Human mesenchymal stem/stromal cells (hMSCs), when engrafted into immunodeficient mice, can form ectopic bone organs with hematopoietic stem cell (HSC) supportive functions. However, the ability to do so, through a cartilage intermediate, appears limited to 30% of donor bone marrow samples. In this study, we characterize the heterogeneous nature of hMSCs and their ability to efficiently form humanized ossicles observed in “good donors” to correlate with the frequency and functionality of chondrocyte progenitors. Flow cytometry of putative hMSC markers was enriched in the CD271+CD51+ stromal cell subset, which also possessed enhanced hMSC activity as assessed by single-cell colony-forming unit fibroblast (CFU-F) and undifferentiated mesensphere formation. Transcriptome analysis of CD271+ cells presented upregulation of chondrogenesis-/osteogenesis-related genes and HSC/niche maintenance factors such as C-X-C motif chemokine 12 (CXCL12) and ANGIOPOIETIN 1. Among the candidate genes selected to enrich for subsets with greater chondrogenic ability, cells negative for the actin cross-linker PALLADIN displayed the greatest CFU-F potential. Our study contributes to a better characterization of ossicle-forming hMSCs and their efficient isolation for the optimized engineering of human bone organs.
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