Oligodendrocytes derived from human embryonic stem cells for remyelination and treatment of spinal cord injury

• US 20040009593A1
• Filed: 04/04/2003
• Published: 01/15/2004
• Est. Priority Date: 07/11/2002
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Keirstead, Hans S., Nistor, Gabriel I.
• Granted Patent: US 7,285,415 B2
• Time in Patent Office: Days
• US Class Current: 435/368
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• 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 27/02 Ophthalmic agents
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/395 Thyroid hormones
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0622 Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells
• CPC Class Codes: C12N 5/0623 Stem cells

Abstract

This invention provides populations of neural cells bearing markers of glial cells, such as oligodendrocytes and their precursors. The populations are generated by differentiating pluripotent stem cells such as human embryonic stem cells under conditions that promote enrichment of cells with the desired phenotype or functional capability. Various combinations of differentiation factors and mitogens can be used to produce cell populations that are over 95% homogeneous in morphological appearance, and the expression of oligodendrocyte markers such as GalC. The cells are capable of forming myelin sheaths, and can be used therapeutically improve function of the central nervous system.

Claims

1. A differentiated cell population as part of a system for generating glial cells,

wherein at least ˜

80% of cells in the differentiated cell population are progeny of primate pluripotent stem (pPS) cells, and have at least one of the following properties;

they stain with 04 or RIP antibody, or with antibody specific for NG2 or galactocerebroside (GalC);

they express GaIC or MBP after being cultured for 3 days on poly-L-lysine and laminin in the absence of mitogens;

or

following implantation in or around the spinal cord in contusion-injured rats, they cause improvement in overground locomotion;

and wherein the system further comprises the line of pPS cells from which the differentiated cells were produced.

2. The differentiated cell population of claim 1, wherein at least 90% of cells express antibody-detectable GalC.

3. The differentiated cell population of claim 1, wherein at least 90% of cells express antibody-detectable MBP after being cultured for 3 days on poly-L-lysine and laminin in the absence of mitogens.

4. The differentiated cell population of claim 1, wherein implantation of the population in or around the spinal cord in contusion-injured rats causes improvement in overground locomotion.

5. The differentiated cell population of claim 1, obtained by a process in which the undifferentiated pPS cells are cultured in a medium containing a mitogen and at least two oligodendrocyte differentiation factors.

6. The differentiated cell population of claim 1, obtained by a process in which the undifferentiated pPS cells are cultured in suspension so as to form cell aggregates in the presence of basic fibroblast growth factor (FGF), triiodothyronine (T3), and retinoic acid.

7. The differentiated cell population of claim 1, obtained by a process in which glial cells are separated from non-glial cells.

8. The differentiated cell population of claim 1, obtained by a process in which the cells are cultured in a medium comprising thyroid hormone and selenium.

9. The differentiated cell population of claim 1, obtained by a process in which the cells are caused to differentiate further by culturing in the absence of mitogens.

10. The differentiated cell population of claim 1, wherein the pPS cells are human embryonic stem (hES) cells.

17. The method of claim 11, wherein the cells obtained have the characteristic of differentiated cells according to claim 1.

18. A method of screening a compound for its effect on glial cells, comprising:

a) obtaining a population of differentiated cells according to claim 1;

b) combining the cell population with the compound;

c) determining any change to cells in the population or their activity that results from being combined with the compound; and

d) correlating the change with the effect of the compound on glial cells.

19. A method of myelinating an axon, comprising combining a differentiated cell obtained from the population according to claim 1 with a neuronal cell from which the axon extends.

20. A method of improving spinal cord function in a subject, comprising administering to the subject a differentiated cell population according to claim 1.

11. A method for producing glial cells from undifferentiated primate pluripotent stem (pPS) cells, comprising culturing the undifferentiated pPS cells in a medium containing a mitogen and at least two oligodendrocyte differentiation factors.

12. The method of claim 11, comprising culturing undifferentiated pPS cells in suspension culture so as to form cell aggregates in the presence of basic FGF, T3, and retinoic acid.

13. The method of claim 11, comprising separating glial cells from non-glial cells.

14. The method of claim 13, wherein the separating is performed by plating the cells onto a solid surface, and harvesting cells that adhere to the surface.

15. The method of claim 11, comprising culturing the cells in a medium comprising thyroid hormone and selenium.

16. The method of claim 11, wherein glial cells are caused to differentiate further by culturing in the absence of mitogens.

21. (New) A method for producing glial cells from undifferentiated primate pluripotent stem (pPS) cells, comprising culturing undifferentiated pPS cells so as to form cell aggregates in the presence of a growth factor, a ligand for a thyroid hormone receptor, and a ligand for a retinoic acid receptor.

22. (New) The method of claim 21, wherein the growth factor is basic fibroblast growth factor.

23. (New) The method of claim 22, wherein the cells are cultured in a medium comprising thyroid hormone T3 and retinoic acid.

24. (New) The method of claim 21, wherein the cells are cultured in a medium comprising the thyroid hormone T3 and selenium.

25. (New) The method of any of claim 21, wherein the cells are cultured in suspension so as to form yellow spheres.

26. (New) The method of claim 21, further comprising separating glial cells from non-glial cells.

27. (New) The method of claim 26, wherein the separating is performed by plating the cells onto a solid surface, and harvesting cells that adhere to the surface.

28. (New) The method of claim 21, further comprising harvesting from the culture a cell population in which at least ˜

80% of cells are positive for NG2 proteoglycan, but negative for NeuN.

29. (New) The method of claim 21, further comprising culturing the glial cells in the absence of mitogens so as to produce a population containing cells that have numerous processes characteristic of mature oligodendrocytes.

30. (New) The method of claim 21, further comprising formulating the glial cells in a composition suitable for administration into a spinal cord.

31. (New) The method of claim 21, further comprising screening the effect of a compound by combining the glial cells with the compound, and determining any resulting phenotypic or functional change in the cells.