Differential traction forces underlie spatial heterogeneity in early differentiation of human embryonic stem cell clones

Mechanical responses are critical to elucidating the early-stage differentiation of human embryonic stem cells (hESCs), as these cells typically form cohesive clones and exhibit heterogeneous differentiation states. But mechano-regulatory mechanisms underlying the spatial heterogeneity of hESC clones during early differentiation remain poorly understood. Here, we investigated the role of cell-substrate adhesion and associated mechanotransductive pathways in governing the regional differentiation of hESCs into definitive endoderm (DE). At this early differentiation stage, H1 hESCs displayed spatial heterogeneity, with elevated DE marker expression at the periphery of individual clones compared to the interior. This pattern aligned well with a differential distribution of cellular traction forces, which was peaked at the periphery. Correlative spatial distributions of β₁-integrin, phosphorylated FAK (p-FAK) and vinculin were observed, supporting the mechanical dominance of peripheral regions. Inhibition of β₁-integrin or disruption of F-actin suppressed both traction force and DE differentiation capacity, primarily by inhibiting YAP nuclear translocation and thereby reducing spatial heterogeneity between peripheral and interior regions. The positive correlation between traction force and differentiation capacity was further validated by increasing cellular traction forces via culture on stiff substrates. This work highlights the pivotal role of biomechanical cues in early fate decisions of hESCs and provides insights into optimizing early differentiation through mechanical modulation. STATEMENT OF SIGNIFICANCE: This study defines a mechano-regulatory mechanism underlying spatial heterogeneity during early definitive endoderm (DE) differentiation of human embryonic stem cells (hESCs). Within self-organized H1 colonies, peripheral cells exhibit enhanced DE commitment driven by elevated traction forces transmitted through β₁-integrin-F-actin-YAP axis. This spatial bias emerges intrinsically, even on mechanically uniform substrates, and is further modulated by substrate stiffness. By integrating single-cell transcriptomics with traction force microscopy and high-resolution imaging, we demonstrate that colony-scale mechanical compartmentalization governs early lineage specification. These findings advance understanding of integrin-mediated mechanotransduction in stem cell early differentiation and provide a mechanobiological framework for optimizing directed differentiation through controlled mechanical microenvironments.