(I-322) miR29 Loaded Antifibrotic Extracellular Vesicles Derived from Mesenchymal Stem Cells

Introduction:: Fibrotic processes contribute to ~45% of all deaths in the developed world [1].  Fibrosis involves aberrant deposition of extracellular matrix molecules and one pathway that has been implicated in this process is the mechanosensitive YAP/TAZ pathway. Fibrotic remodeling results in the stiffening of the local tissue environment, driving a self-amplifying positive feedback loop of disease progression [2]. Recent work has identified several miRNAs that target fibrosis related genes in response to fibrotic stimuli, indicating their potential to slow or reverse fibrotic processes. In particular, miR29 has been identified as a potential anti-fibrotic miRNA [4]. Additionally, mesenchymal stem cells and their therapeutically relevant extracellular vesicles (EVs) could provide a means to deliver these miRNAs therapeutically.  The current work seeks to load and test MSC derived extracellular vesicles loaded with an anti-fibrotic miR29a mimic.

Materials and Methods:: Human mesenchymal stem cells (MSCs) were cultured in αMEM supplemented with 10% FBS and 1% pen-strep. NIH/3T3 fibroblasts were cultured in high glucose DMEM supplemented with 10% FBS and 1% pen-strep. Initial EV modification began with transfection of cultured MSCs with either a scrambled miRNA sequence (Scr.) or miR29a mimic using INTERFERin transfection reagent with a non-transfected control (Neg.). Cells were switched to αMEM made with EV-free FBS, then media and cells were harvested after 48 hrs. A sample of cells were seeded on fibronectin coated glass coverslips for 24hrs, fixed with 4% PFA, and stained with DAPI, Alexa-488 Phalloidin, and YAP primary antibody then imaged at 20x. The nuclear to cytoplasmic ratio of YAP was quantified using MATLAB. For EV isolation, MSC culture media was enriched with 24% PEG and spun following a differential centrifugation protocol [5]. EV size distribution and count was determined using nanoparticle tracking analysis (NTA). Cultured 3T3 cells were transfected with LSB-hsa-miR-29a-3p [6, Addgene 103391] miRNA sensor using jetPRIME transfection reagent and seeded on fibronectin coated glass coverslips for 24hrs. Cells were treated with isolated MSC EVs for 24 hrs at various dosages (1e6, 5e6, 1e7; 1e6 for Scr) and fixed with 4% PFA. Cells were stained with BioTracker 650 Red Nuclear Dye and imaged at 20x. mKate expression level is regulated by a miRNA29 miRNA response element such that the ratio of mKate to BFP was quantified using MATLAB and indicates the relative activity of miRNA (low ratio= high targeting and activity).

Results, Conclusions, and Discussions:: Results and Discussion: Mimic transfection of miR29a resulted in significantly lower YAP nuclear localization when directly transfected into cultured MSCs (Fig. 1A,B). Isolated EVs from transfected MSC culture followed an expected size distribution for EVs and showed a slight decrease but no significant changes in the amount of EVs produced after transfection (Fig. 1C,F). Sensor experiments in the 3T3 reporter cell line show that with increasing dose of miRNA29a containing EVs there is a decrease in the mKate/BFP ratio, indicating delivery of miRNA29a and targeting of the mKate reporter (Fig 1D,E).

Conclusions: The current results indicate the potential role of miR29a in regulating fibrotic signaling when directly supplied and when delivered by engineered MSC EVs. This is shown through the reduction of YAP activation in MSCs and reduced mKate/BFP ratio in reporter cells. These engineered EVs could have potential uses for therapeutic applications in a variety of fibrotic diseases. Future work will test their ability to influence cellular contractility and collagen gel remodeling in human dermal fibroblasts.

Acknowledgements (Optional): : LSB-hsa-miR-29a-3p was a gift from Ron Weiss (Addgene plasmid # 103391)

References (Optional): : [1] Wynn+ Journal of Pathology (2008), [2] Distler+ Nat Rev Rheum (2019), [3] O’Reily+ Arthritis Res and Ther (2016), [4] Rooij+ PNAS (2008), [5] Rider+ Sci Rep (2016), [6] Gam+ (2018).