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bioRxiv · 10.1101/2025.01.09.631479

Modeling the human limb skeletal development using human pluripotent stem cell-derived skeletal assembloid

Takao, T.·Osone, T.·Sato, K.·Yamada, D.·Fujisawa, Y.·Hagiwara, M.·Nakata, E.·Ozaki, T.·Toguchida, J.·Takarada, T.
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Abstract

Despite recent advances in pluripotent stem cell-based approaches to induce skeletal cells, recapitulating human limb skeletal development in terms of structure and longitudinally oriented growth remains an unresolved challenge. Here, we report a method to differentiate human pluripotent stem cells into region-specific skeletal organoids harboring GDF5+PRG4+ interzone/articular chondrocyte progenitors (IZ/ACPs) and SP7+ growth plate chondrocytes (GPCs) via PRRX1+ limb-bud mesenchymal cells. Comparative analysis demonstrated marked similarities of IZ/ACP and GPC organoids to the human embryonic limb, and graft fate and regenerative capacity in vivo were further characterized. We also mimicked the limb skeletal developmental process in a spatially structured manner by vertically positioning two IZ/ACP organoids at both ends of a GPC organoid to generate a human skeletal assembloid. Notably, this human skeletal assembloid recapitulated endochondral ossification with longitudinal skeletal growth upon transplantation. In summary, our study provides a novel research platform for human limb skeletal development and disease.

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Takao, T., Osone, T., Sato, K., Yamada, D., Fujisawa, Y., Hagiwara, M., Nakata, E., Ozaki, T., Toguchida, J., Takarada, T.. 2025-01-10. Modeling the human limb skeletal development using human pluripotent stem cell-derived skeletal assembloid. https://doi.org/10.1101/2025.01.09.631479

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