Abstract

◼ fibroblasts ◼ induced pluripotent stem cells ◼ microRNA ◼ secretome C ardiac-derived exosomes have received intense interest for their roles in paracrine communications and regenerative therapies.However, current understanding of how exosomes mediate cellular signaling is incomplete, in part because the contents of exosomes from different cardiac cell types are poorly defined.To learn what signals cardiac cells release, we examined the microRNA (miR-NA) compositions secreted in exosomes from human induced pluripotent stem cells (iPSCs) and 3 major iPSC-derived cardiac cell types.With approval by the Stanford University Institutional Review committee and informed consent, human iPSC lines from 2 healthy donors were reprogrammed in the Stanford Cardiovascular Institute Biorepository and differentiated into cardiomyocytes (iPSC-CMs), endothelial cells (iPSC-ECs), and cardiac fibroblasts (iPSC-CFs) using established protocols. 1Low yield has been a bottleneck in elucidating the composition and function of extracellular exosomes.To boost isolation yields, we applied a mechanical-sorting device (ExoTIC) that isolates exosomes from small volumes of culture medium. 2,3Briefly, the device is an engineered fluidic system that delivers culture medium through nanoporous membranes to selectively capture vesicles at 50 to 200 nm in diameter.We validated the extracted exosomes using nanoparticle tracking analysis and immunoblots, and confirmed superior yield over conventional precipitation methods (Figure A).We then isolated total RNA (≥18 nt) from exosomes of 2 biological replicate lines for library generation and small RNA sequencing on an Illumina NextSeq platform.Sequencing reads were mapped to GRCh38 human reference genome after adaptor clipping and annotated against miRBase v.22.1 coordinates using conventional pipelines.From the data, we identified 120 miRNAs (mature or stem loop) from 94 miRNA genes to be secreted in the analyzed cell types (normalized read counts ≥10 in both lines).The exosomal miRNA profiles revealed that (1) let-7 miRNA precursors, which are suppressed by Lin28, are depleted in iPSC secretomes as expected; (2) only a subset of total cellular miRNAs are secreted (eg, both miR-155 and miR-143 are preferentially found in intracellular over exosomal pools in iPSCs); (3) a common core of miRNAs is secreted by all 3 cardiac cell types (eg, miR-320); and (4) importantly, we found distinct enrichment or depletion of different mature miR-NAs in each exosome type (Figure B).For instance, we found that miR-1, critical for cardiac development and pathology, is only secreted by iPSC-CMs in our data.Using a published human miRNA atlas, 4 we next compared total tissue expression of miR-1 in 17 different tissues, which showed miR-1 to be specific to striated muscles (Figure C).Similarly, miR-302c, known to be specific to pluripotent cells, is enriched in iPSC-derived vesicles 5 and also appears to be enriched in the bone, likely reflecting bone marrow hematopoietic stem cells. 4Finally, miR-155 is primar-