Recapitulation of human endochondral ossification by SOX9+ sclerotomal progenitors.

Endochondral ossification generates most of the load-bearing bones during skeletal development. Fully recapitulating it in human cells remains a challenge. Here, we established a toolkit to derive near uniform (>99%) SOX9 ⁺ sclerotomal progenitors (scl-progenitors), a key bipotent mesenchymal precursor at the pre-condensation stage, from human pluripotent stem cells and accompanying highly efficient osteochondral induction methods. Upon lineage-specific induction, SOX9 ⁺ scl-progenitors could not only regenerate articular cartilage but also undergo spontaneous condensation, cartilaginous anlagen formation, chondrocyte hypertrophy, vascular invasion, and finally bone formation with stroma, thus fully recapitulating the endochondral ossification. Moreover, self-organized growth plate-like structures could also be induced using SOX9 ⁺ scl-progenitor-derived fusion constructs with chondro- and osteo-spheroids, mimicking in vivo metaphysis. Furthermore, we identified ITGA9 as a specific surface marker to enable reporter-independent isolation of SOX9 ⁺ scl-progenitors and established a culture system to continuously expand them without compromising their osteochondral bipotential. Our work highlights SOX9 ⁺ scl-progenitors as a promising tool for modeling human skeletal development and bone/cartilage bioengineering.