Abstract
Purpose :
Central retinal arterial occlusion amplifies cellular damage characterized by inflammation of retinal cells like microglia by inducing activation ultimately leading to vision loss with no current therapy. Extracellular vesicles (EV) from mesenchymal stem cells (MSC) derived from bone marrow (BMSC) reduced inflammation in the retina by mediating paracrine effects of BMSCs while avoiding the complications. Here, we characterize the anti-inflammatory role of MSCs derived from dental pulp stem cells (DPSCs), which is reported to contain more anti-inflammatory factors vs BMSCs. However, the anti-inflammatory capacity of DPSC EVs vs BMSC EVs in microglia is unexplored. We hypothesize that DPSC EVs reduce inflammation from microglia superior to BMSC EVs.
Methods :
To isolate EVs, MSCs are cultured in serum-free media for 48 h then centrifuged (3,000 x g) at 4C for 30 min to clear cellular debris. EVs are isolated using ExoQuick-TC EV precipitation solution overnight then resuspended in PBS. First, EVs were categorized for size and concentration via nanoparticle tracking analysis (NTA) and will be characterized for EV membrane markers (CD9 and CD63) via western. Then, qualitative illustration of labeled BMSC EVs and DPSC EVs uptake by microglia will be assessed using confocal microscopy. Internal protein of EVs is labeled with ExoGlow solution. To measure anti-inflammatory capacity, SIM-A9 murine microglia were stimulated with “TII,” (10 ng/uL TNF-α, 10 ng/uL IL-1β, and 5 ng/uL IFNg) for 6 h, rinsed with PBS, then treated with EVs from BMSC and DPSC for 18 h. Levels of pro-inflammatory mediators including nitrite will be assessed via Griess reagent, and expression of iNOS, COX2, and MMP-2 will be measured via immunoblotting. Pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 will be measured via ELISA. For statistical analysis, a one-way ANOVA will be used.
Results :
For EV characterization, NTA shows EVs from BMSCs and DPSCs have the same concentration of 1.6 x 108 particles/ mL. Majority of BMSC EVs and DPSC EVs are at characteristic EV size of 200 nm. Next, EVs will undergo immunoblotting for expression of EV membrane markers. Currently, EV uptake as well as anti-inflammatory activity of BMSC EVs and DPSC EVs in microglia are on-going.
Conclusions :
NTA results suggests there is no difference in size and concentration between EVs from BMSCs and DPSCs.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.