Reduced Cell–ECM Interactions in the EpiSC Colony Center Cause Heterogeneous Differentiation

Abstract

Mechanoregulation of cell–extracellular matrix (ECM) interactions are crucial for dictating pluripotent stem cell differentiation. However, not all pluripotent cells respond homogeneously which results in heterogeneous cell populations. When cells, such as mouse epiblast stem cells (EpiSCs), are cultured in clusters, the heterogeneity effect during differentiation is even more pronounced. While past studies implicated variations in signaling pathways to be the root cause of heterogeneity, the biophysical aspects of differentiation have not been thoroughly considered. Here, we demonstrate that the heterogeneity of EpiSC differentiation arises from differences in the colony size and varying degrees of interactions between cells within the colonies and the ECM. Confocal imaging demonstrates that cells in the colony periphery established good contact with the surface while the cells in the colony center were separated by an average of 1–2 µm from the surface. Traction force measurements of the cells within the EpiSC colonies show that peripheral cells generate large tractions while the colony center cells do not. A finite element modeling of EpiSC colonies shows that tractions generated by the cells at the colony periphery lift off the colony center preventing the colony center from undergoing differentiation. Together, our results demonstrate a biophysical regulation of heterogeneous EpiSC colony differentiation.

Ground-state pluripotent stem cells are characterized by Rac1-dependent Cadherin-enriched F-actin protrusions

“…As summarized in Fig. 5c, these include cadherin-enriched cadheropodia in ground-state pluripotent mESCs described in this study, the cortical asters analyzed earlier in naïve SMLIF mESCs (Xia et al, 2019a), and the peripheral stress fibers in EpiSCs (this study and (Amar et al, 2023;Narva et al, 2017;Rosowski et al, 2015)). Interestingly, these actin-based structures have been shown to perform functions related to the unique requirement of distinct pluripotent states.…”

“…Interestingly, these actin-based structures have been shown to perform functions related to the unique requirement of distinct pluripotent states. For example, the cortical asters in SMLIF mESCs are associated with the maintenance of sparse isotropic cortex that maintain the soft mechanics of mESCs, while the peripheral stress fibers in EpiSCs are thought to impart mechanical compression that maintain the pluripotency of the colony interior (Amar et al, 2023;Narva et al, 2017;Rosowski et al, 2015). As described above, the cadheropodia is observed primarily in the 2iLIF ground state but not in SMLIF mESCs.…”