P1229Massive expansion of native human atrial cardiomyocytes through immortogenetics: generation of the hiAM cell lines

Abstract

Abstract Background Preclinical cardiac research greatly depends on animal-derived cellular models, thereby hampering clinical translation. While upcoming human pluripotent stem cell technology seems to decrease this gap between bench and bedside, its complex/multi-step protocol to produce cardiac muscle cells, its required expertise, and its trouble to produce large numbers of phenotypically homogeneous cardiomyocytes so far has limited broad application. Purpose We aimed to conditionally immortalize native human atrial cardiomyocytes to produce natural and standardized lines of these cells by gaining full control over their proliferation and differentiation. Methods Human fetal atria (gestational age 18 weeks) were dissociated and transduced with a lentiviral vector directing myocyte-specific and doxycycline-inducible expression of simian virus 40 large T antigen (here defined as immortogenetics). Addition of doxycycline to the culture medium pushed cardiomyocytes towards a proliferative phenotype. In total, 125 proliferating monoclones were isolated, expanded and screened for their cardiomyogenic differentiation capacity upon doxycycline removal. Selected clones were characterised using various molecular biological and electrophysiological assays. Results Upon doxycycline removal (i.e. under differentiation conditions), cells spontaneously reacquired a cardiomyocyte-like appearance as judged by phase-contrast microscopy and were observed contracting. Simultaneously, these cells stopped proliferating, which was accompanied by a drop in large T level, loss of Ki67 expression and the development of sarcomeres with striated α-actinin and troponin T staining patterns. These cells were tagged conditionally immortalized human atrial cardiomyocytes (hereinafter called hiAMs). Optical voltage mapping of hiAM monolayers revealed excitable cells showing homogeneous spreading of action potentials at 22,5±3,1 cm/s following 1-Hz point stimulation, with a mean APD80 of 139±22 ms. Monolayers of hiAMs could easily be created as big as 10cm2 while continuing to display homogenous conduction throughout the culture. Single-cell patch clamp recordings of a hiAM clone in current-clamp mode confirmed excitability with a resting membrane potential of −62,2±4,3 mV, peak potential of 39,4±3,9 mV and APD80 of 339±9 ms. Excitable monolayer of hiAMs Conclusion We have generated first-of-a-kind lines of natural human atrial cardiomyocytes through immortogenetics, allowing massive cell expansion under proliferation conditions and robust formation of cross-striated, contractile and excitable cardiomyocytes after differentiation. Thereby, a user-friendly, clinically-relevant and much-anticipated research model has been produced, which application could range from multi-scale electrophysiological studies and drug response studies to disease modelling and myocardial regeneration.