Epithelial stem cells in the premenopausal human vagina

The vagina undergoes physiologic changes across the menstrual cycle, pregnancy, birth and menopause. Most women will experience vaginal dysfunction at some point during their lives and treatment options are limited. The cyclic regeneration of the vaginal epithelium during each menstrual cycle suggests it is a stem-cell based tissue; however, human vaginal epithelial stem cells (veSCs) have not been identified. We utilized in vitro colony forming and organoid assays to confirm that cells in the human vaginal epithelium have self-renewal and differentiation potential. Specifically, we determined that stem cell activity resides in the ITGA6+ and NGFR+ fractions of the basal epithelium. We performed single-cell RNA sequencing to identify the distinct cellular compartments of the full thickness human vagina, including spatially distinct populations comprising layers of the stratified vaginal epithelium. Markers of these cells within the vaginal epithelium were validated by immunohistochemistry. CD9+ITGA6+NGFR+ and CD9+ITGA6+NGFR- cells were capable of efficient colony formation but only the CD9+ITGA6+NGFR+ fraction produced organoids containing basal, intermediate and superficial layers of the vaginal epithelium. We characterized the premenopausal human vagina at single cell resolution, validated markers and assays to test veSC developmental potential, and identified putative stem cells that may open new avenues for treating vaginal dysfunction.