Neural stem cell having increased passage ability, method for manufacturing neural stem cell having said increased passage abiility, and method for culturing neural stem cells for increasing passage ability of neural stem cells

Patent metadata

Field	Value
Publication number	US-9574176-B2
Application number	US-201314436206-A
Country	US
Kind code	B2
Filing date	Oct 29, 2013
Priority date	Oct 31, 2012
Publication date	Feb 21, 2017
Grant date	Feb 21, 2017

Abstract

The present invention provides a neural stem cell having increased passage ability and a method for manufacturing a neural stem cell having increased passage ability. A neural stem cell in which the N-type calcium channel gene is knocked out or the influx of Ca2+ via the N-type calcium channel is substantially absent can be passaged for at least 4 generations and maintains the differentiation potential into a nerve cell even after passage for 4 generations.

First claim

The invention claimed is:

1. A neural stem cell having the following characteristics:
(a) function or expression of the N-type calcium channel is knocked out in the cell,
(b) influx of Ca2+ via the N-type calcium channel is substantially absent when function or expression of the N-type calcium channel is knocked out in the cell,
(c) the cell can be passaged for at least 4 generations, and
(d) the cell maintains differentiation potential into a nerve cell after passage for 4 generations.

2. The neural stem cell according to claim 1 , wherein the gene encoding the α1B subunit of the N-type calcium channel is knocked out.

3. The neural stem cell according to claim 1 , wherein the neural stem cell is nestin-positive after passage for 4 generations.

4. The neural stem cell according to claim 1 , wherein the neural stem cell has high proliferation ability and high sphere-forming ability after passage for 4 generations.

5. The neural stem cell according to claim 1 , wherein function or expression of the N-type calcium channel is knocked out by contacting the neural stem cell with an agent that inhibits function or expression of the N-type calcium channel.

6. The neural stem cell according to claim 5 , wherein the agent is ω-conotoxin GVIA.

7. A method for manufacturing a neural stem cell, wherein the method comprises:
(A) a step of preparing a neural stem cell in vitro, and
(B) a step of knocking out function or expression of the N-type calcium channel of the neural stem cell in vitro,
wherein the manufactured neural stem cell has the following characteristics:
(a) function or expression of the N-type calcium channel is knocked out in the cell,
(b) influx of Ca2+ via the N-type calcium channel is substantially absent when function or expression of the N-type calcium channel is knocked out in the cell,
(c) the cell can be passaged for at least 4 generations, and
(d) the cell maintains differentiation potential into a nerve cell after passage for 4 generations.

8. The method for manufacturing a neural stem cell according to claim 7 , wherein the neural stem cell in step (A) is a cell derived from a human.

9. The method for manufacturing a neural stem cell according to claim 7 , wherein the neural stem cell in step (A) is a neural stem cell prepared by differentiation induction of an embryonic stem (ES) or induced pluripotent stem (iPS) cell into a neural stem cell.

10. The method for manufacturing a neural stem cell according to claim 7 , wherein the gene encoding the α1B subunit of the N-type calcium channel is knocked out.

11. The method for manufacturing a neural stem cell according to claim 7 , wherein function or expression of the N-type calcium channel is knocked out by contacting the neural stem cell with an agent that inhibits function or expression of the N-type calcium channel.

12. The method for manufacturing a neural stem cell according to claim 11 , wherein the agent is ω-conotoxin GVIA.

Assignees

• Eisai R&D Man Co Ltd

Inventors

• Miyamoto Norimasa
• Ono Yuichi
• Namiki Kana
• Kasuya Yoshitoshi

Classifications

• C12N2501/405

  Cell cycle regulated proteins, e.g. cyclins, cyclin-dependant kinases · CPC title

• C12N2500/14

  Calcium; Ca chelators; Calcitonin · CPC title

• C12N2506/00

  Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells · CPC title

• C12N15/85

  for animal cells · CPC title

• C12N5/0623 Primary

  Stem cells · CPC title