US5981165A
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In vitro induction of dopaminergic cells
Assignee: Neurospheres Holdings Ltd
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Filing: 1995-06-07
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Granted: 1999-11-09
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Expires: 2015-06-07
Abstract
Abstract A culture method for inducing the expression of tyrosine hydroxylase in neural cells is provided. Mammalian CNS neural cells are cultured in the presence of a fibroblast growth factor and at least one selected from a member of the transforming growth factor beta family, a feeder layer bed of cells, and cell conditioned medium. Cells cultured as provided above may be transplanted to provide dopaminergic cells to a patient. The cells may also be used in methods for drug screening.
Inventors
- 👤 Samuel Weiss
- 👤 Brent Reynolds
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 11:10
CPC Classifications
C; C12; C12N; C12N5/00; C12N5/06; C12N5/0602; C12N5/0618; C12N5/0619; A; A61; A61K; A61K35/00; A61K35/12; A61K35/30; A61P; A61P25/00; A61P25/14; A61P25/16; C07; C07K; C07K14/00; C07K14/435; C07K14/46; C07K14/47; C07K14/475; C07K14/82; C12N5/0623; C12N5/0676; C12N9/00; C12N9/0004; C12N9/0071; C12Y; C12Y114/00; C12Y114/16; C12Y114/16002; A01; A01K; A01K2217/00; A01K2217/05; A61K48/00; C12N2500/00; C12N2500/05; C12N2500/10; C12N2500/20; C12N2500/24; C12N2500/25; C12N2500/90; C12N2501/00; C12N2501/10; C12N2501/11; C12N2501/115; C12N2501/148; C12N2501/155; C12N2501/16; C12N2502/00; C12N2502/08; C12N2510/00
Claims Analysis
Total Claims: 45
Claim 1
Independent Claim
62 words
A method for inducing the expression of tyrosine hydroxylase in neural cells in vitro comprising culturing mammalian CNS neural cells derived from normally non-dopaminergic, non-tumorigenic tissue in a culture medium to which has been added at least one member of the fibroblast growth factor family and at least one member of the transforming growth factor beta family wherein tyrosine hydroxylase is expressed.
Claim 2
Independent Claim
62 words
A method for inducing the expression of tyrosine hydroxylase in neural cells in vitro comprising culturing mammalian CNS neural cells derived from normally non-dopaminergic, non-tumorigenic tissue in a culture medium to which has been added at least one member of the fibroblast growth factor family and at least one member of the transforming growth factor beta family wherein tyrosine hydroxylase is expressed.
Claim 3
Dependent Claim
46 words
The method of claim 1 wherein said culture medium is defined, said member of the fibroblast growth factory family is FGF-2 and said culture medium comprises a member of the transforming growth factor beta family selected from the group consisting of activin and bone morphogenic protein-2.
Claim 4
Dependent Claim
24 words
The method of claim 1 wherein said normally non-dopaminergic tissue is neural tissue selected from the group consisting of the striatum and the cortex.
Claim 5
Dependent Claim
12 words
The method of claim 1 wherein said culture medium further comprises serum.
Claim 6
Dependent Claim
18 words
The method of claim 1 wherein said neural cells are cultured in the absence of a feeder layer.
Claim 7
Dependent Claim
16 words
The method of claim 1 wherein said neural cells are primary cells obtained from embryonic tissue.
Claim 8
Dependent Claim
11 words
The method of claim 1 wherein said culture medium is defined.
Claim 9
Dependent Claim
28 words
The method of claim 1 wherein said neural cells are cultured on an ionically charged surface selected from the group consisting of poly-D-lysine, poly-L-ornithine, MATRIGEL™, laminin, and fibronectin.
Claim 10
Dependent Claim
11 words
The method of claim 8 wherein said charged surface is poly-L-ornithine.
Claim 11
Dependent Claim
36 words
The method of claim 1 wherein said neural cells are the progeny of at least one multipotent neural stem cell which was induced to proliferate in vitro by the presence of one or more growth factors.
Claim 12
Dependent Claim
19 words
The method of claim 10 wherein said multipotent neural stem cell is derived from juvenile or adult neural tissue.
Claim 13
Dependent Claim
12 words
The method of claim 1 wherein said neural cells are primary cells.
Claim 14
Dependent Claim
38 words
The method of claim 12 wherein said member of the fibroblast growth factor family is FGF-2 and said member of the transforming growth factor beta family is selected from the group consisting of activin and bone morphogenic protein-2.
Claim 15
Independent Claim
71 words
A method of providing dopaminergic cells to a patient comprising transplanting neural cells which are mammalian CNS neural cells derived from normally non-dopaminergic, non-tumorigenic tissue in a culture medium to which has been added at least one member of the fibroblast growth factor family and at least one member of the transforming growth factor beta family to induce the expression of tyrosine hydroxylase in said neural cells into said patient's CNS.
Claim 16
Independent Claim
71 words
A method of providing dopaminergic cells to a patient comprising transplanting neural cells which are mammalian CNS neural cells derived from normally non-dopaminergic, non-tumorigenic tissue in a culture medium to which has been added at least one member of the fibroblast growth factor family and at least one member of the transforming growth factor beta family to induce the expression of tyrosine hydroxylase in said neural cells into said patient's CNS.
Claim 17
Dependent Claim
21 words
The method of claim 14 wherein said patient has Parkinson's disease and said neural cells are administered to said patient's striatum.
Claim 18
Dependent Claim
38 words
The method of claim 14 wherein said member of the fibroblast growth factor family is FGF-2 and said member of the transforming growth factor beta family is selected from the group consisting of activin and bone morphogenic protein.
Claim 19
Dependent Claim
11 words
The method of claim 14 wherein said culture medium is defined.
