The role of mechanotransduction in signalling and pluripotent stem cell fate
Early embryonic development is a dynamic process in which cells are exposed to soluble and mechanical cues in the microenvironment in a spatially and temporally organised manner. Although increasing evidence suggests that the regulation of pluripotent stem cell fate is strongly influenced by external mechanical factors, the mechanisms of interplay between soluble factor signalling and mechanical cues remain unclear. Further understanding of how mechanical factors of the extracellular matrix (ECM) affect pluripotency and cell fate will not only improve the differentiation of pluripotent stem cells in vitro, but also advance the understanding of development. In this study, we used StemBond, a hydrogel chemistry in which stiffness and ECM-to-gel tethering can be independently controlled, to investigate how these two properties influence pluripotent stem cell behaviour. Our results showed that in mouse embryonic stem cells, soft StemBond gels partially compensated for chemical factors to maintain naive pluripotency by reducing ERK signalling, a major initiator of differentiation. Additionally, the WNT pathway did not appear to be mechanosensitive. Our results in naive human pluripotent stem cells suggested that while the lack of mechanosensitivity in WNT signalling was conserved, ERK signalling was reduced on both soft and stiff gels compared to plastic. This indicated that ERK signalling responds to differences in ECM-to-gel tethering between StemBond gels and plastic rather than stiffness. We subsequently investigated how mechanical factors regulated early lineage differentiation using human primed pluripotent stem cells, and showed that compared to tissue culture plastic, both soft and stiff StemBond gels significantly enhanced the response to BMP4 and increased mesoderm differentiation. Activation of SMAD1/5, the effector of the BMP4 pathway, was found to be enhanced and differentially patterned between gels and plastic. To further dissect the mechanisms by which StemBond gels upregulated BMP/SMAD signalling, we explored the roles of integrins and signalling receptors. Our results suggested that activation of integrin β 1 on StemBond gels was necessary for enhanced p-SMAD1/5 levels on stiff gels. Together, our results shed light on how properties of the ECM can regulate major signalling pathways involved in regulating the cell fate of pluripotent stem cells.