Regulation And Direction Of NSCs To Adopt Neuronal Fate By Hyperthermia Conditioned OECs Serum-free Conditioned Medium

Objective Recent studies provide solid evidence for the importance to delineate the combined transplantation of neural stem cells (NSCs) and olfactory ensheathing cells (OECs) for the repair of central nervous system injury. One of the limitations of this approach is that the proportion of neurons differentiated from NSCs still remains at low level.Thus,how to induce NSCs to differentiate into neurons more efficiently by OECs is an attractive problem, which needs attention to be resolved. In the present study, we investigated the effects of hyperthermia-conditioned OEC-conditioned medium (OCM) on induction of NSCs into neurons.Methods3-day-old postnatal mouse were killed, and the olfactory bulbs(OBs) were dissected. The conditioned media, named370CM,400CM, and400CM+R (treated with HIF-1a inhibitor),were collected after OECs had been incubated at37℃or40℃for6hr, followed by incubation at37℃for42hr. Cell viabilities of OECs were measured using an MTT assay. Then, these conditioned medium were collected and used for NSC induction. The effects of hyperthermic preconditioning on OECs HIF-1a and binding activity of HIF-1expression using RT-PCR and Western blot analysis.The content of VEGF and EPO that were released were detected using ELISA kit.Results Results of the present study demonstrated for the first time that6hr of hyperthermia (40℃) could induce OECs upregulation and the expression of hypoxia-inducible factor-1alpha (HIF-1a). Moreover, compared with370CM (23.60+1.36%for Tuj+cells、22.80±1.66%for MAP-2+cells) and400CM+R (22.60±1.50%for Tuj+cells、24.00±2.30%for MAP-2+),400CM (31.40±1.08%for Tuj+cells、31.80±1.60%for MAP-2+) could induce NSCs differentiation into neurons more efficiently, and this phenomenon is associated with the upregulation of HIF-1a after hyperthermia conditioning.Conclusion The data implied that the secretory protein in OECs-cultured medium and upregulation of HIF-1a expression and binding activity induced by hyperthermia-conditioned OECs have synergistic effect to induce NSCs differentiation into a neural lineage. The up-regulation of HIF-1a and binding activity is a latent mechanism involved in the differentiation role of hyperthermic preconditioned OECs.