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

Mesenchymal stromal/stem cells (MSCs) are 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 toward soft or stiff tissue lineages. Even under controlled conditions, MSC differentiation exhibits substantial cell-to-cell variation that remains poorly characterized. By single-cell transcriptional profiling of naïve, matrix-conditioned, and early differentiation state cells, we identified distinct MSC subpopulations with distinct mechanosensitivities, differentiation capacities, and cell cycling. We showed that soft matrices support adipogenesis of multipotent cells and endochondral ossification of non-adipogenic cells, whereas intramembranous ossification and pre-osteoblast proliferation are enhanced by stiff matrices. Using diffusion pseudotime mapping, we delineated hierarchical matrix-directed differentiation and identified mechanoresponsive genes. We found that tropomyosin-1 (TPM1) is highly sensitive to stiffness cues both at RNA and protein levels and that changes in expression of TPM1 determine adipogenic or osteogenic fates. Thus, cell-to-cell variation in tropomyosin-mediated matrix-sensing contributes to impaired differentiation with implications to the biomedical potential of MSCs.