Research Of Induced Pluripotent Cells Fused With Cardiomyocytes

[Objective]To construct fusion cells with induced pluriopotent stem cells andcardiomyocytes in vitro,and to investigate biological features of fusioncells.[Methods]1. iPSCs cell lines (IP14D-102) purchased from the Institute ofZoology, Chinese Academy of Sciences (stem cell Bank of Beijing). iPSCswas cultured on the MEF in advance of Mitomycin c-treated. Culturemedium for DMEM-H, containing10%FBS,1000IU/ml LIF,0.1mmol/Lβ-mercaptoethanol, l-glutamine,1%0.1mmol/L non-essential amino acids.Cell suspension was distinction attachment1hour to removal MEF beforepassaged, then suction the supernatant and vaccinate on new MEF feederlayer.2.0.1%Ⅱ type collagenase and0.1%trypsin were used to digestmyocardial tissue, respectively. to obtain neonatal mice cardiomyocytes.Observation biological characteristics of cardiomyocytes, and identifiedcardiomyocytes by immunofluorescence method in vitro.3. iPSCs and primary cardiomyocytes were fused with polyethylene glycol（PEG-4000） to construct fusion cells. Fusion cells whether or notexpressed stem cells and cardiomyocytes specific protein was identified byimmunofluorescence method. Fusion Cell cultivation in accordance withstandard culture conditions of iPSCs. Growth characteristic changes offusion cells were observed dynamically, and detect the ability of formationof embryoid bodies in vitro.4. Stem cells and cardiomyocytes specific gene and protein weredetected by RT-PCR and immunofluorescence method in fusion cells.Chromosome karyotype analysis were performed to deremine whether theoccurrence of nuclear fusion and degree of integration. And alkalinphosphatase（AKP） staining of fusion cells were performed.[Results]1. The feeder layer cells was Long spindle-shape. The growthmorphological of iPSCs colony was round or oval island-shape on feederlayer cells, refraction is strong around the colony. Among of cells arrangetightly in colony, and the delimitation and morphological not easydistinguish. It is clear that green colony(Oct-4-GFP+) under fluorescentmicroscope corresponding light microscope2. Cardiomyocytes were triangles, polygons, or flat irregular forms.Cardiomyocytes out of the pseudopod contact with each other andinterwoven into a network, and local synchronization contractioncardiomyocytes were observed from3days, the beating frequency between 30-50/min. On12days, most cardiomyocytes stopped beating, cytoplasmicvacuole appears, cell shrinkage, and defluvium defluxion. cTnT of redfluorescence were observed within the cytoplasmic, after cells immunefluorescent staining.3. Fusion cells were constructed successfully between iPSCs andcardiomyocytes by polyethylene glycol4000mediation. Colony-like cellclusters occurred4days after fusion, throughout the test period, thecardiomyocyte-like fusion cells did not observed. Karyotype analysisshows that more than65%of fusion cells had tetraploid karyotype orresemble tetraploid karyotype.4. At the different time point, AKP positive rate of fusion cellsincompletion identity. But at the same time point, the AKP positive rate offusion cells significantly lower than those of iPSCs. Stem cell-specificgenes (Oct-4, Nanog) and cardiomyocytes-specific genes (α-MHC,beta--MHC) were detected at the same time in fusion cells before passage5,but there was only stem cell-specific genes was detected in fusion cellsafter passage5. Oct-4protein and cTnT protein were detected at the sametime in early fusion cells, but there was only Oct-4protein express waspositive in fusion cells after passage4, and cTnT protein was not seen inthose of fusion cells.[Conclusions]1. There were can happen cell fused between diploid iPSCs and diploid primary cardiomyocytes by PEG-4000. Fusion cells showingbidirectional reprogramming in early age, along with cultivation timeextension, fusion cells only displayed one-way reprogramming to stemcells characteristics after passage5.2. This research results can not yet explained that the origin of stemcell-specific genes and stem cell-specific proteins in fusion cells:Continuation expressed of the parents stem cell genome, or that wasoriginally silent stem cell genes in parents cardiomyocytes was activatedand expressed again.