Human CNS neural stem cells

Claim 1

A cell culture, comprising: (a) a culture medium containing one or more predetermined growth factors effective for inducing multipotent central nervous system (CNS) neural stem cell proliferation; and (b) suspended in the culture medium, human multipotent CNS neural stem cells, wherein (i) the cells are derived from primary CNS neural tissue by growth in culture medium containing one or more predetermined growth factors effective for inducing multipotent CNS neural stem cell proliferation; (ii) the population comprises cells which stain positive for nestin; (iii) in the presence of differentiation-inducing conditions, the cells produce progeny cells that differentiate into neurons, astrocytes, and oligodendrocytes, and (iv) the cells have a doubling rate faster than 30 days.

Claim 10

A method for proliferating human multipotent central nervous system (CNS) neural stem cells, comprising: proliferating multipotent CNS human neural stem cells in serum-free culture medium containing one or more predetermined growth factors effective for inducing multipotent CNS neural stem cell proliferation and at least 0.1 ng/ml LIF, wherein: (a) the cells are derived from primary CNS neural tissue by growth in serum-free culture medium containing one or more predetermined growth factors effective for inducing multipotent CNS neural stem cell proliferation; (b) the population comprises cells which stain positive for nestin; and (c) in the presence of differentiation-inducing conditions, the cells produce progeny cells that differentiate into neurons, astrocytes, and oligodendrocytes.

Claim 11

A method for proliferating human multipotent central nervous system (CNS) neural stem cells, comprising: proliferating multipotent CNS human neural stem cells in serum-free culture medium containing one or more predetermined growth factors effective for inducing multipotent CNS neural stem cell proliferation and at least 0.1 ng/ml LIF, wherein: (a) the cells are derived from primary CNS neural tissue by growth in serum-free culture medium containing one or more predetermined growth factors effective for inducing multipotent CNS neural stem cell proliferation; (b) the population comprises cells which stain positive for nestin; and (c) in the presence of differentiation-inducing conditions, the cells produce progeny cells that differentiate into neurons, astrocytes, and oligodendrocytes.

Claim 13

A method for proliferating human multipotent self-renewing central nervous system (CNS) neural stem cells, comprising: proliferating human multipotent CNS neural stem cells, wherein: (a) the cells are suspended in a culture medium containing one or more growth factors that stimulate proliferation of multipotent self-renewing CNS neural stem cells proliferation, and at least 0.1 ng/ml LIF; (b) the cells are derived from primary CNS neural tissue by growth in serum-free culture medium containing one or more predetermined growth factors effective for inducing multipotent CNS neural stem cell proliferation; (c) the population comprises cells which stain positive for nestin; (d) in the presence of differentiation-inducing conditions, the cells produce progeny cells that differentiate into neurons, astrocytes, and oligodendrocytes; and (e) the cells have a doubling rate faster than 30 days.

Claim 14

A method for proliferating human multipotent self-renewing central nervous system (CNS) neural stem cells, comprising: proliferating human multipotent CNS neural stem cells, wherein: (a) the cells are suspended in a culture medium containing one or more growth factors that stimulate proliferation of multipotent self-renewing CNS neural stem cells proliferation, and at least 0.1 ng/ml LIF; (b) the cells are derived from primary CNS neural tissue by growth in serum-free culture medium containing one or more predetermined growth factors effective for inducing multipotent CNS neural stem cell proliferation; (c) the population comprises cells which stain positive for nestin; (d) in the presence of differentiation-inducing conditions, the cells produce progeny cells that differentiate into neurons, astrocytes, and oligodendrocytes; and (e) the cells have a doubling rate faster than 30 days.

