Matrix stiffness regulates telocyte differentiation into fibroblasts in vitro

Primary telocytes from ankle joints of Efhd1CreER x Ai9 mice were purified by FACS based on tdTomato (tdT) expression. These tdT? telocytes were cultured on collagen-coated CytoSoft® plates of varying stiffness (0.2, 8, and 64 kPa). After 24 hours, cells were harvested for bulk RNA sequencing using Illumina NovaSeq X Plus. The expression of telocyte markers and fibroblast/myofibroblast markers showed stiffness-dependent changes. Notably, stiff ECM induced a telocyte-to-myofibroblast transition, evidenced by downregulation of Efhd1 and Myoc and upregulation of myofibroblast markers(eg. Acta2 and Tagln). Additional analysis revealed significant changes in expression of Timp, Mmp, and Collagen genes. These findings suggest that ECM stiffness is a key regulator of telocyte fate and function at the PLV surface. Overall design: Telocytes were isolated from ankle synovium of Efhd1CreER x Ai9 mice, and tdTomato? cells were purified by flow cytometry. Purified cells were cultured for 24 hours on collagen-coated CytoSoft® 24-well plates with three levels of stiffness: 0.2 kPa (soft), 8 kPa (intermediate), and 64 kPa (stiff). Total RNA was extracted and subjected to bulk RNA sequencing. Gene expression changes were analyzed with significance set at p < 0.05.