Gli1⁺ cell aggregates promote type H vessel formation and orchestrate bone defect regeneration

Rapid and stable regeneration of bone defects remains a pressing clinical challenge. We previously fabricated stem cell aggregates (CA) by mimicking developmental condensation and demonstrated their efficacy in promoting bone defect repair. Endogenous Gli1 + skeletal stromal/progenitor cells (SSPCs) are a pivotal SSPC subtype known to maintain bone homeostasis and enhance bone regeneration; however, the functional properties and translational potential of CA derived from these cells (Gli1 + CA) remain largely elusive. Single-cell RNA sequencing was performed to characterize differential gene expression profiles between Gli1 high and Gli1 low SSPCs. The spatial relationship among Gli1 + cells, RUNX2 + cells, and type H vessels in vivo was further validated. Gli1-CreER T2 ; mT/mG transgenic mice were generated to enable the isolation of Gli1 + SSPCs and the subsequent fabrication of Gli1 + CA. The pro-angiogenic potential of Gli1 + CA was assessed in vitro, and the underlying regulatory mechanisms were further explored. Finally, Gli1 + CA were implanted into a mouse femoral defect model, and bone regenerative efficacy was evaluated by micro-CT and immunofluorescence staining. Single-cell RNA sequencing revealed that, compared with Gli1 low SSPCs, Gli1 high SSPCs highly expressed genes associated with osteogenesis, angiogenesis, and extracellular matrix synthesis. In vivo validation demonstrated robust enrichment of Gli1 + cells in the metaphysis; these cells exhibited a tight spatial correlation with the osteogenic master transcription factor RUNX2 and type H vessels. We subsequently sorted Gli1 + SSPCs via flow cytometry and fabricated CA, and in vitro analysis confirmed that their expression profiles were consistent with the sequencing data. Functional assays further revealed that Gli1 + CA promoted endothelial tube formation through paracrine signaling. Ultimately, Gli1 + CA markedly accelerated bone regeneration in a mouse femoral defect model compared with unsorted CA and Gli1⁻ CA, likely by inducing type H vessel formation. This study not only addresses a critical knowledge gap in Gli1 + CA-mediated bone regeneration, but also proposes a novel strategy termed “precision screening of endogenous SSPC subsets coupled with targeted aggregate fabrication”. This approach offers a more precise therapeutic direction for the regenerative treatment of bone defects.