Cell population balance of cardiovascular spheroids derived from human induced pluripotent stem cells

Stem cell-derived cardiomyocytes and vascular cells can be used for a variety of applications such as studying human heart development and modelling human disease in culture. In particular, protocols based on modulation of Wnt signaling were able to produce high quality of cardiomyocytes or vascular cells from human pluripotent stem cells (hPSCs). However, the mechanism behind the development of 3D cardiovascular spheroids into either vascular or cardiac cells has not been well explored. Hippo/Yes-associated protein (YAP) signaling plays important roles in the regulation of organogenesis, but its impact on cardiovascular differentiation has been less evaluated. In this study, the effects of seeding density and a change in YAP signaling on 3D cardiovascular spheroids patterning from hPSCs were evaluated. Compared to 2D culture, 3D cardiovascular spheroids exhibited higher levels of sarcomeric striations and higher length-to-width ratios of α-actinin⁺ cells. The spheroids with high seeding density exhibited more α-actinin⁺ cells and less nuclear YAP expression. The 3D cardiovascular spheroids were also treated with different small molecules, including Rho kinase inhibitor (Y27632), Cytochalasin D, Dasatinib, and Lysophosphatidic acid to modulate YAP localization. Nuclear YAP inhibition resulted in lower expression of active β-catenin, vascular marker, and MRTF, the transcription factor mediated by RhoGTPases. Y27632 also promoted the gene expression of MMP-2/-3 (matrix remodeling) and Notch-1 (Notch signaling). These results should help our understanding of the underlying effects for the efficient patterning of cardiovascular spheroids after mesoderm formation from hPSCs.