Feeder layer and serum independent embryonic stem cells

• US 20060172414A1
• Filed: 11/23/2005
• Published: 08/03/2006
• Est. Priority Date: 11/23/2004
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Inventors: Weissman, Irving L., Yamane, Toshiyuki, Dylla, Scott
• Granted Patent: US 7,641,897 B2
• Time in Patent Office: Days
• US Class Current: 435/354
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2501/48 Regulators of apoptosis
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• 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

Abstract

Undifferentiated primordial stem cells are manipulated to permit their long term growth in defined media lacking serum and feeder layer cells by shifting the apoptotic balance of the cells, through increasing the activity of Bcl-2 family anti-apoptotic proteins or decreasing the activity of Bcl-2 family pro-apoptotic proteins. In some embodiments of the invention, the Bcl family protein is Bcl-2. The ES cells sustain the characteristics of undifferentiated, pluripotent stem cells during long-term serum- and feeder layer cell-free conditions, including the ability to be expanded in vitro, but maintain their potential to differentiate into mature cell types.

Claims

1. A cellular composition comprising:

undifferentiated primordial stem cells having a shifted apoptotic balance to allow proliferation of said cells in a medium free of feeder layer cells while maintaining differentiative capacity; and

culture medium free of feeder layer cells.

2. The cellular composition of claim 1, said apoptotic balance is shifted by increased activity of an anti-apoptotic Bcl-2 family member.

3. The cellular composition of claim 1, wherein said anti-apoptotic Bcl-2 family member is Bcl-2.

4. The cellular composition according to claim 3, wherein said undifferentiated primordial stem cells comprise a genetic construct encoding Bcl-2 operably linked to a promoter active in said undifferentiated cells.

5. The cellular composition according to claim 1, wherein said undifferentiated primordial stem cells are embryonic stem cells.

6. The cellular composition according to claim 5, wherein said embryonic stem cells are mouse cells.

7. The cellular composition according to claim 5, wherein said embryonic stem cells are human cells.

8. The cellular composition according to claim 1, wherein said culture medium further comprises LIF.

9. The cellular composition according to claim 1, wherein said medium is serum-free.

10. A method of expanding undifferentiated primordial stem cells in the absence of feeder layer cells, the method comprising:

shifting apoptotic balance of said cells to allow proliferation of said cells in a medium free of feeder layer cells while maintaining differentiative capacity.

11. The method of claim 10, said apoptotic balance is shifted by increased activity of an anti-apoptotic Bcl-2 family member.

12. The method of claim 11, wherein said anti-apoptotic Bcl-2 family member is Bcl-2.

13. The method according to claim 12, comprising the step of introducing into said stem cells a genetic construct encoding Bcl-2 operably linked to a promoter active in said cells.

14. The method according to claim 11, comprising the step of introducing into said stem cells a native or modified or encapsulated bcl2 family member protein such as bcl2, or an active portion of a bcl2 anti-apoptotic family member.

15. The method according to claim 11, comprising the step of introducing into said stem cells a molecule mimicking an anti-apoptotic bcl2 family member.

16. The method according to claim 10, wherein said undifferentiated primordial stem cells are embryonic stem cells.

17. The method according to claim 16, wherein said embryonic stem cells are mouse cells.

18. The method according to claim 16, wherein said embryonic stem cells are human cells.

19. The method according to claim 10, wherein said culture medium further comprises LIF.

20. The method according to claim 10, wherein said medium is serum-free.