Abstract 217: TBX5 Clover2 /NKX2-5 TagRFP hiPSCs for Simultaneously Isolating Lineage-Specific Cardiovascular Cells

Abstract

In vertebrate, the heart is the first organ to develop, comprises multiple highly specialized cell types arising from different lineages. Early animal studies have identified two cardiac lineages: the first and second heart fields which contribute to the four-chamber heart formation. These cardiac progenitor cells develop into different parts of the heart and any perturbation of them frequently leads to congenital heart disease. However, given the difficulty in studying human embryology, current understanding of the hierarchy of cardiac lineages is primarily based on mouse models. The recent breakthrough in generation of human induced pluripotent stem cells (hiPSCs) from somatic cells and de novo differentiation into cardiomyocytes offers an unprecedented opportunity to investigate the genetic control of the cellular process of the heart development and investigate cardiac lineages under human genetic background. Despite the prominent advantages of hiPSCs in cardiac research, heterogeneity of hiPSC-derived cardiomyocytes (hiPSC-CMs) significantly impede their applications in regenerative medicine, disease modeling, and personalized drug testing. Using CRISPR/Cas9 genome-editing technology, I have generated a dual-color iPSC reporter line by targeting fluorescent protein sequences to two transcription factors (TBX5 and NKX2.5) which play a critical role in cardiac differentiation. Using this novel reporter line, I was able to simultaneously isolate four distinct subpopulations which exhibited different gene expression patterns. Continuous culture after isolation, it was found that TBX5 + NKX2.5 + subpopulation primarily differentiated into ventricular myocytes (90% purity), whereas TBX5 - NKX2.5 + subpopulation differentiated into atrial myocytes (80% purity). Moreover the TBX5 + NKX2.5 - subpopulation exhibited the epicardial lineage characteristics and contributed to nodal myocytes (80% purity). However, TBX5 - NKX2.5 - subpopulation developed into endothelial cells. In conclusion, using this reporter line, different lineage subpopulations can be identified and isolated from a heterogeneous pool of cardiac cells. These purified myocyte subtypes hold the potential for precise disease modeling and drug testing.