DNA Methylation Status During Islet-1Promotes Cardiac Specific Differentiation Of Mesenchymal Stem Cells

ObjectiveIslet-1(Insulin gene enhancer binding protein isl-1) expressionlentiviral vectors (Lenti-Islet-1) are intended to construct and use forC3H10T1/2cells infection. Improved Islet-1expression could induceC3H10T1/2cells to differentiate specifically into cardiomyocyte-like cells.The DNA methylation status changes of cardiac relative gene Nkx2.5andMef2c promoter are intended to investigate during this process. DNAmethylation status is intended to study during the process Islet-1promotescardiac specific differentiation of MSCs.Materials and methods1. Construction of lenti-Islet-1vectors: Islet-1gene sequence wassynthesized by PTDS method. Islet-1gene sequence and lentiviral vectorpLenOR-THM were double enzyme digested by DNA incision enzymes.DNA fragments were purified and incubated by DNA ligase. The bacterialcells are treated by calcium chloride to render competent. Products of the connected reaction were transduced into the bacterial cells renderedcompetent. Plasmids from the bacterial colony were extracted and doubleenzyme digested by DNA incision enzymes. Products of the double enzymedigestion were purified and identified by PCR. The positive bacterial colonywas chose to sequencing. Sequencing result was compared with Islet-1sequence to verify the Islet-1-pLenOR-THM plasmid.Islet-1-pLenOR-THM plasmid, pMD2G plasmid, pRsv-REV plasmid andpMD1g-pRRE plasmid were purified and co-transduced to produce thelentiviral vectors by293T cells. The culture supernatant was collected andconcentrated. Virus titer of lenti-Islet-1vectors was detected by dilutionmethod of counting.2. Lentiviral vectors infection of C3H10T1/2cells: C3H10T1/2cellswere transplanted in to24hole plate. Make sure the cell number in each holeis about4×104. Polybrene, with a final concentration8mg/L, was added intoeach hole while the plates were overspread60%-70%by C3H10T1/2cells.Lentiviral vectors were added into the plate with a multiplicity of infection(MOI)=20. DMEM/F12culture medium with10%FBS was used to replacethe mixture liquid in each hole24hours after lentiviral vectors infection.GFP expression was observed72hours after the replacement of culturemedium. Islet-1expression in each experimental group was detected byindirect immunofluorescence.3. C3H10T1/2cells were divided into three groups: C3H10group (untreated C3H10T1/2cells， blank control), negative control group(C3H10T1/2cells infected by blank lentiviral vectors) and experimentalgroup (C3H10T1/2cells infected by Lenti-Islet-1)。4. Detection of Nkx2.5and Mef2c promoter methylation status:Genome DNA of cells in each experimental group was extracted two weeksafter the lentiviral vectors infection. DNA of each experimental group wastreated by sulphite. DNA concentration was detected before sulphitetreatment to make sure there was exactly350ng DNA treated each time. TheMS-PCR used DNA treated by sulphite as template.Results1. Construction of lenti-Islet-1vectors: Islet-1gene was verified toconnect to the pLenOR-THM plasmid by PCR. DNA sequencing showsIslet-1-pLenOR-THM recombinant plasmid contained the target Islet-1gene precisely. Virus titer of lenti-Islet-1vectors was2×108TU/mL。2. GFP expressions in cells of the negative control group andexperimental group could be observed five days after the lentiviral vectorsinfection; The infection efficiency of the negative control group is90.09%,The infection efficiency of the experimental group is87.71%； Four weeksafter lentiviral vectors infection, there were no morphologic changesobserved in C3H10group and negative control group, while cellmorphologic changes to cardiomyocyte-sharped cells were observed inmultiple regions in the experimental group; The expression of Islet-1protein, particularly in the nucleus, in the experimental group cells was observed.3. DNA methylation of cardiac relative gene Nkx2.5and Mef2c in theexperiment goup is much lower than C3H10group and negative controlgroup two weeks after the Lenti-Islet-1infection.ConclusionsDNA methylation of cardiac relative genes reduced to promote thesegenes’ expressions during the process mesenchymal stem cells are specificdifferentiated into cardiomyocyte-like cells.