Effects Of Reporter Gene On Mitochondrial Morphology And Function Of I Pscs-derived Cardiomyocytes

ObjectiveInduced pluripotent stem cells（i PSCs）have the potential to differentiate into cardiomyocytes（CMs）,which has been a research hotspot in the treatment of myocardial infarction（MI）.Triple fusion（TF）reporter gene succeeds in monitoring the biological behaviors of transplanted cells in vivo.Mitochondria play a crucial role in the differentiation of i PSCs,but there is little known about the effects of TF reporter gene on the mitochondrial morphology and function of i PSCs-derived cardiomyocytes.The purpose of this study was to investigate the effects of TF reporter gene on the mitochondrial morphology and function of i PSCs-derived cardiomyocytes.MethodsThe i PSCs were divided into two groups:i PSCs group and TF-i PSCs group transduced with TF reporter gene,which were respectively differentiated into i-CMs group and TF-CMs group.γ-counter,positron emission tomography（PET）and small animal in vivo imaging system were used to test the expression of TF reporter gene in vitro.After the TF-CMs transplantation,small animal in vivo imaging system,¹⁸F-FDG and ¹⁸F-FHBG micro-PET were performed one week later to evaluate the in vivo expression of TF reporter gene in a rat model of myocardial infarction.The effects of TF reporter gene on the viability,proliferation and differentiation of i PSCs were further tested using trypan blue assay,Cy QUANT cell proliferation assay and immunofluorescence.And the effects of TF reporter gene on the mitochondrial morphology and function of i PSCs-derived cardiomyocytes were finally detected by in vivo fluorescence,transmission electron microscopy（TEM）,fluorescent quantitative PCR and MTT assay.ResultsIn experiments to test the expression of TF reporter gene,γcounter and micro-PET analysis showed that TF-i PSCs or TF-CMs had specific high radiation signal,while i PSCs or i-CMs showed no signal;meanwhile,quantification analysis showed a strong positive correlation between TF-i PSCs cell number and bioluminescence imaging（BLI）signal（R²=0.92）.In vivo imaging showed that transplanted TF-CMs can be detected by BLI and micro PET one week after transplantation.Compared with i PSCs,TF-i PSCs cell viability,proliferation and differentiation ability were not adversely affected by TF reporter gene.Compared with i PSCs,TF-i PSCs mitochondrial morphology and function,including the morphology and quantity of mitochondria,reactive oxygen species（ROS）,mitochondrial membrane potential,mitochondrial DNA copy number and mitochondria activity,were not affected by TF reporter gene.P<0.05 was statistically significant.ConclusionThis study established a rat model of myocardial infarction,transplanted differentiated cardiomyocytes into the injured area and combined the use of multiple in vivo and in vitro monitoring methods to confirm that the TF reporter gene can achieve in vivo and noninvasive monitoring of cardiomyocytes after transplantation and it had no adverse effect on mitochondrial morphology and function of i PSCs-derived cardiomyocytes.This study demonstrated the safety of TF reporter gene in multimodality imaging of transplanted i PSCs in mitochondria level.