Method for inducing differentiation of insulin-producing cells

• US 10,457,916 B2
• Filed: 05/19/2015
• Issued: 10/29/2019
• Est. Priority Date: 05/20/2014
• Status: Active Grant

• Current Assignee: Tokyo Institute of Technology (National University Corporation Tokyo Institute of Technology)
• Original Assignee: Tokyo Institute of Technology (National University Corporation Tokyo Institute of Technology)
• Inventors: Kume, Shoen, Shiraki, Nobuaki
• Primary Examiner(s): Ton, Thaian N
• Assistant Examiner(s): Montanari, David A.
• Application Number:
  US15/312,273
• Publication Number:
  US 20170081639A1
• Time in Patent Office: 1,624 Days
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C12N 15/09 Recombinant DNA-technology
• CPC Class Codes: C12N 2500/92 Medium free of human- or animal-derived components
• CPC Class Codes: C12N 2500/98 Xeno-free medium and culture conditions
• CPC Class Codes: C12N 2501/01 Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• 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 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2501/73 Hydrolases (EC 3.)
• 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
• CPC Class Codes: G01N 33/507 Pancreatic cells
• CPC Class Codes: G01N 33/5073 : Stem cells
• CPC Class Codes: G01N 33/74 : involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors

Abstract

It is an object of the present invention to provide a method for efficiently directing differentiation into insulin-producing cells in a xeno-free culture system. According to the present invention, there is provided a method for directed differentiation into insulin-producing cells, comprising culturing stem cells in the following steps (1) to (5): (1) a step of culturing stem cells in a medium comprising an activator of activin receptor-like kinase-4/-7 and a GSK3 inhibitor and then culturing in a medium comprising an activator of activin receptor-like kinase-4/-7; (2) a step of culturing the cells obtained in step (1) in a medium comprising a hedgehog signaling inhibitor and an FGF; (3) a step of culturing the cells obtained in step (2) in a medium comprising a retinoic acid receptor agonist, a hedgehog signaling inhibitor and a BMP signaling inhibitor; (4) a step of culturing the cells obtained in step (3) in a medium comprising a TGF-β type I activin receptor-like kinase-4/-5/-7 inhibitor and a BMP signaling inhibitor; and (5) a step of culturing the cells obtained in step (4) in a medium comprising a phosphodiesterase inhibitor.

Claims

1. A method for directed differentiation into insulin-producing cells, comprising culturing endodermal cells in the following steps (a) to (d):

(a) culturing the endodermal cells in a medium comprising a hedgehog signaling inhibitor and an FGF;

(b) culturing the cells obtained in step (a) in a medium comprising a retinoic acid receptor agonist, a hedgehog signaling inhibitor and noggin;

(c) culturing the cells obtained in step (b) in a medium comprising a TGF-β

type I activin receptor-like kinase-4/-5/-7 inhibitor and noggin; and

(d) culturing the cells obtained in step (c) in a medium comprising GLP-1 receptor agonist and nicotinamide to produce insulin-producing cells,

wherein the concentration of the noggin in steps (b) and (c) is at least 200 ng/ml or more.

2. The method of claim 1, wherein the concentration of the noggin in steps (b) and (c) is 200-500 ng/ml.

3. The method of claim 1, wherein step (c) further comprises a protein kinase C activator.

4. The method of claim 1, wherein the retinoic acid receptor agonist in step (b) is retinoic acid and the hedgehog signaling inhibitor in steps (a) and (b) is KAAD-cyclopamine.

5. The method of claim 3, wherein the protein kinase C activator is indolactam V.

6. The method of claim 1, which is characterized by conducting all of the steps (a) to (d) in a xeno-free culture system.

7. The method of claim 1, wherein the endodermal cells are differentiated from pluripotent stem cells or embryonic stem cells.

8. The method of claim 1, further comprising, before step (a), (1-1) culturing human or mouse pluripotent stem cells in a medium comprising an activator of activing receptor-like kinase-4/-7 and a GSK3 inhibitor and then (1-2) culturing in a medium comprising an activator of activing receptor-like kinase-4/-7 to effect differentiation into endodermal cells.

9. The method of claim 8, wherein the medium in step (1-2) does not comprise a GSK3 inhibitor.

10. The method of claim 8, all of the steps (1-1) to (d) are in a xeno-free culture system.

11. The method of claim 1, wherein the hedgehog signaling inhibitor is SANT-1.

12. The method of claim 1, wherein FGF is FGF10.

13. The method of claim 1, wherein the BMP signaling inhibitor is LDN193189.

14. The method of claim 1, wherein the retinoic acid receptor agonist is retinoic acid.

15. The method of claim 1, wherein the TGF-β

type I activin receptor-like kinase-4/-5/-7 inhibitor is Alk5 inhibitor.

16. The method of claim 1, wherein the GLP-1 receptor agonist is exendin-4.

17. The method of claim 1, further comprising isolating the cells from step (d).

18. The method of claim 1, wherein the medium of step (d) excludes a growth factor.

19. A method for directed differentiation into mature insulin-producing cells, comprising culturing endodermal cells in the following steps (a) to (d):

(a) culturing the endodermal cells in a medium comprising SANT-1 and FGF10;

(b) culturing the cells obtained in step (a) in a medium comprising retinoic acid, SANT-1 and noggin;

(c) culturing the cells obtained in step (b) in a medium comprising Alk5 inhibitor, noggin, and indolactam V; and

(d) culturing the cells obtained in step (c) in a medium comprising nicotinamide and exendin-4 to produce insulin-producing cells,

wherein the concentration of the noggin in steps (b) and (c) is at least 200 ng/ml or more.

21. The method of claim 19, wherein the concentration of noggin in steps (b) and (c) is 200-500 ng/ml.

20. A method for directed differentiation into mature insulin-producing cells, the method consisting of:

(a) culturing human or mouse pluripotent stem cells in a medium comprising an activator of activing receptor-like kinase-4/-7 and a GSK3 inhibitor;

(b) culturing the cells of step (a) in a medium comprising an activator of activing receptor-like kinase-4/-7 to effect differentiation into endodermal cells;

(c) culturing the endodermal cells of step (b) in a medium comprising a hedgehog signaling inhibitor and an FGF;

(d) culturing the cells obtained in step (c) in a medium comprising a retinoic acid receptor agonist, a hedgehog signaling inhibitor and a BMP signaling inhibitor;

(e) culturing the cells obtained in step (d) in a medium comprising a TGF-β

type I activin receptor-like kinase-4/-5/-7 inhibitor and a BMP signaling inhibitor; and

(f) culturing the cells obtained in step (e) in a medium comprising GLP-1 receptor agonist and nicotinamide to produce insulin-producing cells.

22. The method of claim 20, wherein the endodermal cells are differentiated from pluripotent stem cells or embryonic stem cells.