In vitro growth and proliferation of genetically modified multipotent neural stem cells and their progeny
DCFirst Claim
1. A method of producing genetically modified neural cells comprising:
- (a) obtaining cells derived from mammalian neural tissue containing at least one multipotent neural stem cell capable of producing progeny that are capable of differentiating into neurons, astrocytes, and oligodendrocytes, wherein said obtained cells have not been cultured in a serum-containing medium;
(b) preparing a substantially serum-free culture medium containing one or more predetermined growth factors capable of inducing multipotent neural stem cell proliferation, and(c) preparing a cell culture by combining the cells obtained in (a) with the culture medium prepared in (b) to induce proliferation of said multipotent neural stem cell to produce proliferating multipotent neural stem cells and proliferating multipotent neural stem cell progeny which includes daughter multipotent neural stem cells and introducing exogenous DNA that does not contain oncogenes to genetically modify said proliferating multipotent neural stem cells and/or said multipotent neural stem cell progeny to produce genetically modified neural stem cell progeny.
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Abstract
A method for producing genetically modified neural cells comprises culturing cells derived from embryonic, juvenile, or adult mammalian neural tissue with one or more growth factors that induce multipotent neural stem cells to proliferate and produce multipotent neural stem cell progeny which include more daughter multipotent neural stem cells and undifferentiated progeny that are capable of differentiating into neurons, astrocytes, and oligodendrocytes. The proliferating neural cells can be transfected with exogenous DNA to produce genetically modified neural stem cell progeny. The genetic modification can be for the production of biologically useful proteins such as growth factor products, growth factor receptors, neurotransmitters, neurotransmitter receptors, neuropeptides and neurotransmitter synthesizing genes. The multipotent neural stem cell progeny can be continuously passaged and proliferation reinitiated in the presence of growth factors to result in an unlimited supply of neural cells for transplantation and other purposes. Culture conditions can be provided that induce the genetically modified multipotent neural stem cell progeny to differentiate into neurons, astrocytes, and oligodendrocytes in vitro.
567 Citations
40 Claims
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1. A method of producing genetically modified neural cells comprising:
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(a) obtaining cells derived from mammalian neural tissue containing at least one multipotent neural stem cell capable of producing progeny that are capable of differentiating into neurons, astrocytes, and oligodendrocytes, wherein said obtained cells have not been cultured in a serum-containing medium; (b) preparing a substantially serum-free culture medium containing one or more predetermined growth factors capable of inducing multipotent neural stem cell proliferation, and (c) preparing a cell culture by combining the cells obtained in (a) with the culture medium prepared in (b) to induce proliferation of said multipotent neural stem cell to produce proliferating multipotent neural stem cells and proliferating multipotent neural stem cell progeny which includes daughter multipotent neural stem cells and introducing exogenous DNA that does not contain oncogenes to genetically modify said proliferating multipotent neural stem cells and/or said multipotent neural stem cell progeny to produce genetically modified neural stem cell progeny. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 28, 29, 31, 32, 33)
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24. A method of producing genetically modified neural cells comprising:
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(a) obtaining cells derived from juvenile or adult mammalian neural tissue containing at least one multipotent neural stem cell capable of producing progeny that are capable of differentiating into neurons, astrocytes, and oligodendrocytes; (b) preparing a culture medium containing one or more predetermined growth factors capable of inducing multipotent neural stem cell proliferation; and (c) preparing a cell culture by combining the cells obtained in (a) with the culture medium prepared in (b) to induce proliferation of said multipotent neural stem cell to produce multipotent neural stem cell progeny which includes daughter multipotent neural stem cells and introducing exogenous DNA that does not contain oncogenes to genetically modify said multipotent neural stem cell progeny.
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25. A method of producing genetically modified neural cells comprising:
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(a) obtaining cells derived from human neural tissue containing at least one multipotent neural stem cell capable of producing progeny that are capable of differentiating into neurons, astrocytes, and oligodendrocytes; (b) preparing a culture medium containing one or more growth factors capable of inducing multipotent neural stem cell proliferation; and (c) preparing a cell culture by combining the cells obtained in (a) with the culture medium prepared in (b) to induce proliferation of said multipotent neural stem cell to produce multipotent neural stem cell progeny which includes daughter multipotent neural stem cells and introducing exogenous DNA that does not contain oncogenes to genetically modify said multipotent neural stem cell progeny.
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30. A method of producing genetically modified differentiated neural cells comprising:
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(a) obtaining cells derived from mammalian neural tissue containing at least one multipotent neural stem cell capable of producing progeny that are capable of differentiating into neurons, astrocytes, and oligodendrocytes, wherein said obtained cells have not been cultured in a serum-containing medium; (b) preparing a substantially serum-free culture medium containing one or more predetermined growth factors capable of inducing multipotent neural stem cell proliferation, (c) preparing a cell culture by combining the cells obtained in (a) with the culture medium prepared in (b) to induce proliferation of said multipotent neural stem cell to produce proliferating multipotent neural stem cells and multipotent neural stem cell progeny which includes daughter multipotent neural stem cells, and (d) subjecting the cells proliferated in (c) to culture conditions that induce neural cell differentiation to produce differentiating and differentiated neural cells, and introducing exogenous DNA that does not contain oncogenes to genetically modify said differentiated and/or differentiating neural cells to produce genetically modified differentiated neural stem cell progeny. - View Dependent Claims (34, 35, 36)
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- 37. A composition comprising a population of non-primary neural cells which are derived from a primary cell culture, said population of non-primary neural cells having a greater percentage of multipotent neural stem cells compared to that of said primary culture, wherein a single multipotent neural stem cell is capable of producing progeny that are capable of differentiating into neurons, and glia, including astrocytes, and wherein said multipotent neural stem cells are genetically modified and do not contain exogenous oncogenes.
Specification