(D-144) Extracellular vesicles from human right atrial appendage stromal cells are cardioprotective

Introduction:: Extracellular vesicles (EVs) from bone marrow (BM), adipose, umbilical and other mesenchymal stromal cells (MSCs) have been explored for use as therapeutics in myocardial infarction (MI). We isolated an MSC population from the right atrial appendage (RAA) of human heart tissues and previously showed their efficacy in mouse MI (Czosseck, 2022 j.jconrel.2022.10.057). Here we evaluate the cardioprotective effects of their EVs on hypoxic human cardiomyocytes, and examine their cargo and mechanisms of action.

Materials and Methods:: Cardiac MSC-like cells (CMSCLCs) were isolated from the RAA (N = 6 separate donors) and cultured in aMEM with FGF2. Flow cytometry was used to confirm surface marker expression. EVs were isolated by ultracentrifugation of conditioned culture media for 100,000 g for 16 hours at 4 ˚C. EV count and size was quantified by nanoparticle tracking analysis (NTA) and cryoEM. EVs were temporarily added (67ng/µl, 48h) to hypoxic (1% O2), human induced pluripotent cell-derived cardiomyocytes (iPSC-CMs) to assess their protective effects. LDH release was used to measure the degree of iPSC-CM injury at early (48h) and late (8d post-reoxygenation and EV removal) timepoints. CMSCLC EV miRNA content was profiled using Qiagen miRNome and RNA-seq was used to compare hypoxic EV-treated iPSC-CMs against hypoxic controls. IRB approval was obtained from Taipei Medical University, protocol N201910027.

Results, Conclusions, and Discussions:: A summary of the experimental design is shown in (a). CMSCLCs were isolated from human donor cardiac tissues (a). Cells were positive for CD44, CD166 and CD105, and negative for CD19 and CD45, comparable to BM-MSCs (b). EVs were isolated and profiled. NTA (c) showed a mean particle size of 104.7 ± 11.2 nm, and cryoEM (d) showed spherical vesicles with lipid bilayer membranes, confirming EVs. miRNA cargo was analysed (e), revealing several highly expressed miRNAs including miR-21-5p, 202-5p, 1260a and others. miRNA target gene pathway analysis (f) revealed several highly significant pathways including reduction of cardiomyocyte apoptosis, anti-inflammation, ERK1/ERK2 activation, and angiogenesis. Hypoxic human iPSC-derived CMs incubated with EVs showed strong preservation of viability, with no increase in LDH after 48h hypoxia, or 8 days after reoxygenation injury (g). EVs were removed after the hypoxia period. RNA-seq of iPSC-CMs showed several significantly affected biological pathways (h, upper) and KEGG pathways (h, lower) with EV incubation compared to hypoxia and vehicle control. Analysis of GO:0010667 (i) confirmed that EVs preserved more normal expression of CM survival/apoptosis-related genes, including higher GATA4, HAND2 and NOL3.

Conclusions: The significant preservation of iPSC-CM viability observed in our study suggests that these EVs could be a promising therapeutic strategy for treating MI in the future. We are currently confirming activity of key miRNAs, activation of downstream anti-apoptotic, pro-survival pathways, and examining improved methods for EV delivery to the myocardium.

Acknowledgements (Optional): :

References (Optional): : https://doi.org/10.1016/j.jconrel.2022.10.057