Claim 15

A method for producing a culture of differentiated human CNS neural stem cells having greater than 10% neurons, wherein, of the neurons present, at least 20% are GABA positive, comprising: (a) proliferating human multipotent CNS neural stem cells, wherein: (i) the cells are suspended in a culture medium containing one or more growth factors that stimulate proliferation of multipotent self-renewing CNS neural stem cells, (ii) the cells are derived from primary CNS neural tissue by growth in serum-free culture medium containing one or more predetermined growth factors effective for inducing multipotent CNS neural stem cell proliferation, (iii) the population comprises cells which stain positive for nestin, and (iv) in the presence of differentiation-inducing conditions, the cells produce progeny cells that differentiate into neurons, astrocytes, and oligodendrocytes; and (b) exposing the proliferated cells to a differentiation-inducing culture medium, to induce in vitro differentiation of the neural stem cells to a culture of differentiated cells having greater than 10% neurons, wherein, of the neurons present, at least 20% are GABA positive.

Claim 16

A method for producing a culture of differentiated human CNS neural stem cells having greater than 10% neurons, wherein, of the neurons present, at least 20% are GABA positive, comprising: (a) proliferating human multipotent CNS neural stem cells, wherein: (i) the cells are suspended in a culture medium containing one or more growth factors that stimulate proliferation of multipotent self-renewing CNS neural stem cells, (ii) the cells are derived from primary CNS neural tissue by growth in serum-free culture medium containing one or more predetermined growth factors effective for inducing multipotent CNS neural stem cell proliferation, (iii) the population comprises cells which stain positive for nestin, and (iv) in the presence of differentiation-inducing conditions, the cells produce progeny cells that differentiate into neurons, astrocytes, and oligodendrocytes; and (b) exposing the proliferated cells to a differentiation-inducing culture medium, to induce in vitro differentiation of the neural stem cells to a culture of differentiated cells having greater than 10% neurons, wherein, of the neurons present, at least 20% are GABA positive.

Claim 17

A method for producing a culture of human glioblasts, the method comprising: (a) proliferating human multipotent CNS neural stem cells in a culture medium, wherein: (i) the culture medium contains one or more growth factors that stimulate proliferation of multipotent self-renewing CNS neural stem cells and at least 0.1 ng/ml LIF, (ii) the cells are derived from primary CNS neural tissue by growth in serum-free culture medium containing one or more predetermined growth factors effective for inducing multipotent CNS neural stem cell proliferation, (iii) the population comprises cells which stain positive for nestin, and (iv) in the presence of differentiation-inducing conditions, the cells produce progeny cells that differentiate into neurons, astrocytes, and oligodendrocytes; (b) introducing a polynucleotide into the proliferated cells, wherein the polynucleotide comprises an inducible promoter operably linked to a cell-immortalizing polynucleotide to produce transfected cells; (c) culturing the transfected cells in a differentiation-inducing culture medium, to induce the differentiation of the transfected cells to glial phenotypes; and (d) exposing the transfected cells to culture conditions that induce the expression of the cell-immortalizing polynucleotide in the differentiated neural stem cells, to produce immortalized glioblasts.

Claim 18

A method for producing a culture of human glioblasts, the method comprising: (a) proliferating human multipotent CNS neural stem cells in a culture medium, wherein: (i) the culture medium contains one or more growth factors that stimulate proliferation of multipotent self-renewing CNS neural stem cells and at least 0.1 ng/ml LIF, (ii) the cells are derived from primary CNS neural tissue by growth in serum-free culture medium containing one or more predetermined growth factors effective for inducing multipotent CNS neural stem cell proliferation, (iii) the population comprises cells which stain positive for nestin, and (iv) in the presence of differentiation-inducing conditions, the cells produce progeny cells that differentiate into neurons, astrocytes, and oligodendrocytes; (b) introducing a polynucleotide into the proliferated cells, wherein the polynucleotide comprises an inducible promoter operably linked to a cell-immortalizing polynucleotide to produce transfected cells; (c) culturing the transfected cells in a differentiation-inducing culture medium, to induce the differentiation of the transfected cells to glial phenotypes; and (d) exposing the transfected cells to culture conditions that induce the expression of the cell-immortalizing polynucleotide in the differentiated neural stem cells, to produce immortalized glioblasts.