57 Massive Expansion of Functional Human iPSC-derived Cardiomyocytes 3 INTRODUCTION Adult mammalian heart has limited capacity for regeneration. Cardiac injuries such as myocardial infarction lead to significant cardiomyocyte (CM) loss and subsequent heart failure with significant morbidity and mortality. Cell-based therapeutic approaches such as injection of stem cell-derived cells or transplantation of engineered cardiac tissue patches have shown promise to re-muscularize the damaged myocardium and enhance cardiac functions (Chong et al., 2014; Grego-Bessa et al., 2007; Liu et al., 2018; Ogle et al., 2016; Senyo et al., 2014; Shiba et al., 2016) . Since the myocardium is densely packed with CMs, cell-based therapy requires introductions of billions of human CMs (Chong et al., 2014; Liu et al., 2018) . In principle, pluripotent stem cells provide an unlimited supply of human CMs, however, in practice the generation of therapeutically relevant number of human CMs still remains an extremely labor-and time-intensive process, despite robust and highly efficient directed differentiation protocols. To overcome this hurdle, several studies have focused on identifying molecules for promoting proliferation of differentiated CMs from pluripotent stem cells (PSCs) (Mills et al., 2019; Sharma et al., 2018; Titmarsh et al., 2016; Uosaki et al., 2013). However, when these molecules were applied to induce expansion of human PSC-derived CMs in vitro, the extent of proliferation has generally been modest (3-4-fold), which limits the use of these cells for cell-based therapeutic applications. This modest extent of proliferation in vitro may result from contact inhibition of proliferation, which is a natural regulatory process of normal tissue morphogenesis, homeostasis, and regeneration. Contact inhibition of proliferation has been well-described in many cell types other than cardiomyocytes including epithelial cells and endothelial cells (Grazia Lampugnani et al., 2003; Kim and Asthagiri, 2011; Puliafito et al., 2012) , where cell proliferation is cell-density dependent. When human induced PSCs (hiPSCs) undergo directed cardiac specification and differentiation in vitro, the differentiated CMs become fully confluent in a dish. It is in this densely-packed cardiomyocytes condition that screening of mitogen candidates have been performed (Mills et al., 2017, 2019; Sharma et al., 2015; Titmarsh et al., 2016). Hence, we asked whether the removal of contact inhibition would further promote hiPSC-CM proliferation beyond that achieved in the presence of mitogen. In fact, a few previous studies including our earlier work on compensation of CM loss in the fetal heart by residual unablated CMs and another study on targeted disruption of cell-cell contacts in the mouse heart show evidence of cell cycle re-entry and enhanced CM proliferation in vivo (Li et al., 2015; Sturzu et al., 2015) . Therefore, we hypothesize that combined inhibition of cell-cell contacts and treatment with CM mitogen would enable continuous CM proliferation and expansion. Here, we demonstrate for the first time the ability to massively expand functional hiPSC-CMs by continuous treatment of GSK-3β inhibitor CHIR-99021and concomitant removal of cellcell contact. We chose CHIR-99021 (CHIR) as the hiPSC-CM mitogen because several studies
RkJQdWJsaXNoZXIy MTk4NDMw