BS30 A scaffold for heart repair: using human embryonic stem cell derived (HESC) epicardial extracellular matrix to enhance hesc-cardiomyocyte function

IntroductionIschemic heart disease (IHD) is the most common cause for heart failure (HF) worldwide. However, current HF therapies fall short of addressing the irreversible loss of cardiomyocytes after a heart attack. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) has emerged as a promising strategy to remuscularise the failing heart, but this approach remains inefficient. hESC-Epicardium (hESC-Epis) has been shown to improve hESC-CM function when co-transplanted into animal IHD models, though its mechanism of action remains unclear. Bulk RNA-sequencing of hESC-Epis implicate extracellular matrix (ECM) remodelling as a key mediator of these beneficial effects. We therefore hypothesise that hESC-Epi derived ECM can be leveraged to improve hESC-CM cell therapy in heart failure.Study ObjectiveTo examine the unique impact of hESC-Epi derived ECM (dEpi-ECM) on hESC-CM function and to dissect out potential ECM factors responsible for these effects.MethodsdEpi-ECM was isolated through decellularisation of hESC-Epi cultures in vitro. hESC-CMs were then seeded onto the dEpi-ECM and changes in calcium handling, sarcomeric alignment and cell morphology were examined. A multi-omics approach (Single cell RNA sequencing and mass spectroscopy) was then used to further characterise the composition of dEpi-ECM and the respective interactions between its components and hESC-CMs.ResultsDecellularisation of hESC-Epis resulted in a network-like ECM with a dense three-dimensional structure compared of ECM obtained from other cell types. hESC-CMs seeded on dEpi-ECM resulted in an elongation in cell shape, improved sarcomeric length and calcium handling properties in 2D culture. Single cell-RNA sequencing of hESC-Epis and hESC-CMs in isolation and in co-culture demonstrates that several ECM related genes are enriched in hESC-Epis within the co-culture setting compared to monoculture, these include laminin subunit gamma 1(LAMC1), fibronectin (FN1) and several collagen subtypes (COL18A1, COL1A1, COL4A1, COL4A2). Mass spectroscopy analysis of dEpi-ECM revealed the composition of the hESC-Epi matrisome and was able to capture majority of the ECM components found to be enriched in hESC-Epis from the transcriptomic data.ConclusionsWe have demonstrated that dEpi-ECM is capable of improving the structural and functional maturity of hESC-CMs in vitro. These findings recapitulate the hESC-CM maturation signature from our transcriptomic data showing that hESC-CMs contractile fun