Endotome as an Unrecognized Source of Human Peri-Aortic Brown Adipocytes

Brown adipose tissue (BAT) uncouples nutrient catabolism from ATP synthesis to dissipate energy as heat, making it a promising regenerative medicine approach for metabolic disorders. In mice and humans, classical BAT is thought to arise developmentally from a PAX3+/MYF5+ dermomyotome precursors, yet lineage tracing studies reveal that the majority of cervical and all peri-aortic brown adipocytes derive from PAX3+/MYF5− somitic origins, suggesting an alternative, poorly understood developmental trajectory. Recent single-cell RNA sequencing (scRNA-seq) of mouse organogenesis and human trunk embryoid models identified a previously unrecognized somite compartment, the endotome, as a precursor pool for vascular endothelium and smooth muscle cells. Here, by interrogating scRNA-seq datasets from mouse somite development alongside our in-house human trunk embryoid model (hTEM), we characterize the EBF2+ endotome as an MYF5-independent source of UCP1+ brown adipocytes in a spatiotemporal manner. Mechanistically, we show that TGF-β-induced epithelial-to-mesenchymal transition (EMT) is required for endotome commitment, and that sequential BMP inhibition followed by WNT activation, rather than the BMP activation and WNT inhibition used for dermomyotome-derived BAs, fully potentiates a BA fate. Consequently, endotome-derived BAs resemble in vivo peri-aortic BAT. The developmental authenticity of these endotome precursors is further validated by differentiation into smooth muscle and vascular endothelial cells in response to HGF and VEGFA, respectively. Collectively, we demonstrate that recapitulating the developmental sequence of signaling cues governing endotome plasticity enables efficient brown adipocyte production from human pluripotent stem cells. Our work identifies a novel source of BAT and establishes a streamlined, rapid, and cost-efficient method to generate metabolic active brown adipocytes, with broad implications for regenerative medicine.