Cultures of GFAP+ nestin+ cells that differentiate to neurons

• US 6,878,543 B1
• Filed: 10/24/2000
• Issued: 04/12/2005
• Est. Priority Date: 10/25/1999
• Status: Expired due to Term

• Current Assignee: BOCO Silicon Valley, Inc.
• Original Assignee: Nsgenee A/S
• Inventors: Skogh, Charlotta, Wictorin, Klas, Campbell, Kenneth, Wahlberg, Lars, Eriksson, Cecilia
• Primary Examiner(s): BRUMBACK, BRENDA G
• Assistant Examiner(s): BRUMBACK, BRENDA G
• Application Number:
  US09/696,530
• Time in Patent Office: 1,631 Days
• Field of Search: 435/325, 435/366, 435/368, 435/378, 435/69.1, 435/69.52, 435/172.3, 435/240.2, 435/377, 435/384, 435/392, 435/395, 424/520, 424/563
• US Class Current: 435/325
• 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: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 5/0619 Neurons
• CPC Class Codes: C12N 5/0622 Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells
• CPC Class Codes: C12N 5/0623 Stem cells

Abstract

Cultures of cells immunoreactive for glial fibrillary acidic protein (GFAP), as well as for the intermediate filament marker nestin were grown in a medium including epidermal growth factor (EGF) and serum. The cultured cells had the morphology of astroglial cells. The cells can be proliferated in adherent or suspension cultures. Depending on the culture conditions, the cells can be induced to differentiate to neurons or glial cells. The cultures can be expanded over a large number of passages during several months, and survive, express an astroglial phenotype and integrate well after transplantation into both neonatal and adult rat forebrain.

Claims

1. An in vitro adhesion cell culture comprising at least 90% GFAP⁺ cells, wherein

a) one or more cells in the culture have the capacity to differentiate into neurons;

b) the cell culture divides in a culture medium containing serum and at least one proliferation-inducing growth factor, wherein the at least one proliferation-inducing growth factor is selected from the group consisting of EGF, amphiregulin, aFGF, bFGF, TGFα

, and combinations thereof;

c) one or more cells in the culture differentiate into neurons upon withdrawal of both serum and the proliferation-inducing growth factor;

d) wherein greater than 50% of the cells in the culture are nestin⁺ under proliferation-promoting culture conditions; and

e) wherein the cell culture differentiates into at least 10% neurons under differentiation-inducing culture conditions.

2. An in vitro adhesion cell culture comprising at least 90% GFAP⁺ cells, wherein

a) one or more cells in the culture have the capacity to differentiate into neurons;

b) the cell culture divides in a culture medium containing serum and at least one proliferation-inducing growth factor, wherein the at least one proliferation-inducing growth factor is selected from the group consisting of EGF, amphiregulin, aFGF, bFGF, TGFα

, and combinations thereof;

c) one or more cells in the culture differentiate into neurons upon withdrawal of both serum and the proliferation-inducing growth factor;

d) wherein greater than 50% of the cells in the culture are nestin⁺ under proliferation-promoting culture conditions; and

e) wherein the cell culture differentiates into at least 25% neurons under differentiation-inducing culture conditions.

3. An in vitro adhesion cell culture comprising at least 90% GFAP⁺ cells, wherein

a) one or more cells in the culture have the capacity to differentiate into neurons;

b) the cell culture divides in a culture medium containing serum and at least one proliferation-inducing growth factor wherein the at least one proliferation-inducing growth factor is selected from the group consisting of EGF, amphiregulin, aFGF, bFGF, TGFα

, and combinations thereof;

c) one or more cells in the culture differentiate into neurons upon withdrawal of both serum and the proliferation-inducing growth factor;

d) wherein greater than 50% of the cells in the culture are nestin⁺ under proliferation-promoting culture conditions; and

e) wherein the cell culture is capable of at least 6 doublings.

