Gestational Age, Maternal Age, and Culture Dynamics Shape the Pluripotency Marker Profile of Primary Amniocytes: Insights for Stem Cell Biology and Regenerative Applications

INTRODUCTION: Amniotic Fluid Cells (AFCs) have emerged as a promising source for regenerative medicine due to their multipotent properties and accessibility. This study aimed to quantify pluripotency-associated transcript profiles in primary amniocyte cultures and evaluate their associations with gestational age, maternal age, and culture dynamics. METHODS: This study analyzed the expression of ten pluripotency-associated genes (OCT3/OCT4, SOX2, KLF4, MYC, KIT, NANOG, DPPA3, DPPA5, FUT4, and SALL4) in primary AFC cultures derived from 17 pregnancies using quantitative real-time PCR (2-ΔΔCt; ACTB-normalized). Samples were evaluated according to gestational age, maternal age, and fetal sex; culture expandability was assessed based on the maximum passage achieved. AFC cultures were serially subcultured, and RNA was isolated independently at each available passage to quantify longitudinal passage-associated expression changes. Spearman's rank correlation assessed associations between gestational age and maternal age (continuous variables) and expression of SOX2, DPPA5, KIT, NANOG, and SALL4, with Benjamini-Hochberg FDR adjustment (q = 0.05). RESULTS: All investigated markers were detectable across the AFC cultures. In group-based analyses, SOX2, DPPA3, and DPPA5 expression levels were higher in cultures derived from lower gestational ages, while SOX2 and DPPA5 were higher in younger maternal-age groups (p < 0.05). Culture expandability showed positive associations with KIT and selected markers. Serial-passaging analyses suggested that KIT expression was maintained in cultures achieving extended passaging, whereas SOX2 and DPPA5 expression tended to decrease with increasing passage number. DISCUSSION: These findings indicate that developmental timing and in vitro culture dynamics are associated with variability in pluripotency-marker expression in primary amniocyte cultures. Transcript- level profiling may therefore help contextualize inter-individual heterogeneity and passagerelated shifts relevant to stem cell biology and standardized culture selection. CONCLUSION: AFC pluripotency-marker transcripts vary with gestational age, maternal age, and passaging. These transcript-level findings provide a quantitative reference but require protein/functional validation in larger, standardized cohorts.