Mechanisms integrating mechanical and biochemical signalling in human pluripotent stem cells

Abstract

Understanding how stem cell differentiation is regulated is of key importance for developmental biology and regenerative medicine. Increasing evidence indicates that, alongside biochemical signalling, mechanical signals contribute to the regulation of fate acquisition in different stem cell types. For instance, in human pluripotent stem cells (hPSCs), which have been extensively used to study the biochemical regulation of fate transitions, several recent studies show that substrate stiffness can also regulate the extent of mesendodermal fate acquisition in the presence of mesoderm-inducing signals. Yet, while feedbacks between mechanical and biochemical signals have been shown in various systems, the mechanisms that integrate the two types of signalling remain elusive. In this thesis, I used hPSCs to investigate the mechanisms by which mechanical and biochemical signalling are integrated to regulate mesendodermal fate acquisition. I cultured hPSCs on hydrogels of different stiffnesses and analysed their response to the mesoderm-inducing signal BMP4. I observed that while on stiff substrates, only cells at the colony edge responded to BMP signalling, on soft substrates the BMP4 response was increased and observed throughout the colony, leading to enhanced mesendodermal differentiation. I found that this increased response to BMP4 on soft substrates stems from reduced activity of the FAK-PI3K cascade, a key mechanotransduction pathway. Crucially, this mechanical signal impacts the epithelial organisation of hPSC colonies. On soft substrates hPSCs exhibited reduced epithelial integrity, which increased accessibility of basolaterally localised BMP receptors (BMPRs) to apically applied BMP4. In addition, hPSCs on soft substrates lost apical identity, which led to mislocalisation of the conventionally basolateral BMPRs to the apical membrane, further increasing receptor accessibility. Furthermore, I discovered that hPSCs on soft substrates retained secreted laminin on their apical surfaces. Strikingly, supplementing soluble laminin to hPSCs on stiff substrates was sufficient to increase their response to BMP4. This increased response occurred via loss of apical identity and epithelial integrity; the same mechanisms responsible for increased response on soft substrates. Finally, I asked whether the mechanisms of integration between mechanical and biochemical signalling I identified in cultured hPSCs play a role in fate acquisition \textit{in vivo}. In pre-gastrulating mouse embryos, hPSC-equivalent epiblast cells are exposed to BMP4 but do not differentiate before the start of gastrulation. Gastrulation is initiated when remodelling of Collagen IV-rich basement membrane occurs. I hypothesised that this remodelling results in local substrate softening. Strikingly, I showed that softening the basement membrane by inhibiting Collagen IV crosslinking induced premature mesendodermal differentiation in mouse embryos. This suggests that change in substrate stiffness could influence BMP responsiveness \textit{in vivo} and thereby contribute to the timing of gastrulation. This thesis reveals a mechanism by which substrate stiffness regulates the response of hPSCs to BMP4. It also provides new insight into the role of soluble ECM in tuning biochemical signalling. Together, these findings highlight the importance of understanding how mechanical and biochemical signals integrate in dynamic contexts where ECM composition and mechanics are changing, such as during embryonic development and in diseases like cancer.