Rytel, K. | bioRxiv Science

bioRxiv2026

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.

cell biology↗

bioRxiv2024

Comparative study of enriched dopaminergic neurons from siblings with Gaucher disease discordant for parkinsonism

Inducible pluripotent stem cells (iPSCs) derived from patient samples have significantly enhanced our ability to model neurological diseases. Comparative studies of dopaminergic (DA) neurons differentiated from iPSCs derived from siblings with Gaucher disease discordant for parkinsonism provides a valuable avenue to explore genetic modifiers contributing to GBA1-associated parkinsonism in disease-relevant cells. However, such studies are often complicated by the inherent heterogeneity in differentiation efficiency among iPSC lines derived from different individuals. To address this technical challenge, we devised a selection strategy to enrich dopaminergic (DA) neurons expressing tyrosine hydroxylase (TH). A neomycin resistance gene (neo) was inserted at the C-terminus of the TH gene following a T2A self-cleavage peptide, placing its expression under the control of the TH promoter. This allows for TH+ DA neuron enrichment through geneticin selection. This method enabled us to generate comparable, high-purity DA neuron cultures from iPSC lines derived from three sisters that we followed for over a decade: one sibling is a healthy individual, and the other two have Gaucher disease (GD) with GBA1 genotype N370S/c.203delC+R257X (p.N409S/c.203delC+p.R296X). Notably, the younger sister with GD later developed Parkinson disease (PD). A comprehensive analysis of these high-purity DA neurons revealed that although GD DA neurons exhibited decreased levels of glucocerebrosidase (GCase), there was no substantial difference in GCase protein levels or lipid substrate accumulation between DA neurons from the GD and GD/PD sisters, suggesting that the PD discordance is related to of other genetic modifiers.

neuroscience↗