Establishment and properties of a growth factor-dependent, perpetual neural stem cell line from the human CNS.
The ready availability of unlimited quantities of neural stem cells derived from the human brain holds great interest for basic and applied neuroscience, including therapeutic cell replacement and gene transfer following transplantation. We report here the combination of epigenetic and genetic procedures for perpetuating human neural stem cell lines. Thus we tested various culture conditions and genes for those that optimally allow for the continuous, rapid expansion and passaging of human neural stem cells. Among them, v-myc (the p110 gag-myc fusion protein derived from the avian retroviral genome) seems to be the most effective gene; we have also identified a strict requirement for the presence of mitogens (FGF-2 and EGF) in the growth medium, in effect constituting a conditional perpetuality or immortalization. A monoclonal, nestin-positive, human neural stem cell line (HNSC.100) perpetuated in this way divides every 40 h and stops dividing upon mitogen removal, undergoing spontaneous morphological differentiation and upregulating markers of the three fundamental lineages in the CNS (neurons, astrocytes, and oligodendrocytes). HNSC.100 cells therefore retain basic features of epigenetically expanded human neural stem cells. Clonal analysis confirmed the stability, multipotency, and self-renewability of the cell line. Finally, HNSC.100 can be transfected and transduced using a variety of procedures and genes encoding proteins for marking purposes and of therapeutic interest (e.g., human tyrosine hydroxylase I).
Villa
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Department of Molecular Biology, Center of Molecular Biology Severo Ochoa, Autonomous University of Madrid-CSIC, Campus Cantoblanco, Madrid, 28049, Spain.
Snyder
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Vescovi
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Martínez-Serrano
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D015139:Blotting, Southern; D001921:Brain / Q000166:cytology; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D002455:Cell Division / Q000187:drug effects / Q000235:genetics; D002461:Cell Line, Transformed; D002490:Central Nervous System / Q000166:cytology; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D015967:Gene Expression Regulation, Viral; D015750:Genes, gag / Q000502:physiology; D016259:Genes, myc / Q000502:physiology; D015316:Genetic Therapy; D005904:Glial Fibrillary Acidic Protein / Q000032:analysis; D006801:Humans; D007381:Intermediate Filament Proteins / Q000032:analysis; D008869:Microtubule-Associated Proteins / Q000032:analysis; D009410:Nerve Degeneration; D009416:Nerve Regeneration; D009419:Nerve Tissue Proteins; D064231:Nestin; D009474:Neurons / Q000737:chemistry / Q000166:cytology* / Q000187:drug effects; D010641:Phenotype; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis; D012190:Retroviridae / Q000235:genetics; D013234:Stem Cells / Q000737:chemistry / Q000166:cytology* / Q000187:drug effects; D014162:Transfection; D014404:Tubulin / Q000032:analysis; D014746:Vimentin / Q000032:analysis
D005904:Glial Fibrillary Acidic Protein; D007381:Intermediate Filament Proteins; D008869:Microtubule-Associated Proteins; C577484:NES protein, human; D009419:Nerve Tissue Proteins; D064231:Nestin; D018809:Proliferating Cell Nuclear Antigen; D014404:Tubulin; D014746:Vimentin; D016222:Fibroblast Growth Factor 2; D004815:Epidermal Growth Factor