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

Shlomi Brielle, Danny Bavli, Alex Motzik, Yoav Kan-Tor, Xue Sun, Chen Kozulin, Batia Avni, Oren Ram*, Amnon Buxboim*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

25 Scopus citations


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 toward soft or stiff tissue lineages. Even under controlled culture conditions, MSC differentiation exhibits substantial cell-to-cell variation that remains poorly characterized. By single-cell transcriptional 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 nonadipogenic cells, whereas intramembranous ossification and preosteoblast proliferation are directed by stiff matrices. Using diffusion pseudotime mapping, we outline hierarchical matrix-directed differentiation and perform whole-genome screening of mechanoresponsive 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 differentiation. Knockdown of tropomyosin-1 reversed YAP1 nuclear localization consistent with relaxation of cellular contractility, suppressed osteogenesis, activated early endochondral ossification genes after 3 d of culture in induction medium, and facilitated adipogenic differentiation after 1 wk. Our results delineate cell-to-cell variation of matrix-directed MSC differentiation and highlight tropomyosin-mediated matrix sensing.

Original languageAmerican English
Article numbere2016322118
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number19
StatePublished - 11 May 2021

Bibliographical note

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© 2021 National Academy of Sciences. All rights reserved.


  • Cell heterogeneity
  • Mechanobiology
  • Mesenchymal stem cells
  • Single-cell analysis
  • Tropomyosin


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