NextSeq 500 sequencing; Extracellular Vesicles Derived from Amniotic Fluid Stem Cells Rescue Impaired Fetal Lung Development via the Release of Small RNAs
Extracellular Vesicles Derived from Amniotic Fluid Stem Cells Rescue Impaired Fetal Lung Development via the Release of Small RNAs
At E14.5, a single cell suspension was obtained from pooled lungs from either control or nitrofen exposed rat fetuses by trypsinization (0.25% Trypsin-EDTA, ThermoFisher, Waltham, MA) for 20 minutes at 37Â°C humidified chamber. Cells were spun down by centrifugation for 5 minutes at 800g and the pellet was resuspended in DMEM supplemented with 10% FBS and subjected to three serial depletion of fibroblasts by incubation for 1 hour each following an established protocol [Caniggia 1991]. Cells were used to test epithelial homeostasis and to generate fetal lung organoids for testing epithelial differentiation. For epithelial homeostasis experiments, cells were grown for 5 days in Bronchial Epithelial Cell Growth Medium (BEGM; Lonza, Basel Switzerland), which was changed every other day. Cells were checked daily for proper epithelial morphology using a light microscope. On the fifth day, cells were confirmed to be positive for SPC and negative for vimentin via immunofluorescence staining assays.  Rat bone-marrow derived mesenchymal stem cells (MSCs) were purchased (CellBiologics, Chicago, IL) and grown in supplier recommended medium until 90% confluence.  Total RNA extraction was conducted using mirVana miRNA isolation kit with phenol (ThermoFisher, Waltham, MA), following supplier recommended protocols. Purified RNA was quantified using NanoDrop and Bioanalyzer was used to assess the quality of RNA. Samples with RNA integrity number >9 were used to construct libraries.  10ng of input RNA from three replicates of each condition was synthesized into sequencing libraries using the Capture and Amplification by Tailing and Switching (Diagenode Inc., Denville, NJ).
Experimental Factor: organism part
amniotic fluid
Experimental Factor: strain
AFSC-EVs