In vitro generation of GABAergic neurons from pluripotent stem cells

• US 7,820,439 B2
• Filed: 08/31/2004
• Issued: 10/26/2010
• Est. Priority Date: 09/03/2003
• Status: Expired due to Fees

• Current Assignee: Reliance Life Sciences Pvt. Ltd.
• Original Assignee: Reliance Life Sciences Pvt. Ltd.
• Inventors: Alam, Khan Firdos, Mahadeorao, Totey Satish
• Primary Examiner(s): Crouch; Deborah
• Application Number:
  US10/930,675
• Publication Number:
  US 20050095702A1
• Time in Patent Office: 2,247 Days
• Field of Search: 435/375, 435/377
• US Class Current: 435/377
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/08 Antiepileptics; Anticonvulsants
• CPC Class Codes: A61P 25/14 for treating abnormal movements, e.g. chorea, dyskinesia
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• 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: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0619 Neurons

Abstract

The present disclosure is directed to improved methods for efficiently producing neuroprogenitor cells and differentiated neural cells such as GABAergic neurons from pluripotent stem cells, for example embryonic stem cells. Using the disclosed methods, cell populations containing a high proportion of GABAergic neurons have been isolated. The neuroprogenitor cells and terminally differentiated cells of the present disclosure can be generated in large quantities, and therefore may serve as an excellent source for cell replacement therapy in neurodegenerative disorders and neuronal diseases such as stroke, ischemia, epilepsy, and Huntington'"'"'s disease.

Claims

1. A method of generating a differentiated neural cell population from mammalian pluripotent stem cells comprising the following steps:

(a) expanding a culture of pluripotent stem cells;

(b) culturing the pluripotent stem cells to select for neuroprogenitor cells that are positive for nestin;

(c) isolating the nestin-positive cells;

(d) expanding the nestin-positive neuroprogenitor cells; and

(e) differentiating the nestin-positive cells to generate a differentiated cell population by culturing the cells in a differentiation media which comprises cytosine β

-d-Arabino furanoside (Ara-C).

2. The cell population of claim 1, wherein the mammalian pluripotent stem cells are murine embryonic stem cells.

3. The cell population of claim 1, wherein the mammalian pluripotent stem cells are human embryonic stem cells.

4. The method of claim 1, wherein the differentiated cell population comprises at least about 60% GABAergic neurons.

5. The method of claim 1, wherein the differentiated cell population comprises at least about 15% dopaminergic neurons.

6. The method of claim 1, wherein the differentiated cell population comprises at least about 10% glutamatergic neurons.

7. The method of claim 1, wherein the differentiated cell population comprises at least about 5% serotonergic neurons.

8. The method of claim 1, wherein the differentiated cell population comprises at least about 5% oligodendrocytes.

9. The method of claim 1, wherein the differentiated cell population comprises at least about 5% astrocytes.

10. The method of claim 1, wherein the neuroprogenitor cells that are positive for nestin are selected by culturing the stem cells in serum-free medium.

11. The method of claim 10, wherein the serum-free medium is ITSFn serum-free defined medium.

12. The method of claim 11, wherein the cells are grown in the ITSFn serum-free defined medium for 6-10 days.

13. The method of claim 10, wherein the serum-free medium comprises one or more soluble factors selected from the group consisting of insulin, sodium selenite, transferrin, and fibronectin.

14. The method of claim 1, further comprising culturing the mammalian pluripotent stem cells of step (b) to form embryoid bodies.

15. The method of claim 14, wherein the embryoid bodies are cultured to select for neuroprogenitor cells that are positive for nestin.

16. The method of claim 15, wherein the neuroprogenitor cells comprise at least about 90% nestin-positive cells.

17. The method of claim 14, wherein the neuroprogenitor cells that are positive for nestin are selected by culturing the embryoid bodies in serum-free medium.

18. The method of claim 17, wherein the serum-free medium is ITSFn serum-free defined medium.

19. The method of claim 17, wherein the serum-free medium comprises one or more soluble factors selected from the group consisting of insulin, sodium selenite, basic fibroblast growth factor, transferrin, and fibronectin.

20. The method of claim 1, further comprising expanding the nestin-positive neuroprogenitor cells of step (c) in CNS expansion media.

21. The method of claim 20, wherein the nestin-positive neuroprogenitor cells are plated on a culture dish pre-coated with poly-L-ornithine or laminin.

22. The method of claim 20, wherein the CNS expansion media comprises one or more soluble factors selected from the group consisting of N2 supplement, B27 supplement, and a neural-inducing agent.

23. The method of claim 22, wherein the neural-inducing agent is basic fibroblast growth factor (bFGF).

24. The method of claim 20, wherein the cells are grown in the CNS expansion media for 6-10 days.

25. The method of claim 1, wherein the differentiation media comprises N2 supplement, B27 supplement, or both, but not basic fibroblast growth factor (bFGF).

26. The method of claim 1, wherein the cells are grown in the differentiation media for 2 or more days.

27. The method of claim 1, further comprising step (e), wherein the differentiated cell population of step (d) are further differentiated by culturing the cells in a second differentiation media that does not contain cytosine β

-d-Arabino furanoside (Ara-C).

28. The method of claim 27, wherein the differentiated cell population is grown in the second differentiation media for 8-16 days.

29. A method of generating GABAergic neurons from mammalian neuroprogenitor cells, comprising enriching the neuroprogenitor cells for cells that are positive for nestin, isolating the nestin-positive cells, and differentiating the nestin-positive cells to generate GABAergic neurons by culturing the cells in the presence of cytosine β

-d-Arabino furanoside (Ara-C).