Promoter-based isolation, purification, expansion, and transplantation of neuronal progenitor cells, oligodendrocyte progenitor cells, or neural stem cells from a population of embryonic stem cells

• US 20040029269A1
• Filed: 05/06/2003
• Published: 02/12/2004
• Est. Priority Date: 05/07/2002
• Status: Abandoned Application

• Current Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Original Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Inventors: Goldman, Steven A., Roy, Neeta Singh
• Application Number:
  US10/430,822
• Publication Number:
  US 20040029269A1
• Time in Patent Office: Days
• US Class Current: 435/368
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0623 Stem cells

Abstract

The present invention relates to a method of isolating neuronal progenitor cells, oligodendrocyte progenitor cells, or neural stem cells from a population of embryonic stem cells. This method comprises selecting a promoter which functions only in neuronal progenitor cells, oligodendrocyte progenitor cells, or neural stem cells and introducing a nucleic acid molecule encoding a marker protein under control of said promoter into the population of embryonic stem cells. The population of embryonic stem cells are then differentiated to produce a mixed population of cells comprising neuronal progenitor cells, oligodendrocyte progenitor cells, or neural stem cells. The neuronal progenitor cells, oligodendrocyte progenitor cells, or neural stem cells are then allowed to express the marker protein. Cells expressing the marker protein are separated from the mixed population of cells, where the separated cells are neuronal progenitor cells, oligodendrocyte progenitor cells, or neural stem cells. In an alternative embodiment, the embryonic stem cells are differentiated before the nucleic acid is introduced. The present invention also relates to the resulting neuronal progenitor cells, oligodendrocyte progenitor cells, or neural stem cells themselves.

Claims

1. A method of isolating neuronal progenitor cells, oligodendrocyte progenitor cells, or neural stem cells from a population of embryonic stem cells comprising:

selecting a promoter which functions only in said neuronal progenitor cells, oligodendrocyte progenitor cells, or neural stem cells;

introducing a nucleic acid molecule encoding a marker protein under control of said promoter into the population of embryonic stem cells;

differentiating the population of embryonic stem cells to produce a mixed population of cells comprising neuronal progenitor cells, oligodendrocyte progenitor cells, or neural stem cells;

allowing the neuronal progenitor cells, oligodendrocyte progenitor cells, or neural stem cells to express the marker protein; and

separating the cells expressing the marker protein from the mixed population of cells, wherein said separated cells are said neuronal progenitor cells, oligodendrocyte progenitor cells, or neural stem cells.

2. The method of claim 1, wherein said introducing comprises viral mediated transduction of the population of embryonic stem cells.

3. The method of claim 2, wherein said viral mediated transduction comprises adenovirus-mediated transduction, retrovirus-mediated transduction, lentivirus-mediated transduction, or adeno-associated virus-mediated transduction.

4. The method of claim 1, wherein said introducing comprises electroporation.

5. The method of claim 1, wherein said introducing comprises biolistic transformation.

6. The method of claim 1, wherein said introducing comprises liposomal mediated transformation.

7. The method of claim 1, wherein the marker protein is a fluorescent protein and said separating comprises fluorescence activated cell sorting.

8. The method of claim 1, wherein the marker protein is either lacZ/beta-galactosidase or alkaline phosphatase.

9. The method of claim 1, wherein neuronal progenitor cells are separated from the mixed population and said promoter is a Tα

1 tubulin promoter, a Hu promoter, an ELAV promoter, a MAP-1B promoter, or a GAP-43 promoter.

10. The method of claim 1, wherein oligodendrocyte progenitor cells are separated from the mixed population and said promoter is a CNP promoter, an NCAM promoter, a myelin basic protein promoter, a JC virus minimal core promoter, a myelin-associated glycoprotein promoter, or a proteolipid protein promoter.

11. The method of claim 1, wherein neural stem cells are separated from the mixed population and said promoter is the musashi promoter or the nestin enhancer.

12. The method according to claim 1 further comprising:

identifying the cells of said mixed population of cells that express the marker protein, wherein the identifying step is after the allowing step.

