A Circuit of Mechanically Regulated Transcription Factors Balances Regenerative and Fibrotic Memory of Mesenchymal Stromal Cells

ABSTRACT Therapeutic mesenchymal stromal cells (MSCs) promote healing in severe injuries like skin burns. However, expansion on stiff culture surfaces activates MSCs into scar‐promoting myofibroblasts. We previously introduced ‘mechanical memory’ to describe how MSCs primed on scar‐stiff surfaces retain myofibroblast traits even after switching to softer, skin‐like surfaces. Now, we identify mechanisms and factors that suppress myofibroblast activation during priming in soft cultures. These ‘soft memory’ factors are poised to preserve MSC regenerative features while preventing fibrogenesis. Mechanically primed MSCs were compared via RNA‐ and ATAC‐sequencing to co‐analyze gene transcription and chromatin accessibility. Highly accessible promoters of genes upregulated after soft priming, which retained this pattern after transitioning to stiff surfaces, were enriched for HOXA11 transcription factor binding motifs. Knocking down HOXA11 increased osteogenic gene expression in soft‐primed MSCs and reduced anti‐fibrotic factors, including the transcription factor SALL1, which suppresses pro‐fibrotic genes like Postn , Col8a1 , Grem2 , Thbs1 , Thbs2 , and Gata6 . We identify GATA6 as a keeper of stiff‐induced myofibroblast memory after switching to soft surfaces. Manipulating the SALL1‐GATA6 circuit yielded therapeutic MSCs that suppressed fibrosis in a hypertrophic skin‐scarring animal model. Therefore, controlling myofibroblast memory could improve MSC‐based organ repair therapies.