Delineating the heterogeneity of matrix-directed differentiation toward soft and stiff tissue lineages via single-cell profiling

Mesenchymal stromal/stem cells (MSCs) form a heterogeneous population of multipotent progenitors that contribute to tissue regeneration and homeostasis. MSCs assess extracellular elasticity by probing resistance to applied forces via adhesion, cytoskeletal, and nuclear mechanotransducers that direct differentiation to-ward soft or stiff tissue lineages. Even under controlled culture conditions, MSC differentiation exhibits substantial cell-to-cell var-iation that remains poorly characterized. By single-cell transcrip-tional profiling of nonconditioned, matrix-conditioned, and early differentiating cells, we identified distinct MSC subpopulations with distinct mechanosensitivities, differentiation capacities, and cell cycling. We show that soft matrices support adipogenesis of multipotent cells and early endochondral ossification of nonadipo-genic cells, whereas intramembranous ossification and preosteo-blast proliferation are directed by stiff matrices. Using diffusion pseudotime mapping, we outline hierarchical matrix-directed dif-ferentiation and perform whole-genome screening of mechanor-esponsive genes. Specifically, top-ranked tropomyosin-1 is highly sensitive to stiffness cues both at RNA and protein levels, and changes in TPM1 expression determine the differentiation toward soft versus stiff tissue lineage. Consistent with actin stress fiber stabilization, tropomyosin-1 overexpression maintains YAP1 nuclear localization, activates YAP1 target genes, and directs osteogenic dif-ferentiation. Knockdown of tropomyosin-1 reversed YAP1 nuclear localization consistent with relaxation of cellular contractility, sup -pressed osteogenesis, activated early endochondral ossification genes after 3 d of culture in induction medium, and facilitated adi-pogenic differentiation after 1 wk. Our results delineate cell-to-cell variation of matrix-directed MSC differentiation and highlight tropomyosin-mediated matrix sensing.

Automated summary

The study identified distinct subpopulations of MSCs with different mechanosensitivities and differentiation capacities under various matrix conditions. Tropomyosin-1 was found to play a key role in MSC differentiation, responding sensitively to stiffness cues and guiding differentiation toward soft or stiff tissue lineages.