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High School Student with a Traditionally Underrepresented Background

HS Poster

Poster 74 - Exploration of Mesenchymal Stem Cell-Derived Extracellular Vesicle Effect on Endothelial Colony-Forming Cells

Saturday, October 26, 2024
10:00 AM - 12:00 PM EST
Location: Exhibit Hall E, F & G

Introduction: Tissue-engineered vascular grafts (TEVGs) are a researched alternative to autologous bypass grafts for occluded blood vessels. Ultimately a TEVG must recellularize with endothelial cells (ECs), resulting in a native-like endothelium. In long grafts, one method for accomplishing this is “fallout endothelialization," where endothelial colony-forming cells (ECFCs) from the blood attach to the lumenal surface, differentiate into ECs, and grow to form a monolayer. Various ways to differentiate ECFCs into endothelial cells have been studied, including shear stress and vascular endothelial growth factor (VEGF). Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) are nanoparticles secreted from parent cells that contain proteins and RNA, one protein of note contained in the EVs being VEGF. This study investigates the differentiation of ECFCs into ECs with co-culture of MSC-EVs. We hypothesize that ECFCs treated with MSC-EVs will exhibit reduced ECFC markers and increased EC markers and form more capillary-like structures than untreated ECFCs.

Materials and

Methods: Human ECFCs and human coronary artery ECs were cultured in the supplier-recommended media during expansion but cultured in ECFC media for all assays for consistency. A 1e9 particles/mL MSC-EV concentration was used for treatment based on our lab's previous success with other cell types. Immunofluorescent staining assays were run to identify CD31, CD34, and von Willebrand factor (vWF) presence in adherent cells in three different groups: the ECFCs with and without EVs, and the ECs. Western blotting was used to determine the expression of VEGF receptor (KDR), vWF, and beta actin using 40ug of cell lysate per lane. Treatment for each assay was added for 24hrs before paraformaldehyde fixation or lysis, respectively. 2-D tube formation assays on 2.5% collagen gel were used as functional assays to observe the amount of tubes formed by the cells and the rate at which they formed; imaging was performed using 10K or 35K cells/well at 8hr or 24hrs. Micro bicinchoninic acid assays (BCA) were run to quantify protein concentration within different particle concentration of EVs (1e9, 2e9, 3e9 particles/mL) with or without lysis buffer treatment.

Results, Conclusions, and Discussions: Immunofluorescent staining of the ECFCs, ECFCs + EVs and ECs showed the presence of vWF in all groups but was inconclusive for CD34 and CD31. In contrast, the western blot showed an increase in high molecular weight vWF levels in the EC group compared to both ECFC groups. KDR was present at higher levels in ECFCs, but no EV effect was noted; CD34 was not shown in any group. Tube formation was minimal in any group, but ECs showed a qualitative increase versus both ECFC groups. MSC-EVs demonstrated a significant increase in protein for lysed samples in the 2e9 particles/mL (p = 0.0043) and 3e9 particles/mL (p = < 0.0001) concentrations but not for the 1e9 particle/mL sample.
The study did not support our hypothesis. There was no effect of EVs on the ECFCs that suggested a differentiation towards an EC state, as would be needed in the TEVG context. Areas of future effort in this study (beyond the short time that the presenting author had available to perform the work as a high school intern) will include an improved protocol for use of the immunofluorescent antibodies, troubleshooting of the western blot procedure, and adoption of a different hydrogel system for the tube formation assay. Increasing EV dosage may also be needed since the lysis + BCA assay showed no protein release in the 1e9 particle/mL concentration after lysis. Longer and/or higher concentration EV treatment may be needed to effect an ECFC transition to ECs. Lastly, a more quantitative experimental approach, such as flow cytometry, may be needed.

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