Abstract 1067: Controlled Expression of SV40T Antigen in Rat Cardiomyocytes Promotes Switching from Proliferation to Differentiation
Background: Large numbers of well-differentiated cardiomyocytes are needed for cell therapies. Several investigators have progressesd cell cycle to induce proliferation of cardiomyocytes. However, these cells are not well-differentiated. Here, we report the amplification of the cardiomyocyte population using retrovirus-mediated transfer of simian virus 40 large T (SV40T) antigen. SV40T was subsequently removed by Cre-loxP-based site-specific recombination. After elimination of SV40T, proliferation was stopped and differentiation was promoted.
Methods and Results: Cultured neonatal rat cardiomyocytes were transduced with a retroviral vector expressing SV40T which is flanked by a pair of loxP recombination targets. Transduced cardiomyocytes yielded clones with greatly extended life spans up to population doubling level 142. Laser scanning cytometry analysis revealed that the transduction caused an induction of cell cycle progression. Complete elimination of the transferred SV40T gene was achieved after infection with a recombinant adenovirus expressing the Cre recombinase. Loss of SV40T caused G1-phase cell cycle arrest and discontinuance of cellular proliferation. DNA microarray analysis of cells that had lost SV40T showed increases in expression levels of cardiomyocyte-specific genes (cardiac troponin T, ventricular myosin light chain 3, cardiac alpha actin) and decreases in expression levels of genes associated with cellular proliferation (cyclin B1, C, D1, D2 and D3, cdk2, cdk7 and proliferating cell nuclear antigen).
Conclusion: This study provides a means to obtain large numbers of well-differentiated cardiomyocytes.