Urine-derived induced pluripotent stem cells from Klinefelter Syndrome patients exhibit compromised capacity to differentiate into human primordial germ cell-like cells

Background: Klinefelter syndrome (KS) represents the most prevalent sex chromosome aneuploidy associated with male infertility. However, the underlying pathogenic mechanisms remain largely unexplored, primarily due to the lack of appropriate disease-relevant cell models and the inaccessibility of early primordial germ cell (PGC) development in vivo. This study aimed to generate induced pluripotent stem cells (iPSCs) from urine samples of KS patients and investigate their capacity to differentiate into PGC-like cells (PGCLCs). Methods: Urine-derived iPSCs (UiPSCs) were generated from two KS patients and two healthy controls (one male, one female) using a non-integrating reprogramming method, and characterized by karyotyping, immunocytochemistry, quantitative PCR (qPCR) and teratoma formation. These UiPSCs were subsequently differentiated into PGC-like cells (PGCLCs) via a two-step protocol involving incipient mesoderm-like cell (iMeLC) induction. Mesodermal gene expression in iMeLCs was examined by qPCR. PGCLC aggregates were identified for early PGC gene expression by qPCR and immunocytochemistry, and for PGCLC induction efficiency by flow cytometry using the PGC surface markers—epithelial cell adhesion molecule (EpCAM) and integrin subunit alpha 6 (INTEGRIN-α6). Results: Both KS-UiPSCs and control-UiPSCs displayed chromosomal stability, expressed canonical pluripotency markers, and exhibited trilineage differentiation capacity in vivo. The optimal duration for iMeLC induction time was 48 h. The iMeLCs induced from KS-UiPSCs showed lower expression of mesodermal genes than those from control-UiPSCs. Upon differentiation into PGCLCs, aggregates from all groups displayed increased expression of early PGC genes relative to their corresponding iMeLCs. The KS-PGCLC aggregates yielded a decreased ratio of EpCAM/ INTEGRIN-α6 double-positive cells than those from healthy controls. Conclusions: This study represents the first investigation using UiPSCs from KS patients, demonstrating that KS-UiPSCs exhibit compromised capacity for PGCLC differentiation. These findings provide a valuable platform to model early germ cell defects in KS and may facilitate the development of future therapeutic strategies.