Human Neural Stem Cells Isolated From Different Embryonic Brains And Transplanted Into Non-neurogenic Region Of Adult Brain

Part 1: Isolation, culture and identification of human neural stem cells from different embryonic brainsObjective: To isolate, culture and identify human neural stem cells from 7-week to 13-week embryonic brains and to study different suitable culture conditions.Methods: By using serum-free cell culture and single cell clone test, the neural stem cells were isolated and cultured from 7-week human embryonic forebrains and 13-week human embryonic cerebral cortex in different conditions with combination of EGF, bFGF, and LIF. The cells could be expanded with mechanical method. Immunofluorescence tests were used to identify the cells with neural stem cells marker Nestin andmature neural cells markers NSE, GFAP and CNP. Results: The human neural stem cells with the abilities of self-renewal and multipotency were successfully isolated and cultured from different embryonic brains. In 7-week brain, the cells could form clones in different ways except in high concentration suspension culture; in 13-week brain, the cells could form clones only in middle concentration suspension culture and high concentration adhere-wall culture; in coated petri dish, the cells couldn't form clones.Conclusion: There are neural stem cells in human embryonic brains With gestational age increasing, they become more and more difficult to be isolated, the different concentrations and methods should be selected. High concentration adherent culture can be used in embryonic brains with different gestation age. The cells from different embryonic brains could express same neural markers.Part 2: Human neural stem cells transplanted into non-neurogenic region of adult rat brainObjective: To study the survival .migration and integration of the long-term expanded human neural stem cells transplanted into the striatum and cortex of adult rat brain.Methods: The human neural stem cells were isolated from 7-week embryonic forebrain with a combination of EGF, bFGF and LIF, the cellscould be expanded for a long term. The P10-P15 cells labeled with BrdU were transplanted into the striatum and cortex of adult rat brain through a micro-needle on stereotaxic apparatus. Eight animals were sacrificed Iweek postgrafting, and the rest of the animals were left to survive for 6 weeks. Brains were removed cut on a freezing microtome at a thickness of 30 m. Immunofluorescence technique was used to detect BrdU and Nestin, NSE and GFAP were detected by immunohistochemistry. Results: A combination of EGF, bFGF, and LIF could be used to expand human neural stem cells for a long time, in order to get enough cells for transplantation. 1 week and 6 week after transplantation, BrdU-positive cells could be detected in the animal brains. The cells were observed to have migrated into the surrounding host striatum, without any preferential direction, to a distance of 1-1.5 mm from the graft core, in cortex, the distance was 2-2.5mm. Nestin downregulation and multipotency in vivo were observed, the main differentiated cells were astrocytes, a few NSE-positive cells were detected in cortex.Conclusion: Human embryonic neural stem cells can survive, migrate and integrate into non-neurogenic regions of adult rat brain, their migration and differentiation display site-specific properties, and have limited migration and multipotency.