Single-cell and spatial transcriptomics reveal Thbs1-CD36 crosstalk between Kupffer and hepatic stellate cells following mesenchymal stem cell-derived small extracellular vesicles transplantation, alleviating liver fibrosis

Small extracellular vesicles (sEVs) derived from mesenchymal stem cells represent a novel regenerative therapeutic strategy for various liver injuries. Menstrual blood-derived mesenchymal stem cells (MenSCs)-derived EVs may have therapeutic potential in treating liver fibrosis. In this study, systemically injected MenSCs-derived sEVs migrated into the injured liver, ameliorated serological indices, and decreased collagenous fiber accumulation in carbon tetrachloride (CCl 4 )-induced liver fibrosis mouse models. We aimed to explore the underlying mechanism for the therapeutic effect of MenSC-sEVs. Single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics were combined to investigate the mechanism by which classical fibrosis-related genes are reduced to inhibit liver fibrosis, targeting Kupffer cells and hepatic stellate cells (HSCs). Fourteen major cellular clusters were determined in the livers of the control (or con), CCl 4 , and MenSC-sEV groups through scRNA-seq data. Thrombospondin 1 (Thbs1) and cluster of differentiation 36 (CD36) pathways were identified as important links between Kupffer cells and HSCs in the CellChat analyses. Additionally, spatial transcriptomics further confirmed the reduced Thbs1 and CD36 expression after MenSC-sEVs injection in fibrotic livers. We investigated the relationship between the Kupffer cells and HSCs, highlighting the role of Thbs1/CD36-mediated molecular crosstalk following transplantation with MenSC-sEVs. Furthermore, the integration of scRNA-seq and spatial transcriptomics data revealed eight genes, C1qa , C1qb , C1qc , Ctsd , Cd5l , Ctss , Wfdc17 , and Gpnmb , that were upregulated in the CCl₄ group across platforms. Thbs1 expression was reduced in primary mouse Kupffer cells stimulated with MenSC-sEVs, and the culture medium of primary mouse Kupffer cells stimulated by MenSC-sEVs reduced the expression of CD36 in primary mouse HSCs in vitro. In this study, we identified interacting target cells (Kupffer cells and HSCs), signaling molecules (Thbs1-CD36), and underlying therapeutic targets of MenSC-sEVs in treating liver fibrosis.