Abstract: TH-PO0039
miRNA Content of Induced Nephron Progenitor Cell Extracellular Vesicles
Session Information
- Bioengineering: MPS, Flow, and Delivery
November 06, 2025 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Bioengineering
- 400 Bioengineering
Authors
- Farry, Justin M., Vanderbilt University, Nashville, Tennessee, United States
- Bejoy, Julie, Vanderbilt University Medical Center, Nashville, Tennessee, United States
- Cartailler, Jean-Philippe, Vanderbilt University, Nashville, Tennessee, United States
- Woodard, Lauren Elizabeth, Department of Veterans Affairs, Nashville, Tennessee, United States
Background
Extracellular vehicles (EVs), including exosomes, have emerged as a promising therapeutic and diagnostic tool. EVs can contain diverse cargo of RNA, protein, and lipids. The cargo varies based on cell origin and environmental conditions. We studied EVs derived from an induced nephron progenitor (iNP) cell line. iNPs are a line of engineered HK-2 cells containing a doxycycline-inducible piggyBac transposon that expresses transcription factors SNAI2, EYA1, and SIX1 to reprogram the cells to a nephron progenitor phenotype. We sought to investigate the miRNA contents of iNP-derived EVs (iNP-EVs) to understand their potential to treat acute kidney injury and chronic kidney disease.
Methods
iNPs were cultured in standard HK2 cell culture media supplemented with doxycycline for five days, followed by three days in nepron progenitor-specific media (NPSR). We isolated EVs from the NPSR media using a polyethylene glycol gradient technique. miRNA was isolated from iNP-EVs, and the RNA library was prepared from 1 µg of RNA using TruSeq Small RNA Sample Prep Kits. Sequencing was done using an Illumina Hiseq 4000 at 50bp single-end sequencing and 7-10 million reads per sample by LC Science. miRNA-seq data were processed using the TIGER pipeline. miRNA target and pathway analysis using normalized TPM counts and the multiMiR database, focusing on targets validated via luciferase assays. miRNAs demonstrating significant expression levels (above 1000 TPM) underwent over-representation analysis with Gene Ontology categories for biological process (BP), cellular component (CC), and molecular function (MF).
Results
iNP-EVs contain a diverse array of miRNA, with 25 miRNAs accounting for 82% of the total miRNA expression. The most abundant miRNA, mir21-5p, accounted for 48% of the total miRNA. Analysis of mir21-5p go terms shows an enrichment in the BP terms epithelial cell proliferation, the CC term for collagen containing extracellular matrix, and the MF terms involved in DNA transcription factor binding.
Conclusion
iNP-EVs carry a diverse cargo of miRNA with the most abundant miRNA being mir21-5p accounting for 48% of the total miRNA expression. GO analysis of mir21-5p indicated it could lead to increased cellular proliferation and extracellular matrix remodeling.
Funding
- Veterans Affairs Support