The Study Of Angiopoietin 2-induced Differentiation Of Fetal Mouse Cortex-derived Neural Stem Cells And Its Molecular Mechanism

Objective With the capabilities in compensating neuronal damages and reconstructing disrupted neuronal connections, multipotent neural stem cell(NSC) is considered to be the most potential candidate for the regeneration of central nervous system(CNS) injury, especially spinal cord injury(SCI). Howevr, it is still in urgent need to regulate the differentiation of NSCs into neurons to guarantee efficient regenerative effects. Recent studies indicate that angiopoietin2(Ang2) plays an important role not only in angiogenesis, but also in neurogenesis during the development and regeneration of central nervous system(CNS). Nevertheless, the underlying mechanism is still unknown. This study investigated the effects of Ang2 on the PI3K-AKT-m TOR signaling pathway in regulating the differentiation of NSCs. NSCs were isolated from the cortex of C57BL/6J mice on embryonic day 12.5 and cultured with recombinant human Ang2. Furthermore, specific inhibitors of PI3K-AKT-m TOR signaling pathway were used to study the molecular mechanism and its key factor of Ang2 in the regulation of NSCs differentiation.Methods NSCs were isolated from the cortex of C57BL/6J mice on embryonic day 12.5. The purified NSCs were observed under light microscope, identified by Nestin, and immuostained with specific markers of neurons, astocytes and oligodendrocytes to demonstrate its capacity in multilinage differentiation. Besides, we improved the cryopreservation and recovery protocol of NSCs, and the thawed NSCs after different freezing duration(0, 1, 6, and 12 months) were assayed by morphorlogy observation, Nestin identification, and differentiation capacity evaluation. After in vitro cultue in serum-free medium for two passages, the purified NSCs were cultured in plates coated with Poly-L-lysine(PLL) and cultured with recombinant human Ang2. By using Reverse-transcription polymerase chain reaction(RT-PCR), immunocytochemistry and Western blot, this study evaluated the differentiaton diection and efficiency of cultured NSCs with Image-Pro Plus 6.0 and Quantity One. By blocking PI3K-AKT-m TOR signaling pathway using specific inhibitors, LY294002 and rapamycin, we evaluated the alered differentiation effects and efficiency of Ang2-induced NSCs with immunocytochemistry, Western blot, andFlow cytometry.Results NSCs were successfully isolated from the cortex of C57BL/6J mice on embryonic day 12.5, and identified with morphology observation, Nestin immuostaining, and specific markers immuostaining of neurons, astocytes and oligodendrocytes. The improved cryopreservation and recovery protocol was demonstrated feasible for NSCs by morphorlogy observation, Nestin identification, and differentiation capacity evaluation. No significant changes were found in the differentiation capacity of thawed NSCs after different freezing duration(0, 1, 6, and 12 months)(p > 0.05). Immunofluorescent staining showed a significant increase in the percentages of βⅢ-tubulin-positive cells and microtubule associated protein2(MAP2)-positive cells(p < 0.001; p < 0.001), both of which are markers of neurons, while no significant alteration were observed in the percentage of glial fibrillary acidic protein(GFAP)-positive cells, a marker of astrocytes, nor the percentage of cyclic nucleotide 3’phosphohydrolase(CNPase)-positive cells(p > 0.05;p > 0.05), a marker of oligodendrocytes. It was further confirmed by Western blot results showing the significant improvement in the βⅢ-tubulin and MAP2 expression(p < 0.001;p < 0.001) without obvious disruption in the GFAP or CNPase expression(p > 0.05;p > 0.05). The phosphorylation of m TOR was demonstrated to be up-regulated through the PI3K–AKT signaling pathway during the neuronal induction of NSCs. The blockage of PI3K-AKT-m TOR signaling pathway using specific inhibitors, LY294002 and rapamycin, abrogated the promoted neuronal differentiation of NSCs by Ang2.Conclusion This study revealed the novel effects of Ang2 on the neuronal differentiation of NSCs, which were isolated from the cortex of C57BL/6J mice on embryonic day 12.5. It further uncovered the critical roles of m TOR in a PI3K-AKT dependent manner during the regulation of Ang2 in NSC differentiation. This study provides the experimental evidences of Ang2 on the aspect of neurogenesis and neural regerneration, and reveals the potential of Ang2 in promoting the regeneratve effects of NSCs for central nervous system injury, especiall SCI.