Experimental study of the differentiation induction of adult human bone marrow-derived neural stem cells

doi:10.3321/j.issn:1673-8225.2003.22.013

Abstract：AIM:To study the feasibility of isolating and culturing neural stem cells (NSCs) from adult human bone marrow for potential clinical application of NSCs autotransplantation for treating cerebral degenerative diseases. METHODS:Ilium puncture was performed in 20 adult voluntary donors of either genders (aged between 28 and 48 years) to obtain the bone marrow specimens,from which bone marrow stromal cells were harvested by means of density gradient centrifugation and cultured in " Cytokine@ NSCs medium" (a patent medium prepared by our lab) to ascertain the optimal in vitro culture conditions of the cells.The proliferation of NSCs was confirmed by observing the cell clone formation, and the NSCs,neurons and glial cells were identified by immunocytochemical detection of the expressions of nestin,neuron specific -endase (NSE),and glial fibrillary acidic protein (GFAP) respectively.The cytokines employed in the cell culture mainly included brain derived neurotrophic factor (BDNF),leukemia inhibitory factor (LIF, 10 μ g/L) and retinoic acid (RA, 0.5 mg/L). RESULTS:When proper culture condition was satisfied, the bone marrow stromal cells proliferated rapidly and differentiated into NSCs,some of which that contained rough and large cytoplasmic granules merged into islet like spherical cell cluster (nerve bulb) which were positive of nestin.After the cell clusters were separated into single cells for further culture,the islet like pellets again appeared. Further differentiation of the NSCs clusters gave rise to the formation of small buds in some cells that developed into the long projects with the potential to connect with each other.The cells with long projects were positive for NSE or GFP expression. CONCLUSION:It is possible to induce the differentiation of adult human bone marrow stromal cells into NSCs under proper experimental conditions.