Objective: Since the last decade, organoids have been generated from pluripotent stem cells (PSCs), adult stem cells (ASCs) and some progenitor cells. These organoids aim to mimic tissue structures at the molecular level and in terms of cell composition and 3D organization. Thanks to organoids, considerable efforts are currently being made to study the (re)generation and aging of tissues, close to in vivo context. We described previously in vitro methods to obtain highly immature bone marrow (BM) MSCs (hiMSCs) exhibiting stem cell-like properties. Here we address the question of their abilities to generate human BM organoids thanks to easy-to-use processes.

Methods: HiMSCs were lineage-negative with strong abilities to proliferate and differentiate into several types of mature cells both in vitro and in vivo. After their expansion in 2D, hiMSCs were cultured in low attachment plates allowing them to aggregate, forming sphere-like structures. Then, we performed phenotypic and functional studies on these 3D structures.

Results: Along the first week of 3D culture, spheroids were able to self-organize and form endothelial networks leading to vascularized spheres with spontaneous activation of osteogenic program. Interestingly, their comparison by GSEA with DLX5-induced osteogenesis model showed significant similarities in the commitment of these two models towards skeletogenesis. GSEA analyses with more public data further confirmed these results. Interestingly, these analyzes emphasized ciliogenesis as key activated mechanism in these spheroids. Then, we sought to examine the functions of primary cilia by activating or inhibiting it and its related functional molecular pathways like TGFβ1 and Hedghog pathways. Our results showed that the cell organization and differentiation processes are influenced by cilium. Finally, these spheroids were able to form human bone with osteoblasts, stromal and perivascular cells of human origin when transplanted in NSG mice.

Conclusion: hiMSCs self-organized to form vascularized multicellular 3D organoids resembling primary osteogenic units that were functioned both in vitro and in vivo. Therefore, based on these general data and the relative simplicity of the culture process, these new osteogenic organoids should be considered as relevant models for studying bone regeneration, aging and diseases like ciliopathies in human context.