Study Of Differentiation Of Rat Marrow Mesenchymal Stem Cells Into Neuron-like Cells In Vitro

Objective: To study the effects of two treatments, antioxidants (DMSO&BHA) and BDNF/bFGF/ RT-RA, on inducing the marrow mesenchymal stem cells derived from Wistar rat to differentiate into neurons.Methods: The MSCs were cultured and purified by their characteristic of plastic adhesion in vitro. To characterize these cells, fluorescent cell sorting was performed using antibodies against cell surface marker by Flow cytometry and the percentage of positive cells for CD90, CD31, CD34 were determined respectively.In order to promote the differentiation of MSCs into neuron cells, MSCs were treated with antioxidants (2% DMSO&200 umol/L BHA) and some cytokines (10 ug/L BDNF, 20 ug/L bFGF)/RT-RA(lumol/L)respectively. The morphological changes were observed by inverted microscope, with the fetal rat skin fibroblast as the negative control. At different times post induction (0 min, 15 min, 30 min, 45 min, 60 min, 90 min), the morphological change of MSCs and fetal rat skin fibroblast were recorded by time-lapse microscopy. Then cells were transferred into normal media 2.5h later and observed for the morphological changes at 0 min, 15 min, 30 min, 60 min.Immunocytochemical staining was conducted for the neural stem cells(NSC), neuron and neuroglia specific cell surface marker such as nestin, MAP-2, NSE and GFAP.Whole-cell patch clamp technique was used to detect the electrophysiological properties of untreated MSCs and differentiated neuron-like cells, as well as the acute isolated rat hippocampal neurons as positive control group.Results: Assayed by flow cytometry, the third passage of cultured MSCs showed homogeneity as high as 95%. The percentage of positive cells for CD90,CD31, CD34 was 99%, 3.4%, 0.3%, respectively.In antioxidants(DMSO&BHA)-treated cells, neuron-like cells were present by 0.5 h, and peaked at 4 h with a percentage of 86.47%+5.56%;while in the other treatment, neuron-like cells can not be seen until induced 24 h and the percentage reached a maximum of 24.01%±3.76% at 72h, significantly lower than that from antioxidants treatment(P<0.01)oAntioxidants (DMSO&BHA) could also induce fetal rat skin fibroblast to differentiate into neuron-like cells, while some cytokines (bFGF& BDNF) / RT-RA could not, at least within 72 hours after induction. Time-lapse microscopy results showed: cell body retracted gradually to form the neuron-like "process", with increasing time from 0 to 90 min. Neuron-like cells were restored to the uninduced cell morphology after transferred into normal media.Nestin maker was present in both induced and uninduced MSCs. Upon both treatments, an increase staining intensity was registered for the expression of NSE, and the immunocytochemical staining results of MAP-2 and GFAP shifted from negative to weakly positive.The percentage of cells that had currents of neuron-like cells induced by antioxidants (DMSO&BHA) was significantly lower than the results of MSCs and some cytokines (bFGF& BDNF) / RT-RA (i><0.01). Unlike hippocampal neurons, the positive control, no inward sodium current was found in neuron-like cells from the two treatments. The peak currents of outward currents in neuron-like cells induced by were significantly higher than that in antioxidants(DMSO? BHA) treated and untreated MSCs (PO.01), while no significant differences was observed between neuron-like cells induced by antioxidants(DMSOr BHA) and MSCs about the peak currents of outward currents(P>0.05).Conclusions: MSCs were successfully isolated and cultured in vitro, and the percentage of CD90 positive cells reached 99%. Some cytokines (bFGF& BDNF) / RT-RA could induced the MSCs to differentiate into neuron-like cells, while the antioxidants treatment could not. The MSCs induced by some cytokines (bFGF& BDNF) / RT-RA generated neuron-like cells expressed neuron specific markers, and showed the tendency to differentiate into mature neurons, though having no electrophysiological properties of mature neurons.Patch-clamp recording technique has a vital significance in evaluating the electro- physiological properties of MSCs derived neuron-like cells.