Claim 20
Dependent Claim
17 words
The method of claim 14 wherein said neural cells are obtained from the CNS of said patient.
Claim 21
Independent Claim
96 words
A method of screening the effects of a drug on dopaminergic cells comprising culturing mammalian CNS neural cells derived from normally non-dopaminergic, non-tumorigenic tissue in a culture medium to which has been added at least one member of the fibroblast growth factor family and at least one member of the transforming growth factor beta family to induce the expression of tyrosine hydroxylase in said neural cells contacting said neural cells with a drug and measuring the response, selected from the group consisting of a dopaminergic response and cell survival, of said dopaminergic cells to said drug.
Claim 22
Independent Claim
96 words
A method of screening the effects of a drug on dopaminergic cells comprising culturing mammalian CNS neural cells derived from normally non-dopaminergic, non-tumorigenic tissue in a culture medium to which has been added at least one member of the fibroblast growth factor family and at least one member of the transforming growth factor beta family to induce the expression of tyrosine hydroxylase in said neural cells contacting said neural cells with a drug and measuring the response, selected from the group consisting of a dopaminergic response and cell survival, of said dopaminergic cells to said drug.
Claim 23
Dependent Claim
22 words
The method of claim 19 wherein said effects are determined by measuring the ability of said cells to produce or metabolize dopamine.
Claim 24
Dependent Claim
13 words
A cell culture comprising tyrosine-hydroxylase-expressing cells prepared by the method of claim 1.
Claim 25
Dependent Claim
44 words
The cell culture of claim 21, wherein said culture medium is defined, said member of the fibroblast growth factor family is FGF-2 and said member of the transforming growth factor beta family is selected from the group consisting of activin and bone morphogenic protein-2.
Claim 26
Dependent Claim
26 words
The cell culture of claim 21 which is cultured on an ionically charged surface selected from the group consisting of poly-D-lysine, poly-L-ornithine, MATRIGEL™, laminin, and fibronectin.
Claim 27
Dependent Claim
24 words
The cell culture of claim 21 wherein said normally non-dopaminergic CNS tissue is selected from the group consisting of the striatum and the cortex.
Claim 28
Dependent Claim
28 words
The cell culture of claim 21 wherein said culture medium further comprises conditioned medium selected from the group consisting of astrocyte conditioned medium and glial cell conditioned medium.
Claim 29
Dependent Claim
45 words
The cell culture of claim 21 wherein said neural cells are the progeny of at least one multipotent neural stem cell proliferated in vitro in the presence of one or more growth factors which induced proliferation of said at least one multipotent neural stem cell.
Claim 30
Dependent Claim
18 words
The cell culture of claim 26 wherein said multipotent neural stem cell is derived from adult neural tissue.
Claim 31
Dependent Claim
31 words
The method of claim 1 wherein said culture medium further comprises at least one member selected from the group consisting of a feeder layer bed of cells and cell conditioned medium.
Claim 32
Dependent Claim
38 words
The method of claim 28 wherein said member of the fibroblast growth factor family is FGF-2 and said member of the transforming growth factor beta family is selected from the group consisting of activin and bone morphogenic protein-2.
Claim 33
Dependent Claim
27 words
The method of claim 28 wherein said culture medium comprises cell conditioned medium selected from the group consisting of astrocyte conditioned medium and glial cell conditioned medium.
Claim 34
Dependent Claim
18 words
The method of claim 30 wherein said cell conditioned medium is derived from rat B49 glial cell line.
Claim 35
Independent Claim
90 words
A method for inducing the expression of tyrosine hydroxylase in neural cells in vitro comprising preparing a cell culture comprising mammalian CNS neural cells derived from normally non-dopaminergic neural tissue and a culture medium to which has been added at least one member of the fibroblast growth factor family and wherein said culture medium additionally comprises a member selected from the group consisting of a feeder layer bed of cells, cell conditioned medium, and members of the TGFβ family to induce the expression of tyrosine hydroxylase in said neural cells.
Claim 36
Independent Claim
90 words
A method for inducing the expression of tyrosine hydroxylase in neural cells in vitro comprising preparing a cell culture comprising mammalian CNS neural cells derived from normally non-dopaminergic neural tissue and a culture medium to which has been added at least one member of the fibroblast growth factor family and wherein said culture medium additionally comprises a member selected from the group consisting of a feeder layer bed of cells, cell conditioned medium, and members of the TGFβ family to induce the expression of tyrosine hydroxylase in said neural cells.
Claim 37
Dependent Claim
12 words
The method of claim 32 wherein said culture medium further comprises serum.
Claim 38
Dependent Claim
44 words
The method of claim 32 wherein said neural cells are the progeny of at least one multipotent neural stem cell proliferated in vitro in the presence of one or more growth factors which induced proliferation of said at least one multipotent neural stem cell.
Claim 39
Dependent Claim
22 words
The method of claim 32 wherein said normally non-dopaminergic tissue is selected from the group consisting of the striatum and the cortex.
Claim 40
Dependent Claim
20 words
The method of claim 32 wherein said culture medium comprises a feeder layer bed derived from normally non-dopaminergic neural tissue.
Claim 41
Dependent Claim
12 words
The method of claim 36 wherein said culture medium further comprises serum.
Claim 42
Dependent Claim
13 words
The method of claim 32 wherein said culture medium comprises cell conditioned medium.
Claim 43
Dependent Claim
15 words
The method of claim 38 wherein said cell conditioned medium is derived from glial cells.
Claim 44
Dependent Claim
14 words
The method of claim 38 wherein said cell conditioned medium is derived from astrocytes.
Claim 45
Dependent Claim
18 words
The method of claim 38 wherein said cell conditioned medium is derived from rat B49 glial cell line.
References Cited (Backward Citations)
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