4. An in vitro adhesion cell culture comprising at least 90% GFAP⁺ cells, wherein

a) one or more cells in the culture have the capacity to differentiate into neurons;

b) the cell culture divides in a culture medium containing serum and at least one proliferation-inducing growth factor, wherein the at least one proliferation-inducing growth factor is selected from the group consisting of EGF, amphiregulin, aFGF, bFGF, TGFα

, and combinations thereof;

c) one or more cells in the culture differentiate into neurons upon withdrawal of both serum and the proliferation-inducing growth factor;

d) wherein greater than 50% of the cells in the culture are nestin⁺ under proliferation-promoting culture conditions; and

e) wherein the cell culture is capable of at least 12 doublings.

5. An in vitro adhesion cell culture comprising at least 90% GFAP⁺ cells, wherein

a) one or more cells in the culture have the capacity to differentiate into neurons;

b) the cell culture divides in a culture medium containing serum and at least one proliferation-inducing growth factor, wherein the at least one proliferation-inducing growth factor is selected from the group consisting of EGF, amphiregulin, aFGF, bFGF, TGFα

, and combinations thereof;

c) one or more cells in the culture differentiate into neurons upon withdrawal of both serum and the proliferation-inducing growth factor;

d) wherein greater than 50% of the cells in the culture are nestin⁺ under proliferation-promoting culture conditions; and

e) wherein the cell culture is capable of at least 18 doublings.

6. An in vitro adhesion cell culture comprising at least 90% GFAP⁺ cells, wherein

a) one or more cells in the culture have the capacity to differentiate into neurons;

b) the cell culture divides in a culture medium containing serum and at least one proliferation-inducing growth factor, wherein the at least one proliferation-inducing growth factor is selected from the group consisting of EGF, amphiregulin, aFGF, bFGF, TGFα

, and combinations thereof;

c) one or more cells in the culture differentiate into neurons upon withdrawal of both serum and the proliferation-inducing growth factor;

d) wherein greater than 50% of the cells in the culture are nestin⁺ under proliferation-promoting culture conditions; and

e) wherein the cells are derived from the lateral ganglionic eminence (LGE) or medial ganglionic eminence (MGE) of the mammal.

7. An in vitro adhesion cell culture comprising at least 90% GFAP⁺ cells, wherein

a) one or more cells in the culture have the capacity to differentiate into neurons;

b) the cell culture divides in a culture medium containing serum and at least one proliferation-inducing growth factor, wherein the at least one proliferation-inducing growth factor is selected from the group consisting of EGF, amphiregulin, aFGF, bFGF, TGFα

, and combinations thereof;

c) one or more cells in the culture differentiate into neurons upon withdrawal of both serum and the proliferation-inducing growth factor;

d) wherein greater than 50% of the cells in the culture are nestin⁺ under proliferation-promoting culture conditions; and

e) wherein the doubling rate of the culture is faster than seven days.

8. An in vitro adhesion cell culture comprising at least 90% GFAP⁺ cells, wherein

a) one or more cells in the culture have the capacity to differentiate into neurons;

b) the cell culture divides in a culture medium containing serum and at least one proliferation-inducing growth factor, wherein the at least one proliferation-inducing growth factor is selected from the group consisting of EGF, amphiregulin, aFGF, bFGF, TGFα

, and combinations thereof;

c) one or more cells in the culture differentiate into neurons upon withdrawal of both serum and the proliferation-inducing growth factor;

d) wherein greater than 50% of the cells in the culture are nestin⁺ under proliferation-promoting culture conditions; and

e) wherein the cells in the cell culture are murine.

9. An in vitro adhesion cell culture comprising at least 90% GFAP⁺ cells, wherein

a) one or more cells in the culture have the capacity to differentiate into neurons;

b) the cell culture divides in a culture medium containing serum and at least one proliferation-inducing growth factor, wherein the at least one proliferation-inducing growth factor is selected from the group consisting of EGF, amphiregulin, aFGF, bFGF, TGFα

, and combinations thereof;

c) one or more cells in the culture differentiate into neurons upon withdrawal of both serum and the proliferation-inducing growth factor;

d) wherein greater than 50% of the cells in the culture are nestin⁺ under proliferation-promoting culture conditions; and

e) wherein the cells in the cell culture are human.

