Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Reexamination Certificate
1998-04-14
2004-12-28
Falk, Anne-Marie (Department: 1632)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of regulating cell metabolism or physiology
C435S004000, C435S325000, C435S353000, C435S366000, C435S368000, C435S455000
Reexamination Certificate
active
06835567
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to PNS cell lines. The invention is more particularly related to conditionally-immortalized neural crest stem cell lines and dorsal root ganglion 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 PNS cell development, death and abnormalities.
BACKGROUND OF THE INVENTION
The peripheral nervous system (PNS), which comprises the autonomic nervous system and the sensory nervous system, can be affected by a variety of disorders that are currently difficult to treat. For example, there are no generally effective therapies for PNS disorders such as chronic pain, diabetic neuropathy, chemotherapy-induced neuropathy and Charcot-Marie Tooth, a genetic form of peripheral neuropathy. The development of therapies for PNS disorders has been considerably hampered by the lack of sufficient cells for research and development.
The molecular and cellular properties of sensory neurons have generally been studied in primary dorsal root ganglion (DRG) cultures. Such cultures contain neurons that exhibit many of the specialized features of sensory neurons. However, the limited number of cells in such cultures represents a substantial drawback for most biochemical and molecular studies, which require more material that such cultures can readily provide.
To overcome this limitation, some researchers have generated immortalized cell lines that can be differentiated into neurons exhibiting certain sensory characteristics. For example, sensory neuronal-like cell lines have been established by fusion of post-mitotic embryonic (F-11 cell line; Platika et al.,
Proc. Natl. Acad. Sci. USA
82:3499-3503, 1985) or neonatal (ND cell lines; Wood et al.,
Proc. R. Soc. Lond. B
241:187-194, 1990) rat DRG neurons with mouse N18Tg2 neuroblastoma cells. These hybrid cell lines exhibit some DRG-selective properties, including transcription factors, cytoskeletal proteins, neurotransmitters and neurotransmitter receptors. In addition, voltage-gated currents, synaptic proteins, glycosylphosphatidylinositol-linked molecules and apoptotic responses in the cells have been reported. Such properties have made these cell lines beneficial for research and drug development, but the use of these cell lines has been limited by the low percentage of cells that differentiate and by the loss of the ability to differentiate upon passaging. Furthermore, some cell lines exhibit both neural and glial characteristics, and human cells are presently unavailable.
Accordingly, there is a need in the art for stable PNS lines that can be readily differentiated. 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 PNS progenitor cell lines capable of differentiation into neurons. In one aspect, the present invention provides methods for producing a conditionally-immortalized rat neural crest stem cell, comprising: (a) transfecting rat neural crest cells plated on a first surface and in a first growth medium that permit proliferation with DNA encoding a selectable marker and regulatable growth-promoting gene; and (b) passaging the transfected cells onto a second surface and in a second growth medium that permit attachment and proliferation; and therefrom producing a conditionally-immortalized rat neural crest stem cell. Suitable surfaces include substrates comprising one or more of a polyamino acid, fibronectin, laminin, collagen or tissue culture plastic. Within certain embodiments, the growth-promoting gene is an oncogene such as v-myc.
Within related aspects, the present invention provides conditionally-immortalized rat neural crest stem cells capable of differentiation into neurons.
In further aspects, methods are provided for producing a conditionally-immortalized dorsal root ganglion progenitor cell, comprising: (a) transfecting dorsal root ganglion progenitor cells plated on a first surface and in a first growth medium that permit proliferation with DNA encoding a selectable marker and regulatable growth-promoting gene; and (b) passaging the transfected cells onto a second surface and in a second growth medium that permit attachment and proliferation; and therefrom producing a conditionally-immortalized dorsal root ganglion progenitor cell Within certain embodiments, the dorsal root ganglion progenitor cells are rat or human cells. Suitable surfaces include substrates comprising one or more of a polyamino acid, fibronectin, laminin, collagen or tissue culture plastic. Within certain embodiments, the growth-promoting gene is an oncogene such as v-myc.
Within related aspects, the present invention provides conditionally-immortalized dorsal root ganglion progenitor cells capable of differentiation into neurons. Within certain embodiments, the conditionally-immortalized dorsal root ganglion progenitor cells are transfected rat or human cells. Such cells may be capable of differentiation into sensory neurons and/or nociceptive sensory neurons.
Within further aspects, the present invention provides methods for producing neurons, comprising culturing a cell as described above under conditions inhibiting expression of the growth-promoting gene. Within certain embodiments, the cells are conditionally-immortalized rat or human dorsal root ganglion progenitor cells, and wherein the cells are cultured on a substrate in the presence of one or more differentiating agents.
Within related aspects, the present invention provides neurons produced as described above.
The present invention further provides methods for determining whether conditionally-immortalized dorsal root ganglion progenitor cells are capable of differentiation into neurons, comprising the step of determining the presence or absence of &bgr;III-tubulin positive cells in the proliferative growth condition, and therefrom determining whether the cells are capable of differentiation into neurons.
Within other aspects, methods are provided for transplanting a PNS cell into a mammal, comprising administering to a mammal a cell as described above.
Within further aspects, methods are provided for treating a patient, comprising administering to a patient a cell as described above. Within certain embodiments, the patient may be afflicted with chronic pain and/or a pathological condition characterized by neurodegeneration (e.g., a neuropathy).
Methods are also provided for screening for an agent that modulates activity of a protein produced by a PNS 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 activity of a protein produced by the cell.
The present invention further provides methods for detecting the presence or absence of a protein in a sample, comprising: (a) contacting a sample with a cell as described above; and (b) subsequently detecting a response in the cell, and therefrom detecting the presence of a protein in the sample.
Methods are further provided for identifying a human PNS gene or protein, comprising detecting the presence of a gene or protein within a culture of cells as described above.
Within further aspects, the present invention provides methods for screening for an agent that affects PNS cell death, comprising: (a) contacting a cell 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 further provides methods for screening for a protein that regulates PNS cell death, comprising: (a) altering the level of expression of a protein within a cel
Raymon Heather K.
Sah Dinah W. Y.
Falk Anne-Marie
Signal Pharmaceuticals Inc.
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