Human CNS cell lines and methods of use therefor
AssigneeSignal Pharmaceuticials, Inc.
InventorsDinah W. Y. Sah, Fred H. Gage, Jasodhara Ray
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Abstract
Conditionally-immortalized human CNS progenitor cell lines are provided. Such cell lines, which may be clonal, may be used to generate neurons and/or astrocytes. Such cell lines and/or differentiated cells may be used for the development of therapeutic agents to prevent and treat a variety of CNS-related diseases. Such cell lines and/or differentiated cells may also be used in assays and for the general study of CNS cell development, death and abnormalities.
Claims23
A method for producing a conditionally-immortalized human CNS cell, comprising:
- (a) plating human CNS progenitor cells on a surface that permits proliferation, said surface being tissue culture plastic or a surface treated with fibronectin;
- (b) adding serum-free growth medium to the cells;
- (c) allowing the CNS progenitor cells to proliferate in the serum-free medium;
- (d) transfecting the cells with DNA encoding a selectable marker and regulatable growth-promoting gene, wherein the growth-promoting gene encodes a protein selected from the group consisting of SV40 large T antigen, v-myc, N-myc, c-myc, p53, polyoma large T antigen, E1a adenovirus and E7 protein of human papilloma virus;
- (e) passaging the transfected cells onto a substrate;
- and
- (f) adding serum-free growth medium containing one or more proliferation-enhancing factors to the transfected cells, wherein said proliferation-enhancing factors are selected from the group consisting of FGF-2, PDGF, EGF, medium conditioned by perpetualized adult rat hippocampal progenitor cells, and a combination thereof;
- and therefrom producing a conditionally-immortalized human CNS progenitor cell that differentiates only upon suppression of the growth-promoting protein.
A conditionally-immortalized clonal human CNS progenitor cell that is transfected with an externally-regulated gene encoding a growth-promoting protein, wherein said cell differentiates into neurons and astrocytes, but not into oligodendrocytes, following suppression of production or growth-promoting activity of the growth-promoting protein.
A method for producing astrocytes and neurons, wherein the number of neurons produced predominates over the number of astrocytes produced, comprising culturing a conditionally-immortalized clonal human CNS progenitor cell that is transfected with an externally-regulated growth promoting gene in serum-free medium under conditions inhibiting expression of the growth promoting gene.
A method for screening for an agent that modulates activity of a protein produced by a CNS cell, comprising:
- (a) contacting a conditionally-immortalized clonal human CNS progenitor cell that is transfected with an externally-regulated growth promoting gene with a candidate agent;
- and
- (b) subsequently determining whether said candidate agent detectable suppresses or enhances an activity of a protein produced by said cell, wherein the protein is an ion channel, a growth factor receptor, a neurotransmitter receptor, a transcription factor, a MAP kinase, a caspase, a presinillin or an amyloid precursor protein;
- and therefrom determining whether the candidate agent modulates an activity of a protein produced by a CNS cell.
A method for detecting the presence or absence of a protein in a sample, comprising
- (a) contacting a sample with a conditionally-immortalized clonal human CNS progenitor cell that is transfected with an eternally-regulated growth promoting gene;
- and
- (b) subsequently detecting a response in said cell, wherein the response is indicative of the presence or absence of a protein in said sample, wherein the protein is an ion channel, a growth factor receptor, a neurotransmitter receptor, a transcription factor, a MAP kinase, a caspase, a presinillin or an amyloid precursor protein.
A method for screening for an agent that affects CNS cell death, comprising:
- (a) contacting a conditionally-immortalized clonal human CNS progenitor cell that is transfected with an externally-regulated growth promoting gene with a candidate agent under conditions that, in the absence of candidate agent, result in death of said cell;
- and
- (b) subsequently measuring the ability of said candidate agent to affect the death of said cell.
A method for screening for a protein that regulates CNS cell death, comprising:
- (a) altering the level of expression of a protein within a conditionally-immortalized clonal human CNS progenitor cell that is transfected with an externally-regulated growth promoting gene;
- and
- (b) subsequently measuring the affect of said alteration on the death of said cell, and thereby identifying a protein that regulates CNS cell death.
Dependencies are inferred from the claim text.