10. An in vitro adhesion cell culture comprising at least 90% GFAP⁺ cells, wherein

a) one or more cells in the culture have the capacity to differentiate into neurons;

b) the cell culture divides in a culture medium containing serum and at least one proliferation-inducing growth factor, wherein the at least one proliferation-inducing growth factor is selected from the group consisting of EGF, amphiregulin, aFGF, bFGF, TGFα

, and combinations thereof;

c) one or more cells in the culture differentiate into neurons upon withdrawal of both serum and the proliferation-inducing growth factor;

d) wherein greater than 50% of the cells in the culture are nestin⁺ under proliferation-promoting culture conditions; and

e) wherein fewer than 5% of the cells in the culture are β

-tubulin III immunoreactive (β

-tubulin III⁺) under proliferation-promoting culture conditions and between 10-40% of the cells in the culture are β

-tubulin III immunoreactive (β

-tubulin III⁺) under differentiation-inducing culture conditions.

11. An in vitro cell culture consisting essentially of:

(a) a culture medium containing serum and at least one proliferation-inducing growth factor; and

(b) cells derived from the central nervous system of a mammal, wherein;

(i) at least 90% of the cells are glial fibrillary acidic protein immunoreactive (GFAP⁺);

(ii) the cells are capable proliferating in a culture medium containing serum and at least one proliferation-inducing growth factor, wherein the at least one proliferation-inducing growth factor is selected from the group consisting of EGF, amphiregulin, aFGF, bFGF, TGFα

, and combinations thereof;

(iii) the cells are capable of differentiating into at least 10% neurons in the absence of both the serum and the proliferation-inducing growth factor from the culture medium;

(iv) wherein greater than 50% of the cells in the culture are nestin immunoreactive (i.e., nestin⁺) under proliferation-promoting culture conditions,

wherein the culture is an adhesion culture.

12. An in vitro adhesion cell culture comprising at least 90% GFAP⁺ cells, wherein

a) one or more cells in the culture have the capacity to differentiate into neurons;

b) the cell culture divides in a culture medium containing serum and at least one proliferation-inducing growth factor, wherein the at least one proliferation-inducing growth factor is selected from the group consisting of EGF, amphiregulin, aFGF, bFGF, TGFα

, and combinations thereof;

c) one or more cells in the culture differentiate into neurons upon withdrawal of both serum and the proliferation-inducing growth factor;

d) wherein greater than 50% of the cells in the culture are nestin⁺ under proliferation-promoting culture conditions; and

e) wherein at least a portion of the cells in culture differentiate into radial glia in the absence of serum from the culture medium.

13. The cell culture of claim 12, wherein the radial glia are both GFAP⁺ and vimentin positive.

14. The cell culture of claim 12, wherein the morphology of the radial glia is:

(a) bipolar;

(b) elongated; and

(c) non-fibrillary.

15. An in vitro adhesion cell culture comprising at least 90% GFAP⁺ cells, wherein

a) one or more cells in the culture have the capacity to differentiate into neurons;

b) the cell culture divides in a culture medium containing serum and at least one proliferation-inducing growth factor, wherein the at least one proliferation-inducing growth factor is selected from the group consisting of EGF, amphiregulin, aFGF, bFGF, TGFα

, and combinations thereof;

c) one or more cells in the culture differentiate into neurons upon withdrawal of both serum and the proliferation-inducing growth factor;

d) wherein greater than 50% of the cells in the culture are nestin⁺ under proliferation-promoting culture conditions; and

e) wherein at least some of the cells in culture, under differentiation-inducing culture conditions, differentiate into neurons that exhibit;

(i) axon-dendrite polarity,

(ii) synaptic terminals, and

(iii) localization of proteins involved in synaptogenesis and synaptic activity including

(1) neurotransmitter receptors,

(2) transporters, and

(3) processing enzymes.

