METHOD OF GENERATING MYELINATING OLIGODENDROCYTES

• US 20100159595A1
• Filed: 12/18/2008
• Published: 06/24/2010
• Est. Priority Date: 12/20/2007
• Status: Active Grant

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Du, Zhong-Wei, Hu, Baoyang, Zhang, Su-Chun
• Granted Patent: US 8,227,247 B2
• Time in Patent Office: Days
• US Class Current: 435/377
• CPC Class Codes: C12N 2501/01 Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/13 Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
• CPC Class Codes: C12N 2501/135 Platelet-derived growth factor [PDGF]
• CPC Class Codes: C12N 2501/395 Thyroid hormones
• CPC Class Codes: C12N 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0622 Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells

Abstract

A method of differentiating embryonic stem cells into oligodendroglial precursor cells and oligodendroglial cells by culturing a population of cells comprising a majority of cells that are characterized by a neural tube-like rosette morphology and are Pax6+/Sox1+ into a population of cells that are PDGFRα+.

Claims

1. A method of generating a population of oligodendroglial precursor cells, the method comprising the steps of:

(a) differentiating isolated pluripotent stem cells into a population of cells having a neural tube-like rosette morphology that are Pax6+/Sox1+;

(b) culturing neural tube-like rosette cells in the presence of retinoic acid and an agent that activates a sonic hedgehog pathway to obtain Olig2-expressing progenitor cells; and

(c) culturing the Olig2-expressing progenitors cells in the presence of the agent that activates the sonic hedgehog pathway, but in the absence of retinoic acid, to obtain PDGFRα

+/SOX10+/OLIG2+/NKX2.2+ oligodendroglial precursor cells.

2. A method as recited in claim 1, wherein the agent that activates the sonic hedgehog is added within one week after the addition of retinoic acid.

3. A method as recited in claim 1, wherein the agent that activates the sonic hedgehog pathway is selected from sonic hedgehog and purmorphamine.

4. A method as recited in claim 3, wherein sonic hedgehog is between 50 to 500 ng/ml (±

10%).

5. A method as recited in claim 3, wherein purmorphamine is between 0.5 to 1.2 μ

M (±

10%).

6. A method as recited in claim 1, wherein the retinoic acid is between 0.05 to 0.3 μ

M (±

10%).

7. A method as recited in claim 1, further comprising culturing the neural tube-like rosette cells in step (b) with FGF2.

8. A method as recited in claim 7, wherein the FGF-2 is between 2 to 30 ng/ml (±

10%).

9. A method as recited in claim 1, wherein the FGF-2 is not present after day 35.

10. A method as recited in claim 1, wherein the pluripotent stem cells are primate embryonic stem cells.

11. A method as recited in claim 1, wherein the pluripotent stem cells are human embryonic stem cells.

12. A method as recited in claim 1, wherein the pluripotent stem cells are primate induced pluripotent cells (iPS cells).

13. A method as recited in claim 1, wherein the pluripotent stem cells are human induced pluripotent cells (iPS cells).

14. Cells generated by the method of claim 1.

16. A method as recited in claim 11, wherein the extracellular matrix material is selected from a basement membrane preparation, collagen, hyaluronic acid, gelatin, elastin, fibronectin, laminin and mixtures thereof.

17. Cells generated by the method of claim 13.

15. A method of generating a population of oligodendroglial cells, the method comprising the steps of:

(a) obtaining cells having a neural tube-like rosette morphology that are Pax6+/Sox1+;

(b) culturing the neural tube-like rosette cells in the presence of retinoic acid and an agent that activates a sonic hedgehog pathway to obtain Olig2-expressing progenitor cells;

(c) culturing the progenitor cells in the presence of an agent that activates a sonic hedgehog pathway to obtain Olig2/Nkx2.2-expressing glial progenitor cells;

(d) culturing the Olig2/Nkx2.2-expressing glial progenitors in the presence of the agent that activates the sonic hedgehog pathway, but in the absence of retinoic acid, to obtain PDGFR+ oligodendroglial precursor cells; and

(e) culturing the PDGFRα

+ oligodendroglial precursor cells on an extracellular matrix material in the absence of the agent that activates the sonic hedgehog pathway to obtain oligodendroglial cells.

18. A method of generating a population of oligodendroglial precursor cells, the method comprising the steps of:

(a) obtaining neural tube-like rosette cells (Pax6+/Sox1+);

(b) culturing the neural tube-like rosette cells in the presence of retinoic acid and an agent that activates a sonic hedgehog pathway to obtain Olig2-expressing progenitor cells; and

(c) culturing the Olig2-expressing progenitors cells in the presence of the agent that activates the sonic hedgehog pathway, but in the absence of retinoic acid, to obtain PDGFRα

+/SOX10+/OLIG2+/NKX2.2+ oligodendroglial precursor cells.

19. The method of claim 18 wherein the agent of step (b) is selected from sonic hedgehog and purmorphamine.

20. A population of cells generated by the method of claim 18.