Method for proliferation of pancreatic progenitor cells

• US 10,655,105 B2
• Filed: 08/03/2015
• Issued: 05/19/2020
• Est. Priority Date: 08/04/2014
• Status: Active Grant

• Current Assignee: Orizuru Therapeutics Incorporated
• Original Assignee: Takeda Pharmaceutical Company Limited
• Inventors: Kunisada, Yuya
• Primary Examiner(s): Fox, Allison M
• Application Number:
  US15/501,703
• Publication Number:
  US 20170233700A1
• Time in Patent Office: 1,751 Days
• Field of Search: None
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• 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 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2501/998 Proteins not provided for elsewhere
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: C12N 5/0678 Stem cells; Progenitor cells; Precursor cells

Abstract

The present invention relates to a method for preparing highly pure pancreatic progenitor cells by using pluripotent stem cells such as ES cells or iPS cells as a source, inducing their differentiation into pancreatic progenitor cells, and culturing and proliferating the pancreatic progenitor cells. Specifically, the present invention relates to a method for proliferation of pancreatic progenitor cells, comprising the step of culturing the pancreatic progenitor cells in a medium containing (i) an EGF signal transduction activator and/or an FGF signal transduction activator and (ii) a ROCK inhibitor.

Claims

1. A method for proliferation of pancreatic progenitor cells, comprising:

culturing the pancreatic progenitor cells in a medium comprising (i) an EGF signal transduction activator and/or an FGF signal transduction activator, and (ii) a Wnt signal inhibitor, wherein the Wnt signal inhibitor is selected from the group consisting of IWP2, IWP3, IWP4, 2-(4-trifluoromethylphenyl)-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-4(3H)-one (XAV939), IWR1, G-CSF, IGFBP4, Dkk1, and Frzb-1.

2. The proliferation method according to claim 1, wherein the pancreatic progenitor cells are pancreatic progenitor cells induced by culturing PDX1-positive cells in a medium comprising (a) the EGF signal transduction activator and/or the FGF signal transduction activator and (b) the Wnt signal inhibitor.

3. The proliferation method according to claim 1, wherein the EGF signal transduction activator is selected from the group consisting of EGF, TGFalpha, HB-EGF, Amphiregulin, Betacellulin, and Epiregulin, and wherein the FGF signal transduction activator is selected from the group consisting of aFGF (FGF1), bFGF (FGF2), and any one of FGF3 to FGF23.

4. The proliferation method according to claim 1, wherein the medium further comprises (iii) a ROCK inhibitor.

5. The proliferation method according to claim 4, wherein the ROCK inhibitor is selected from the group consisting of N-(4-pyridinyl)-4β

-[(R)-1-aminoethyl]cyclohexane-1α

-carboxamide (Y-27632), Fasudil (HA1077), (2S)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]hexahydro-1H-1,4-diazepine (H-1152), 4β

-[(1R)-1-aminoethyl]-N-(4-pyridyl)benzene-1α

-carboxamide (Wf-536), N-(1H-pyrrolo[2,3-b]pyridin-4-yl)-4PER(R)-1-aminoethyl]cyclohexane-1α

-carboxamide (Y-30141), N-(3-{[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4,5-c]pyridin-6-yl]oxy}phenyl)-4-{[2-(4-morpholinyl)ethyl]-oxy}benzamide (GSK269962A), and N-(6-fluoro-1H-imidazol-5-yl)-6-methyl-2-oxo-4-[4-(trifluoromethyl)phenyl]-3,4-dihydro-1H-pyridine-5-carboxamide (GSK429286A).

6. A method for production of pancreatic progenitor cells, comprising:

culturing and proliferating pancreatic progenitor cells in a medium comprising (i) an EGF signal transduction activator and/or an FGF signal transduction activator, and (ii) a Wnt signal inhibitor, wherein the Wnt signal inhibitor is selected from the group consisting of IWP2, IWP3, IWP4, 2-(4-trifluoromethylphenyl)-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-4(3H)-one (XAV939), IWR1, G-CSF, IGFBP4, Dkk1, and Frzb-1; and

collecting the pancreatic progenitor cells from the cultures.

7. The production method according to claim 6, further comprising culturing PDX1-positive cells in a medium comprising (a) the EGF signal transduction activator and/or the FGF signal transduction activator and (b) the Wnt signal inhibitor to induce differentiation thereof into the pancreatic progenitor cells.

8. The production method according to claim 6, wherein the EGF signal transduction activator is selected from the group consisting of EGF, TGFalpha, HB-EGF, Amphiregulin, Betacellulin, and Epiregulin, and wherein the FGF signal transduction activator is selected from the group consisting of aFGF (FGF1), bFGF (FGF2), and any one of FGF3 to FGF23.