Lozano, J.; Lees, J.; Cross, J.; rai, a.; Lim, S. Y.; Greening, D.

Human cardiac microtissues are a promising model to study cardiac biology and disease, but their application is constrained by therapeutic remodelling strategies and limited knowledge of their functional protein expression profiles. Here, we define the use of human cardiac microtissue (hCMT) model generated by assembling iPSC-derived endothelial cells, cardiac fibroblasts, and cardiomyocytes to model ischemia-reperfusion injury (IRI) through a model of hypoxia and reoxygenation and nanovesicle-mediated functional remodelling. Engineered nanovesicles (NVs), generated directly from human stem cells, have been shown to influence cardiac tissue and cell repair, and provide a platform for scalable and reproducible cell free-mediated therapy. We show the functional regulation of the hCMT model and define that administration of NVs (from human induced pluripotent stem cell origin) during reoxygenation significantly increase cardiomyocyte survival and preserve contractility function (contractile duration, relaxation time, relaxation:contraction velocity). Quantitative proteomics was applied to decipher the cell proteome dynamics and molecular mechanisms of IRI in our in vitro model following NV treatment, linked with networks associated with cell survival, energy production, and stress response regulation. Conserved proteome dynamics in NVs from different iPSC source reveal conserved upregulation of cellular protein networks involved in tissue repair (HSP70, CYFIP1), cardiac function (XIRP1, SLMAP, MYH6, CTNNA1, NDUFS2, GPD2), response to stress (CANX, PDCD6,), pro-survival (MDH2, LRPPRC, NIPSNAP1) and pro-angiogenic (FARSA, ECE1, RRAS) relative to vehicle treatments in context of IRI. Finally, we show that NVs also mediate differential remodelling in hCMT in response to IRI based on their cell origin, including altered wound healing and tissue repair response. Our findings provide an advanced human stem cell-based platform to understand underlying mechanisms of IRI and assess cell-free therapeutic cardioprotective strategies.