Method for inducing differentiation of pluripotent stem cells into neural precursor cells

• US 10,119,120 B2
• Filed: 08/12/2010
• Issued: 11/06/2018
• Est. Priority Date: 08/12/2009
• Status: Active Grant

• Current Assignee: Kyoto University
• Original Assignee: Kyoto University
• Inventors: Takahashi, Jun, Morizane, Asuka
• Primary Examiner(s): Kim, Taeyoon
• Application Number:
  US13/390,225
• Publication Number:
  US 20120142093A1
• Time in Patent Office: 3,008 Days
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• 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 5/0618 Cells of the nervous system

Abstract

The present invention provides a method for inducing differentiation of pluripotent stem cells into neural precursor cells, comprising culturing the pluripotent stem cells in the presence of a small molecule BMP inhibitor, and induced neural precursor cells prepared by this method.

Claims

1. A method for inducing differentiation of a pluripotent cell into a neural precursor cell, the method comprising:

treating the pluripotent stem cell with a p160-Rho-associated coiled-coil kinase (ROCK) inhibitor; and

culturing the treated pluripotent stem cell for inducing the differentiation into the neural precursor cell in a culture medium comprising substances consisting of 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline (LDN-193189) and a TGFβ

family inhibitor selected from the group consisting of 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (SB431542) and 3-(6-methylpyridin-2-yl)-1-phenylthiocarbamoyl-4-quinolin-4-ylpyrazole (A-83-01), wherein the concentration of LDN-193189 is 50 nM or higher.

2. The method of claim 1, wherein the culture medium further comprises a feeder cell, which is a stromal cell.

3. The method of claim 2, wherein the stromal cell is a mouse stromal cell.

4. The method of claim 1, wherein the culture medium is serum free and prior to the culturing of the differentiation induction an embryoid body is formed from the pluripotent stem cell.

5. The method of claim 1, wherein the culture is performed on a Matrigel coated dish without feeder cells.

6. The method of claim 1, wherein the pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell.

7. The method of claim 1, wherein the TGFβ

family inhibitor is 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (SB431542).

8. The method of claim 1, wherein the TGFα

family inhibitor is 3-(6-methylpyridin-2-yl)-1-phenylthiocarbamoyl-4-quinolin-4-ylpyrazole(A-83-01).

9. The method of claim 1, wherein the pluripotent stem cell is an embryonic stem cell.

10. The method of claim 1, wherein the pluripotent stem cell is an induced pluripotent stem cell.