Differentiation Of Rat Bone Mesenchymal Stem Cells Into Neuron-like Cells Induced By Mecobalamin In Vitro

Objective: To study the feasibility of differentiating bone marrow mesenchymalstem cells (BMSCs) into neuron-like cell induced by the different doses ofMecobalamin in vitro and observe the cell viability and proliferation of differentiatedcells. To provide the preliminary research basis for cell transplantation and effectivetreatment of spinal cord injury using BMSCs induced by Mecobalamin.Methods:1. Rat bone marrow mesenchymal stem cells were isolated, cultured andpurified by density gradient centrifugation and adherent culture. The cell growth,biological activity and morphological changes were observed under an invertedmicroscope. The specific surface markers of BMSCs, CD44were detected byimmunofluorescence cytochemistry.2. The fourth to fifth generation of BMSCs were made into single cell suspension. Thecells were divided into control group and the experimental group (25ug/ml,50ug/mland100ug/ml concentration group). The experimental groups were treated withdifferent concentrations of Mecobalamin in10%FBS L-DMEM culture medium andcontrol group treated with10%FBS L-DMEM without any inducer. After treatmentfor24h,48h and72h, the morphological changes and cell growth were observedunder an inverted microscope. The viability and proliferation of induced cell wasdetected by MTT assay. The expression of neuroepithelial stem cell protein (Nestin), aneural precursor cells specific marker and neuron-specific enolase protein (NSE), anerve cell-specific marker, were identified by RT-PCR and western blotting.Results:1. Adherent cells were single scattered and the majority of cells were smallround or oval after the primary cells inoculated for24-48hours. The spindle cellswere scattered in72hours and the cell colonies were formed after cultured for a week, and the cell morphology were spindle-shaped, triangular or star. Cell proliferation wassignificantly accelerated after primary culture for about two weeks, and theproliferated cells form the colony as "swirling" with a typical fibroblast-likemorphology. Immunofluorescence cytochemistry analysis showed that the cell surfacemarkers of BMSCs, CD44were positive, and CD34, a surface antigen ofhematopoietic stem cell, was negative.2. The refraction of cells were enhanced and the neuron-like changes, such as the longprotruding, stereoscopic and enhanced refraction, in each experimental groupappeared in BMSCs after treated with25ug/ml,50ug/ml and100ug/ml Mecobalaminfor24,48and72hours. While there were not significant changes in the cellmorphology of control group. MTT assay showed that the biological activity in allexperimental groups was not significant difference when compared with control groupafter24h,48h and72h (P>0.05). RT-PCR and western blotting results showed thatthe levels of Nestin and NSE mRNA and protein were significantly increased aftertreated with different doses of Mecobalamin in48h. Comparing with the controlgroup, differences are statistically significant（P＜0.05）. Similarly, the levels ofNestin and NSE mRNA and protein were significantly increased after treated with100ug/ml Mecobalamin for24h,48h and72h. Comparing with the control group,differences are statistically significant（P＜0.05）.Conclusions:1. Mecobalamin can induce BMSCs into neuron-like cells, and thedifferentiated cells have proliferative capacity. Mecobalamin did not have apparentinhibition to the differentiated cells.2. In vitro experimental groups showed that100ug/ml Mecobalamin is the betterinduced concentration, which can efficiently induce of BMSCs into the neuron-likecells.