13. The method of claim 1, wherein the population of embryonic stem cells is in a cell culture.

14. The method of claim 1 further comprising:

transplanting the separated cells into a subject.

15. The method of claim 1, wherein the neuronal progenitor cells, oligodendrocyte progenitor cells, and neural stem cells are of human origin.

16. An enriched population of neuronal progenitor cells isolated by the method of claim 1.

17. An enriched population of oligodendrocyte progenitor cells isolated by the method of claim 1.

18. An enriched population of neural stem cells produced by the method of claim 1.

19. A method of isolating neuronal progenitor cells, oligodendrocyte progenitor cells, or neural stem cells from a population of embryonic stem cells comprising:

providing a population of embryonic stem cells;

differentiating the population of embryonic stem cells to produce a mixed population of cells comprising neuronal progenitor cells, oligodendrocyte progenitor cells, or neural stem cells;

selecting a promoter which functions only in said neuronal progenitor cells, oligodendrocyte progenitor cells, or neural stem cells;

introducing a nucleic acid molecule encoding a marker protein under control of said promoter into the mixed population of cells;

allowing the neuronal progenitor cells, oligodendrocyte progenitor cells, or neural stem cells to express the marker protein; and

separating the cells expressing the marker protein from the mixed population of cells, wherein said separated cells are said neuronal progenitor cells, oligodendrocyte progenitor cells, or neural stem cells.

20. The method of claim 19, wherein said introducing comprises viral mediated transduction of the mixed population of cells.

21. The method of claim 20, wherein said viral mediated transduction comprises adenovirus-mediated transduction, retrovirus-mediated transduction, lentivirus-mediated transduction, or adeno-associated virus-mediated transduction.

22. The method of claim 19, wherein said introducing comprises electroporation.

23. The method of claim 19, wherein said introducing comprises biolistic transformation.

24. The method of claim 19, wherein said introducing comprises liposomal mediated transformation.

25. The method of claim 19, wherein the marker protein is a fluorescent protein and said separating comprises fluorescence activated cell sorting.

26. The method of claim 19, wherein the marker protein is either lacZ/beta-galactosidase or alkaline phosphatase.

27. The method of claim 19, wherein neuronal progenitor cells are separated from the mixed population and said promoter is a Tα

1 tubulin promoter, a Hu promoter, an ELAV promoter, a MAP-1B promoter, or a GAP-43 promoter.

28. The method of claim 19, wherein oligodendrocyte progenitor cells are separated from the mixed population and said promoter is a CNP-P2 promoter, a CNP-P1+P2 promoter, an NCAM promoter, a myelin basic protein promoter, a JC virus minimal core promoter, a myelin-associated glycoprotein promoter, or a proteolipid protein promoter.

29. The method of claim 19, wherein neural stem cells are separated from the mixed population and said promoter is the musashi promoter or the nestin enhancer.

30. The method according to claim 19, further comprising:

identifying the cells of said mixed population of cells that express the marker protein, wherein the identifying step is after the allowing step.

31. The method of claim 19 further comprising:

transplanting the separated cells into a subject.

32. The method of claim 19, wherein the neuronal progenitor cells, oligodendrocyte progenitor cells, and neural stem cells are of human origin.

33. An enriched population of neuronal progenitor cells produced by the process of claim 19.

34. An enriched population of oligodendrocyte progenitor cells produced by the process of claim 19.

35. An enriched population of neural stem cells produced by the process of claim 19.

36. An enriched or purified preparation of isolated oligodendrocyte progenitor cells derived from embryonal stem cells.

37. The enriched or purified preparation of isolated oligodendrocyte progenitor cells of claim 36 which are human.

38. An enriched or purified preparation of isolated neuronal progenitor cells derived from embryonal stem cells.

39. The enriched or purified preparation of isolated neuronal progenitor cells of claim 38 which are human.

40. An enriched or purified preparation of isolated neural stem cells derived from embryonal stem cells.

41. The enriched or purified preparation of isolated neural stem cells of claim 40 which are human.