Administration of Amniotic Fluid Stem Cell Extracellular Vesicles to Hypoplastic CDH Fetal Lungs Induces Epithelial Proliferation and Differentiation via a miRNA Mediated Mechanism

Background/Purpose:
Impaired fetal lung development is the recognized cause of poor outcome for babies with congenital diaphragmatic hernia (CDH). We recently showed that administration of extracellular vesicles derived from amniotic fluid stem cells (AFSC-EVs) restores lung branching morphogenesis in fetal rats with CDH. Herein, we aimed to evaluate whether AFSC-EV administration could promote maturation of hypoplastic fetal lungs in experimental CDH.

Methods:
AFSC-EVs were isolated from conditioned medium by ultracentrifugation and characterized for size (nanoparticle tracking analysis), morphology (electron microscopy), and canonical protein markers (Western blot). AFSC-EV RNA cargo was isolated with SeraMir and sequenced with NextSeq.
Lung explants were harvested at E14.5 from fetuses of dams that received nitrofen at E9.5. Explants were treated with vehicle (nitrofen group) or AFSC-EVs (nitrofen+AFSC-EV group) for 72h. Explants were compared for cell proliferation (EdU) and density of distal lung progenitor cells (Sox9) using Mann-Whitney test.
Lung primary epithelial cells were isolated from nitrofen exposed lungs treated with vehicle or with AFSC-EVs. RNA was extracted using miRvana and sequenced with NextSeq. Pathway enrichment analysis was performed using g:Profiler.

Results:
Nitrofen exposed lungs treated with AFSC-EVs had more proliferating cells than lungs treated with vehicle (Figure A&B). The latter instead had more Sox9+ cells compared to AFSC-EV treated lungs. Most proliferating cells in AFSC-EV treated lungs were differentiated epithelium, contrarily to untreated lungs whose proliferating cells were Sox9+. AFSC-EV cargo was abundant in mir-17~92 cluster that controls lung cell proliferation and differentiation. At gene-set enrichment analysis, primary epithelial cells from lungs treated with AFSC-EVs were highly enriched for biological processes related to cell proliferation and its regulation.

Conclusions:
In experimental CDH, nitrofen exposure retains fetal lungs at an immature state. Conversely, AFSC-EV administration stimulates epithelial proliferation and differentiation, possibly through a miRNA-mediated mechanism. AFSC-EVs contain microRNA that are essential for normal fetal lung development.