Human umbilical cord mesenchymal stem cell-derived small extracellular vesicles delivering CERS1 promote squamous differentiation in HPV16-positive cervical intraepithelial neoplasia via remodeling sphingomyelin metabolism.

Cervical intraepithelial neoplasia (CIN), a precursor of human papillomavirus (HPV)-driven cervical cancer, poses a severe threat to women's health. High-risk HPV infection drives CIN progression, whereas well-defined intervention targets and effective non-invasive therapies remain lacking. Herein, Using New Approach Methodologies (NAMs), specifically HPV16-positive ectocervical organoids as a human-relevant disease model, we evaluated the therapeutic potential of engineered cell-free small extracellular vesicles derived from human umbilical cord mesenchymal stem cells (hucMSC-sEV). We found that hucMSC-sEV significantly promoted squamous differentiation in S12 cells and HPV16-positive ectocervical organoids. Mechanistically, integrated multi-omics analyses within this NAMs platform revealed that hucMSC-sEV delivered ceramide synthase 1 (CERS1) to upregulate C18 ceramide (C18 Cer) synthesis, activating the non-canonical p53 signaling to drive squamous differentiation. In NCG mice bearing subcutaneous xenografts of S12 cells, hucMSC-sEV effectively inhibited S12 xenograft growth and modulated squamous differentiation via regulation of C18 ceramide metabolism, while this therapeutic effect was abrogated in CERS1-knockdown hucMSC-sEV. This study delineates the molecular mechanism by which hucMSC-sEV remodel sphingolipid metabolism to promote cervical squamous epithelial differentiation and suppress S12 xenograft development, providing novel potential targets and therapeutic strategies for CIN intervention.