Specifying the Anterior Primitive Streak by Modulating YAP1 Levels in Human Pluripotent Stem Cells

Specifying the primitive streak (PS) guides stem cell differentiation in vitro, however much remains to be learned about the transcription networks that direct anterior and posterior PS cells (APS and PPS, respectively) to differentiate to distinct mesendodermal subpopulations. Here, we show that APS genes are predominantly induced in YAP1-/- hESCs in response to ACTIVIN. This finding establishes the Hippo effector YAP1 as a master regulator of PS specification, functioning to repress ACTIVIN-regulated APS genes in hESCs. Moreover, transient exposure of wild-type hESCs to dasatinib, a potent C-SRC/YAP1 inhibitor, enables differentiation to APS-derived endoderm and cardiac mesoderm in response to ACTIVIN. Importantly, these cells can differentiate efficiently to normal beating cardiomyocytes without the cytoskeletal defect seen in YAP1-/- hESC-derived cardiomyocytes. Overall, we uncovered an induction mechanism to generate APS cells using a cocktail of ACTIVIN and YAP1i molecules that holds practical implications for hESC and iPSC differentiation into distinct mesendodermal lineages. Our data strongly indicate that combined treatment of ACTIVIN and YAP1i during the first 24h of differentiation in WT hESCs and hiPSCs is sufficient to generate APS cells that efficiently differentiate into cardiac mesoderm. Thus, we reasoned that transient ACTIVIN+YAP1i exposure might provide an alternative strategy to generate cardiomyocytes in WT hESCs.We then compared the transcriptomes of cardiomyocytes generated from WT H1 hESC by YAPi+ACTIVIN treament to the GiWi protocol at Day 30 of differentiation.