Differentiation Of Human Umbilical Cord Blood Mesenchymal Stem Cells Into Myoblasts In Vitro

Background and objective human umbilical cord blood mesenchymal stem cells (hUCB-MSCs),which were studied from this early century,have the biological characteristic similar to those from other organs.There is no standard culture system of hUCB-MSCs,with a low culture efficiency.Otherwise the study on the differentiation of hUCB-MSCs into myoblasts was not enough. So we selected the hUCB-MSCs as seed cell to study,established optimal culture systems,discussed the mechanism of myogenesis.Methods①Optimal selection of culture systems for hUCB-MSCs:Mononuclear cells were isolated from umbilical cord blood using the lymphocyte isolation method,hydroxyethyl starch sedimentation,hydroxyethyl starch sedimentation+lymphocyte isolation method.Following incubation,cells were separately cultured in DMEM/F12 containing 10%fetal bovine serum, L-DMEM and MesencultTM,at various fetal bovine serum(5%,10%and 20%) and various cell densities(5×10~6,1×10~7,5×10~7,1×10~8).At the third passage, hUCB-MSCs were identified using the surface marker by flow cytometry and osteoblasts were identified by Von Kossa staining.②Differentiation of hUCB-MSCs into myoblasts induced by 5-Azacytidine:the differentiation of the 3rd passage of hUCB-MSCs was induced by the reasonable concentration of 5-azacytidine,which was selected by MTT.After 7 days and 14 days induced by 5-azacytidine,the expressions of MyoD1,myogenin and myosin heavy chain were analyzed with immunofluorescence stain and real-time PCR.Results Compared with the lymphocyte isolation method,the number of mononuclear cells significantly increased using the hydroxyethyl starch sedimentation and hydroxyethyl starch sedimentation+lymphocyte isolation method(P＜0.05,P＜0.01).Primary culture time of cells was significantly shorter using the hydroxyethyl starch sedimentation+lymphocyte isolation method compared with the hydroxyethyl starch sedimentation,and the lymphocyte isolation method(P＜0.01).Cells at 5×107 were incubated in DMEM/F12 or MesencultTM medium,containing 10%fetal bovine serum in the T25 culture flask.Culture efficiency was higher than other condition media (P＜0.01).Adherent cells were positively for CD29 and CD105,but negatively for CD34,CD45,CD90 and CD133.After 21 days osteogenic inductive culture, black mineralization nodules appeared following Von Kossa staining. immunofluorescence stain showed that After 7 days treated by 5μmmol/L 5-azacytidine,the cells expressed MyoD1 positively,and after 14 days, MyoD1 and myogenin positively,but no myosin heavy chain positively all thte time.Real-time PCR had the same result.Otherwise,uninduced the HUCB-MSCs expressed MyoD1 spontaneously.Conclusions Plenty of umbilical cord blood mononuclear cells are obtained using the hydroxyethyl starch sedimentation+lymphocyte isolation method. Cells at 5×107 in DMEM/F12 or MesencultTM medium,containing 10%fetal bovine serum can elevate the culture efficiency of hUCB-MSCs.hUCB-MSCs possess a potential of myogenic differentiation,5μmmol/L 5-azacytidine can induce HUCB-MSCs differentiated into myoblast in vitro.