Rhee and Wu CCND2-Overexpressing iPSC-CM for Cell Therapy 15 sustained proliferative activity of the transplanted iPSC-CMs resulting in extensive myocardial regeneration in vivo. Another approach to promote cell cycle activity is by treating cardiac cells with hormones, growth factors, microRNAs, or small molecules to enhance cell cycle signaling or to induce reentry of differentiated myocytes (Figure). 10 For example, treatment of embryonic stem cell–derived CMs with insulin-like growth factor was shown to enhance their proliferation by activating the phosphoinositide 3-kinase/Akt signaling pathway. 11 Via a different pathway of JAK/STAT (janus tyrosine kinasesignal transducer and activator of transcription), treatment of iPSC-CMs and embryonic stem cell–derived CMs with granulocyte colony-stimulating factor resulted in significant proliferation of the derived cardiomyocytes. 12 Many microRNAs were also reported to enhance cardiac cell cycle activity when they were overexpressed or inhibited/knocked down in cardiomyocytes in vivo. 10 More recently, using a high-throughput combinatorial screening approach, several small molecules enhancing iPSC-CM proliferation have also been identified. 13

Significant research efforts have been made to discover genetically modifiable or druggable targets to enhance cardiac cell cycle activity. Although promising, we still know relatively little about the mechanism underlying cardiac cell cycle regulation. Because of the complex molecular circuits involved in this process, it is difficult to fully understand how the treatment of the proproliferative molecules affects overall cell cycle activity and function. Rather than targeting upstream signaling pathways, overexpression of direct cell cycle regulators may provide a more reliable and consistent method to activate the cell cycle. In particular, with advances in genome-editing technologies such as CRISPR/Cas9, genetic engineering of iPSCs has become highly efficient, precise, and convenient. 14 This ability to genetically modify iPSCs is particularly attractive because it does not require temporal and spatial regulation of drug treatments. The continued myocardial regeneration of iPSC-CMs observed by Zhu et al6 also supports the durable effectiveness of genetically engineered cell cycle activation. Although more research is needed to