Abstract
Neural stem cells are a potential source of cells for cell therapy of neurodegenerative diseases or drug screening. Ethical and practical considerations limit the application of neural stem cells derived from human embryonic stem cells or adult brain tissue. Therefore, alternative sources of adult human neural stem cells are of high interest for basic research as well as potential clinical use. These sources have to satisfy the demands of easy accession, rapid expansion in serum-free media and reliable induction to a neural fate.CD133 positive hematopoietic stem cells (HSCs) isolated from mobilized leukapheresis were cultured and expanded in the presence of epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2/bFGF) in serum-free medium. After two weeks of culture, neurosphere formation was observed. Subsequent culture in serum-free, EGF and FGF-2 containing medium resulted in large numbers of CD133−, Nestin+ and Sox-2+ neural stem cells. Neural stem cells derived from HSCs (hNSCs) are highly proliferative and are able to migrate in response to chemotactic stimuli described to induce migration of neural stem cells. We used immunocytochemical techniques and PCR to assess neural differentiation. Adherence to poly-D-lysine/laminin and growth factor deprivation resulted in cells of neuronal morphology and high expression of neuronal differentiation markers. We furthermore show that after retinoic acid treatment neuronal induction was greatly enhanced with more than 90% of cells expressing neuronal differentiation markers as beta-III-tubulin and neurofilaments. In addition, hNSCs were able to differentiate into cells of the glial lineage as shown by expression of glial fibrillary acidic protein (GFAP). Our novel method provides nearly limitless numbers of neural stem cells from an easily accessible autologous adult human source, which could be used as a starting point for further experimental studies and potential therapeutic use.
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