METHOD FOR MANUFACTURING PANCREATIC-HORMONE-PRODUCING CELLS

• US 20130022986A1
• Filed: 12/28/2010
• Published: 01/24/2013
• Est. Priority Date: 12/29/2009
• Status: Active Grant

• Current Assignee: Takeda Pharmaceutical Company Limited
• Original Assignee: Takeda Pharmaceutical Company Limited
• Inventors: Hosoya, Masaki, Kunisada, Yuya, Shoji, Masanobu, Yamazoe, Noriko
• Granted Patent: US 8,932,853 B2
• Time in Patent Office: Days
• US Class Current: 435/6.13
• CPC Class Codes: A61K 35/39 Pancreas; Islets of Langerhans Langerhans cells of epidermis A61K35/36
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• 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 2501/727 Kinases (EC 2.7.)
• 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
• CPC Class Codes: G01N 33/507 Pancreatic cells

Abstract

The present invention provides a method of more efficiently producing pancreas cells, particularly pancreatic hormone-producing cells, a method of stably producing pancreas cells in a large amount by more efficiently inducing differentiation of stem cells into pancreas cells, a medicament containing a pancreas cells and a screening method using the cells.

A method of producing pancreatic hormone-producing cells, including subjecting stem cells to the following steps (1)-(4):

• (1) a step of cultivating stem cells in a medium containing an activator of activin receptor-like kinase-4,7 and a GSK3 inhibitor
• (2) a step of cultivating the cells obtained in the aforementioned step (1) in a medium containing an activator of activin receptor-like kinase-4,7
• (3) a step of cultivating the cells obtained in the aforementioned step (2) in a medium containing any one or more kinds selected from the group consisting of (a) retinoic acid receptor agonists, (b) at least one kind selected from the group consisting of inhibitors of AMP-activated protein kinase and/or activin receptor-like kinase-2,3,6, and BMP antagonists, and (c) inhibitors of activin receptor-like kinase-4,5,7
• (4) a step of cultivating the cells obtained in the aforementioned step (3).

Claims

1. A method of producing a pancreatic hormone-producing cell(s), comprising subjecting a stem cell(s) to the following steps (1)-(4):

(1) a step of cultivating a stem cell(s) in a medium containing an activator of activin receptor-like kinase-4,7 and a GSK3 inhibitor

(2) a step of cultivating the cell(s) obtained in the aforementioned step (1) in a medium containing an activator of activin receptor-like kinase-4,7

(3) a step of cultivating the cell(s) obtained in the aforementioned step (2) in a medium containing any one or more kinds selected from the group consisting of (a) retinoic acid receptor agonists, (b) at least one kind selected from the group consisting of inhibitors of AMP-activated protein kinase and/or activin receptor-like kinase-2,3,6, and BMP antagonists, and (c) inhibitors of activin receptor-like kinase-4,5,7

(4) a step of cultivating the cell(s) obtained in the aforementioned step (3).

2. The production method according to claim 1, wherein the activator of activin receptor-like kinase-4,7 in steps (1) and (2) is activin, and step (3) is a step of cultivating the cell(s) obtained in step (2) in a medium containing any one or more kinds selected from the group consisting of (a) retinoic acid receptor agonists, (b′

) inhibitors of AMP-activated protein kinase and/or activin receptor-like kinase-2,3,6 and (c) inhibitors of activin receptor-like kinase-4,5,7.

3. The production method according to claim 1, wherein step (4) is performed in a medium containing any one or more kinds selected from the group consisting of (i) at least one kind selected from the group consisting of adenylate cyclase activators, cAMP phosphodiesterase inhibitors and cAMP analogs, (ii) steroids and (iii) inhibitors of activin receptor-like kinase-4,5,7.

8. The production method according to claim 1, wherein the medium in step (3) contains the following (a)-(c):

(a) a retinoic acid receptor agonist

(b) at least one kind selected from the group consisting of inhibitors of AMP-activated protein kinase and/or activin receptor-like kinase-2,3,6, and BMP antagonists, and

(c) an inhibitor of activin receptor-like kinase-4,5,7.

9. The production method according to claim 1, wherein the GSK3 inhibitor in step (1) is 6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]nicotinonitrile.

10. The production method according to claim 1, wherein the inhibitor of activin receptor-like kinase-4,5,7 in step (3) is 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide or a hydrate thereof.

