US5750376A — In vitro growth and proliferation of genetically …

Claim 1

Independent Claim 156 words

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.

Claim 2

Independent Claim 156 words

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.

Claim 3

Dependent Claim 42 words

The method of claim 1 wherein the growth factor in the culture medium prepared in (b) is selected from the group consisting of acidic fibroblast growth factor, basic fibroblast growth factor, epidermal growth factor, amphiregulin, transforming growth factor alpha, and combinations thereof.

Claim 4

Dependent Claim 25 words

The method of claim 1 wherein said growth factor in the culture medium prepared in (b) is epidermal growth factor or transforming growth factor alpha.

Claim 5

Dependent Claim 21 words

The method of claim 1 wherein said growth factor in the culture medium prepared in (b) is a fibroblast growth factor.

Claim 6

Dependent Claim 17 words

The method of claim 4 wherein said culture medium prepared in (b) additionally contains epidermal growth factor.

Claim 7

Dependent Claim 20 words

The method of claim 1 wherein the growth factor in the culture medium prepared in (b) is epidermal growth factor.

Claim 8

Dependent Claim 14 words

The method of claim 1 wherein the culture medium prepared in (b) is defined.

Claim 9

Dependent Claim 21 words

The method of claim 1 wherein said multipotent neural stem cell progeny are genetically modified to produce a growth factor product.

Claim 10

Dependent Claim 53 words

The method of claim 8 wherein said growth factor product is selected from the group consisting of nerve growth factor, brain-derived neurotrophic factor, neurotrophins, ciliary neuroatrophic factor, amphiregulin, basic fibroblast growth factor, acidic fibroblast growth factor, epidermal growth factor, transforming growth factor-alpha, transforming growth factor-beta, platelet-derived growth factor, insulin-like growth factors, and interleukins.

Claim 11

Dependent Claim 19 words

The method of claim 1 wherein said multipotent neural stem cell progeny are genetically modified to produce a neuropeptide.

Claim 12

Dependent Claim 30 words

The method of claim 10 wherein said neuropeptide is selected from the group consisting of substance-P, neuropeptide-γ, enkephalin, vasopressin, vasoactive intestinal peptide, cholecystokinin, glucagon, bombesin, somatostatin, and calcitonin gene-related peptide.

Claim 13

Dependent Claim 21 words

The method of claim 1 wherein said multipotent neural stem cell progeny are genetically modified to express a growth factor receptor.

Claim 14

Dependent Claim 40 words

The method of claim 12 wherein said growth factor receptor is selected from the group consisting of low affinity nerve growth factor receptor, ciliary neurotrophic factor receptor, neurotrophin receptors, epidermal growth factor receptor, fibroblast growth factor receptor, and amphiregulin receptor.

Claim 15

Dependent Claim 19 words

The method of claim 1 wherein said multipotent neural stem cell progeny are genetically modified to express a neurotransmitter.

Claim 16

Dependent Claim 32 words

The method of claim 14 wherein said neurotransmitter is selected from the group consisting of serotonin, L-dopa, dopamine, norepinephrine, epinephrine, tachykinin, endorphin, histamine, N-methyl D-aspartate, glycine, glutamate, γ-amino butyric acid, and acetylcholine.

Claim 17

Dependent Claim 21 words

The method of claim 1 wherein said multipotent neural stem cell progeny are genetically modified to contain a neurotransmitter synthesizing gene.

Claim 18

Dependent Claim 34 words

The method of claim 16 wherein said neurotransmitter synthesizing gene is selected from the group consisting of tyrosine hydroxylase, dopa decarboxylase, dopamine-β-hydroxylase, phenylethanolamine N-methyltransferase, glutamic acid decarboxylase, tryptophan hydroxylase, choline acetyltransferase, and histidine decarboxylase.

Claim 19

Dependent Claim 20 words

The method of claim 1 wherein said multipotent neural stem cell progeny are genetically modified to express a neurotransmitter receptor.

Claim 20

Dependent Claim 21 words

The method of claim 1 wherein said multipotent neural stem cell progeny are genetically modified to express chromaffin granule amine transporter.

Claim 21

Dependent Claim 24 words

The method of claim 1 wherein the multipotent neural stem cell progeny produced in (c) grow in the form of clonally-derived clusters of cells.

