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A circuit of mechanically regulated transcription factors balances regenerative and fibrotic memory of mesenchymal stromal cells [ATAC-Seq]

ABSTRACT: Therapeutic mesenchymal stromal cells (MSCs) are used to support the healing of severe injuries such as skin burn wounds. However, the pivotal expansion from donor biopsies on conventionally stiff culture surfaces activates MSCs into scar-promoting myofibroblasts. We previously coined the term mechanical memory to describe that priming of MSCs on scar-stiff culture substrates imprints myofibroblast features that persist even after a switch to skin-soft substrates. We now unravel mechanisms and factors that, conversely, suppress myofibroblast activation upon priming in skin-soft expansion culture. Such ‘soft memory’ factors are poised to preserve MSC regenerative features while suppressing fibrogenesis. Mechanically primed MSCs were compared by RNA- and assay for transposase-accessible chromatin (ATAC)-sequencing. The promoters of genes that were highly accessible and upregulated upon soft priming and memorized after switch to stiff substrates were significantly enriched for motifs predicted to bind the transcription factor HOXA11. Knockdown of HOXA11 resulted in enhanced expression of fibrogenic and osteogenic genes in soft-primed MSCs and reduced expression of anti-fibrotic factors, including the transcription factor SALLl1. SALL1 is a potent suppressor of pro-fibrotic genes such as Postn, Col8a1, Grem2, Thps1 and Thps2, and the transcription factor Gata6. GATA6, in turn, emerges as a keeper of stiff-induced myofibroblast memory by maintaining the accessibility of pro-fibrotic genes even after switch to soft substrates. By manipulating the SALL1-GATA6 transcriptional circuit in culture, we produced therapeutic MSCs that suppress fibrosis in an animal model of hypertrophic skin scarring. We propose that similar control over myofibroblast memory will enhance the success of therapeutic MSC applications in organ repair.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE310502 | GEO | 2026/04/09

REPOSITORIES: GEO

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Project description:Background: The differentiation of pericytes into myofibroblasts causes microvascular degeneration, extracellular matrix (ECM) accumulation, and tissue stiffening, characteristics of fibrotic diseases. It is unclear how pericyte-myofibroblast differentiation is regulated in the microvascular environment. Our previous study established a novel two-dimensional platform for coculturing microvascular endothelial cells (ECs) and pericytes derived from the same tissue. This study investigated how ECM stiffness regulated microvascular ECs, pericytes, and their interactions. Methods: Primary microvessels were cultured in the TGM2D medium. Stiff ECM was prepared by incubating ECM solution in regular culture dishes for one hour followed by PBS wash. Soft ECM with Young’s modulus of approximately 6 kPa was used unless otherwise noted. Bone grafts were prepared from the rat skull. Immunostaining, RNA sequencing, qRT-PCR, western blotting, and knockdown experiments were performed on the cells. Results: Primary microvascular pericytes differentiated into myofibroblasts (NG2+αSMA+) on stiff ECM, even with the TGFβ signaling inhibitor A83-01. Soft ECM and A83-01 cooperatively maintained microvascular stability while inhibiting pericyte-myofibroblast differentiation (NG2+αSMA-/low). We thus defined two pericyte subpopulations: primary (NG2+αSMA-/low) and activated (NG2+αSMA+) pericytes. Soft ECM promoted microvascular regeneration and inhibited fibrosis in bone graft transplantation in vivo. As Integrins are the major mechanosensor, we performed qRT-PCR screening of Integrin family members selected from RNA sequencing data. We found that Integrin β1 (Itgb1) was the major subunit downregulated by soft ECM and A83-01 treatment. Knocking down Itgb1 suppressed myofibroblast differentiation on stiff ECM. Interestingly, ITGB1 phosphorylation (Y783) was mainly located on microvascular ECs on stiff ECM, which promoted EC secretion of paracrine factors, including CTGF, to induce pericyte-myofibroblast differentiation. CTGF knockdown or monoclonal antibody treatment partially reduced myofibroblast differentiation, implying the participation of multiple pathways in fibrosis formation. Conclusions: Microvascular ECs mediate ECM stiffness-induced pericyte-myofibroblast differentiation through paracrine signaling.

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A circuit of mechanically regulated transcription factors balances regenerative and fibrotic memory of mesenchymal stromal cells [ATAC-Seq]

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