Super‐Enhancer‐Mediated <scp> <i>DLX5</i> </scp> Activation Defines Regulatory Mechanisms in Human Embryonic Stem Cell‐Derived Osteoblasts

Osteoblast differentiation is essential for skeletal development and homeostasis. Although bone marrow-derived mesenchymal stem/stromal cells (BM-MSCs) are commonly used to study osteoblast differentiation in the context of bone homeostasis, their relevance to osteoblast differentiation during human skeletal development remains unclear. To understand the regulatory mechanisms underlying osteoblast differentiation in a human developmental context, we performed Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq) and RNA-seq analyses on osteoblasts isolated from an in vivo implantation system using induced sclerotome derived from Col2.3-GFP reporter human embryonic stem cells (hESCs). The resulting datasets revealed skeletal development-associated chromatin accessibility and transcriptional profiles. Comparative analysis with BM-MSC-derived osteoblasts revealed that hESC-derived osteoblasts were enriched for regulatory gene sets associated with ossification. Notably, we identified a super-enhancer associated with DLX5, a known osteoblast regulator, consisting of multiple cooperative enhancer elements to drive transcription. Taken together, this study provides a valuable resource for examining cis-trans regulatory mechanisms in human skeletal development and highlights DLX5 as a key transcriptional regulator controlled by an osteoblast super-enhancer.