Distinct Intrinsic and Extrinsic Factors Differentially Regulate Skeletal Stem Cells in Calvaria Versus Long Bones During Bone Regeneration

Calvarial suture skeletal stem cells (Su-SSCs) are a distinct stem cell population for craniofacial bone formation by intramembranous ossification, compared to long bone periosteal SSCs (LB-PSSCs) with endochondral (osteochondrogenic) ossification. However, whether SSC intrinsic or extrinsic factors affect their differentiation process has not been well elucidated. Here, using an inducible <i>Prx1</i>-CreER-EGFP^+/-;Rosa26-tdTomato mouse model, we observed that endogenous <i>Prx1</i>⁺ Su-SSCs and their orthotopic transplantation into calvarial injury do not form cartilage intermediates at the injury sites, while the transplantation of <i>Prx1</i>⁺ LB-PSSCs into LB injury induces osteochondrogenic differentiation, respectively. However, the heterotopic transplantation of <i>Prx1</i>⁺ Su-SSCs (Su-SSCs into LB injury) showed some surprising findings that the transplanted Su-SSCs acquire new chondrocyte differentiation properties at the LB injury sites, although the heterotopic-transplanted <i>Prx1</i>⁺ LB-PSSCs maintained their endochondral ossification properties at the calvarial injury sites. Further, a comparative single-cell transcriptomic analysis of LB-PSSCs and Su-SSCs revealed that Su-SSCs express a higher set of anti-chondrogenic genes, such as <i>Wnt5b</i>, <i>Twist1</i> while LB-PSSCs highly express chondrogenic <i>Hoxa-9</i>, <i>Hoxc-9</i>, <i>Hoxa-10</i>, <i>Hoxc-10,</i> and <i>Comp</i> genes. We also found that the heterotopic transplantation of LB-PSSCs into calvarial injury enhances bone healing in vivo. Taken together, these findings suggest that LB-PSSCs have high regenerative capability with invariable endochondral ossification even after the heterotopic transplantation but Su-SSCs are more flexible and regulated by the local bone environment. The transplantation of periosteal SSCs will be a promising method for large craniofacial bone defects.