Abstract: Regenerative medicine using cardiomyocytes derived from human-induced pluripotent stem cells (hiPSCs) has already entered clinical trials. However, to establish this approach as a standard treatment, several challenges remain, including improving cell engraftment rates and reducing production costs.
Cardiomyocytes were differentiated from hiPSCs through monolayer culture. On day 12 of culture, cells were passaged and treated with a GSK-3β inhibitor to generate expanded cardiomyocytes (E-CM). Non-expanded cardiomyocytes (N-CM) were prepared in parallel by simply exchanging the culture medium, without passage or GSK-3β inhibitor treatment. Both cell types were harvested on day 20. Myocardial infarction models were created using athymic rats, and 2 × 10⁷ N-CM or E-CM were transplanted one week after infarction. Four weeks post-transplantation, the hearts were excised for histological analysis.
Compared to N-CM, E-CM exhibited approximately a threefold increase in cell numbers at the time of harvest. However, E-CM showed a decreased Ki67-positive rate and significantly fewer viable cells 12 hours post-seeding. Moreover, E-CM exhibited increased expression of senescence markers, including SA-β-galactosidase, p16, and p21. The graft size in the E-CM transplantation group was significantly smaller, with a higher rate of apoptotic cells.
This study demonstrated that cardiomyocytes proliferated using GSK-3β inhibitors underwent senescence, leading to reduced cell engraftment capacity post-transplantation. To advance myocardial regenerative therapy as a routine treatment, it is crucial to suppress cardiomyocyte senescence prior to transplantation.
Funding Source: This work was supported by JST SPRING, Grant Number JPMJSP2144 (Shinshu University).
