Screening cross-injury responses in skin organoids identifies a THBS2-enriched pro-repair fibroblast state during regeneration.

INTRODUCTION: Skin injuries impose a substantial global health burden. Conventional wound healing models fail to distinguish evolutionarily conserved regenerative mechanisms from injury specific responses because they cannot capture microenvironmental complexity. OBJECTIVES: This study established a high-fidelity mouse skin organoid platform to simulate clinically relevant stressors and identify the core regulatory components of the regenerative program. METHODS: Mature skin organoids were individually exposed to hypoxia, hyperthermia, or hyperinflammation. Transcriptomic analysis identified both divergent and convergent responses across diverse injury types. Single-cell RNA sequencing (scRNA-seq) was utilized to characterize fibroblast states in physiological and diabetic wounds. RESULTS: A conserved 85-gene injury-responsive module was identified across all stressors, with thrombospondin 2 (Thbs2) emerging as a highly connected candidate hub within the extracellular matrix (ECM)-remodeling network. Integration with scRNA-seq datasets further showed that Thbs2 expression was enriched in an independently defined pro-repair fibroblast state characterized by ECM remodeling and repair-associated transcriptional features. In the diabetic wound microenvironment, this Thbs2-associated repair signature was attenuated, accompanied by accumulation of intermediate-state fibroblasts. CONCLUSION: These findings highlight that the acquisition of a specific functional cellular state is closely linked to regeneration. This multi stress platform facilitates the development of universal pro-healing strategies by shifting the focus from molecular targeting to restoring functional cellular states.