Cultures of GFAP+ nestin+ cells that differentiate to neurons
First Claim
1. An in vitro adhesion cell culture comprising at least 90% GFAP+ cells, whereina) 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.
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Accused Products
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.
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Citations
46 Claims
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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11. An in vitro cell culture consisting essentially of:
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(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.
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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. - View Dependent Claims (13, 14)
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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.
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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. - View Dependent Claims (17)
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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. - View Dependent Claims (19)
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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. - View Dependent Claims (21)
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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. - View Dependent Claims (32, 33)
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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.
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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. - View Dependent Claims (36)
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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. - View Dependent Claims (38)
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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. - View Dependent Claims (40)
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41. A method of producing a neuronal cell in vitro comprising the steps of:
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(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. - View Dependent Claims (42)
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43. A method of producing a neuronal cell in vitro comprising the steps of:
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(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. - View Dependent Claims (44)
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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. - View Dependent Claims (46)
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Specification