Cell culture substrate, and cell culturing method using the substrate and method for inducing differentiation of pluripotent stem cells using the substrate

• US 9,745,549 B2
• Filed: 04/18/2013
• Issued: 08/29/2017
• Est. Priority Date: 10/19/2012
• Status: Active Grant

• Current Assignee: Somar Pty Limited, Toshihiro Akaike
• Original Assignee: Somar Pty Limited, Toshihiro Akaike
• Inventors: Haque, Amranul, Nagaoka, Masato, Akaike, Toshihiro
• Primary Examiner(s): Alstrum Acevedo, James H
• Assistant Examiner(s): Coffa, Sergio
• Application Number:
  US13/865,642
• Publication Number:
  US 20140113372A1
• Time in Patent Office: 1,594 Days
• Field of Search: None
• 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 2533/30 Synthetic polymers thermoreactive polymers, e.g. PNIPAm, C12N2539/10
• CPC Class Codes: C12N 2533/50 Proteins
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0618 Cells of the nervous system
• CPC Class Codes: C12N 5/0623 Stem cells
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

[Problem] To provide a cell culture substrate, and a cell culturing method using the substrate and a method for inducing differentiation of pluripotent stem cells using the substrate, which allow culturing of pluripotent stem cells and allow differentiation of pluripotent stem cells into a specified cell species, particularly neural and neural progenitor cells, at a high purity.

[Means for Solution] A cell culture substrate, characterized in that, onto the surface, one or more selected from the group consisting of N-cadherin, a fusion protein comprising an entire or partial region of N-cadherin, and a fusion protein comprising an entire or partial region of a protein homologous to N-cadherin are immobilized or coated.

Claims

1. A cell culture substrate, which is used for culturing for induction into neural progenitor cells or neural cells, characterized in that, onto the surface, at least two proteins are immobilized or coated, wherein said at least two proteins comprise:

a first protein which is selected from the group consisting of a fusion protein comprising an entire or partial region of N-cadherin, and a fusion protein comprising an entire or partial region of a protein homologous to N-cadherin; and

a second protein which is selected from the group consisting of a protein belonging to cadherin family other than N-cadherin, a fusion protein comprising at least one extracellular (EC) domain selected from EC1 domain, EC2 domain, EC3 domain, EC4 domain and EC5 domain of a protein belonging to cadherin family other than N-cadherin, and a fusion protein comprising at least one extracellular (EC) domain selected from EC1 domain, EC2 domain, EC3 domain, EC4 domain and EC5 domain of a protein homologous to a protein belonging to cadherin family other than N-cadherin, wherein said protein belonging to cadherin family other than N-Cadherin is E-cadherin wherein the concentration of the first protein is 3-1000 μ

g/mL and the concentration of the second protein is 0.01-1000 μ

g/mL;

ac4 wherein the substrate comprises polystyrene, polyethylene or polypropylene; and

wherein said protein homologous to N-cadherin is a protein which comprises one or more of the EC1 domain, EC2 domain, EC3 domain, EC4 domain and EC5 domain, and which has hemophilic binding ability with N-cadherin.

2. The cell culture substrate of claim 1, wherein a fusion protein comprising at least one extracellular (EC) domain selected from EC1 domain, EC2 domain, EC3 domain, EC4 domain and EC5 domain of E-cadherin or of a protein homologous to E-cadherin, and a fusion protein comprising an entire or partial region of N-cadherin or a protein homologous to N-cadherin are immobilized or coated onto the surface.

3. The cell culture substrate of claim 1, wherein said protein homologous to E-cadherin is a protein which comprises one or more of the EC1 domain, EC2 domain, EC3 domain, EC4 domain and EC5 domain, and which has homophilic binding ability with E-cadherin.

4. The cell culture substrate of claim 1, wherein the fusion protein comprising an entire or partial region of N-cadherin or a protein homologous to N-cadherin is a fusion protein of an entire or partial region of N-cadherin or a protein homologous to N-cadherin and an immunoglobulin Fc region.

5. The cell culture substrate of claim 1, wherein the fusion protein comprising at least one extracellular (EC) domain selected from EC1 domain, EC2 domain, EC3 domain, EC4 domain and EC5 domain of E-cadherin or of a protein homologous to E-cadherin is a fusion protein of at least one extracellular (EC) domain selected from EC1 domain, EC2 domain, EC3 domain, EC4 domain and EC5 domain of E-cadherin or of a protein homologous to E-cadherin and an immunoglobulin Fc region.

7. A cell culturing method, characterized by growing pluripotent stem cells using the cell culture substrate of claim 1 and a liquid medium while maintaining their undifferentiated state and pluripotency.

8. A method for inducing differentiation of pluripotent stem cells, characterized by differentiating pluripotent stem cells using the cell culture substrate of claim 1 and a liquid medium containing a differentiation-inducing factor(s).

9. The method for inducing differentiation of pluripotent stem cells of claim 8, by which pluripotent stem cells are differentiated into neural progenitor cells or neural cell.

10. A method of producing neural progenitor cells or neural cells, characterized by culturing ES cells or iPS cells on the cell culture substrate of claim 1 using a liquid medium containing a differentiation-inducing factor(s).

11. The cell culture substrate of claim 1, wherein a fusion protein comprising at least one extracellular (EC) domain selected from EC1 domain, EC2 domain, EC3 domain, EC4 domain and EC5 domain of E-cadherin or of a protein homologous to E-cadherin, and a fusion protein comprising an entire or partial region of N-cadherin or a protein homologous to N-cadherin are immobilized or coated onto the surface.

12. The cell culture substrate of claim 2, wherein said protein homologous to N-cadherin is a protein which comprises one or more of the EC1 domain, EC2 domain, EC3 domain, EC4 domain and EC5 domain, and which has homophilic binding ability with N-cadherin.

13. The cell culture substrate of claim 2, wherein said protein homologous to E-cadherin is a protein which comprises one or more of the EC1 domain, EC2 domain, EC3 domain, EC4 domain and EC5 domain, and which has homophilic binding ability with E-cadherin.

14. The cell culture substrate of claim 1, wherein said protein homologous to E-cadherin is a protein which comprises one or more of the EC1 domain, EC2 domain, EC3 domain, EC4 domain and EC5 domain, and which has homophilic binding ability with E-cadherin.

15. The cell culture substrate of claim 1, wherein the first fusion protein and the second fusion protein are immobilized or coated onto the surface.

6. A cell culture substrate, characterized in that, onto the surface, two or more selected from the group consisting of a protein belonging to cadherin family and a fusion protein comprising an entire or partial region of a protein belonging to cadherin family are immobilized or coated.