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

Nefertiti Chaves-Solano; Silvio Kau-Strebinger; Johannes Oesterreicher; Marianne Pultar; Wolfgang Holnthoner; Johannes Grillari; Simone Hennerbichler; Andreas Brandstetter; Andreas Spittler; Matthias Hackl; Susanne Wolbank; Asmita Banerjee; Adelheid Weidinger

doi:10.3389/fcell.2025.1692501

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

Front Cell Dev Biol. 2025 Oct 29:13:1692501. doi: 10.3389/fcell.2025.1692501. eCollection 2025.

Authors

Nefertiti Chaves-Solano^{1

2}, Silvio Kau-Strebinger^{2

3}, Johannes Oesterreicher^{1

2}, Marianne Pultar^{2

4}, Wolfgang Holnthoner^{1

2}, Johannes Grillari^{1

2

5}, Simone Hennerbichler^{2

6}, Andreas Brandstetter⁷, Andreas Spittler^{2

8}, Matthias Hackl^{2

4}, Susanne Wolbank^{1

2}, Asmita Banerjee^#^{1

2}, Adelheid Weidinger^#^{1

2}

Affiliations

¹ Ludwig Boltzmann Institute for Traumatology, The Research Center in Cooperation with AUVA, Vienna, Austria.
² Austrian Cluster for Tissue Regeneration, Vienna, Austria.
³ Department of Biomedical Sciences and Pathobiology, Centre of Pathobiology, Unit of Morphology, University of Veterinary Medicine, Vienna, Austria.
⁴ TAmiRNA GmbH, Vienna, Austria.
⁵ Institute for Molecular Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria.
⁶ Red Cross Blood Transfusion Service of Upper Austria, Linz, Austria.
⁷ Department of Gynecology and Obstetrics, St. Josef Krankenhaus GmbH Vienna, Vienna, Austria.
⁸ Core Facilities, Unit Flow Cytometry and Surgical Research Laboratories, Medical University of Vienna, Vienna, Austria.

^# Contributed equally.

Abstract

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.

Keywords: extracellular vesicles; human amniotic membrane; miRNA; non-vesicular secretome; perinatal tissue; tissue regeneration; vesicular secretome.