Hydrogels with enhanced protein conjugation efficiency reveal stiffness-induced YAP localization in stem cells depends on biochemical cues.

Polyacrylamide hydrogels have been widely used in stem cell mechanotransduction studies. Conventional conjugation methods of biochemical cues to polyacrylamide hydrogels suffer from low conjugation efficiency, which leads to poor attachment of human pluripotent stem cells (hPSCs) on soft substrates. In addition, while it is well-established that stiffness-dependent regulation of stem cell fate requires cytoskeletal tension, and is mediated through nuclear translocation of transcription regulator, Yes-associated protein (YAP), the role of biochemical cues in stiffness-dependent YAP regulation remains largely unknown. Here we report a method that enhances the conjugation efficiency of biochemical cues on polyacrylamide hydrogels compared to conventional methods. This modified method enables robust hPSC attachment, proliferation and maintenance of pluripotency across varying substrate stiffness (3 kPa-38 kPa). Using this hydrogel platform, we demonstrate that varying the types of biochemical cues (Matrigel, laminin, GAG-peptide) or density of Matrigel can alter stiffness-dependent YAP localization in hPSCs. In particular, we show that stiffness-dependent YAP localization is overridden at low or high density of Matrigel. Furthermore, human mesenchymal stem cells display stiffness-dependent YAP localization only at intermediate fibronectin density. The hydrogel platform with enhanced conjugation efficiency of biochemical cues provides a powerful tool for uncovering the role of biochemical cues in regulating mechanotransduction of various stem cell types.