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Human CNS cell lines and methods of use therefor

a technology of human cns and cell lines, which is applied in the field of cns cell lines, can solve the problems of affecting the development of central nervous system (cns) disorders, difficult to obtain human cns tissue, and limited life span of cultures, and achieve the effect of promoting expression of the growth-promoting gen

Inactive Publication Date: 2005-02-17
SIGNAL PHARMA LLC
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Benefits of technology

[0007] Briefly stated, the present invention provides conditionally-immortalized human CNS progenitor cell lines capable of differentiation into astrocytes and / or neurons. In one aspect, the present invention provides a method for producing a conditionally-immortalized human CNS progenitor cell, comprising (a) plating human progenitor cells on a surface that permits proliferation; (b) adding growth medium to the cells; (c) transfecting the cells with DNA encoding a selectable marker and regulatable growth-promoting gene under conditions promoting expression of the growth-promoting gene; (d) passaging the transfected cells onto a substrate; and (e) adding growth medium supplemented with one or more proliferation-enhancing factors to the transfected cells.

Problems solved by technology

The development of therapies for central nervous system (CNS) disorders has been hampered by the lack of human cells for research and development.
Human CNS tissue is difficult to obtain and is not available on a regular basis.
Primary human CNS cultures derived from such tissue generally express neuronal markers and contain functional ion channels and neurotransmitter receptors, but such cultures have a limited life span (about one month), necessitating frequent dissections and plating.
Accordingly, neither tissue nor primary cultures are capable of supplying the cells needed for extensive research and development.
However, in v-myc immortalized cells, the mitotic activity of the oncogene is always present; cells do not undergo differentiation, and channels and receptors are not functional.
A combination of factors and substrates is needed to differentiate the cells further in vitro, and complete differentiation of these cells has not been achieved.
In addition, species differences continue to render the use of rat CNS cells problematic.
Cloned human CNS channels and receptors provide a potential solution to this problem, but such cloned proteins are not present in their native environment (typically they are expressed in cell lines derived from non-CNS tissue, such as kidney (HEK 293) or cervical (HeLa) cells) and downstream signaling pathways are abnormal.
Expressed receptor subunit combinations are artificial and may not reflect those in vivo, and attempts to match native receptors with heterologously expressed subunits have not been successful in a number of cases.
Consequently, expressed channels and receptors differ fundamentally from their native counterparts and are not optimal for drug development.
This lengthy time period is inconvenient for basic research and prohibitive for use in high-throughput screens of thousands of compounds.
Thus far, however, it has not been possible to generate such cell lines, and the techniques for generating the rat progenitor cell lines have been largely unsuccessful when applied to human cells.

Method used

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  • Human CNS cell lines and methods of use therefor
  • Human CNS cell lines and methods of use therefor
  • Human CNS cell lines and methods of use therefor

Examples

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example 1

5 Preparation and Characterization of Human CNS Progenitor Cell Lines

[0072] This Example illustrates the preparation of a representative conditionally-immortalized human CNS progenitor cell line.

[0073] A. Primary Cell Culture

[0074] Proliferating primary cultures of human fetal CNS progenitor cells were established from whole human fetal brain tissue of 13 weeks gestational age, which was obtained through Advanced Bioscience Resources, Inc. (Alameda, Calif.). The tissue was procured in compliance with state and federal laws and regulations, including those set forth by the Uniformed Anatomical Gift Act and National Organ Transplant Act, and appropriate consent forms were used. Brain tissue fragments were kept for approximately 24 hours at 4° C. in Hank's balanced salt solution (with no calcium or magnesium) containing penicillin (50 I.U. / mL) and streptomycin (50 μg / mL) before dissociation.

[0075] The tissue was dissociated enzymatically and maintained in culture for 5 days before ...

example 2

Differentiation of Human CNS Progenitor Cell Lines

[0083] This Example illustrates the preparation of differentiated CNS cells from the human CNS progenitor cell line described in Example 1, and the characterization of the differentiated cells.

[0084] A. Differentiation of Immortalized Cultures

[0085] To differentiate the immortalized cells, cultures (clone B4, described below) were switched to DMEM / F12 containing N2 supplements and tetracycline (1 μg / mL to suppress transcription of the oncogene). After approximately 7 days of tet addition (tet+RA for 1 day, followed by tet, high K+, NT-3 and BDNF for 6 days) neuronal and glial morphological differentiation occurred (FIG. 2, right panel). Phase-bright cells with long, thin processes were present (suggestive of neuronal morphology), as well as larger phase-dark cells with wider processes (suggestive of astrocyte morphology). In the proliferative growth condition (FIG. 2, left panel), cells exhibited progenitor cell morphology (small ...

example 3

Preparation and Characterization of Clonal Human CNS Progenitor Cell Lines

[0095] This Example illustrates the generation of clonal human CNS progenitor cell lines, and the characterization of such cell lines.

[0096] A. Isolation of Clonal Cell Lines

[0097] Clones were isolated from the human CNS progenitor cell line described in Example 1 by limit dilution in 96-well plates. Clones were fed and passaged as above. Of the distinct clonal cell lines generated, four are described in detail below and are referred to herein as clones B4, C2, E5 and C10.

[0098] EGF and PDGF Enhance Proliferation Rate and Cell Survival. To facilitate the expansion of the human CNS clones, we identified factors that increase the proliferation rate and / or cell survival. To examine the effects of EGF and PDGF on the proliferation and survival of the human CNS clones, 40 ng / mL EGF, 20 ng / mL PDGF or both were added to the standard growth medium, which already contained FGF-2 and 50% CM, and total cell number an...

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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.

Description

TECHNICAL FIELD [0001] The present invention relates generally to CNS cell lines. The invention is more particularly related to conditionally-immortalized human CNS progenitor cell lines and to differentiated cells derived from such cell lines. Such cell lines and / or differentiated cells may be used in the development of therapeutic agents for the prevention and treatment of neurological diseases and other conditions. The present invention is also related to the use of such cell lines and / or differentiated cells in assays and for the study of CNS cell development, death and abnormalities. BACKGROUND OF THE INVENTION [0002] The development of therapies for central nervous system (CNS) disorders has been hampered by the lack of human cells for research and development. Human CNS tissue is difficult to obtain and is not available on a regular basis. Primary human CNS cultures derived from such tissue generally express neuronal markers and contain functional ion channels and neurotransm...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/50A61K45/00A61K48/00A61P25/00C12N5/0797C12N5/10C12N15/09C12Q1/02G01N33/15G01N33/68
CPCA61K48/00C12N5/0623C12N2501/135C12N2501/13C12N2501/11A61P25/00
Inventor SAH, DINAHGAGE, FREDRAY, JASODHARA
Owner SIGNAL PHARMA LLC
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