Real-Time Cell Evaluation System Based On Human Induced Pluripotent Stem Cell Derived Cardiomyocytes Establishment Of A Method For Evaluating The Cardiac Toxicity Of Drugs

Background:Advances in bioengineering and in vitro culture technologies have led to a rapid expansion of myocardial model development for use in drug efficacy/toxicity testing(Navarrete et al.,2013),disease modeling(Moretti et al.,2010,Wang et al.,2014),and mechanistic studies of cardiac development(Paige et al.,2012).However,the widespread adoption of such techniques for generating engineered human cardiac constructs that accurately model the in vivo tissue is predicated on the establishment of reliable sources of human cardiomyocytes.To that end,much recent effort has been devoted to the development of novel engineered biomimetic cardiac tissue platforms that accurately recapitulate the structure and function of the human myocardium.Within the field of cardiac engineering,induced pluripotent stem cells(iPSCs)are an exciting tool that offer the potential to advance the current state of the art,as they are derived from somatic cells,enabling the development of personalized medical strategies and patient specific disease models.Other methods used for cardiac modeling so far include the use of Sendai virus vectors(Churko et al.,2013),which obviate translational issues associated with transgene integration into the host cell’s genome.Additionally,the use of episomal plasmids(Burridge et al.,2011),co-MIP(Diecke et al.,2015),microRNAs(Li et al.,2011),and direct protein delivery(Zhou et al.,2009)have all been shown to be capable of producing iPSCs that can be differentiated into beating cardiomyocytes.Enthusiasm for the use of iPSC lines in advancing clinical and basic science research,concerns have been raised regarding whether such cells are identical to ESCs.On the one hand,it has been argued that iPSCs and ESCs are nearly identical,since both cell types exhibit similar phenotypes,dependencies,and behavior in vitro(Kong et al.,2010).Additionally,analysis performed over a significant number of clones highlights a considerable overlap in terms of cellular properties between iPSC and ESC sources,making it difficult to distinguish them without in-depth testing(Yamanaka,2012).On the other hand,microarray research has demonstrated that hundreds of genes,as well as DNA methylation patterns,are differentially expressed between iPSCs and ESCs(Chin et al.,2009,Newman and Cooper,2010).Overall,measurement of a range of properties of iPSC and ESC lines,including gene expression,DNA methylation,microRNA expression,differentiation propensity,and complementation activity in embryos,suggest that their properties do vary(Chin,Mason,2009,Wilson et al.,2009).Although specific differences have been reported between iPSC and ESC lines,there is little conclusive evidence that cardiomyocytes produced from these cell sources differ in any meaningful way,once differentiated.Therefore,despite distinctive dissimilarities in undifferentiated stem cell sources,the high degree of overall comparability between iPSC-and ESC-derived cardiomyocytes and the reproducibility of the cardiac differentiation methods routinely employed,coupled with the advantages of iPSCs in terms of disease modeling and personalized medicine applications,make iPSCs exciting candidates for application in both clinical and basic cardiac research applications.Objective:To establish human induced pluripotent stem cell-derived cardiomyocytes(hiPSC-CM)for cardiotoxicity assessment.Method:Four cardiotoxic drugs were selected as references.They were doxorubicin(anthracycline anticancer drug,10 nmol·L-1、100 nmol·L-1、1μmol·L-1 and 10μmol·L-1),amiodarone(class Ⅲ antiarrhythmic drug,10 nmol·L-1、100 nmol· L-1、300 nmol·L-1、1μmol·L-1 and 10 μ mol·L-1),and verapamil(calcium channel blocker,10 nmol·L-1、100 nmol·L-1、1μmol·L-1 and 10 μ mol·L-1).hiPSC-CMs were incubated with these drugs.The real-time cell analysis system was used to monitor the changes in cardiomyocytes’ beat rate,amplitude,cell index and