Method for inducing dopaminergic neuron progenitor cells

Patent metadata

Field	Value
Publication number	US-2016215260-A1
Application number	US-201414916696-A
Country	US
Kind code	A1
Filing date	Sep 4, 2014
Priority date	Sep 5, 2013
Publication date	Jul 28, 2016
Grant date	—

Abstract

The present invention provides a method for producing dopaminergic neuron progenitor cells from pluripotent stem cells, which method comprises the steps of: (i) performing adherent culture of pluripotent stem cells on an extracellular matrix in a medium containing a reagent(s) selected from the group consisting of BMP inhibitor, TGFβ inhibitor, SHH signal-stimulating agent, FGF8, and GSK3β inhibitor; (ii) collecting Corin- and/or Lrtm1-positive cells from the cells obtained in Step (i) using a substance which binds to Corin and/or a substance which binds to Lrtm1; and (iii) performing suspension culture of the cells obtained in Step (ii) in a medium containing a neurotrophic factor.

First claim

What is claimed is:

1 . A method for producing dopaminergic neuron progenitor cells from pluripotent stem cells, said method comprising the steps of:
(i) performing adherent culture of pluripotent stem cells on an extracellular matrix in a medium containing a reagent(s) selected from the group consisting of BMP inhibitor, TGFβ inhibitor, SHH signal-stimulating agent, FGF8, and GSK3β inhibitor; (ii) collecting Corin- and/or Lrtm1-positive cells from the cells obtained in Step (i) using a substance which binds to Corin and/or a substance which binds to Lrtm1; and (iii) performing suspension culture of the cells obtained in Step (ii) in a medium containing a neurotrophic factor.

2 . The method according to claim 1 , wherein Step (i) comprises the steps of:
(a) performing adherent culture of pluripotent stem cells on an extracellular matrix in a medium containing BMP inhibitor and TGFβ inhibitor; (b) performing adherent culture of the cells obtained in Step (a) on an extracellular matrix in a medium containing BMP inhibitor, TGFβ inhibitor, SHH signal-stimulating agent, and FGF8; (c) performing adherent culture of the cells obtained in Step (b) on an extracellular matrix in a medium containing BMP inhibitor, TGFβ inhibitor, SHH signal-stimulating agent, FGF8, and GSK3β inhibitor; and (d) performing adherent culture of the cells obtained in Step (c) on an extracellular matrix in a medium containing BMP inhibitor and GSK3β inhibitor.

3 . The method according to claim 1 , wherein said neurotrophic factor is BDNF and GDNF.

4 . The method according to claim 1 , wherein the medium in Step (iii) further comprises B27 supplement, ascorbic acid, and dibutyryl cyclic AMP.

5 . The method according to claim 1 , wherein said Step (i) is carried out for at least 10 days.

6 . The method according to claim 1 , wherein said Step (i) is carried out for 12 days to 21 days.

7 . The method according to claim 1 , wherein said Step (iii) is carried out for at least 7 days.

8 . The method according to claim 1 , wherein said Step (iii) is carried out for 14 days to 30 days.

9 . The method according to claim 1 , wherein said extracellular matrix is laminin or a fragment(s) thereof.

10 . The method according to claim 1 , wherein said extracellular matrix is laminin 511E8.

11 . The method according to claim 1 , wherein said substance which binds to Corin or said substance which binds to Lrtm1 is an antibody or an aptamer which binds to Corin or Lrtm1.

12 . Dopaminergic neuron progenitor cells obtained by the method according to claim 1 .

13 . A method for treating Parkinson's disease, comprising administering to a subject in need thereof dopaminergic neuron progenitor cells obtained by the method according to claim 1 .

14 . A kit for preparing dopaminergic neuron progenitor cells from pluripotent stem cells, said kit comprising BMP inhibitor, TGFβ inhibitor, SHH signal-stimulating agent, FGF8, GSK3β inhibitor, extracellular matrix, and neurotrophic factor.

15 . The kit according to claim 14 , further comprising an anti-Corin antibody and/or anti-Lrtm1 antibody.

16 . The kit according to claim 14 , wherein said BMP inhibitor is LDN193189; said TGFβ inhibitor is A83-01; said SHH signal-stimulating agent is purmorphamine; said GSK3β inhibitor is CHIR99021; said extracellular matrix is laminin 511E8; and said neurotrophic factor is BDNF and GDNF.

Assignees

• Univ Kyoto
• Univ Osaka
• Eisai R&D Man Co Ltd

Inventors

• Takahashi Jun
• Doi Daisuke
• Samata Bumpei
• Sekiguchi Kiyotoshi
• Ono Yuichi

Classifications

• A61P25/16

  Anti-Parkinson drugs · CPC title

• C12N2501/13

  Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins · CPC title

• C12N2501/727

  Kinases (EC 2.7.) · CPC title

• C12N2533/52

  Fibronectin; Laminin · CPC title

• C12N2506/02

  from embryonic cells · CPC title