Human CNS neural stem cells

• US 5,968,829 A
• Filed: 09/05/1997
• Issued: 10/19/1999
• Est. Priority Date: 09/05/1997
• Status: Expired due to Term

• Current Assignee: BOCO Silicon Valley, Inc.
• Original Assignee: Cytotherapeutics, Inc.
• Inventors: Carpenter, Melissa
• Primary Examiner(s): RAILEY, JOHNNY F
• Application Number:
  US08/926,313
• Time in Patent Office: 774 Days
• Field of Search: 435/368, 435/377, 435/467, 424/93.7
• US Class Current: 435/467
• CPC Class Codes: A61K 2035/126 Immunoprotecting barriers, e.g. jackets, diffusion chambers
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61K 35/30 Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/34 Sugars
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2501/392 Sexual steroids
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 5/0623 Stem cells
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025

Abstract

Isolation, characterization, proliferation, differentiation and transplantation of mammalian neural stem cells is disclosed.

Claims

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.

2. The cell culture of claim 1, wherein the cells have a doubling rate of 5-10 days.

3. The cell culture of claim 1, wherein the cells comprise human forebrain-derived CNS neural stem cells.

4. A cell culture comprising differentiated human central nervous system (CNS) neural stem cells, wherein greater than 10% of the differentiated human neural stem cells are neurons and wherein, of the neurons present, at least 20% are GABA positive.

5. The culture of claim 4 wherein the cell culture comprises at least 20% neurons.

6. A cell culture, comprising differentiated human neural stem cells, wherein said differentiated cells comprise immortalized glioblasts, produced by:

(a) proliferating a population of human multipotent CNS neural stem cells in a culture medium, wherein;

(i) the cells are derived from primary CNS neural tissue by growth in a 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; and

(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.

7. 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.

8. The method of claim 7, further comprising the step of:

exposing the proliferated cells of claim 7 to differentiation-inducing conditions or to an appropriate tissue environment, to produce progeny cells that differentiate into neurons, astrocytes, and oligodendrocytes.

13. The method of claim 7 or 9, wherein the culture medium contains at least 10 ng/ml LIF.

9. 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.

10. 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.

11. 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.

12. The method of claim 11, wherein the coding polynucleotide codes for SV40 large T antigen.