Since the advent of the generation of human induced pluripotent stem cells (hiPSCs), numerous protocols have been developed to differentiate hiPSCs into cardiomyocytes and then subsequently assess their ability to recapitulate the properties of adult human cardiomyocytes. However, hiPSC-derived cardiomyocytes (hiPSC-CMs) are often assessed in single-cell assays. A shortcoming of these assays is the limited ability to characterize the physiological parameters of cardiomyocytes, such as contractile force, due to random orientations. This protocol describes the differentiation of cardiomyocytes from hiPSCs, which occurs within 14 d. After casting, cardiomyocytes undergo 3D assembly. This produces fibrin-based engineered heart tissues (EHTs)-in a strip format-that generate force under auxotonic stretch conditions. 10-15 d after casting, the EHTs can be used for contractility measurements. This protocol describes parallel expansion of hiPSCs; standardized generation of defined embryoid bodies, growth factor and small-molecule-based cardiac differentiation; and standardized generation of EHTs. To carry out the protocol, experience in advanced cell culture techniques is required.

Summary The cellular signals controlling the formation of cardiomyocytes, vascular smooth muscle and endothelial cells from stem cell-derived mesoderm are poorly understood. To identify these signals, a mouse embryonic stem cell (ESC)-based differentiation assay was screened against a small molecule library resulting in a novel 1,4-dihydropyridine inducer of type II TGFβ receptor (TGFBR2) degradation-1 (ITD-1). ITD analogs enhanced proteasomal degradation of TGFBR2, effectively clearing the receptor from the cell surface and selectively inhibiting intracellular signaling (IC50 ~ 0.4-0.8μM). ITD-1 was used to evaluate TGFβ involvement in mesoderm formation and cardiopoietic differentiation, which occur sequentially during early development, revealing an essential role in both processes in ESC cultures. ITD-1 selectively enhanced the differentiation of uncommitted mesoderm to cardiomyocytes, but not to vascular smooth muscle and endothelial cells. Together, ITD-1 is the first selective TGFβ inhibitor and reveals an unexpected role for TGFβ signaling in controlling cardiomyocyte differentiation from multipotent cardiovascular precursors.

Cardiac induction requires stepwise integration of BMP and WNT pathway activity. Human embryonic stem cells (hESCs) are developmentally and clinically relevant for studying the poorly understood molecular mechanisms downstream of these cascades. We show that BMP and WNT signaling drive cardiac specification by removing sequential roadblocks that otherwise redirect hESC differentiation toward competing fates, rather than activating a cardiac program per se. First, BMP and WNT signals pattern mesendoderm through cooperative repression of SOX2, a potent mesoderm antagonist. BMP signaling promotes miRNA-877 maturation to induce SOX2 mRNA degradation, while WNT-driven EOMES induction transcriptionally represses SOX2. Following mesoderm formation, cardiac differentiation requires inhibition of WNT activity. We found that WNT inhibition serves to restrict expression of anti-cardiac regulators MSX1 and CDX2/1. Conversely, their simultaneous disruption partially abrogates the requirement for WNT inactivation. These results suggest that human cardiac induction depends on multi-stage repression of alternate lineages, with implications for deriving expandable cardiac stem cells.