Apelin Promotes Cardiomyogenic Differentiation In Wharton’s Jelly-derived Mesenchymal Stem Cells Of Human Umbilical Cord In Vitro

The cardiovascular morbidity was constantly rising in our country. According toincomplete statistics, the total population of patients who suffered from heart diseases wasup to230millions which was roughly equivalent to one of every five Chinese adults.About3.5millions patients died of heart diseases each year and acute myocardialinfarction(AMI) was the major cause of death. Although the development of medicine,cardiac interventions technique, bypass surgery treatment reduced myocardial infarctionmortality, there were still more than30%of total patients may have a ventricularremodeling after myocardial infarction, heart failure and other complications. Stem celltransplantation therapy provided therapeutic potential for these patients. As the new idealseed cells for cell transplantation, the biological property of Wharton’s jelly-derivedmesenchymal stem cells(WJ-MSCs) isolated from human umbilical was consideredbetween embryonic stem cells(ESCs) and somatic stem cells(SSCs), which presentedpromising therapeutic potential for clinical application. Meanwhile, growing evidenceshown that apelin/APJ system functions as a critical mediator of cardiac development aswell as endothelial angiogenic processes in several animal models. However, there was notany studies reporting the role of apelin in the cardiomyogenic(CMG) differentiation ofmesenchymal stem cells yet.Part1The regulation study of apelin gene expressed inWharton’s jelly-derived mesenchymal stem cells of humanumbilical cordObjectiveRecombinant lentivirus vectors which up/down-regulated expression of apelin genewere prepared by genetic engineering technology and applied to control the expression of apelin gene in WJ-MSCs by transfection technique. This method provided foundation formechanism study of cardiomyogenic differentiation in WJ-MSCs in vitro in subsequentexperiments.Methods:1. WJ-MSCs were isolated from human umbilical cord and cultured by tissueadherence method. MTT assay was employed to detect the proliferation of WJ-MSCs. Theimmune-phenotypes were detected by flow cytometry. The cells were induced todifferentiate into osteoblasts and lipoblasts by different culture systems.2. Genetic engineering technology were used to produce lentiviral vectors to upgradeor knockdown expression of apelin gene WJ-MSCs. The third passage WJ-MSCs weretransfected by lentiviral vectors. Then infected cells labeled EGFP was observed byfluorescence microscope and the transfection efficiency was calculated. Apelin mRNA andprotein in the transfected cells were detected by RT-PCR and Western blot analysis.3. All in vitro experiments were representative of three independent experiments. Datawere expressed as mean±SE(n≥3) and analyzed by SPSS18.0software package. Thestatistical methods were used according to different characteristics of data. Values ofP<0.05was considered statistically significant.Results:1. About7days after attachment, the primary passage cells were shown asspindle-like morphology, gradually spread out swirly and proliferated quickly. FACSanalysis showed that WJMSCs expressed the surface markers of MSCs, didn’t express thehematopoietic lineage markers. Immunohistochemistry showed that the MSCs werepositive for alizarin red, alkaline phosphatase and oil red-O staining．2. Fluorescence microscopy revealed that the WJ-MSCs transfected by lentiviralvector presented bright green fluorescence48hours later and kept stable96hours later. Thetransfection efficiency were over90%when MOI was over20. The results of RT-PCR andWestern Blot showed that the expression levels of both apelin mRNA and protein weresignificantly regulated as primarily expected (P<0.05).Conclusions:The molecular and immunophenotype of WJ-MSCs cultured in our experiments aresimilar to other MSCs. WJ-MSCs are induced osteogenic and adipogenic differentiation inspecific culture medium. Expression of apelin in P3-P5WJ-MSCs is effectively regulatedafter infecting by recombinant lentivirus vectors. The optimal multiple of infection is20 and the transfection efficiency is stably above90%after4days. The infected cells can beused in subsequent induction experiments. Part2Apelin Promotes Cardiomyogenic Differentiation inMesenchymal Stem Cells Derived from Wharton’s Jelly ofhuman Umbilical Cord in VitroObjectiveWJ-MSCs were used as tool cells in our study and the role of apelin in the CMGdifferentiation of WJ-MSCs was investigated in vitro, which established foundation forfurther application of WJ-MSCs genetically modified by apelin in cardiac regenerationmedicine and cardiovascular tissue engineering.Methods:1.Apelin-overexpression WJ-MSCs, apelin-silenced WJ-MSCs and WJ-MSCs wascultured in complete medium containing5-azacytidine(10μmol/L) and bFGF (10μg/L).After incubating for another24hrs, the medium was changed to complete mediumcontaining bFGF (10μg/L). The medium was changed three times a week thereafter untilthe experiment was terminated4weeks after the drug treatment. WJ-MSCs without anytreatment were served as the control group.2. The dynamic changes in morphology were detected by inverted phase-contrastmicroscopy. The mRNA expression of pre-mesodermal marker Mesp1, cardiac progenitortranscription factor Mef2c, NKX2.5and cardiac specific proteins β-myosin heavychain(β-MHC), myosin light chain(MLC), cTnT were detected at1,4,7,14and28daysafter induction by RT-PCR. The protein expressions of α-actin, MHC, cTnT andconnexin-43were detected by immunofluorescence method.3. All in vitro experiments were representative of three independent experiments.Data were expressed as mean±SE(n≥3) and analyzed by SPSS18.0software package.The statistical methods were used according to different characteristics of data. Values ofP<0.05was considered statistically significant.Results: The morphology of apelin-treated cells gradually increased in size and formed a longstick-like appearence at about1week. Within3-4weeks, many cells formed myotube-likestructures. RT-PCR analysis showed that mRNA expression of Mesp-1reached the peakafter4days of induction, then began to decrease s in all groups except for theapelin-silenced WJ-MSCs and the control group. Positive relationship between apelinexpression levels and the mRNA expression of Mef2c、NKX2.5、β-MHC、MLC、cTnTwere dynamically existed throughout the induction period except for the apelin-silencedWJ-MSCs and the control group. Immunofluorescence analysis revealed that proteinexpression of α-actin, MHC, cTnT and connexin-43were strongly positive, especially inapelin-overexpression WJ-MSCs compared with other groups (P<0.05).Conclusions:WJ-MSCs can be induced to differentiate into CMG cells by5-aza and bFGF andpossessed characterization of cardiomyocytes in structure as well as function, which isconfirmed by morphology, mRNA and protein detection technology. Meanwhile, apelinpromotes cardiac differentiation of WJ-MSCs induced by5-azacytidinecytidine in vitro.Loss of apelin expression in WJ-MSCs may inhibit CMG differentiation.