Stable neural stem cell lines
First Claim
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1. A method for producing a stable cell line of human neural precursor cells in vitro, comprising the steps of:
- a) preparing a culture of neural precursor cells in a serum-free medium;
b) culturing the neural precursor cells in the presence of a first mitogen, wherein said first mitogen is selected from the group consisting of aFGF, bFGF, EGF, TGFα and
combinations thereof;
c) introducing into the neural precursor cells a recombinant DNA construct comprising a receptor ligand-regulated c-myc gene, wherein the c-myc gene is fused with DNA encoding a ligand-binding domain of a nuclear receptor;
d) contacting the neural precursor cells with an agent that binds the ligand-binding domain of the nuclear receptor, wherein the agent comprises a myc-activating chemical selected from the group consisting of β
-estradiol, RU38486, dexamethasone, thyroid hormones, retinoids, and ecdysone;
e) further culturing the neural precursor cells in a medium containing the first mitogen and a second mitogen to produce a stable cell line of neural precursor cells, wherein said second mitogen is selected from the group consisting of aFGF, bFGF, EGF, TGFα
, serum and combinations thereof, with the proviso that the second mitogen is other than the first mitogen, andf) expanding the neural precursor cells of the stable cell line beyond thirty cell doublings in the medium containing the first mitogen and the second mitogen,wherein the expanded neural precursor cells of the stable cell line of neural precursor cells are capable of differentiating into neurons, astrocytes, and oligodendrocytes after removal of the first and second mitogen from the medium.
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Abstract
A systematic and efficient method for establishing stable neural stem cell lines and neuronal progenitor lines is described. The resulting cell lines provide robust, simple, and reproducible cultures of human and other mammalian neurons in commercially useful mass quantities while maintaining normal karyotypes and normal neuronal phenotypes.
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Citations
19 Claims
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1. A method for producing a stable cell line of human neural precursor cells in vitro, comprising the steps of:
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a) preparing a culture of neural precursor cells in a serum-free medium; b) culturing the neural precursor cells in the presence of a first mitogen, wherein said first mitogen is selected from the group consisting of aFGF, bFGF, EGF, TGFα and
combinations thereof;c) introducing into the neural precursor cells a recombinant DNA construct comprising a receptor ligand-regulated c-myc gene, wherein the c-myc gene is fused with DNA encoding a ligand-binding domain of a nuclear receptor; d) contacting the neural precursor cells with an agent that binds the ligand-binding domain of the nuclear receptor, wherein the agent comprises a myc-activating chemical selected from the group consisting of β
-estradiol, RU38486, dexamethasone, thyroid hormones, retinoids, and ecdysone;e) further culturing the neural precursor cells in a medium containing the first mitogen and a second mitogen to produce a stable cell line of neural precursor cells, wherein said second mitogen is selected from the group consisting of aFGF, bFGF, EGF, TGFα
, serum and combinations thereof, with the proviso that the second mitogen is other than the first mitogen, andf) expanding the neural precursor cells of the stable cell line beyond thirty cell doublings in the medium containing the first mitogen and the second mitogen, wherein the expanded neural precursor cells of the stable cell line of neural precursor cells are capable of differentiating into neurons, astrocytes, and oligodendrocytes after removal of the first and second mitogen from the medium. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 19)
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10. A method for producing a stable human neural precursor cells clone in vitro, comprising the steps of:
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a) preparing a culture of neural precursor cells in a serum-free medium; b) culturing the neural precursor cells in the presence of a first mitogen, wherein said first mitogen is selected from the group consisting of aFGF, bFGF, EGF, TGFα and
combinations thereof;c) introducing into the neural precursor cells a recombinant DNA construct comprising a retroviral vector containing the neomycin-resistance gene and a fusion gene of human c-myc cDNA and human estrogen receptor cDNA; d) further culturing the neural precursor cells in a medium containing the first mitogen and a second mitogen, wherein said second mitogen is selected from the group consisting of aFGF, bFGF, EGF, TGFα
, serum and combinations thereof, with the proviso that the second mitogen is other than the first mitogen, wherein the medium includes G418 to select for the neural precursor cells containing the recombinant DNA construct, wherein the medium includes β
-estradiol;e) plating the neural precursor cells obtained from step d) at a cell density of 0.5×
106 to 1.0×
106 cells per 100 mm plate by supplementing the neural precursor cells with unmodified primary cells in the continued presence of G418 in a medium containing the first mitogen and the second mitogen, andf) expanding the neural precursor cells of the stable cell line beyond thirty cell doublings in the continued presence of G418 in the medium containing the first mitogen and the second mitogen, and g) isolating a stable neural precursor clone from the plated neural precursor cells. - View Dependent Claims (11, 12, 13, 14, 15, 16, 17, 18)
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Specification