Expression dynamics of HAND1/2 in in vitro human cardiomyocyte differentiation

Hand1 and Hand2 are transcriptional factors, and knockout mice of these genes show left and right ventricular hypoplasia, respectively. However, their function and expression in human cardiogenesis are not well studied. To delineate their expressions and assess their functions in human cardiomyocytes (CMs) in vitro, we established two triple reporter human induced pluripotent stem cell lines, HAND1mCherry, HAND2EGFP, with MYH6-driven iRFP670 or constitutive tagBFP expression and investigated their expression dynamics during cardiac differentiation. On day 5 of the differentiation, HAND1 expression marked cardiac progenitor cells. We profiled the CM subpopulations on day 20 with RNA-sequencing and found that mCherry+ CMs showed higher proliferative ability than mCherry- CMs and identified CD105 as a surface marker of highly proliferative CMs. Finally, we revealed that LEF1 is a key regulator of proliferation and is repressively regulated by HAND1 and HAND2. Overall design: HAND1-mCherry, HAND2-EGFP and MYH6-iRFP670 triple reporter human induced pluripotent stem cells (hiPSCs) were differentiated into cardiomyocytes. RNA-seq were performed with samples of day 0, 1 , 3 , 5 (purified mCherry+/-), 9 (purified iRFP670+/-), 20 (purified mCherry+EGFP-, mCherry-EGFP+, mCherry+EGFP- and mCherry+EGFP+ in ventricular protocol, and mCherry-EGFP+ in atrial protocol). All day 20 samples were isolated from iRFP670+ cardiomyocytes. For knockdown experiments, cardiomyocytes were isolated on day 15 and transfected with siRNAs of negative control (siNC), HAND1 (siHAND1), HAND2 (siHAND2), LEF1 (siLEF1) using MYH6-EGFP hiPSCs. RNAs were sampled on day 20. The transcriptomes of three/four independent samples were analyzed.