Multi-omics profiling links epigenetic and lncRNA changes to early human endochondral ossification priming

The complexity of stem cell differentiation programs remains incompletely understood across stem cell types, including for human bone marrow mesenchymal stromal/stem (BM-MSCs) cells, a heterogeneous cell population orchestrating bone formation and establishing a functional hematopoietic niche in the bone marrow. BM-MSCs form and repair bone through the evolutionarily conserved process of endochondral ossification (EO), initiated by deposition of a transient cartilage template subsequently remodeled into bone and bone marrow tissues. Despite their considerable potential for skeletal regeneration, the early molecular and cellular events underlying BM-MSCs commitment to endochondral ossification remain elusive. To overcome donor-dependent variability in chondrogenic potential that limits mechanistic studies, we here exploit OssiGel as a potent chondro-inductive extracellular matrix offering robust recapitulation of endochondral ossification by BM-MSCs. Through multi-omics profiling of OssiGel-primed BM-MSCs, we identify rapid chromatin remodeling at chondrogenic enhancer regions as a prerequisite for lineage commitment. The emergence of a chondro-progenitor population is detected as early as 3 days in vivo, and correlates with successful EO recapitulation. Mechanistically, we identify LINC02511 as a novel enhancer-associated element involved in the onset of EO. We confirm presence of LINC02511 in human skeletal atlases, and its CRISPR-mediated silencing was shown to significantly impair EO. By integrating tissue engineering with single cell multi-omics profiling, our study provides a framework for deciphering BM-MSCs fate decisions, highlighting the role of enhancers and non-coding elements as key determinants of early lineage specification. These findings advance our understanding of BM-MSCs biology and will prompt their translational exploitation in regenerative medicine.