Glial cells and oligodendrocytes produced by reprogramming somatic cells with Sox10, Olig2 and Nkx6.2

Patent metadata

Field	Value
Publication number	US-9487757-B2
Application number	US-201113280562-A
Country	US
Kind code	B2
Filing date	Oct 25, 2011
Priority date	Oct 26, 2010
Publication date	Nov 8, 2016
Grant date	Nov 8, 2016

Abstract

The present invention relates to the reprogramming of differentiated somatic cells, such as those differentiated cells that arise from embryonic mesoderm, into glial cells. Glial cells produced from this reprogramming are functionally equivalent to glial cells that arise from ectodermal origins.

First claim

We claim:

1. A method for producing glial cells comprising:
providing somatic cells from a patient;
culturing said somatic cells;
infecting somatic cells with a retroviral vector to deliver reprogramming factors Sox 10, Olig2, and Nkx6.2;
inducing expression of reprogramming factors Sox 10, Olig2, and Nkx6.2 in said cultured somatic cells under conditions to reprogram the somatic cells into a population of glial cells, wherein the glial cells express endogenous gene Olig2.

2. The method of claim 1 , further comprising infecting somatic cells with a retroviral vector to deliver or more reprogramming factors selected from Olig1, Nkx2.2, ST18, MYT1 or GM98; and inducing expression of one or more reprogramming factors selected from Olig1, Nkx2.2, ST18, MYT1 or GM98 in said cultured somatic cells.

3. The method of claim 2 , further comprising infecting somatic cells with a retroviral vector to deliver at least three reprogramming factors selected from Olig1, Nkx2.2, ST18, MYT1 or GM98; and inducing expression of at least three of reprogramming factors selected from Olig1, Nkx2.2, ST18, MYT1 or GM98 in said somatic cells.

4. The method of claim 1 , wherein the vector comprises an inducible promoter.

5. The method of claim 4 , wherein said vector further includes a selectable marker.

6. The method of claim 1 , wherein said somatic cells are fibroblasts, hematopoietic cells, adipocytes or chondrocytes.

7. The method of claim 6 , wherein the somatic cells are fibroblasts.

8. The method of claim 1 , further comprising a sorting step after said expressing step to sort said population of glial cells from said cultured somatic cells to produce an enriched population of glial cells.

9. The method of claim 8 , wherein said sorting step is accomplished by using one or more markers specific for glial cells.

10. The method of claim 9 , wherein said one or more markers is selected from PLP-1, myelin basic protein (MBP), A2B2, A2B5, nextin, platelet-derived growth factor alpha receptor, SOX10, Olig1, chondroitin sulphate proteoglycan NG2, myelin-associated glycoprotein, myelin oligodendrocyte glycoprotein, or galactocerebrosides O1 or GalC.

11. The method of claim 1 , further comprising a step of expanding said population of glial cells after said expressing step.

12. The method of claim 1 , wherein said glial cells are oligodendrocyte progenitor cells.

13. The method of claim 1 , wherein one or more of the reprogramming factors are expressed from one or more exogenous genes in the somatic cells.

14. The method of claim 1 , wherein at least 5% of said population of cells are glial cells.

15. The method of claim 14 , wherein at least 10% of said population of cells are glial cells.

16. A method for producing oligodendrocyte progenitor cells comprising:
(a) providing fibroblasts from a patient;
(b) culturing said fibroblasts;
(c) infecting said fibroblasts with a retroviral vector expressing Sox 10, Nkx6.2, and Olig2 and one or more reprogramming factors of Olig1, Nkx2.2, ST18, MYT1, or GM98; and
(d) inducing expression of reprogramming factors Sox 10, Nkx6.2, and Olig2, and one or more of reprogramming factors Olig1, Nkx2.2, ST18, MYT1 or GM98 in said fibroblasts to produce a population of induced fibroblasts, where said induced fibroblasts are reprogrammed into oligodendrocyte progenitor cells in a supplemented oligodendrocyte progenitor cell-promoting medium, wherein the oligodendrocyte progenitor cells express endogenous gene Olig2.

