Am80 activates cardiomyocytes cell cycle via retinoic acid receptor for efficient transplantation

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-cardiomyocytes) are promising sources for the regenerative therapy to treat heart failure. To realize this therapy, the engraftment potential of hiPSC-cardiomyocytes following the injection into the host heart should be improved. Here, we established the efficient method to analyze the cell cycle activity of hiPSC-cardiomyocytes using Fluorescence Ubiquitination-based Cell Cycle Indicator (FUCCI) system. High-throughput screening analysis using FUCCI-expressing hiPSC-cardiomyocytes identified a retinoic acid receptor (RAR) agonist, Am80, as an effective cell cycle activator in hiPSC-cardiomyocytes. The transplantation of hiPSC-cardiomyocytes treated by Am80 prior to the injection significantly enhanced the engraftment into the damaged mouse heart for 6 months. RNA sequencing analysis in Am80-treated iPSC-cardiomyocytes revealed that both RARA and RARB played important roles in the Am80-mediated cell cycle activation in hiPSC-cardiomyocytes. Collectively, this study highlights the effectiveness of FUCCI system to easily analyze the cell cycle status in hiPSC-cardiomyocytes and the cell cycle activation by Am80 is the possible strategy to increase the graft size following the cell transplantation into the damaged heart. Overall design: Fluorescent Ubiqutination-based Cell Cycle Indicator (FUCCI) reporter human induced pluripotent stem cells (201B7) were differentiated into cardiomyocytes (CMs). All cardiomyocytes were isolated from lineage (CD31, CD90, CD49a, CD140b) negative and SIRPA (CD172a/b) positive population by fluorescence activated cell sorting (FACS). Three FUCCI subpopulations (Red:Kusabira-Orange(+) and Azami-Green(-), White:Kusabira-Orange(-) and Azami-Green(-), Green:Kusabira-Orange(-) and Azami-Green(+)) were collected on day 20 (N=2). For Am80/DMSO treated samples, Am80 and DMSO were administered to embryonic bodies in culture medium from day 18 and CMs were purified on day 20 (N=3). For knockdown experiments, purified CMs were transfected with siRNA for negative control (siNC), RARA (siRARA), RARB (siRARB) and seeded onto fibronectin coated plates on day 17. Am80 and DMSO were administered from day 18 and RNA was collected on day 20 (N=3).