Research Of In Vitro Cultivation Of Rat Bone Marrow Mesenchymal Stem Cells And Differentiation To Sensory Neuron-like Cells

Objective:This experiment aims to optimize the culture and induce condition and to establish an operation procedure of gaining rat BMSCs and inducing them into sensory neuron-like cells. The research results lay a foundation for further study of BMSCs in the treatment of sensorineural deafness.Method:BMSCs were isolated from healthy rats and cultured in vitro by density gradient centrifugation. To make sure that the cultured cells were BMSCs, flow cytometry and direct differentiation were carried out. Then we used two-step induction. Frist, BMSCs were induced to otic progenitors by EGF and IGF-1combined effect, immunofluorescence assay was used to detect the expression of SOX2and PAX8in otic progenitors. Second, we induced otic progenitors to sensory neuron-like cells in UN-BMP4group and BMP4group, realtime-PCR assay was used to comparative differences between groups. The treated cells were identified by the cell morphological observation, cell growth curve and immunofluorescence.Results:1、We gained uniform adherent cells through cell cultivation techniques. Flow cytometry assay showed positive expression of CD44、 CD90、CD106and negative expression of CD34, CD45by obtained cells. After adipogenic induction and osteogenic induction, obtained cells performed adipocyte and osteobast morphology and can be satined by oil red O and Alizarin Red. It confirmed that we gain high-purity BMSCs.2、Realtime-PCR results showed that BMP4, SHH and RA synergistically improve neuronal specific mRNA level of rat BMSCs.The treated cells had a special form of neuron,which is in the growth plateau with sensory neurons associated protein positive. It confirmed we have successfully induced BMSCs into sensory neuron-like cells.Conclusion:1、We successfully established the technical standards for process of rat bone marrow mesenchymal stem cells collection, isolation, cultivation and identification, provided a theoretical and practical reference to obtain high quality BMSCs.2、We successfully established the technical standards that induced BMSCs to sensory neuron-like cells.It is proved that BMP4, SHH and RA synergistically promote sensory fate specification from BMSCs by the realtime-PCR, cell morphological observation and immunofluorescence.