Study On The Cellular Mechanisms And Signaling Pathways Underlying The Differentiation Of Human Umbilical Cord Derived Mesenchymal Stem Cells Into Cardiomyocytes Induced By5-azacytidine

Part Ⅰ Establishment of method to culture and induce humanumbilical cord derived mesenchymal stem cell to differentiate intocardiomyocytes in vitroObjective To establish the method to culture and induce human umbilical cordderived mesenchymal stem cells (hUC MSCs) to differentiate into cardiomyocytes invitro.Methods hUC MSCs were isolated and purified f rom the umbilical cord ofhealthy full-term delivery fetus after the conset of puerpera by adhering to the culturedishes. The three passage of hUC MSCs was induced by different concentrations(5μmol/L,10μmol/L,20μmol/L,30μmol/L and40μmol/L) of5-azacytidine (5-aza) for fourweeks, and then cultured in new medium. Control group was cultured in complete medium.Cells growth morphology was observed by convert microscope during the whole process.hUC MSCs surface antigens of passages3were measured by flow cytometry. Myocardiumspecific cardiac troponin I (cTn I) was examined with immunohistochemistry4weeksafter induction.Results Spindle-shaped hUC MSCs were presented from human umbilical cordtissue by adhibition culture method. hUC MSCs treated with5-aza had long cytoplasmicprocess1-2weeks after induction, and they connected with adjoining cells formingmyotube-like structures3-4weeks after induction. Flow cytometry ananlysis revealed thatCD13,CD29,CD44,CD90and CD105were highly expressed on the surface of passages3,but there was negative for CD31,CD34,CD45and HLA-DR. the differentiation rates ofhMSCs increased gradually.There were massive death cells in high concentration groups(20μmol/L,30μmol/L,40μmol/L). Immunohistochemical eramination revealed negativeexpression of cTnI in control group and positive expression in induced groups. The expression rates of5,10,20,30and40μmol/L induced groups were (13.20±3.03)%,(32.20±3.35)%,(25.60±2.30)%,(24.40±3.51)%and (22.60±2.41)%. There wassignificantly difference between5μmol/L,10μmol/L induced groups and20μmol/L,30μmol/L,40μmol/L induced groups.Conclusion We can obtain an amount of hUC MSCs from human umbilical cordtissue. hUC MSCs can be induced to differentiate into cardiomyocytes by5-aza in vi tro.10μmol/L of5-aza is the optimal concentration. Part Ⅱ Cellular mechanisms underlying the differentiation ofhuman umbilical cord derived mesenchymal stem cells intocardiomyodytes induced by5-azacytidineObjective: To investigate the cellular mechanism underlying the differentiation ofhuman umbilical cord derived mesenchymal stem cells (hUCMSCs) into cardiomyodytesinduces by5-azacytidine(5-aza).Methods: hUCMSCs were isolated and purified from the umbilical cord of healthyfull-term delivery fetus after the conset of puerpera by adhering to the culture dishes.hUCMSCs was induced by10μmol/L concentration of5-aza for4weeks. mRNAexpression of Nkx2.5and GATA4were analyzed by real time polymerase chain reaction(RT-PCR).Results: After4weeks of induced culturing, statistically higher level expression ofNkx2.5and GATA4mRNA in induced group was observed in comparison with those ofcontrol group. The expression of Nkx2.5and GATA4in induced grouip was increased to4.72±0.58,3.76±0.06times compared with that in control group. There weresignificantly difference of cycle threshold values of Nkx2.5and GATA4between inducedgroup and control group(P<0.05).Conclusions: hUCMSCs can be induced to differentiate into cardiomyocytes by5-azain vitro and the molecular mechanism underlying the differentiation of hUCMSCs intocardiomyodytes induced by5-aza might be through upregulating the expression of Nkx2.5and GATA4. Part Ⅲ Expression and significance of Dll4-Notch signalingpathway in the differentiation of human umbilical cord derivedmesenchymal stem cells into cardiomyodytes induced by5-azacytidineObjective: To study the expression of DLL4and Notch1in the differentiation ofhuman umbilical cord derived mesenchymal stem cells (hUCMSCs)into cardiomyodytesinduces by5-azacytidine(5-aza), investigate the regulation of differentiation of hUCMSCsinto cardiomyocytes by Notch signaling system．Methods: hUCMSCs were isolated and purified from the umbilical cord of healthyfull-term delivery fetus after the conset of puerpera by adhering to the culture dishes.hUCMSCs was induced by5-aza. Then the cultured cells were collected in1,3,5,7dayafter5-aza intervention. The expression of Notch signal was detected by RT-PCR.Results: RT-PCR showed that higher level expression of Notch1gene in5-azainduced group was observed by comparing with that in the control group.There was astable higher expression level in induced group. Compared with control group, theexpression levels of Notch1was respetively increased6.60,7.36,7.595,7.805times at1,3,5,7days after intervention of5-aza. Statistically higher CT value of Notch1mRNA ininduced group was observed in comparison with that of the control group(0.51±0.21vs7.85±0.35,t=35.98, P<0.01). The expression level of Dll4increased stablely comparedwith the control group. Compared with control group, the expression levels of Dll4wasrespetively increased11.53,10.1,10.17,11.46times at1,3,5,7days after intervention of5-aza. There was a significantly difference of Dll4CT value between the5-aza inducedgroup and the control group(1.60±0.49vs12.42±0.73, t=11.71, P<0.01).Conclusions: The expression of Dll4-Notch signaling pathy protein has apparentchange, Notch signaling pathy may be involed in the differentiation of hUCMSCs intocardiomyodytes induced by5-aza.