Claim 22

Dependent Claim 71 words

The method of claim 1 wherein prior to genetically modifying said proliferating multipotent neural stem cell progeny, a subsequent cell culture is prepared by combining said multipotent neural stem cell progeny with fresh substantially serum-free culture medium containing one or more predetermined growth factors which induces multipotent neural stem cell proliferation to proliferate said daughter multipotent neural stem cells to produce more progeny which include more daughter multipotent neural stem cells.

Claim 23

Dependent Claim 17 words

The method of claim 1 wherein said mammalian neural tissue is obtained from a juvenile or adult.

Claim 24

Dependent Claim 15 words

The method of claim 1 wherein said mammalian neural tissue is obtained from a human.

Claim 25

Independent Claim 123 words

A method of producing genetically modified neural cells comprising: (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.

Claim 26

Independent Claim 123 words

A method of producing genetically modified neural cells comprising: (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.

Claim 27

Independent Claim 119 words

A method of producing genetically modified neural cells comprising: (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.

Claim 28

Independent Claim 119 words

A method of producing genetically modified neural cells comprising: (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.

Claim 29

Dependent Claim 48 words

The method of claim 1 further comprising: (d) subjecting said genetically modified multipotent neural stem cell progeny to culture conditions that induce neural cell differentiation to produce a cell culture comprising genetically modified, differentiated neural cells selected from the group consisting of astrocytes, neurons, oligodendrocytes, and combinations thereof.

Claim 30

Dependent Claim 24 words

The method of claim 26 wherein in (d) the genetically modified multipotent neural stem cell progeny are differentiated in a culture medium containing serum.

Claim 31

Dependent Claim 32 words

The method of claim 26 wherein in (d) the genetically modified multipotent neural stem cell progeny are differentiated on a fixed substrate to which said multipotent neural stem cell progeny can adhere.

Claim 32

Dependent Claim 29 words

The method of claim 26 wherein in (d) the genetically modified multipotent neural stem cell progeny are differentiated in a culture medium containing a growth factor that influences differentiation.

Claim 33

Independent Claim 172 words

A method of producing genetically modified differentiated 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, (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.

Claim 34

Independent Claim 172 words

A method of producing genetically modified differentiated 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, (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.

Claim 35

Dependent Claim 17 words

A composition comprising genetically modified multipotent neural stem cell progeny prepared by the method of claim 1.

Claim 36

Dependent Claim 43 words

A composition according to claim 31 wherein said genetically modified multipotent neural stem cell progeny are genetically modified to express a biologically active substance selected from the group consisting of growth factor products, growth factor receptors, neurotransmitters, neurotransmitter receptors, neuropeptides, and neurotransmitter-synthesizing genes.

Claim 37

Dependent Claim 18 words

A composition comprising genetically modified differentiated multipotent neural stem cell progeny prepared by the method of claim 26.

Claim 38

Dependent Claim 18 words

A composition comprising genetically modified differentiated multipotent neural stem cell progeny prepared by the method of claim 30.

Claim 39

Dependent Claim 15 words

A composition comprising genetically modified differentiated neural cells produced by the method of claim 30.

Claim 40

Dependent Claim 41 words

A composition according to claim 35 wherein said genetically modified differentiated neural cells are genetically modified to express a biologically active substance selected from the group consisting of growth factor products, growth factor receptors, neurotransmitters, neurotransmitter receptors, neuropeptides, and neurotransmitter-synthesizing genes.

Claim 41

Dependent Claim 78 words

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.

Claim 42

Dependent Claim 78 words

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.

Claim 43

Dependent Claim 33 words

The composition of claim 37 wherein said percentage of multipotent neural stem cells of said population of non-primary neural cells is at least ten fold higher than that of said primary cell culture.

Claim 44

Dependent Claim 17 words

The composition of claim 37 wherein said multipotent neural stem cells are derived from human neural tissue.

Claim 45

Dependent Claim 19 words

The composition of claim 37 wherein said multipotent neural stem cells are derived from juvenile or adult neural tissue.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	2001-05-11	2001-11-10	2002-05-12	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	2005-05-11	2005-11-10	2006-05-12	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	2009-05-11	2009-11-10	2010-05-12	$7700.00	Window Closed / Lapsed

In vitro growth and proliferation of genetically modified multipotent neural stem cells and their progeny

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