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

Introduction: The human amniotic membrane (hAM) has largely been used in tissue regeneration and wound healing applications. A promising alternative to decellularized hAM or isolated cells is the usage of native viable hAM which contains and releases cell-derived bioactive factors that are known to enhance tissue regeneration. MicroRNAs (miRNAs) are known regulators of gene expression at post-transcriptional level and are important drivers of regeneration processes in several tissues. In this study, we characterized the miRNA profile of hAM tissue and its vesicular and non-vesicular secretome in the reflected and placental hAM as two spatially and physiologically distinct regions. Methods: Extracellular vesicles were enriched from the secretome by size exclusion chromatography (SEC). Small RNAs were determined by Next Generation Sequencing in the conditioned medium and in tissue. Results: After SEC, we identified predominantly small hAM-derived EVs (≤200 nm) expressing CD81. The highest percentage of miRNA relative to all mapped reads was found in tissue (15%-40%), while 2%-15% were protein-bound and 3%-6% associated with EVs. Unsupervised clustering revealed distinct clusters of miRNA expression according to sample fraction (EV-associated, protein-bound, and tissue) and amniotic regions (reflected, placental). Gene ontology analysis linked EV-associated and tissue miRNAs to (smooth) muscle proliferation, while protein-bound miRNAs were associated with connective tissue development, chondrocyte differentiation and glial cell proliferation. Furthermore, correlation analysis of tissue miRNAs and extracellular expression identified EV-associated and protein-bound miRNAs specifically released from the tissue. Conclusion: These findings support the assumption that native viable hAM could serve as a miRNA source for applications in regenerative medicine.