16. An in vitro adhesion cell culture comprising at least 90% GFAP⁺ cells, wherein

a) one or more cells in the culture have the capacity to differentiate into neurons;

b) the cell culture divides in a culture medium containing serum and at least one proliferation-inducing growth factor, wherein the at least one proliferation-inducing growth factor is selected from the group consisting of EGF, amphiregulin, aFGF, bFGF, TGFα

, and combinations thereof;

c) one or more cells in the culture differentiate into neurons upon withdrawal of both serum and the proliferation-inducing growth factor;

d) wherein greater than 50% of the cells in the culture are nestin⁺ under proliferation-promoting culture conditions; and

e) wherein greater than 50% of differentiated neuronal cells are immunoreactive with striatal neuronal markers.

17. The culture of claim 16 wherein said striatal neuronal markers are DLX1 and/or MEIS2.

18. An in vitro adhesion cell culture comprising at least 90% GFAP⁺ cells, wherein

a) one or more cells in the culture have the capacity to differentiate into neurons;

b) the cell culture divides in a culture medium containing serum and at least one proliferation-inducing growth factor, wherein the at least one proliferation-inducing growth factor is selected from the group consisting of EGF, amphiregulin, aFGF, bFGF, TGFα

, and combinations thereof;

c) one or more cells in the culture differentiate into neurons upon withdrawal of both serum and the proliferation-inducing growth factor;

d) wherein greater than 50% of the cells in the culture are nestin⁺ under proliferation-promoting culture conditions; and

e) wherein greater than 50% of differentiated neuronal cells are not immunoreactive with cortical neuronal markers.

19. The culture of claim 18, wherein the cortical neuronal marker is PAX6.

20. An in vitro adhesion cell culture comprising at least 90% GFAP⁺ cells, wherein

a) one or more cells in the culture have the capacity to differentiate into neurons;

b) the cell culture divides in a culture medium containing serum and at least one proliferation-inducing growth factor, wherein the at least one proliferation-inducing growth factor is selected from the group consisting of EGF, amphiregulin, aFGF, bFGF, TGFα

, and combinations thereof;

c) one or more cells in the culture differentiate into neurons upon withdrawal of both serum and the proliferation-inducing growth factor;

d) wherein greater than 50% of the cells in the culture are nesting⁺ under proliferation-promoting culture conditions; and

e) wherein greater than 50% of differentiated neuronal cells are not immuno reactive with neuronal markers of the medial ganglion eminence.

21. The culture of claim 20 wherein one of said neuronal markers of the medial ganglionic eminence is NKX2.1.

22. An in vitro cell culture consisting essentially of:

(a) a culture medium containing serum and at least one proliferation-inducing growth factor; and

(b) cells derived from the central nervous system of a mammal, wherein;

(i) at least 90% of the cells are glial fibrillary acidic protein immunoreactive (GFAP⁺),

(ii) the cells are capable proliferating in a culture medium containing serum and at least one proliferation-inducing growth factor, wherein the at least one proliferation-inducing growth factor is selected from the group consisting of EGF, amphiregulin, aFGF, bFGF, TGFα

, and combinations thereof,

(iii) the cells are capable of differentiating into at least 10% neurons in the absence of both the serum and the proliferation-inducing growth factor from the culture medium; and

(iv) wherein greater than 50% of the cells in the culture are nestin immunoreactive under proliferation-promoting culture conditions, and

(v) wherein the cell culture differentiations into at least 25% neurons under differentiation-inducing culture conditions.

