The Change Of Surface Marker In The Prosess Of Human Mesenchymal Stem Cells Differentiated Into Neuron-like Cells

Traditional viewpoint believed that neurons lack the ability to regenerate, so they will be replaced by glial cells when injured or degenerated. But recent study have shown that neural stem cells (NSCs) can be induced into neurons, astroglias and oligodendrocytes by cytokines to compensate for the damaged neurons tissue. NSCs have been found widely distributed in embryo, adult brain and peripheral nervous system. However, endogenous NSCs lack necessary stimulating singals so that they are not capable differentiate into neurons automatically when damage occurs. While the utility of NSCs from embryonic and fetal brain is restricted methodically and ethically, so it is of great significance to find a new source of NSCs for clinical use in the field of neuroscience.Mesenchymal stem cell (MSCs) is the portion of adherent cells different from the hemotopoietic stem cells in bone marrow, which is also called the plastic-adherent cells or clone-forming-unit fibroblast. They can be cultured and proliferated easily in vitro and can differentiated into multiple cell lineages. These characteristics of MSCs make them ideal engineering cells in cell and gene therapies. Recent studies have proved that MSCs can give rise to neural cells in vitro.Therefore, the aim of the experiment is to identify the feasibility of bone marrow MSCs to transdifferentiate into neurons and to compare the induction function of twofactors EGF and bFGF, which might provide theoretic and experimental foundationfor clinical application of MSCs.Methods:Human bone marrow MSCs were isolated by centrifugation over Lymphoprep(density 1.077g/ml) and cultivated with 1×106/ml density in T-50 tissue culture fliisks in humidified atmosphere with 5%CO2 at 37 ℃ with DMEM/F12 medium which contain 10% fetal bovine serum. Growth curve was determined with MTT method. Part of the 4th passage cells were induced for 4 day, 7day, 10day and 14day , then the cells of un-induced 4th passage and the cells of the 4th passage induced for 4day,7day,10day and 14day were collected respectively. The expressions of specific markers of NSCs and neurons (nestin, NF-M, MAP2, GFAP) were detected by reverse transcriptase chain reaction(RT-PCR) method. The nsetin, NSE and GFAP were also examined by immunocytochemistry after inducement. The pattern of phosphorylation tau(pSer202) was observed by immunocytochemistry. Telomerase activity were examined by TRAP(PCR)-ELISA after inducement. Additionally, in order to identify the functional differences of the two cytokines (EGF , bFGF), MSCs of the 4th passage were divided into 3 groups applied by different induction conditions:group Ⅰ : DMEM/F12 and 10% fetal bovine serum plus bFGF (with terminalconcentration of 10ng/ml) group Ⅱ: DMEM/F12 and 10% fetal bovine serum plus EGF (with terminalconcentration of 10ng/ml) group Ⅲ: DMEM/F12 and 10% fetal bovine serum and EGF and bFGF(withterminal concentration of 10ng/ml either)After inducing for 7 days, the expression of specific markers of neurons (MAP2, GFAP)were checked by RT-PCR and compared with each other. Results:1.Freshly isolated MSCs were small and round. After cultivated, cells were mainly the spindle-like, sometime appearing cleavage phase. But cells induced by EGF and/or bFGF turned into neuron-like cells.2.The result from immunostaing showed that the ratio of the nestin positive cells were 56% when induced for 7 days and after inducing for 10 days the ratio reduced to 42%. NSE, GFAP positive cells were increased go along with inducement. Positive cells of anti-phosphorylation-tau(pSer202) were detected after cultivated for 4 passages.3. The cells of the un-induced 4th passages expressed nestin, GFAP and NF-M mRNA but the expression of MAP2 mRNA were not detected. With the inducement the expression of nestin, NF-M, GFAP and MAP2 mRNA increased while after inducting for 7days the expression of nestin mRNA finally decrease. Comparison of among the 3 groups, the expression of MAP2 mRNA were higher in group 1 and 3, but in group 2, expression of GFAP were much higher than group 1.4. Low levels of telomerase activity were detected by TRAP-ELISA method. Conclusions:1. bFGF promotes the proliferation of MSCs and induces them to express the specific markers of neurons while EGF accelerates the MSCs turning to glial cells direction.2. Telomerase activity will be enhanced in MSCs induced by EGF combine with bFGF.