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.