Cell size regulates human endoderm specification through actomyosin-dependent AMOT-YAP signaling

Size, as a key physical property of the cell, has enormous impacts on cellular physiology and function. However, whether and how cell size influences stem cell specification is largely unknown. Here, we have analyzed the dynamic changes in cell size during definitive endoderm (DE) differentiation from human pluripotent stem cells. Interestingly, the cell size becomes smaller and with higher stiffness as the differentiation progresses. More importantly, accelerating the cell size diminution with hypertonic pressure significantly improves DE differentiation. Through functional intervention of mechanosensitive elements, we have identified actomyosin activity as a key mediator of DE differentiation and cell size diminution. Mechanistically, the diminution of cell size leads to AMOT nuclear translocation in an actomyosin-dependent manner, which represses YAP activity and thereby permits and facilitates DE differentiation. Together, our study has established a novel connection between cell size diminution and DE differentiation mediated by myosin-dependent AMOT nuclear translocation and suggests application of osmotic pressure an effective facilitator for human endoderm lineage differentiation. human ESC lines, H9, were cultured in mTeSR1 medium (STEMCELL Technologies, #AB217641) on Matrigel-coated plates. The endoderm differentiation protocol of human ESCs was based on the previous reports. IMDM (Gibco, #C12440500BT) and F12 (Gibco, #C11765500BT) mixed at the ratio of 1:1 (IMDM/F12), was used as basal medium, supplemented with 0.2% BSA (YEASEN, #36106ES76), 1% B27 (without Vitamin A, Shanghai BasalMedia Technologies, S441J7), 1% penicillin-streptomycin and Activin A (100 ng/ml, PeproTech, #120-14P) was added for 4 days for endoderm differentiation. When indicated, 10μM Blebbistatin and 2% sucrose was added for 4 days for endoderm differentiation.