Biomechanical forces modulate corneal epithelial stem cell homeostasis and dedifferentiation through YAP – TGFβ pathway The corneal epithelium is the outermost layer covering the eye. Its transparency, high accessibility, and compartmentalization for stem cells (SCs) and differentiated cells make cornea an excellent model for SC research. Recently, we established an in-vivo microscopy approach to identify corneal (limbal) SC using K15-GFP transgene and discovered that depletion of the entire SC pool is restored by committed cells which de-differentiate into bonafide SCs. Contrarily, niche destruction resulted in irreversible SC loss and corneal opacification. However, the mechanism of SC differentiation and reprogramming of committed cells remained unclear. Interestingly, the limbal stromal niche contains a unique extracellular matrix hallmarked by low rigidity, while the differentiation compartment is much stiffer, suggesting that differentiation, as well as the de-differentiation of committed corneal cells, may be linked with mechano-sensing. Indeed, the cultivation of limbal SCs on substrates with physiologically relevant niche stiffness supported stemness while increased stiffness induced cell differentiation. Differentiation was coupled with changes in cytoskeletal organization, translocation of YAP from the nucleus to the cytosol and activation of TGFβ Pathway. Furthermore, pharmacological inhibition of mechanotransduction by blebbistatin or inhibition of TGFβ Pathway significantly attenuated cell differentiation. Finally forced activation of mechanosensing pathways or inhibition of YAP activity induced cell differentiation, significantly delayed wound healing response, and attenuated dedifferentiation process in-vitro/in-vivo. Altogether, we propose that mechano-sensing of matrix rigidity by epithelial cells modulates YAP – TGFβ pathway that in turn plays a vital role in regulating SC fate choices and cell plasticity under homeostasis and injury. Swarnabh Bhattacharya1, Aya Amitai-Lange1, Anna Altshuler1, Abhishek Mukherjee1, Sabrina Pisano2, Waseem Nasser1, Chloe Feral2, Haguy Wolfenson*1, Ruby Shalom-Feuerstein*1 1 Technion - Israel Institute of Technology, Israel; 2 INSERM, U1081, CNRS, Institute for Research on Cancer and Aging of Nice (IRCAN), University of Nice Sophia Antipolis, France.