office: 957 Days
• Field of Search: None
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/117 Keratinocyte growth factors (KGF-1, i.e. FGF-7; KGF-2, i.e. FGF-12)
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/60 Transcription factors
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: C12N 5/0678 Stem cells; Progenitor cells; Precursor cells

Abstract

A method of producing pancreatic cells or pancreatic cell precursors expressing at least 5% PDX1/NKX6.1 double positive, comprising exposing definitive endoderm cells to an effective amount of one or more small molecules, to differentiate the human definitive endoderm cells into the pancreatic cells or pancreatic cell precursors. The present invention also relates to pancreatic endoderm cells produced by said methods and uses of said pancreatic endoderm cells.

Claims

1. A method of producing pancreatic cells or pancreatic cell precursors, comprising a first exposure of definitive endoderm cells to an effective amount of at least one BMP inhibitor in the absence of bFGF, followed by a second exposure to JNK inhibitor II, bFGF, and at least one retinoic acid receptor agonist, wherein at least 5% of the resulting pancreatic cells or pancreatic cell precursors are PDX1NKX6.1 double positive.

2. The method of claim 1, wherein the at least one BMP inhibitor is

3. The method of claim 1, wherein the at least one retinoic acid receptor agonist is

4. The method of claim 1, wherein the at least one retinoic acid receptor agonist is a retinoic acid derivative.

5. The method of claim 1, wherein the at least one BMP inhibitor is

6. The method of claim 1, further comprising exposing the definitive endoderm cells to FGF7 or FGF10.

7. The method of claim 2, further comprising exposing the definitive endoderm cells to at least one compound selected from the group consisting of a wnt inhibitor and a hedgehog inhibitor prior to exposing the definitive endoderm cells to the

8. The method of claim 1, wherein said pancreatic cells or pancreatic cell precursors are at least 10% PDX1/NKX6.1 double positive.

9. A method of producing pancreatic cells or pancreatic cell precursors, comprising exposing definitive endoderm cells to an effective amount of at least one BMP inhibitor, JNK inhibitor II, bFGF, and at least one retinoic acid receptor agonist, wherein at least 5% of the resulting pancreatic cells or pancreatic cell precursors are PDX1NKX6.1 double positive.

10. The method of claim 9, wherein the at least one BMP inhibitor is

11. The method of claim 9, wherein the at least one retinoic acid receptor agonist is

12. The method of claim 9, wherein the at least one BMP inhibitor is