Compositions and methods for propagation of neural progenitor cells

• US 20050118561A1
• Filed: 12/02/2004
• Published: 06/02/2005
• Est. Priority Date: 12/02/2003
• Status: Active Grant

• Current Assignee: Celavie Biosciences, LLC
• Original Assignee: Catholic Healthcare West (CommonSpirit Health (Illinois))
• Inventors: Kopyov, Oleg V.
• Granted Patent: US 7,632,681 B2
• Time in Patent Office: Days
• US Class Current: 435/1.100
• CPC Class Codes: A01N 1/0221 Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
• CPC Class Codes: A01N 1/0226 Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: C12N 2500/14 Calcium; Ca chelators; Calcitonin
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/148 Transforming growth factor alpha [TGF-a]
• CPC Class Codes: C12N 5/0623 Stem cells

Abstract

Compositions and methods for the culturing, propagation, cryopreservation and manipulation of neural progenitor cells (NPC) and pluripotent stem cells (PSC) are provided. The cells exhibit rapid doubling times and can be maintained in vitro for extended periods. Also provided is a method of propagating neural progenitor cells, and a method of transplanting human NPC and/or PSC to a host. The cells can be genetically modified to express a therapeutic agent prior to the transplanting.

Claims

1. A cell culture comprising:

(a) a culture medium, wherein the calcium concentration of the medium is not greater than 0.15 mM;

(b) about 20-100 ng/ml epidermal growth factor (EGF);

(c) about 10-50 ng/ml basic fibroblast growth factor (bFGF);

(d) about 1-150 ng/ml transforming growth factor-alpha (TGFα

);

(e) neural progenitor cells (NPC) and/or pluripotent stem cells (PSC) suspended in the medium.

2. The cell culture of claim 1, further comprising:

(f) about 0.03 to about 0.09 mM calcium chloride, wherein the medium is brought to full volume in a calcium-free minimum essential medium and has a total calcium concentration of less than 0.1 mM.

3. The cell culture of claim 1, further comprising:

(g) about 7-30 ng/ml leukemia inhibiting factor (LIF).

4. The cell culture of claim 2, wherein the total calcium concentration is about 0.05 mM.

5. The cell culture of claim 1, wherein the EGF is about 20 ng/ml.

6. The cell culture of claim 1, wherein the bFGF is about 10 ng/ml.

7. The cell culture of claim 1, wherein the TGFα

is about 10 ng/ml.

8. The cell culture of claim 3, wherein the LIF is about 10 ng/ml.

9. The cell culture of claim 1, wherein the culture medium is serum-free.

10. The cell culture of claim 1, further comprising 2% B27 supplement.

11. The cell culture of claim 1, wherein the growth factors, EGF, bFGF and TGFα

, are recombinant growth factors.

12. The cell culture of claim 1, wherein the cells and the growth factors are human.

13. The cell culture of claim 1, further comprising about 0.11 mg/ml sodium pyruvate.

14. The cell culture of claim 1, wherein the cells have a doubling rate of less than 12 days.

15. The cell culture of claim 1, wherein the cells have a doubling rate of about 5 days.

16. The cell culture of claim 1, wherein the cells continue to proliferate for at least 1 year in vitro.

17. The cell culture of claim 1, wherein the cells are derived from fetal forebrain.

20. A method of transplanting human NPC and/or PSC to a host, comprising:

(a) obtaining a cell culture of claim 1;

and

(b) transplanting the cell culture to the host.

21. The method of claim 20, wherein glutamine (to a concentration 0.5 mM) and LIF (7-30 ng/ml) are added to the culture medium prior to the transplanting.

22. The method of claim 20, wherein the cell culture is transplanted to multiple sites within the host.

23. The method of claim 20, wherein the NPC and/or PSC are genetically modified to express a therapeutic agent prior to the transplanting.

18. A method of propagating NPC and/or PSC, comprising culturing primary human fetal brain tissue in a culture medium, wherein the culture medium comprises:

(a) 0.03 to 0.09 mM calcium;

(b) about 20-100 ng/ml epidermal growth factor (EGF);

(c) about 10-50 ng/ml fibroblast growth factor basic (bFGF); and

(d) about 1-150 ng/ml transforming growth factor-alpha (TGFα

).

19. The method of claim 18, further comprising:

(e) about 7-30 ng/ml leukemia inhibiting factor (LIF).

24. A cryopreservation medium comprising:

(a) 0.03-0.09 mM calcium;

(b) about 20-100 ng/ml epidermal growth factor (EGF);

(c) about 10-50 ng/ml fibroblast growth factor basic (bFGF);

(d) about 1-150 ng/ml transforming growth factor-alpha (TGFα

).

(e) about 2% B27; and

(f) about 10% dimethylsulfoxide (DMSO).

25. A method of cryopreserving NPC and/or PSC comprising:

storing the NPC and/or PSC in the cryopreservation medium of claim 24 at a temperature of below about −

80°

C.;

wherein greater than 50% of the NPC and/or PSC remain viable upon thawing.