23. An in vitro cell culture consisting essentially of:

(a) a culture medium containing serum and at least one proliferation-inducing growth factor; and

(b) cells derived from the central nervous system of a mammal, wherein;

(i) at least 90% of the cells are glial fibrillary acidic protein immunoreactive (GFAP⁺),

(ii) the cells are capable proliferating in a culture medium containing serum and at least one proliferation-inducing growth factor, wherein the at least one proliferation-inducing growth factor is selected from the group consisting of EGF, amphiregulin, aFGF, bFGF, TGFα

, and combinations thereof,

(iii) the cells are capable of differentiating into at least 10% neurons in the absence of both the serum and the proliferation-inducing growth factor from the culture medium; and

(iv) wherein greater than 50% of the cells in the culture are nestin immunoreactive under proliferation-promoting culture conditions, and

(v) wherein the cell culture is capable of at least 6 doublings.

24. An in vitro cell culture consisting essentially of:

(a) a culture medium containing serum and at least one proliferation-inducing growth factor; and

(b) cells derived from the central nervous system of a mammal, wherein;

(i) at least 90% of the cells are glial fibrillary acidic protein immunoreactive (GFAP⁺);

(ii) the cells are capable proliferating in a culture medium containing serum and at least one proliferation-inducing growth factor, wherein the at least one proliferation-inducing growth factor is selected from the group consisting of EGF, amphiregulin, aFGF, bFGF, TGFα

, and combinations thereof,

(iii) the cells are capable of differentiating into at least 10% neurons in the absence of both the serum and the proliferation-inducing growth factor from the culture medium; and

(iv) wherein greater than 50% of the cells in the culture are nestin immunoreactive under proliferation-promoting culture conditions, and

(v) wherein the cell culture is capable of at least 12 doublings.

25. An in vitro cell culture consisting essentially of:

(a) a culture medium containing serum and at least one proliferation-inducing growth factor; and

(b) cells derived from the central nervous system of a mammal, wherein;

(i) at least 90% of the cells are glial fibrillary acidic protein immunoreactive (GFAP⁺),

(ii) the cells are capable proliferating in a culture medium containing serum and at least one proliferation-inducing growth factor, wherein the at least one proliferation-inducing growth factor is selected from the group consisting of EGF, amphiregulin, aFGF, bFGF, TGFα

, and combinations thereof,

(iii) the cells are capable of differentiating into at least 10% neurons in the absence of both the serum and the proliferation-inducing growth factor from the culture medium; and

(iv) wherein greater than 50% of the cells in the culture are nestin immunoreactive under proliferation-promoting culture conditions, and

(v) wherein the cell culture is capable of at least 18 doublings.

26. An in vitro cell culture consisting essentially of:

(a) a culture medium containing serum and at least one proliferation-inducing growth factor; and

(b) cells derived from the central nervous system of a mammal, wherein;

(i) at least 90% of the cells are glial fibrillary acidic protein immunoreactive (GFAP⁺);

(ii) the cells are capable proliferating in a culture medium containing serum and at least one proliferation-inducing growth factor, wherein the at least one proliferation-inducing growth factor is selected from the group consisting of EGF, amphiregulin, aFGF, bFGF, TGFα

, and combinations thereof,

(iii) the cells are capable of differentiating into at least 10% neurons in the absence of both the serum and the proliferation-inducing growth factor from the culture medium; and

(iv) wherein greater than 50% of the cells in the culture are nestin immunoreactive under proliferation-promoting culture conditions, and

(v) wherein the cells are derived from the lateral ganglionic eminence (LGE) or medial ganglionic eminence (MGE) of the mammal.

27. An in vitro cell culture consisting essentially of:

(a) a culture medium containing serum and at least one proliferation-inducing growth factor; and

(b) cells derived from the central nervous system of a mammal, wherein;

(i) at least 90% of the cells are glial fibrillary acidic protein immunoreactive (GFAP⁺),

(ii) the cells are capable proliferating in a culture medium containing serum and at least one proliferation-inducing growth factor, wherein the at least one proliferation-inducing growth factor is selected from the group consisting of EGF, amphiregulin, aFGF, bFGF, TGFα

, and combinations thereof,

(iii) the cells are capable of differentiating into at least 10% neurons in the absence of both the serum and the proliferation-inducing growth factor from the culture medium; and

(iv) wherein greater than 50% of the cells in the culture are nestin immunoreactive under proliferation-promoting culture conditions, and

(v) wherein the doubling rate of the culture is faster than seven days.

