Time-course ATAC-seq and RNA-seq analysis of porcine synovium-derived mesenchymal stem cells under in vitro osteogenic induction

Synovium derived mesenchymal stem cells (SMSCs) are considered promising for orthopedic application due to easy accessibility and strong differentiation potential. However, the transcription factors (TFs) that orchestrate the SMSCs osteogenic commitment, as well as the dynamic landscape of associated cis-regulatory elements, remain largely unclear. In addition, donor-specific epigenetic memory may lead to heterogeneous gene-regulatory profiles. In this study, we isolated porcine SMSCs from two pig breeds (German Saddleback, GS; German Landrace, GL) with distinct intrinsic metabolic characteristics and profiled their dynamic chromatin accessibility and transcriptomes during osteogenic induction. GO terms related to ossification and mesenchymal cell differentiation emerged earlier in the chromatin landscape (ATAC-seq, day 7) than at the transcriptional level (RNA-seq, day 21), indicating that chromatin accessibility captures lineage-specific programs prior to overt gene expression changes. Donor-specific differences in chromatin accessibility were minimal at baseline (day 0), became evident early after induction, and diminished over time. Footprinting analysis showed stronger binding affinity of C/EBP family members in osteogenic-induced SMSCs, whereas the FOS::JUN heterodimer exhibited greater occupancy in control cells. Interestingly, RUNX2 footprints displayed a slight decrease from day 0 to day 21 despite its established role in osteogenesis. De novo motif analysis further revealed TF-binding motif in differentially accessible regions, with RUNX2/RUNX motifs enriched in regions of reduced accessibility and CEBPs enriched in regions of increased accessibility. This study characterizes chromatin accessibility dynamics in SMSCs during osteogenic differentiation, driven mainly by differentiation state and time rather than donor metabolic differences. Integrated ATAC-seq/RNA-seq highlights key transcription factors and networks guiding osteogenic commitment, supporting porcine SMSCs as a translational model for bone regeneration.