11. The production method according to claim 1, wherein said at least one kind selected from the group consisting of inhibitors of AMP-activated protein kinase and/or activin receptor-like kinase-2,3,6, and BMP antagonists in step (3) is dorsomorphin or Noggin.

12. The production method according to claim 1, wherein the medium in step (3) contains retinoic acid, 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide or a hydrate thereof, and dorsomorphin.

13. The production method according to claim 3, wherein at least one kind selected from the group consisting of adenylate cyclase activators, cAMP phosphodiesterase inhibitors and cAMP analogs is forskolin, 3-isobutyl-1-methylxanthine or dibutyl cAMP.

14. The production method according to claim 3, wherein the steroid is dexamethasone.

15. The production method according to claim 3, wherein the inhibitor of activin receptor-like kinase-4,5,7 is 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine, or 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide or a hydrate thereof.

16. The production method according to claim 3, wherein the medium contains nicotinamide.

17. The production method according to claim 1, wherein the stem cell(s) is(are) an induced pluripotent stem cell(s) (iPS cell(s)), an embryonic stem cell(s) (ES cell(s)) or a human somatic stem cell(s).

18. The production method according to claim 1, wherein the pancreatic hormone-producing cell(s) is(are) any selected from the group consisting of insulin-producing cells, glucagon-producing cells, somatostatin-producing cells, pancreatic polypeptide (PP)-producing cells and ghrelin-producing cells.

19. A medicament comprising the pancreatic hormone-producing cell(s) obtained by the production method according to claim 1.

4. A method of producing an endodermal cell(s), comprising cultivating a stem cell(s) in a medium containing at least an activator of activin receptor-like kinase-4,7 and a GSK3 inhibitor.

5. The production method according to claim 4, wherein the activator of activin receptor-like kinase-4,7 is activin.

6. A method of producing a progenitor cell(s) of a pancreatic hormone-producing cell(s), comprising cultivating an endodermal cell(s) in a medium containing any one or more kinds selected from the group consisting of the following (a)-(c):

(a) retinoic acid receptor agonists

(b) at least one kind selected from the group consisting of inhibitors of AMP-activated protein kinase and/or activin receptor-like kinase-2,3,6, and BMP antagonists, and

(c) inhibitors of activin receptor-like kinase-4,5,7.

20. A medicament comprising the progenitor cell(s) of the pancreatic hormone-producing cell(s) obtained by the production method according to claim 6.

7. A method of producing a progenitor cell(s) of a pancreatic hormone-producing cell(s), comprising cultivating an endodermal cell(s) in a medium containing the following (a)-(c):

(a) a retinoic acid receptor agonist

(b) at least one kind selected from the group consisting of inhibitors of AMP-activated protein kinase and/or activin receptor-like kinase-2,3,6, and BMP antagonists, and

(c) an inhibitor of activin receptor-like kinase-4,5,7.

21. A method of screening for a therapeutic drug for diabetes, comprising using the cell(s) obtained by any one or more steps selected from the group consisting of the following steps (1)-(4):

(1) a step of cultivating a stem cell(s) in a medium containing an activator of activin receptor-like kinase-4,7 and a GSK3 inhibitor

(2) a step of cultivating the cell(s) obtained in the aforementioned step (1) in a medium containing an activator of activin receptor-like kinase-4,7

(3) a step of cultivating the cell(s) obtained in the aforementioned step (2) in a medium containing any one or more kinds selected from the group consisting of (a) retinoic acid receptor agonists, (b) at least one kind selected from the group consisting of inhibitors of AMP-activated protein kinase and/or activin receptor-like kinase-2,3,6, and BMP antagonists, and (c) inhibitors of activin receptor-like kinase-4,5,7

(4) a step of cultivating the cell(s) obtained in the aforementioned step (3).

22. The screening method according to claim 21, wherein the activator of activin receptor-like kinase-4,7 in steps (1) and (2) is activin, and step (3) is a step of cultivating the cell(s) obtained in step (2) in a medium containing any one or more kinds selected from the group consisting of (a) retinoic acid receptor agonists, (b′

) inhibitors of AMP-activated protein kinase and/or activin receptor-like kinase-2,3,6, and (c) inhibitors of activin receptor-like kinase-4,5,7.

23. The screening method according to claim 21, wherein step (4) is performed in a medium containing one or more kinds selected from the group consisting of (i) at least one kind selected from the group consisting of adenylate cyclase activators, cAMP phosphodiesterase inhibitors and cAMP analogs, (ii) steroids and (iii) inhibitors of activin receptor-like kinase-4,5,7.