Understanding genomic medicine for thoracic aortic disease through the lens of induced pluripotent stem cells.

Thoracic aortic disease (TAD) is often silent until a life-threatening complication occurs. However, genetic information can inform both identification and treatment at an early stage. Indeed, a diagnosis is important for personalised surveillance and intervention plans, as well as cascade screening of family members. Currently, only 20% of heritable TAD patients have a causative mutation identified and, consequently, further advances in genetic coverage are required to define the remaining molecular landscape. The rapid expansion of next generation sequencing technologies is providing a huge resource of genetic data, but a critical issue remains in functionally validating these findings. Induced pluripotent stem cells (iPSCs) are patient-derived, reprogrammed cell lines which allow mechanistic insights, complex modelling of genetic disease and a platform to study aortic genetic variants. This review will address the need for iPSCs as a frontline diagnostic tool to evaluate variants identified by genomic discovery studies and explore their evolving role in biological insight through to drug discovery.

Extracellular macrostructure anisotropy improves cardiac tissue-like construct function and phenotypic cellular maturation.

Regenerative cardiac tissue is a promising field of study with translational potential as a therapeutic option for myocardial repair after injury, however, poor electrical and contractile function has limited translational utility. Emerging research suggests scaffolds that recapitulate the structure of the native myocardium improve physiological function. Engineered cardiac constructs with anisotropic extracellular architecture demonstrate improved tissue contractility, signaling synchronicity, and cellular organization when compared to constructs with reduced architectural order. The complexity of scaffold fabrication, however, limits isolated variation of individual structural and mechanical characteristics. Thus, the isolated impact of scaffold macroarchitecture on tissue function is poorly understood. Here, we produce isotropic and aligned collagen scaffolds seeded with embryonic stem cell derived cardiomyocytes (hESC-CM) while conserving all confounding physio-mechanical features to independently assess the effects of macroarchitecture on tissue function. We quantified spatiotemporal tissue function through calcium signaling and contractile strain. We further examined intercellular organization and intracellular development. Aligned tissue constructs facilitated improved signaling synchronicity and directional contractility as well as dictated uniform cellular alignment. Cells on aligned constructs also displayed phenotypic and genetic markers of increased maturity. Our results isolate the influence of scaffold macrostructure on tissue function and inform the design of optimized cardiac tissue for regenerative and model medical systems.

Epicardially secreted fibronectin drives cardiomyocyte maturation in 3D-engineered heart tissues.

Ischemic heart failure is due to irreversible loss of cardiomyocytes. Preclinical studies showed that human pluripotent stem cell (hPSC)-derived cardiomyocytes could remuscularize infarcted hearts and improve cardiac function. However, these cardiomyocytes remained immature. Incorporating hPSC-derived epicardial cells has been shown to improve cardiomyocyte maturation, but the exact mechanisms are unknown. We posited epicardial fibronectin (FN1) as a mediator of epicardial-cardiomyocyte crosstalk and assessed its role in driving hPSC-derived cardiomyocyte maturation in 3D-engineered heart tissues (3D-EHTs). We found that the loss of FN1 with peptide inhibition F(pUR4), CRISPR-Cas9-mediated FN1 knockout, or tetracycline-inducible FN1 knockdown in 3D-EHTs resulted in immature cardiomyocytes with decreased contractile function, and inefficient Ca2+ handling. Conversely, when we supplemented 3D-EHTs with recombinant human FN1, we could recover hPSC-derived cardiomyocyte maturation. Finally, our RNA-sequencing analyses found FN1 within a wider paracrine network of epicardial-cardiomyocyte crosstalk, thus solidifying FN1 as a key driver of hPSC-derived cardiomyocyte maturation in 3D-EHTs.

Inducible apelin receptor knockdown reduces differentiation efficiency and contractility of hESC-derived cardiomyocytes.

AIMS: The apelin receptor, a G protein-coupled receptor, has emerged as a key regulator of cardiovascular development, physiology, and disease. However, there is a lack of suitable human in vitro models to investigate the apelinergic system in cardiovascular cell types. For the first time we have used human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and a novel inducible knockdown system to examine the role of the apelin receptor in both cardiomyocyte development and to determine the consequences of loss of apelin receptor function as a model of disease. METHODS AND RESULTS: Expression of the apelin receptor and its ligands in hESCs and hESC-CMs was determined. hESCs carrying a tetracycline-inducible short hairpin RNA targeting the apelin receptor were generated using the sOPTiKD system. Phenotypic assays characterized the consequences of either apelin receptor knockdown before hESC-CM differentiation (early knockdown) or in 3D engineered heart tissues as a disease model (late knockdown). hESC-CMs expressed the apelin signalling system at a similar level to the adult heart. Early apelin receptor knockdown decreased cardiomyocyte differentiation efficiency and prolonged voltage sensing, associated with asynchronous contraction. Late apelin receptor knockdown had detrimental consequences on 3D engineered heart tissue contractile properties, decreasing contractility and increasing stiffness. CONCLUSIONS: We have successfully knocked down the apelin receptor, using an inducible system, to demonstrate a key role in hESC-CM differentiation. Knockdown in 3D engineered heart tissues recapitulated the phenotype of apelin receptor down-regulation in a failing heart, providing a potential platform for modelling heart failure and testing novel therapeutic strategies.

Erratum: Author Correction: A single-cell comparison of adult and fetal human epicardium defines the age-associated changes in epicardial activity.

[This corrects the article DOI: 10.1038/s44161-022-00183-w.].

A single-cell comparison of adult and fetal human epicardium defines the age-associated changes in epicardial activity.

Re-activating quiescent adult epicardium represents a potential therapeutic approach for human cardiac regeneration. However, the exact molecular differences between inactive adult and active fetal epicardium are not known. In this study, we combined fetal and adult human hearts using single-cell and single-nuclei RNA sequencing and compared epicardial cells from both stages. We found that a migratory fibroblast-like epicardial population only in the fetal heart and fetal epicardium expressed angiogenic gene programs, whereas the adult epicardium was solely mesothelial and immune responsive. Furthermore, we predicted that adult hearts may still receive fetal epicardial paracrine communication, including WNT signaling with endocardium, reinforcing the validity of regenerative strategies that administer or reactivate epicardial cells in situ. Finally, we explained graft efficacy of our human embryonic stem-cell-derived epicardium model by noting its similarity to human fetal epicardium. Overall, our study defines epicardial programs of regenerative angiogenesis absent in adult hearts, contextualizes animal studies and defines epicardial states required for effective human heart regeneration.

Modulating hESC-derived cardiomyocyte and endothelial cell function with triple-helical peptides for heart tissue engineering.

In this study, we investigated the role of cardiomyocyte (CM) and endothelial cell (EC) specific interactions with collagen in the assembly of an operational myocardium in vitro. Engineered cardiac patches represent valuable tools for myocardial repair following infarction and are generally constituted of a suitable biomaterial populated by CMs and supportive cell types. Among those, ECs are required for tissue vascularization and positively modulate CM function. To direct the function of human embryonic stem cell (hESC)-derived CM and EC seeded on biomaterials, we replicated cell-collagen interactions, which regulate cellular behaviour in the native myocardium, using triple-helical peptides (THPs) that are ligands for collagen-binding proteins. THPs enhanced proliferation and activity of CMs and ECs separately and in co-culture, drove CM maturation and enabled coordinated cellular contraction on collagen films. These results highlight the importance of collagen interactions on cellular response and establish THP-functionalized biomaterials as novel tools to produce engineered cardiac tissues.