FOXO1 overexpression shifts Wharton’s jelly mesenchymal stromal cells toward a partial germ cell-associated program

Introduction Generating human male germ cell-like cells in vitro could provide tractable models of spermatogenesis, but differentiation from mesenchymal stromal cells generally stalls before meiosis. We investigated whether FOXO1, a regulator of spermatogonial stem cell maintenance, could shift human umbilical cord Wharton’s jelly-derived mesenchymal stromal cells (HuMSCs) toward germ cell-like states. Methods HuMSCs with stable FOXO1 overexpression were evaluated during a 30-day induction using proliferation assays, germ cell-associated marker analyses, single-cell RNA sequencing, and fluorescence in situ hybridization. Pseudotime and correlation analyses were performed to examine differentiation trajectories and predict transcriptional relationships. Results FOXO1 overexpression reduced proliferative activity and increased markers associated with primordial germ cells, spermatogonia, and spermatids. Single-cell RNA sequencing of 22,259 cells revealed enrichment of spermatogenesis-related programs and depletion of DNA replication and mitotic programs in FOXO1-positive cells. Pseudotime and correlation analyses further identified a FOXO1-associated trajectory and a predicted transcriptional network. Fluorescence in situ hybridization detected six rare cells with chromosome-probe patterns compatible with a haploid-like state, although ploidy and gametic function were not independently validated. Discussion FOXO1 overexpression shifts HuMSCs toward a less proliferative, germ cell-like transcriptional state and nominates regulatory relationships for future investigation. These findings do not establish the generation of functional human gametes.