field potential duration.Result1.Purity Identification of Myocardial cellsIn order to verify the accuracy of the real-time evaluation system for cardiomyocytes derived from induced pluripotent stem cells,flow cytometry(FCM)was used to detect troponin(cardiac troponin T,cTnT)of cardiac myocytes,which proved that the purity of cardiac myocytes was 99%.Morphological purity identification indicated that cardiac troponin content was higher in differentiated cardiomyocytes.2.Effect of Amiodarone on hiPSC-CM related IndexesIn order to verify the effect of amiodarone on hiPSC-CM,we measured the contractility,contraction frequency,proliferation curve and field potential duration of hiPSC-CM.The results showed that 10 nmol·L-1、100 nmol·L-1、300 nmol·L-1、1μmol·L-1 and 10 μ mol·L-1 were used to induce myocardial contractility,contractile frequency and proliferation curve after stable pulsatile of 10 nmol PSC-CM,and the results showed that 10 nmol·L-1、100 nmol·L-1、300 nmol·L-1 had a significant effect on myocardial contractility,contractile frequency and proliferation curve.No statistically significant effect was found in the course of field potential.After 1μmol·L-1、10 μmol·L-1 amiodarone was administered,the inhibitory effect of 1μmol·L-1、10 μmol·L amiodarone on the contractility and contractile frequency of Amiodarone 1 μ mol ·L-1 was shown 48 hours after the administration of Amiodarone,which showed a statistically significant decrease in the number of Amiodarone cells.The results showed that the number of cells in 10 μmol·L-1 group decreased slightly more than that in 1 μmol·L-1 group,but there was no significant difference between 10μmol·L-1 group and 12 h after administration of amiodarone,which showed a complete inhibition of hiPSC-CM autonomy,while that of 1μmol·L-1 group was mostly completely inhibited.There are still a few still pulsating.3.Effect of verapamil on hiPSC-CM related indexesTo verify the effect of verapamil on hiPSC-CM,we measured the pulsatility,contractility,contraction frequency,proliferation curve and field potential duration of hiPSC-CM.The results showed that 10 nmol·L-1、100 nmol·L-1、1μ mol·L-1 and 10 μmol·L-1 had no significant effect on cardiac contractility,contractile frequency,proliferation curve and field potential duration after stable pulsatile of hiPSC-CM,and no significant effect was found on the contractility,contractile frequency,proliferation curve and field potential duration of Amiodarone at 10 μ mol·L-1,With the increase of concentration,the inhibition of hiPSC-CM was observed(P<0.05).4.Effect of doxirubicin on hiPSC-CM related indexesTo verify the effect of doxirubicin on hiPSC-CM,we measured the pulsatility,contractility,contraction frequency,proliferation curve and field potential duration of hiPSC-CM.The results showed that 10 nmol·L-1 100 nmol·L-1、1μ mol·L-1 and 10μmol·L-1 had no significant effect on cardiac contractility,contractile frequency,proliferation curve and field potential duration after stable pulsatile of hiPSC-CM,and no significant effect was found on the contractility,contractile frequency,proliferation curve and field potential duration of Amiodarone at 10μmol·L-1.With the increase of concentration,the inhibition of hiPSC-CM was observed(P<0.05).5.Effect of PM2.5 on hiPSC-CM related indexesIn order to verify the effect of PM2.5 on hiPSC-CM related indicators,we detected the contractile force,contraction frequency and proliferation curve of hiPSC-CM.The results showed that after the stability of hiPSC-CM pulsation,the immovable amiodarone(10μg/ml、10μg/ml、1000μg/ml)was given,and the results showed that the two concentrations of 10 μg/ml and 100 μg/ml had no statistically significant effect on myocardial contractility,contraction frequency and proliferation curve,and the inhibitory effect on hiPSC-CM with the concentration of 1000 μg/ml(P<0.05).Conclusions:The establishment of a feasible cardiotoxicity evaluation model based on hiPSC-CMs in vitro,combined with real-time cell analysis can be rapid,sensitive,accurate evaluation of drug cardiotoxicity.