17. The method of claim 16 , further comprising a step of sorting said population of oligodendrocyte progenitor cells after said expressing step.

18. The method of claim 17 , further comprising a step of expanding said population of oligodendrocyte progenitor cells after said sorting step.

19. The method of claim 18 , further comprising culturing said expanded population of oligodendrocyte progenitor cells in the presence of one or more of sonic hedgehog, noggin, insulin-like growth factor, neurotrophin 3 and/or triiodothryonine.

20. The method of claim 19 , comprising culturing said expanded population of oligodendrocyte progenitor cells in the presence of three or more of sonic hedgehog, noggin, insulin-like growth factor, neurotrophin 3 and/or triiodothryonine.

21. The method of claim 16 , wherein one or more of the reprogramming factors are expressed from one or more exogenous genes in the fibroblasts.

22. A method for producing glial cells comprising:
(a) providing fibroblasts from a patient;
(b) culturing said fibroblasts;
(c) infecting said fibroblasts with a retroviral vector driving expression of reprogramming factors Sox 10, Nkx6.2, and Olig2, and one or more of reprogramming factors Olig1, Nkx2.2, ST18, MYT1 or GM98 to said fibroblasts;
(d) inducing expression of said reprogramming factors to produce a population of induced fibroblasts, where said induced fibroblasts are reprogrammed into glial cells, wherein the glial cells express endogenous gene Olig2;
(e) expanding said glial cells;
(f) sorting said glial cells to produce an enriched population of glial cells; and
(g) expanding said enriched population of glial cells.

23. The method of claim 22 , wherein said enriched population of glial cells comprises at least 30% glial cells.

24. The method of claim 23 , wherein said enriched population of glial cells comprises at least 50% glial cells.

25. The method of claim 24 , wherein said enriched population of glial cells comprises at least 80% glial cells.

26. The method of claim 25 , wherein said enriched population of glial cells comprises at least 90% glial cells.

27. The method of claim 22 , further comprising culturing said expanded population of glial cells in the presence of one or more of sonic hedgehog, noggin, insulin-like growth factor, neurotrophin 3 and/or triiodothryonine.

28. The method of claim 27 , further comprising culturing said expanded population of glial cells in the presence of three or more of sonic hedgehog, noggin, insulin-like growth factor, neurotrophin 3 and/or triiodothryonine.

29. The method of claim 16 , further comprising using said oligodendrocyte progenitor cells as a research tool.

30. The method of claim 16 , further comprising using said oligodendrocyte progenitor cells as a diagnostic tool.

31. The method of claim 16 , further comprising using said oligodendrocyte progenitor cells as a pharmaceutical composition.

32. The method of claim 1 , further comprising using said population of glial cells as a research tool.

33. The method of claim 1 , further comprising using said glial cells as a diagnostic tool.

34. The method of claim 1 , further comprising using said glial cells as a pharmaceutical composition.

35. The method of claim 26 , further comprising using said enriched population of glial cells as a research tool.

36. The method of claim 26 , further comprising using said enriched population of glial cells as a diagnostic tool.

37. The method of claim 26 , further comprising using said enriched population glial cells as a pharmaceutical composition.

Assignees

• Tesar Paul J
• Miller Robert H
• Najm Fadi J
• Univ Case Western Reserve

Inventors

• Tesar Paul J
• Miller Robert H
• Najm Fadi J

Classifications

• C12N5/0622

  Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells · CPC title

• C12N2506/1307

  from adult fibroblasts · CPC title

• C12N2501/60

  Transcription factors · CPC title

• C12N2501/135

  Platelet-derived growth factor [PDGF] · CPC title

• C12N5/0696 Primary

  Artificially induced pluripotent stem cells, e.g. iPS · CPC title