Introduction: Senescence can be delayed, but it is an inevitable fate of all cells and organisms. After continuous culturing and propagation, the cells are subjected to progressive telomere attrition, accumulation of oxidative stress and DNA damage, generating rapidly aging cells. Transplanting these heterogeneous mixtures of young and functionally incompetent old (FIC) cells to regenerate injured myocardium leads to a marginal improvement in cardiac function. We hypothesized that selecting long-term repopulating and functionally competent cells from FIC cells and transplanting them into ischemic heart would yield better survival and engraftment rates.
Methods: Transcription factor p53 functions are cellular age dependent: inducing apoptosis in old cells while promoting cell cycle in young cells. Utilizing this discriminating nature of p53, we selected p53-resistant younger (p53RY) iPS cells and induced cardiomyocytes (iCM) from FIC cells using a small-molecule p53 stabilizer nutlin-3a. Differentiation efficiency and functional properties of p53RY cells were compared with FIC cells.
Results and Discussion: The markers for phenotypically young cells such as the accumulation of ROS, superoxide and DNA damage were significantly lesser than FIC cells. However, the telomere and telomerase activity were comparable in both cell groups, indicating p53RY cells are not genotypically younger. When differentiated, p53RY iPS cells generated more beating myocytes with a homogenous beating frequency when compared to FIC iCMs. The differentiated p53RY iCMs are characterized by: high expression of CM markers Nkx2.5, Gata4, TnI, MYL2 and MLYK, myocyte maturation markers KCNH2 and CAMK2B and gap junction proteins CX40 and CX43. Analysis of exosomes isolated from iCMs revealed high density 90 nm size exosomes unique to p53RY iCMs. In vivo study results are due and will be discussed at the meeting.
Conclusion: Our data clearly shows functionally competent cells can be isolated from a population of heterogeneous cells by activating p53. The selected p53RY iCMs showed superior performance over FIC iCMs. Selecting functionally competent cells and transplanting them would better repopulate the ischemic myocardium and improve cardiac performance.