Stem cell research & therapy · 2026
Gli1⁺ cell aggregates promote type H vessel formation and orchestrate bone defect regeneration.
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Ma Chao, Gao Yu-Ru, Wang Hao, Sui Bing-Dong, Huang Shan-Shan, Zhang Yi, Liu Lu, Zhang Xiao-Hui, Xing Shu-Juan, Li Yuan-Yuan, Xiao Yi-Han, Zheng Ya-Nan, Ren Li-Heng, Jin Yan, Zheng Chen-Xi, Xu Hao-Kun, Chen Ji
Аннотация
BACKGROUND: 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.
METHODS: Single-cell RNA sequencing was performed to characterize differential gene expression profiles between Gli1high and Gli1low SSPCs. The spatial relationship among Gli1+ cells, RUNX2+ cells, and type H vessels in vivo was further validated. Gli1-CreERT2;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.
RESULTS: Single-cell RNA sequencing revealed that, compared with Gli1low SSPCs, Gli1high 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.
CONCLUSIONS: 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.
METHODS: Single-cell RNA sequencing was performed to characterize differential gene expression profiles between Gli1high and Gli1low SSPCs. The spatial relationship among Gli1+ cells, RUNX2+ cells, and type H vessels in vivo was further validated. Gli1-CreERT2;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.
RESULTS: Single-cell RNA sequencing revealed that, compared with Gli1low SSPCs, Gli1high 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.
CONCLUSIONS: 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.