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

• US 20070231898A1
• Filed: 12/11/2006
• Published: 10/04/2007
• 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: Nistor, Gabriel, Keirstead, Hans
• Granted Patent: US 7,579,188 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 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• 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 bearing markers of oligodendrocyte precursor cells. Upon further differentiation form complex processes characteristic of mature oligodendrocytes. 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 oligodendrocyte precursors having the following characteristics;

they are progeny of primate pluripotent stem (pPS) cells;

they stain with antibody specific for NG2 proteoglycan; and

they are negative for the neuronal marker NeuN;

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

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

a) obtaining a population of differentiated cells according to claim 1 any of claims 1 14;

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.

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

26. A pharmaceutical composition, comprising the differentiated cell population according to claim 1.

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

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

2. A system for generating glial cells, comprising a line of undifferentiated pPS cells;

and a differentiated cell population in which at least 80% of the cells have the following characteristics;

they are progeny of primate pluripotent stem (pPS) cells;

they stain with antibody specific for NG2 proteoglycan; and

they are negative for the neuronal marker NeuN.

3-23. -23. (canceled)

27-30. -30. (canceled)

33-34. -34. (canceled)

35. A method for producing glial cells from human embryonic stem (hES) cells, comprising

culturing hES cells in suspension in a culture medium comprising basic fibroblast growth factor (bFGF), thyroid hormone T3, and retinoic acid (RA).

36. The method of claim 35, wherein the concentration of thyroid hormone T3 is 20 ng/ml and the concentration of RA is 10 μ

M.

37. The method of claim 35, wherein prior to said culturing, the hES cells are cultured in suspension in a culture medium comprising bFGF and thyroid hormone T3.

38. The method of claim 35, wherein prior to said culturing, the hES cells are cultured in suspension in a culture medium comprising bFGF at a concentration of 4 ng/ml and thyroid hormone T3 at a concentration of 20 ng/ml.

39. The method of claim 35, comprising

following said culturing, replacing the culture medium with a replacement culture medium comprising thyroid hormone T3 and retinoic acid (RA), wherein the replacement culture medium does not contain bFGF;

and

culturing cells in the replacement culture medium.

40. The method of claim 39, wherein thyroid hormone T3 is present in the replacement culture medium in a concentration of 40 ng/ml thyroid hormone T3 and RA is present in the replacement culture medium at a concentration of 10 μ

M.

41. The method of claim 38, wherein the cells are cultured in the culture medium comprising bFGF, thyroid hormone T3, and RA for one day prior to said replacing.

42. The method of claim 35, wherein the cells are cultured in the presence of RA for 8 days.

43. The method of claim 42, wherein RA is present at a concentration of 10 μ

M.

44. The method of claim 39, wherein following said culturing in the replacement culture medium, the method comprises:

plating cells on an adherent surface; and

harvesting cells that adhere to the surface.

45. The method of claim 44, wherein following plating, the plated cells are cultured in a medium comprising epidermal growth factor (EGF) and thyroid hormone T3.

46. The method of claim 45, wherein EGF in the medium following plating is present at a concentration of 20 ng/ml and thyroid hormone T3 is present in the medium following plating at a concentration of 40 ng/ml.

47. The method of claim 44, further comprising separating glial cells from non-glial cells.

48. The method of claim 47, wherein the separating is performed by plating cells from the culture onto a solid surface, and harvesting cells that adhere to the surface.

49. A method for producing glial cells from human embryonic stem (hES) cells, comprising

a) culturing the hES cells in suspension in a culture medium comprising basic fibroblast growth factor (bFGF), and thyroid hormone T3;

b) adding retinoic acid (RA) to the culture medium and continuing said culturing;

c) replacing the culture medium with a replacement culture medium, comprising thyroid hormone T3 and RA, wherein the replacement culture medium does not contain bFGF and culturing cells in the replacement culture medium;

d) plating cells on an adherent surface; and

e) culturing the plated cells in a culture medium comprising epidermal growth factor (EGF) and thyroid hormone T3; and

f) harvesting cells that adhere to the surface.

50. The method of claim 49, wherein in step a) bFGF is present in the culture medium at a concentration of 4 ng/ml and thyroid hormone T3 is present in the culture medium at a concentration of 20 ng/ml.

51. The method of claim 49, wherein in step b), RA is added at a concentration of RA is 10 μ

M.

52. The method of claim 49, wherein the cells are cultured in the presence of RA for one day prior to said replacing.

53. The method of claim 49, wherein EGF in the culture medium following said plating is present at a concentration of 20 ng/ml and thyroid hormone T3 is present at a concentration of 40 ng/ml.

54. A method for producing glial cells from human embryonic stem (hES) cells, comprising

a) culturing the hES cells in suspension in a culture medium comprising basic fibroblast growth factor (bFGF), thyroid hormone T3, and retinoic acid (RA);

and

b) replacing the culture medium with a replacement culture medium comprising thyroid hormone T3 and RA, wherein the replacement culture medium does not contain bFGF and culturing cells in the replacement culture medium;

c) plating cells on an adherent surface; and

d) culturing the plated cells in a culture medium comprising epidermal growth factor (EGF) and thyroid hormone T3; and

e) harvesting cells that adhere to the surface;

wherein the cells are cultured in the presence of RA for 8 days;

55. The method of claim 54, wherein at step b) RA is present at a concentration of 10 μ

M.

56. The method of claim 54, wherein prior to step a), the hES cells are cultured in suspension in a culture medium comprising bFGF and thyroid hormone T3;