Transplanted Neural Stem Cells Or Olfactory Ensheathing Cells Or Their Combined Graft Promote Recovery In Remotely Contused Rat Spinal Cord

Part I : Isolation, culture and identification of rat and human neural stem cellsNeural stem cells(NSCs) have the ability to differentiate into neurons, astrocytes and oligodendrocytes under preferable conditions, therefore, they can repair the diseased or damaged central nervous system theoretically. Before they can be applied clinically, relative pure NSCs must be isolated and cultured. Their proliferation, self-review, clone formation and differentiation should also be understood profoundly. The culture medium and additives should maintain the viability of NSCs as much as possible. E16d SD rat brain cortex, 3d new born SD rat hippocampus, E3m human brain cortex or E4.5-7m human hippocampus were dissociated and cultured with serum-free DMEM/F12 medium, and NSCs were expanded in vitro, their proliferation and differentiation were identified. Results: (1)NSCs form typical neurospheres under adequate concentration in vitro, which are immunoreactive to Vimentin. Typically and terminally differentiated mature neural cells could not be found without the stimulus of mitogen or only under NSCs self-regulation and self-induction; (2)NSCs derived from hippocampus maintain the character of stem cells much longer with better biological behavior; NSCs passed to the 2-3 passage are the best to graft since they have not differentiated; (3)NSCs cultured in vitro could self-regulate and differentiate into neurospheres and progenitors positively immunoreactive to specific antibodies representing neurons, astrocytes, oligodendrocytes and Schwann cells; (4)There are widespread synaptic contacts between various kinds of descendent clones and cells; (5)Neurospheres could be formed without the stimulus of mitogen when NSCs and OECs are cocultured. Many neurospheres and cells immunoreactive to Vimentin, GFAP, MAP2, 02, p75NGFR, GFAP, S-100, Synaptosis, Vimentin, Tau (Tau is only positive in cocultureof HNSCs+HOECs) could be found; (6)The supernatant fluid triturated from adult rat spinal cord stimulates NSCs to differentiate into neurons, but do not terminally differentiate; (7)Fibroblasts and O4 oligodendrocytes are not supported to grow under this culture medium.Part II: Isolation, culture and identification of rat and human olfactory ensheathing cellsOlfactory ensheathing cells/glials(OECs/OEGs) are the most powerful cells to enable the regeneration of axons in the central nervous system. Demyelination and glial scar formation inhibit axonal regeneration after spinal cord injury. However, both experimental and clinical therapy in the past ten years have revealed that transplantation of OECs could achieve excellent result in spinal cord injury. Obtaining relative pure OECs through isolation and culture is the most important. Quantitative culture with serum-free medium results in a much pure population of OECs, whose physiological character have not been altered. Results: (1)Serum-free culture medium satisfies the short-term culture of OECs in vitro. 15 days after cultivation is the most adequate graft time point. Among all the cultures, HOECs grow and expand better in 3-6+106/ml and are valuable for clinical application; (2)OECs form neurosphers and can differentiate into descendent clones. 15 days after cultivation, the OECs grow axons, but still retain some character of stem cells. They do not form typical mature neural cells; (3)To remove fibroblasts by arabinoside(Ara-C) is a simple and effective way to acquire OECs with purity greater than 90%; (4)Adhibited OECs grow out large numbers of axons, which give rise to more branches and form networks;(5) HOECs do not express O4 antigen, whereas ROECs are immunoreactive to O4 antibody; (6)Neurospheres could be formed without the stimulus of mitogen when NSCs and OECs are cocultured. Many neurospheres and cells immunoreactive to Vimenti, GFAP, MP2, 02, p75NGFR, GFAP, S-100, Synaptosis, Vimentin, Tau (Tau is only positive in coculture of HNSCs+HOECs) could be found; (7)Two kinds of OECs:p75NGFRand GFAP ( + ) or S100(+) OECs may be found in the olfactory bulb; (8)Fibrobl...