28. An in vitro cell culture consisting essentially of:

(a) a culture medium containing serum and at least one proliferation-inducing growth factor; and

(b) cells derived from the central nervous system of a mammal, wherein;

(i) at least 90% of the cells are glial fibrillary acidic protein immunoreactive (GFAP⁺),

(ii) the cells are capable proliferating in a culture medium containing serum and at least one proliferation-inducing growth factor, wherein the at least one proliferation-inducing growth factor is selected from the group consisting of EGF, amphiregulin, aFGF, bFGF, TGFα

, and combinations thereof,

(iii) the cells are capable of differentiating into at least 10% neurons in the absence of both the serum and the proliferation-inducing growth factor from the culture medium; and

(iv) wherein greater than 50% of the cells in the culture are nestin immunoreactive under proliferation-promoting culture conditions, and

(v) wherein the cells in the culture are murine.

29. An in vitro cell culture consisting essentially of:

(a) a culture medium containing serum and at least one proliferation-inducing growth factor; and

(b) cells derived from the central nervous system of a mammal, wherein;

(i) at least 90% of the cells are glial fibrillary acidic protein immunoreactive (GFAP⁺),

(ii) the cells are capable proliferating in a culture medium containing serum and at least one proliferation-inducing growth factor, wherein the at least one proliferation-inducing growth factor is selected from the group consisting of EGF, amphiregulin, aFGF, bFGF, TGFα

, and combinations thereof,

(iii) the cells are capable of differentiating into at least 10% neurons in the absence of both the serum and the proliferation-inducing growth factor from the culture medium; and

(iv) wherein greater than 50% of the cells in the culture are nestin immunoreactive under proliferation-promoting culture conditions, and

(v) wherein the cells in the culture are human.

30. An in vitro cell culture consisting essentially of:

(a) a culture medium containing serum and at least one proliferation-inducing growth factor; and

(b) cells derived from the central nervous system of a mammal, wherein;

(i) at least 90% of the cells are glial fibrillary acidic protein immunoreactive (GFAP⁺),

(ii) the cells are capable proliferating in a culture medium containing serum and at least one proliferation-inducing growth factor, wherein the at least one proliferation-inducing growth factor is selected from the group consisting of EGF, amphiregulin, aFGF, bFGF, TGFα

, and combinations thereof;

(iii) the cells are capable of differentiating into at least 10% neurons in the absence of both the serum and the proliferation-inducing growth factor from the culture medium; and

(iv) wherein greater than 50% of the cells in the culture are nestin immunoreactive under proliferation-promoting culture conditions, and

(v) wherein fewer than 5% of the cells in the culture are β

-tubulin III immunoreactive (β

-tubulin III⁺) under proliferation-promoting culture conditions and between 10-40% of the cells in the culture are β

-tubulin III immunoreactive (β

-tubulin III⁺) under differentiation-inducing culture conditions.

31. An in vitro cell culture consisting essentially of:

(a) a culture medium containing serum and at least one proliferation-inducing growth factor; and

(b) cells derived from the central nervous system of a mammal, wherein;

(i) at least 90% of the cells are glial fibrillary acidic protein immunoreactive (GFAP⁺),

(ii) the cells are capable proliferating in a culture medium containing serum and at least one proliferation-inducing growth factor, wherein the at least one proliferation-inducing growth factor is selected from the group consisting of EGF, amphiregulin, aFGF, bFGF, TGFα

, and combinations thereof,

(iii) the cells are capable of differentiating into at least 10% neurons in the absence of both the serum and the proliferation-inducing growth factor from the culture medium; and

(iv) wherein greater than 50% of the cells in the culture are nestin immunoreactive under proliferation-promoting culture conditions, and

(v) wherein at least a portion of the cells in culture differentiate into radial glia in the absence of serum from the culture medium.

