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Loss of myocardial tissue remains a leading cause of disease and death, as the adult heart has insufficient regenerative potential. The (pre-)clinical effects of inducing cardiac regeneration by cardiac cell therapy have been disappointing. This lack of success may result from the fact that it remains largely unclear how the receiving pathological microenvironment affects this process of cardiomyogenic differentiation of implanted cells, and thereby may (negatively) influence the therapeutic outcome. However, the tools to address this lacuna in a proper manner are lacking, as this requires tightly controllable and quantitative models of cardiomyogenic differentiation. Therefore, we have recently generated lines of conditionally immortalized human CMCs (ciCMCs) through doxycycline-dependent expression of SV40 large T antigen after genetic modification and subsequent clonal expansion. In these cells, proliferation and differentiation can be tightly controlled, allowing cardiomyogenic differentiation to be i) induced on cue, ii) precisely monitored and quantified, and iii) manipulated. The aim of this study is to improve our understanding of cardiomyogenic differentiation of guest (transplanted) cells in the context of the host (receiving) microenvironment. To create pathological microenvironments and study the effects on cardiac differentiation, co-culture experiments with human ciCMCs and cardiac fibroblasts were conducted in different ratios (10%, 30%, 60% and 90% ciCMCs). Cardiomyogenic differentiation was determined by immunostaining for cardiac specific markers and electrophysiological analysis by optical voltage mapping. After 12 days of co-culture with cardiac fibroblasts, the amount of ciCMCs that expressed the sarcomeric protein cardiac troponin T was significantly and increasingly reduced (P Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Leiden University Fund

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.

Cardiomyogenic differentiation of stem cells can be accomplished by coculture with cardiomyocytes (CMCs). To facilitate their identification, stem cells are often labeled through viral transduction with a fluorescent protein. A second marker to distinguish stem cell-derived CMCs from native CMCs is rarely used. This study aimed to investigate the occurrence of secondary transduction of unlabeled neonatal rat (nr) CMCs after coculture with human cells that had been transduced 0, 7, or 14 days earlier with a vesicular stomatitis virus (VSV) G protein-pseudotyped lentiviral vector (LV) encoding enhanced green fluorescent protein (GFP). To reduce secondary LV transfer, GFP-labeled cells were incubated with non-heat-inactivated human serum (NHI) or with VSV-neutralizing rabbit serum (αVSV). Heat-inactivated human serum and normal rabbit serum were used as controls. Immunostaining showed substantial GFP gene transfer to nrCMCs in cocultures started at the day of transduction indicated by the presence of GFP-positive/human lamin A/C-negative nrCMCs. The extent of secondary transduction was significantly reduced in cocultures initiated 7 days after GFP transduction, while it was completely abolished when human cells were added to nrCMCs 14 days post-transduction. Both NHI and αVSV significantly reduced the occurrence of secondary transduction compared to their controls. However, under all circumstances, GFP-labeled human cells had to be passaged for 14 days prior to coculture initiation to prevent any horizontal GFP gene transfer to the nrCMCs. This study emphasizes that differentiation experiments involving the use of viral vector-marked donor cells should be interpreted with caution and describes measures to reduce/prevent secondary transduction. STEM CELLS 2012;30:2830–2834