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

Abstract

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.

Evaluation of candidate reference genes for quantitative real-time PCR normalization in blood from red deer developing antlers

“…Finally, TPM1 was found to be a highly potent regulator of cell differentiation in tissue regeneration and immunomodulation 53 .…”

Crimped nanofiber scaffold mimicking tendon-to-bone interface for fatty-infiltrated massive rotator cuff repair

“…Furthermore, the mechanical properties of crosslinked scaffolds were significantly improved compared with those of non-crosslinked scaffolds [ 51 , 52 ]. As suggested, the osteogenic induction of osteoblasts was increased on the stiff scaffolds and more bone matrix was generated at the defect site [ [53] , [54] , [55] ]. Tendon defects and fatty infiltration of the muscle contribute to poorer outcomes for fi-MRCTs than for smaller RCTs.…”

The roles and regulation of Yes-associated protein 1 in stem cells

“…Meanwhile, YAP1 activation by doxycycline can promote the differentiation of pig muscle stem cells at a high cell density (Liu et al, 2021). During osteogenesis, YAP1 is activated by tropomyosin-1 to maintain its nuclear localization and coaxes bone marrow mesenchymal stem cells (BMSCs) to differentiate into osteogenic cells (Brielle et al, 2021). Similarly, YAP1/TAZ is activated by Kindlin-2 signaling to promote the differentiation of BMSCs into osteogenesis and adipocytes (Guo et al, 2018).…”

“…In pluripotent star stem cells, YAP1/TAZ is involved in maintaining cell differentiation and proliferation (Lorthongpanich et al, 2020). It is worth noting that YAP1, activated by tropomyosin-1 overexpression, maintains its nuclear localization and coaxes mesenchymal stem cell differentiation into osteogenic cells (Brielle et al, 2021).…”