32. The cell culture of claim 31, wherein the radial glia are both GFAP⁺ and vimentin positive.

33. The cell culture of claim 31, wherein the morphology of the radial glia is:

(a) bipolar;

(b) elongated; and

(c) non-fibrillary.

34. An in vitro cell culture consisting essentially of:

(a) a culture medium containing serum and at least one proliferation-inducing growth factor; and

(b) cells derived from the central nervous system of a mammal, wherein;

(i) at least 90% of the cells are glial fibrillary acidic protein immunoreactive (GFAP⁺),

(ii) the cells are capable proliferating in a culture medium containing serum and at least one proliferation-inducing growth factor, wherein the at least one proliferation-inducing growth factor is selected from the group consisting of EGF, amphiregulin, aFGF, bFGF, TGFα

, and combinations thereof,

(iii) the cells are capable of differentiating into at least 10% neurons in the absence of both the serum and the proliferation-inducing growth factor from the culture medium; and

(iv) wherein greater than 50% of the cells in the culture are nestin immunoreactive under proliferation-promoting culture conditions, and

(v) wherein at least some of the cells in culture, under differentiation-inducing culture conditions, differentiate into neurons that exhibit;

(1) axon-dendrite polarity,

(2) synaptic terminals, and

(3) localization of proteins involved in synaptogenesis and synaptic activity including

(A) neurotransmitter receptors,

(B) transporters, and

(C) processing enzymes.

35. An in vitro cell culture consisting essentially of:

(a) a culture medium containing serum and at least one proliferation-inducing growth factor; and

(b) cells derived from the central nervous system of a mammal, wherein;

(i) at least 90% of the cells are glial fibrillary acidic protein immunoreactive (GFAP⁺);

(ii) the cells are capable proliferating in a culture medium containing serum and at least one proliferation-inducing growth factor, wherein the at least one proliferation-inducing growth factor is selected from the group consisting of EGF, amphiregulin, aFGF, bFGF, TGFα

, and combinations thereof,

(iii) the cells are capable of differentiating into at least 10% neurons in the absence of both the serum and the proliferation-inducing growth factor from the culture medium; and

(iv) wherein greater than 50% of the cells in the culture are nestin immunoreactive under proliferation-promoting culture conditions, and

(v) wherein greater than 50% of differentiated neuronal cells are immunoreactive with striatal neuronal markers.

36. The culture of claim 35 wherein said striatal neuronal markers are DLX1 and/or MEIS2.

37. An in vitro cell culture consisting essentially of:

(a) a culture medium containing serum and at least one proliferation-inducing growth factor; and

(b) cells derived from the central nervous system of a mammal, wherein;

(i) at least 90% of the cells are glial fibrillary acidic protein immunoreactive (GFAP⁺),

(ii) the cells are capable proliferating in a culture medium containing serum and at least one proliferation-inducing growth factor, wherein the at least one proliferation-inducing growth factor is selected from the group consisting of EGF, amphiregulin, aFGF, bFGF, TGFα

, and combinations thereof,

(iii) the cells are capable of differentiating into at least 10% neurons in the absence of both the serum and the proliferation-inducing growth factor from the culture medium; and

(iv) wherein greater than 50% of the cells in the culture are nestin immunoreactive under proliferation-promoting culture conditions, and

(v) wherein greater than 50% of differentiated neuronal cells are not immunoreactive with cortical neuronal markers.

