Method for generating pancreatic hormone-producing cells

• US 9,796,962 B2
• Filed: 08/06/2014
• Issued: 10/24/2017
• Est. Priority Date: 08/07/2013
• Status: Active Grant

• Current Assignee: Kyoto University
• Original Assignee: Kyoto University
• Inventors: Osafune, Kenji, Inagaki, Nobuya, Toyoda, Taro, Kondo, Yasushi
• Primary Examiner(s): Berke-Schlessel, David
• Application Number:
  US14/909,690
• Publication Number:
  US 20160289642A1
• Time in Patent Office: 1,175 Days
• Field of Search: None
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2501/01 Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• 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 2501/39 Steroid hormones
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2501/82 Histamine
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• 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

Abstract

Provided is a method for inducing pancreatic hormone-producing cells from pancreatic progenitor cells efficiently. The method comprises a step of culturing the cells in a culture medium comprising sodium cromoglicate.

Claims

1. A method for generating pancreatic hormone-producing cells, which comprises culturing pancreas progenitor cells in a medium comprising sodium cromoglicate.

2. The method of claim 1, wherein the medium further comprises at least one agent selected from the group consisting of:

(a) an cAMP analog;

(b) nicotinamide;

(c) a steroid; and

(d) a TGFβ

inhibitor.

3. The method of claim 1, wherein the medium further comprises:

(a) at least one agent selected from the group consisting of an adenylate cyclase activator, a cAMP phosphodiesterase inhibitor and an cAMP analog;

(b) nicotinamide;

(c) a steroid; and

(d) a TGFβ

inhibitor.

4. The method of claim 2, wherein the cAMP analog is forskolin.

5. The method of claim 2, wherein the steroid is dexamethasone.

6. The method of claim 2, wherein the TGFβ

inhibitor is 2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine.

7. The method of claim 1, wherein the pancreatic progenitor cells are the cells derived from a method comprising the following steps:

(1) culturing pluripotent stem cells in a medium comprising an activator of activin receptor-like kinase-4,7 and a GSK3 inhibitor, and

(2) culturing the cells obtained in step (1) in a medium comprising (a) a retinoic acid receptor agonist, (b) a BMP inhibitor and (c) a TGFβ

inhibitor.

8. The method of claim 7, wherein the activator of activin receptor-like kinase-4,7 is activin.

9. The method of claim 7, wherein the GSK3 inhibitor is CHIR99021.

10. The method of claim 7, wherein the TGFβ

inhibitor used in step (2) is SB431542.

11. The method of claim 7, wherein the BMP inhibitor is dorsomorphin.

12. The method of claim 1, wherein the pancreatic hormone-producing cells are selected from the group consisting of insulin producing cells, glucagon producing cells, somatostatin producing cells and pancreatic polypeptide producing cells.

13. The method of claim 12, wherein the pancreatic hormone-producing cells are insulin producing cells and/or glucagon producing cells.

14. The method of claim 1, wherein the pancreatic progenitor cells are human cells.

15. The method of claim 1, wherein the medium further comprises at least one agent selected from the group consisting of:

(a) an adenylate cyclase activator;

(b) nicotinamide;

(c) a steroid; and

(d) a TGFβ

inhibitor.

16. The method of claim 15, wherein the steroid is dexamethasone.

17. The method of claim 15, wherein the TGFβ

inhibitor is 2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine.

18. The method of claim 1, wherein the medium further comprises at least one agent selected from the group consisting of:

(a) a cAMP phosphodiesterase inhibitor;

(b) nicotinamide;

(c) a steroid; and

(d) a TGFβ

inhibitor.

19. The method of claim 18, wherein the steroid is dexamethasone.

20. The method of claim 18, wherein the TGFβ

inhibitor is 2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine.