In vitro generation of GABAergic neurons from embryonic stem cells and their use in the treatment of neurological disorders

• US 20050095702A1
• Filed: 08/31/2004
• Published: 05/05/2005
• Est. Priority Date: 09/03/2003
• Status: Active Grant

• Current Assignee: Reliance Life Sciences Pvt. Ltd.
• Original Assignee: Reliance Life Sciences Pvt. Ltd.
• Inventors: Mahadeorao, Totey Satish, Alam, Khan Firdos
• Granted Patent: US 7,820,439 B2
• Time in Patent Office: Days
• US Class Current: 435/354
• 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 differentiated cell population in an in vitro culture obtained by differentiating murine pluripotent stem cells, wherein at least 60% of the differentiated cells are GABAergic neurons.

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

3. The cell population of claim 1, wherein the GABAergic neurons express GAD65.

4. The cell population of claim 1, wherein the GABAergic neurons express GAD67.

5. The cell population of claim 1, wherein the GABAergic neurons express GABA-A receptor.

6. The cell population of claim 1, wherein the GABAergic neurons express GABA-B receptor.

7. The cell population of claim 1, wherein the GABAergic neurons express GABA-A and GABA-B receptors.

8. A differentiated cell population in an in vitro culture obtained by differentiating murine pluripotent stem cells, wherein at least 60% of the differentiated cells produce gamma aminobutyric acid (GABA).

9. The cell population of claim 8, wherein the murine pluripotent stem cells are murine embryonic stem cells.

10. 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) expanding the nestin-positive neuroprogenitor cells; and

(d) 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).

11. The cell population of claim 10, wherein the mammalian pluripotent stem cells are murine embryonic stem cells.

12. The cell population of claim 10, wherein the mammalian pluripotent stem cells are human embryonic stem cells.

13. The method of claim 10, wherein the differentiated cell population comprises at least about 60% GABAergic neurons.

14. The method of claim 10, wherein the differentiated cell population comprises at least about 15% dopaminergic neurons.

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

16. The method of claim 10, wherein the differentiated cell population comprises at least about 5% serotonergic neurons.

17. The method of claim 10, wherein the differentiated cell population comprises at least about 5% oligodendrocytes.

18. The method of claim 10, wherein the differentiated cell population comprises at least about 5% astrocytes.

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

20. The method of claim 19, wherein the serum-free medium is ITSFn serum-free defined medium.

21. The method of claim 20, wherein the cells are grown in the ITSFn serum-free defined medium for 6-10 days.

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

23. The method of claim 10, further comprising culturing the mammalian pluripotent stem cells of step (b) to form embryoid bodies.

24. The method of claim 23, wherein the embryoid bodies are cultured to select for neuroprogenitor cells that are positive for nestin.

25. The method of claim 24, wherein the neuroprogenitor cells comprise at least about 90% nestin-positive cells.

26. The method of claim 23, wherein the neuroprogenitor cells that are positive for nestin are selected by culturing the embryoid bodies in serum-free medium.

27. The method of claim 26, wherein the serum-free medium is ITSFn serum-free defined medium.

28. The method of claim 26, 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.

29. The method of claim 10, further comprising expanding the nestin-positive neuroprogenitor cells of step (c) in CNS expansion media.

30. The method of claim 29, wherein the nestin-positive neuroprogenitor cells are plated on a culture dish pre-coated with poly-L-ornithine or laminin.

31. The method of claim 29, 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.

32. The method of claim 31, wherein the neural-inducing agent is basic fibroblast growth factor (bFGF).

33. The method of claim 29, wherein the cells are grown in the CNS expansion media for 6-10 days.

34. The method of claim 10, wherein the differentiation media comprises N2 supplement, B27 supplement, or both, but not basic fibroblast growth factor (bFGF).

35. The method of claim 10, wherein the cells are grown in the differentiation media for 2 or more days.

36. The method of claim 10, 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).

37. The method of claim 36, wherein the differentiated cell population is grown in the second differentiation media for 8-16 days.

38. A method of generating GABAergic neurons from mammalian neuroprogenitor cells, comprising enriching the neuroprogenitor cells for cells that are positive for nestin, 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).

39. A method of generating an in vitro transplantation model for neural cells, comprising the following steps:

(a) isolating adult hippocampal cells;

(b) dissociating and culturing the hippocampal cells to generate a hippocampal cell culture; and

(c) culturing neural cells on the hippocampal cell culture;

wherein the survival of the neural cells on the hippocampal cell culture is evaluated.

40. A method of claim 39, wherein the adult hippocampal cells are isolated from a mouse.

41. A method of claim 39, wherein the neural cells are GABAergic neurons.

42. A method of claim 41, wherein greater than 90% of the GABAergic neurons survive after one week of culture.

43. A method of claim 39, further comprising evaluating the synaptic formation between the neural cells and the hippocampal cell culture.