Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1998-04-13
2001-05-01
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S368000, C435S377000, C536S023500, C536S023720
Reexamination Certificate
active
06225122
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to human spinal cord cell lines. The invention is more particularly related to conditionally-immortalized spinal cord cell lines capable of differentiation into neurons, 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 spinal cord-related diseases and injuries, as well as pain. The present invention is also related to the use of such cell lines and/or differentiated cells within various assays and for the study of spinal cord cell development and differentiation.
BACKGROUND OF THE INVENTION
The spinal cord plays a critical role in the functioning of the central nervous system (CNS). Although the spinal cord is the simplest region of the CNS, it contains diverse neural cell types which are interconnected in intricate patterns. During embryonic development, multi-potent stem cells in the neural tube proliferate and then ultimately differentiate into neurons and glia according to extrinsic cues and intrinsic determinants. Diseases and injuries that affect the functioning of spinal cord neural cells are often debilitating, and generally remain difficult to treat effectively.
To develop improved therapies for such disorders, further study of human spinal cord neurons and the differentiation process is needed. To date, such study has been hampered by the difficulty of obtaining sufficient primary human CNS tissue for research and the limited lifespan of primary cultures. In fact, research to date has failed to even demonstrate the existence of neuronal-restricted precursor cells in the human fetal spinal cord. As a result of the difficulties associated with performing human spinal cord research, the differentiation process remains poorly understood and the development of therapies for spinal cord diseases and injuries has been impeded.
Accordingly, there is a need in the art for stable spinal cord lines that can be readily differentiated, and can be used in the development of therapies for spinal cord-related conditions. The present invention fulfills these needs and further provides other related advantages.
SUMMARY OF THE INVENTION
Briefly stated, the present invention provides conditionally-immortalized human spinal cord cell lines capable of differentiation into neurons. In one aspect, the present invention provides methods for producing a conditionally-immortalized human spinal cord neural precursor cell, comprising the steps of: (a) transfecting human spinal cord cells plated on a first surface and in a first growth medium that permit proliferation with DNA encoding a selectable marker and an externally regulatable growth-promoting gene; and (b) selecting the transfected cells on a second surface and in a second growth medium that permit attachment and proliferation, and therefrom producing a conditionally-immortalized human spinal cord neural precursor cell. Within certain embodiments, the first and second surfaces are independently selected from the group consisting of substrates comprising one or more of a polyamino acid (e.g., polylysine or polyornithine), fibronectin, laminin or tissue culture plastic. Similarly, in certain embodiments, the growth-promoting gene may be an oncogene, such as v-myc, and expression of the growth-promoting gene may, but need not, be inhibited by tetracycline.
Within other aspects, the present invention provides conditionally-immortalized human spinal cord neural precursor cells capable of differentiation into neurons such as motoneurons.
The present invention further provides methods for producing neurons, comprising culturing a conditionally-immortalized human spinal cord neural precursor cell as described above under conditions inhibiting expression of the growth-promoting gene. Within certain embodiments, the cell may be cultured in medium comprising tetracycline and/or in the presence of one or more differentiating agents such as NGF, CNTF and BDNF. Within related aspects, the present invention provides neurons, such as motoneurons, produced as described above.
The present invention further provides methods for transplanting a human spinal cord cell into a mammal, comprising administering to a mammal a cell, including neurons, produced as described above. Within related aspects, methods are provided for treating a spinal cord-related condition in a patient, comprising administering to a patient a cell, including neurons, produced as described above.
In other aspects, methods are provided for screening for an agent that modulates an activity of a protein produced by a human spinal cord cell, comprising: (a) contacting a cell produced as described above with a candidate agent; and (b) subsequently measuring the ability of the candidate agent to modulate an activity of a protein produced by the cell.
Within further aspects, the present invention provides methods for detecting the presence or absence of a protein in a sample, comprising: (a) contacting a sample with a cell produced as described above; and (b) subsequently detecting a response in the cell, and therefrom detecting the presence of a protein in the sample.
The present invention also provides methods for identifying a human spinal cord gene or protein, comprising detecting the presence of a gene or protein within a culture of cells produced as described above.
In further aspects, methods are provided for screening for an agent that affects human spinal cord cell death, comprising: (a) contacting a cell produced as described above with a candidate agent under conditions that, in the absence of candidate agent, result in death of the cell; and (b) subsequently measuring the ability of the candidate agent to affect the death of the cell.
The present invention also provides methods for screening for a protein that regulates human spinal cord cell death, comprising: (a) altering the level of expression of a protein within a cell produced as described above; and (b) subsequently measuring the effect of the alteration on the death of the cell, and therefrom identifying a protein that regulates human spinal cord neural precursor cell death.
Within still other aspects, the present invention provides conditionally-immortalized human spinal cord neural precursor cells produced as described above. Such cells may be present within a clonal cell line.
These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.
REFERENCES:
patent: 2 294 946 (1996-05-01), None
patent: WO 97/30168 (1997-08-01), None
patent: WO 98/10058 (1998-03-01), None
Kato et al. Overlapping and additive effects on neurotrophins and CNTF on cultured human spinal cord neurons. Experimental Neurobiology vol. 130 pp. 196-201, 1994.*
Tornatore et al. Expression of tyrosine hydroxylase in an immortalized human fetal astrocyte cell line; in vitro characterization and engraftment into the rodent striatum. Cell Transplantation vol. 5 pp. 145-163, 1996.*
Johe et al. Sincle factors direct the differentiation of stem cells from the fetal and adult central nervous system. Genes and Development vol. 10 pp. 3129-3140, 1996.*
Li et al., “Conditionally Immortalized Neuronal Restricted Cell Lines From Human Spinal Cord,”Society For Neuroscience Abstracts 24(1-2): p. 1529, 1998.
Cashman et al., “Neuroblastoma × Spinal Cord (NSC) Hybrid Cell Lines Resemble Developing Motor Neurons,”Developmental Dynamics 194:209-221, 1992.
Deloulme et al., “Establishment of Pure Neuronal Cultures From Fetal Rat Spinal Cord and Proliferation of the Neuronal Precursor Cells in the Presence of Fibroblast Growth Factor,”Journal of Neuroscience Research 29:499-509, 1991.
Hoshimaru et al., “Differentiation of the immortalized adult neuronal progenitor cell line HC2S2 into neurons by regulatable suppression of the v-myc oncogene,”Proc. Natl. Acad. Sci. USA
Li Rong-hao
Sah Dinah W. Y.
Brusca John S.
Pennie & Edmonds LLP
Signal Pharmaceuticals Inc.
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