In preprints: early cell differentiation and mechanical environment Free

Chemically, naive pluripotency exit can be triggered by activation of extracellular signal-regulated kinase (ERK) signaling and of glycogen synthase kinase 3 (GSK3) (Mulas et al., 2024; Ying et al., 2008). Primed pluripotent stem cells are characterized by lower levels of NANOG and KLF4, and increased levels of OTX2, SOX1 and Brachyury. This transition is also tightly linked with cell mechanics, as endocytosis and ERK levels are regulated by membrane tension (De Belly et al., 2021). Indeed, at the end of blastocyst formation, the membrane tension of epiblast cells decreases simultaneously with the loss of naive pluripotency (De Belly et al., 2021). This drop has been associated with a decrease in membrane-cortex attachment via a β-catenin-dependent loss of the phosphorylation of membrane-cortex attachment proteins (ERM: ezrin, radixin and moesin). Additionally, preventing this reduction of membrane tension also impairs the transition to prime pluripotency (De Belly et al., 2021). Changes in membrane mechanical properties may be associated with cell spreading, which dramatically changes during this transition. At the same time, other studies highlighted that soft substrates modulate cell spreading and maintain naive pluripotency (Chowdhury et al., 2010; Labouesse et al., 2021). Cell spreading results from the action of contractile forces pulling the cell on their substrate. However, how forces exerted on the extracellular matrix act during the naive-to-prime pluripotency transition remained to be investigated.

Viswanadha and colleagues studied naive pluripotent stem cells using traction force microscopy, which tracks movement of the matrix to deduce the force exerted by the cell. Traction force microscopy revealed that the transition to primed pluripotency, taking place when removing ERK and GSK3 inhibitors from the culture medium, was associated with an increase in traction forces.

To investigate whether this increase in traction forces occurs after transition or whether it contributes to pluripotency exit, they affected both the chemical and mechanical environment of naive cells. First, they inhibited myosin contractility, which cells use to pull on the matrix. Both force transmission and the pluripotency marker Rex1-GFP decreased in a manner that depended on the dose of the inhibitor. Second, they used substrates of different stiffness and found that cells in stiffer substrates exhibited higher traction forces, and subsequently primed more rapidly. Finally, they took advantage of the mechanical heterogeneity within a colony and found that cells at the edge of the colony (with higher substrate/cell-cell contact ratio) developed higher traction forces and primed more quickly than cells at the center of the colony. Together, they conclude that exertion of traction force on the substrate is associated with exit from pluripotency.

The authors go on to investigate how traction forces result in nuclear deformation and changes in the import of the co-transcriptional activator YAP into the nucleus. Disrupting the coupling of the nuclear envelope to the cytoskeleton, they only find a minor influence of direct force transmission onto the nuclear envelope during exit from pluripotency.

Finally, the authors question which of the inhibitions of GSK3 or ERK would be most meaningful for traction forces. Seeing that only the removal of GSK3 inhibition led to similar behaviors as the medium containing both inhibitors. They conclude that the exit of naive pluripotency requires the inhibition of β-catenin, leading to a decrease in cell-cell contacts and promoting cell-matrix force transmission. However, as this is only based on chemical inhibition at doses that are empirically determined, much caution should be taken with these final thoughts.

Leveraging traction force microscopy, this new study by Viswanadha and colleagues provides interesting insights and mechanisms into the coordination of chemical and mechanical signals during exit from pluripotency. Previous in vivo studies found that the extracellular matrix acts as a polarity cue for further differentiation of the epiblast (Kim et al., 2022). This polarity cue from the matrix may promote apicobasal polarity establishment, which is thought to act as a checkpoint (Shahbazi et al., 2017). The study by Viswanadha and colleagues suggests that mechanical forces could also regulate the progression of development. A plausible mechanism can be proposed: during embryogenesis, a chemical clock triggers the end of naive pluripotency, but this can only occur when the mechanical environment is stiff enough to accommodate for a decrease in membrane tension. As a basal membrane is actually deposited around the epiblast at the time of exit of naive pluripotency (Bedzhov and Zernicka-Goetz, 2014), this could constitute a checkpoint of mechanical integrity before starting gastrulation. Similarly, neural crest migration in Xenopus embryos is only triggered when the mechanical environment reaches a defined level of stiffness (Barriga et al., 2018). Interestingly, when mouse embryos enter dormancy (diapause), integrin β1 is downregulated (Chen et al., 2024). As integrins are crucial for cell-matrix connectivity, this could be a control mechanism to ensure that embryos remain dormant by preventing the establishment of cell-matrix forces.