PNS cell lines and methods of use therefor
a cell line and cell technology, applied in the field of pns cell lines, can solve the problems of limiting the number of cells in such cultures, unable to generally effective treat pns disorders, and affecting the development of pns disorders therapy,
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example 1
Retroviral Optimization for Immortalization of PNS Cell Lines
[0085] This Example illustrates the optimization of efficiency of retroviral infection of human PNS progenitor cells.
[0086] HEK293 cells (ATCC Accession No. CRL 1573) cultured in DMEM, 10% fetal bovine serum plus pen-strep (100 units / mL penicillin G plus 100 μg / mL streptomycin sulfate) were transiently co-transfected, using the calcium phosphate system (Promega ProFection kit, Promega, Madison, Wis.), with pMD.G (Naldini et al., Science 272:263-267, 1996; Naldini et al., Proc. Natl. Acad. Sci. USA 93:11382-11388, 1996) (VSV-G expression), pBS.CMV.gag.pol (within a Bluescript KS+ backbone, the immediate early CMV promoter is used to express gag / pol from Moloney Leukemia Virus, followed by a bovine polyA sequence) and pLINX v-myc (tetracycline-regulatable v-myc; see Hoshimaru et al., Proc. Natl. Acad. Sci. USA 93:1518-1523, 1996) simultaneously. After approximately 16 hours, the supernatant from the culture was collected a...
example 2
Human PNS Cell Line Development
[0088] This Example illustrates the conditional immortalization of human DRG neural precursors.
[0089] Human fetal DRG cultures were retrovirally infected with VSV-G pseudotyped LINX v-myc, the tetracycline-regulatable v-myc oncogene. A number of putative clones were isolated by limiting dilution. Specifically, DRGs were dissociated with 1 mg / ml collagenase plus 3.6 mg / ml dispase for 20 to 40 minutes at 37° C., with mechanical trituration every 10 minutes. In one case, DNAse (0.1 mg / ml) was included during the last trituration set. The dissociated cells were plated in t-15C (see below for components), FGF-2 (40 ng / ml) plus pen-strep (50 units / mL penicillin G plus 50 μg / mL streptomycin sulfate) onto fibronectin-coated tissue culture plastic at a density of 1.1-2.5×105 cells per 60 mm dish. Cultures were infected for 19 hours with 50-100 μL of VSV-G pseudotyped LINX v-myc retrovirus in the presence of 4 μg / ml polybrene. The retrovirus was then removed b...
example 3
Rat PNS Cell Line Development
[0108] This Example illustrates the conditional immortalization of rat neural crest stem cells and DRG neural precursor cells.
A. Immortalization of Neural Crest Stem Cells
[0109] Cultures of neural crest stem cells were successfully infected with the tetracycline-regulatable v-myc oncogene. It was necessary to modify the published protocol for culturing neural crest stem cells (Stemple and Anderson, Cell 71:973-985, 1992) in order to successfully infect neural crest stem cell cultures with the immortalizing oncogene. Specifically, in order to successfully infect neural crest stem cells, it was necessary to increase the concentration of FGF-2 from 4 ng / ml to 40 ng / ml, and eliminate retinoic acid, EGF and NGF from the published feeding medium. Embryonic day 10.5 rat neural tubes were dissected, dissociated partially with collagenase, and then plated into L-15C plus FGF-2 onto a fibronectin substrate. Cultures were infected for about 24 hours with amphot...
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