Efficient Generation of Neural Stem Cell Lines from Adult Bone Marrow Using Serum Free Defined Media.

Adult bone marrow (BM) derived stem cells may have the ability to differentiate into cells of all three germ layers, including neural cells. However, the efficient and large-scale generation of such cells is still a great challenge. Unfractionated BM cells were cultured at high density (5x10⁶ cells/well) in vitro in serum-free media supplemented with basic fibroblast growth factor and epidermal growth factor. After one month in culture, many cells had died and most of the surviving cells attached to the bottom, where they consistently formed cyst-like structures. Cellular aggregates in the shape of spheres originated from these cysts and started to expand. Comparing these BM-derived spheres (BM-NSC) with neurospheres isolated from fetal brain (NSC), revealed similar morphology and equal expression of the neural precursor marker Nestin. BM-NSC showed a high proliferating capacity, increasing 1000-fold over a period of 50 days. Single cell cultures were initiated to assess clonogenic potential. 35% of single BM-NSC produced a neurosphere colony within 7 days, compared to 21% of NSC. To assess to what extent BM-NSC and NSC were comparable at the molecular level, array analysis, interrogating 5000 cDNAs were performed. A striking overlap in gene expression patterns between the two populations was observed, while comparison of BM-NSC to hematopoietic stem cells resulted in no overlap at all. We have cultured these spheres for more than 1 year in vitro and they have been successfully cryopreserved and thawed. Multilineage neural differentiation was induced by sphere dissociation and plating cells onto poly-L-Lysine/laminin-coated dishes, without FGF and EGF, in the presence of Brain Derived Neurotropic Factor and Ciliary Neurotropic Factor. Immunocytochemistry was performed at day 4 and day 12 and revealed that BM derived NSCs expressed several neuronal markers. Differentiating cells expressed the early neuronal maker Nestin at day 4 and also Map2 and occasionally some cells were positive for GFAP. In some cells Nestin was co-localized with Map2. At day 12 cells where positive for Neun and Map2 and infrequently some cells stained positive for O4. Expression patterns were confirmed by RT-PCR analysis. This indicates that BM-NSC, like their brain-derived counterparts, have neuronal, astrocyte, and pre-oligodendrocyte potential. Collectively, our findings demonstrate that it is feasible to generate a self-renewing population of neural stem cells from BM that are indistinguishable from brain-derived stem cells. We are currently using highly purified BM cell populations to identify the origin of these BM-NSC cells.