Distinct miRNA profiles in human amniotic tissue and its vesicular and non-vesicular secretome

The human amniotic membrane (hAM) is a valuable tissue in regenerative medicine due to its capacity to release cell-derived bioactive factors. Among these, microRNAs (miRNAs) play a critical role in modulating gene expression. This study aimed to comprehensively characterize the miRNA landscape of native viable hAM tissue and its secretome, with a focus on distinguishing between extracellular vesicles (EV)-associated and protein-bound miRNAs. We analyzed two anatomically and functionally distinct regions of the hAM, the reflected and placental amnion. After incubation of tissue biopsies ex vivo for 72 hours, vesicular and non-vesicular components in the conditioned medium were separated by size exclusion chromatography, and small RNA sequencing was performed on tissue and secretome fractions. Our analysis identified three main clusters of miRNA expression corresponding to tissue, EV-associated, and protein-bound fractions. We observed regional differences in miRNA expression between the reflected and placental amnion and identified miRNAs selectively released into EVs or protein-bound fractions. Data of gene ontology analysis suggest distinct biological roles for miRNA depending on the sample type. This dataset provides novel insights into the spatial and functional miRNA release profile of viable hAM and contributes to a better understanding of its regenerative potential. Overall design: This study investigates small RNA expression profiles derived from different regions of the human amniotic membrane. Placental amnion (PA) and reflected amnion (RA) were separated from human placentae obtained after cesarean sections. Two biopsy punches (26 mm diameter) from each region were cultured for 72 hours (Dulbecco Modified Eagle's medium/high glucose, 1 % v/v L-glutamine, 1 % v/v penicillin/streptomycin, 37 °C, 5 % CO2, humidified atmosphere), and extracellular vesicle and protein fractions were isolated from the conditioned media. Small RNAs from EV fractions, protein fractions, and tissue samples were analyzed using next-generation sequencing. Four biological replicates were included for each tissue type and sample fraction to assess differences in small RNA content.