38. The culture of claim 37 wherein the cortical neuronal marker is PAX6.

39. An in vitro cell culture consisting essentially of:

(a) a culture medium containing serum and at least one proliferation-inducing growth factor; and

(b) cells derived from the central nervous system of a mammal, wherein;

(i) at least 90% of the cells are glial fibrillary acidic protein immunoreactive (GFAP⁺),

(ii) the cells are capable proliferating in a culture medium containing serum and at least one proliferation-inducing growth factor, wherein the at least one proliferation-inducing growth factor is selected from the group consisting of EGF, amphiregulin, aFGF, bFGF, TGFα

, and combinations thereof,

(iii) the cells are capable of differentiating into at least 10% neurons in the absence of both the serum and the proliferation-inducing growth factor from the culture medium; and

(iv) wherein greater than 50% of the cells in the culture are nestin immunoreactive under proliferation-promoting culture conditions, and

(v) wherein greater than 50% of differentiated neuronal cells are not immunoreactive with neuronal markers of the medial ganglion eminence.

40. The culture of claim 39 wherein one of said neuronal markers of the medial ganglionic eminence is NKX2.1.

41. A method of producing a neuronal cell in vitro comprising the steps of:

(a) obtaining neural tissue from a mammal, the neural tissue containing at least one GFAP⁺, nestin⁺ cell capable of producing progeny that is a GFAP⁺, nestin⁺ cell;

(b) dissociating the neural tissue to obtain a cell suspension comprising said cell;

(c) culturing the cell suspension in a first culture medium containing serum and at least one proliferation-inducing growth factor to proliferate said GFAP⁺, nestin⁺ cell and produce a GFAP⁺, nestin⁺ cell progeny, wherein the at least one proliferation-inducing growth factor is selected from the group consisting of EGF, amphiregulin, aFGF, bFGF, TGFα

, and combinations thereof; and

(d) differentiating the cell progeny in a second culture medium that is substantially free of both the serum and the proliferation-inducing growth factor;

wherein the majority of differentiated neuronal cells are immunoreactive with striatal neuronal markers.

42. The method of claim 41 wherein said striatal neuronal markers are DLX1 and/or MEIS2.

43. A method of producing a neuronal cell in vitro comprising the steps of:

(a) obtaining neural tissue from a mammal, the neural tissue containing at least one GFAP⁺, nestin⁺ cell capable of producing progeny that is a GFAP⁺, nestin⁺ cell;

(b) dissociating the neural tissue to obtain a cell suspension comprising said cell;

(c) culturing the cell suspension in a first culture medium containing serum and at least one proliferation-inducing growth factor to proliferate said GFAP⁺, nestin⁺ cell and produce a GFAP⁺, nestin⁺ cell progeny, wherein the at least one proliferation-inducing growth factor is selected from the group consisting of EGF, amphiregulin, aFGF, bFGF, TGFα

, and combinations thereof; and

(d) differentiating the cell progeny in a second culture medium that is substantially free of both the serum and the proliferation-inducing growth factor;

wherein greater than 50% of differentiated neuronal cells are not immunoreactive with cortical neuronal markers.

44. The method of claim 43 wherein the cortical neuronal marker is PAX6.

45. A method of producing a neuronal cell in vitro comprising the steps of:

(a) obtaining neural tissue from a mammal, the neural tissue containing at least one GFAP⁺, nestin⁺ cell capable of producing progeny that is a GFAP⁺, nestin⁺ cell;

(b) dissociating the neural tissue to obtain a cell suspension comprising said cell;

(c) culturing the cell suspension in a first culture medium containing serum and at least one proliferation-inducing growth factor to proliferate said GFAP⁺, nestin⁺ cell and produce a GFAP⁺, nestin⁺ cell progeny, wherein the at least one proliferation-inducing growth factor is selected from the group consisting of EGF, amphiregulin, aFGF, bFGF, TGFα

, and combinations thereof; and

(d) differentiating the cell progeny in a second culture medium that is substantially free of both the serum and the proliferation-inducing growth factor;

wherein greater than 50% of differentiated neuronal cells are not immunoreactive with neuronal markers of the medial ganglionic eminence.

46. The method of claim 45 wherein one of said neuronal markers of the medial ganglionic eminence is NKX2.1.