Isolation, Culture And Differentiation Of Human Neural Stem Cells From Elder Embryonic Brain

Objective1. To study the isolation and culture of human neural stem cells (NSCs) from elder embryonic brain.2. To investigate the effects of bone marrow stromal cells (BMSCs) on differentiation of neural stem cells.Methods1. First, the subventricular zone was dissected and separated from embryonic brain of 16-20 weeks. Then, the cells were isolated from the tissue by mechanical dissociation. The cell viability was measured by counting trypan blue-positive cells.2. The NSCs were cultured in DMEM/F12 medium supplemented with 2% B₂₇, 20ng/mL EGF and 20ng/mL bFGF. Morphological changes of cells were observed under inverted microscope. When the cells were passaged, a micro-neurosphere suspension was obtained for inoculum by mechanically dissociating the pellet with a 200μL pipet. The expression of Nestin, NSE, GFAP and CNPase were detected by immunocytochemistry assay.3. Human BMSCs were isolated by gradient centrifligation in lymphocyte separation medium and by their adherence to tissue culture surfaces.4. Human NSCs were co-cultured with BMSCs together in different ways in vitro, natural differentiation group, contact co-culture group and Transwell co-culture group. After 7 days, the expression of NSE and GFAP were detected by immunocytochemistry and western blot assays.