Cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) are functionally immature, but this is improved by incorporation into engineered tissues or forced contraction. Here, we showed that tri-cellular combinations of hiPSC-derived CMs, cardiac fibroblasts (CFs), and cardiac endothelial cells also enhance maturation in easily constructed, scaffold-free, three-dimensional microtissues (MTs). hiPSC-CMs in MTs with CFs showed improved sarcomeric structures with T-tubules, enhanced contractility, and mitochondrial respiration and were electrophysiologically more mature than MTs without CFs. Interactions mediating maturation included coupling between hiPSC-CMs and CFs through connexin 43 (CX43) gap junctions and increased intracellular cyclic AMP (cAMP). Scaled production of thousands of hiPSC-MTs was highly reproducible across lines and differentiated cell batches. MTs containing healthy-control hiPSC-CMs but hiPSC-CFs from patients with arrhythmogenic cardiomyopathy strikingly recapitulated features of the disease. Our MT model is thus a simple and versatile platform for modeling multicellular cardiac diseases that will facilitate industry and academic engagement in high-throughput molecular screening.

Rationale: There are several methods to measure cardiomyocyte (CM) and muscle contraction but these require customized hardware, expensive apparatus and advanced informatics or can only be used in single experimental models. Consequently, data and techniques have been difficult to reproduce across models and laboratories, analysis is time consuming and only specialist researchers can quantify data. Objective: Here we describe and validate an automated, open source software tool (MUSCLEMOTION) adaptable for use with standard laboratory- and clinical imaging equipment that enables quantitative analysis of normal cardiac contraction, disease phenotypes and pharmacological responses. Methods and Results: MUSCLEMOTION allowed rapid and easy measurement of movement from high-speed movies in: (i) 1-dimensional in vitro models such as isolated adult and human pluripotent stem cell-derived CMs (hPSC-CMs); (ii) 2-dimensional in vitro models, such as beating CM monolayers or small clusters of hPSC-CMs; (iii) 3-dimensional multicellular in vitro or in vivo contractile tissues such as cardiac "organoids", engineered heart tissues (EHT), zebrafish- and human hearts. MUSCLEMOTION was effective under different recording conditions (bright field microscopy with simultaneous patch clamp recording, phase contrast microscopy and traction force microscopy). Outcomes were virtually identical to the current gold standards for contraction measurement such as optical flow, pole deflection, edge-detection systems or manual analyses. Finally, we used the algorithm to quantify contraction in in vitro and in vivo arrhythmia models and to measure pharmacological responses. Conclusions: Using a single open source method for processing video recordings, we obtained reliable pharmacological data and measures of cardiac disease phenotype in experimental cell-, animal- and human models.

Human endothelial cells (ECs) and pericytes are of great interest for research on vascular development and disease, as well as for future therapy. This protocol describes the efficient generation of ECs and pericytes from human pluripotent stem cells (hPSCs) under defined conditions. Essential steps for hPSC culture, differentiation, isolation and functional characterization of ECs and pericytes are described. Substantial numbers of both cell types can be derived in only 2–3 weeks: this involves differentiation (10 d), isolation (1 d) and 4 or 10 d of expansion of ECs and pericytes, respectively. We also describe two assays for functional evaluation of hPSC-derived ECs: (i) primary vascular plexus formation upon coculture with hPSC-derived pericytes and (ii) incorporation in the vasculature of zebrafish xenografts in vivo. These assays can be used to test the quality and drug sensitivity of hPSC-derived ECs and model vascular diseases with patient-derived hPSCs.

High‐mobility group box 1 (HMGB1) is released extracellularly upon cell necrosis acting as a mediator in tissue injury and inflammation. However, the molecular mechanisms for the proinflammatory effect of HMGB1 are poorly understood. Here, we define a novel function of HMGB1 in promoting Mac‐1‐dependent neutrophil recruitment. HMGB1 administration induced rapid neutrophil recruitment in vivo . HMGB1‐mediated recruitment was prevented in mice deficient in the β2‐integrin Mac‐1 but not in those deficient in LFA‐1. As observed by bone marrow chimera experiments, Mac‐1‐dependent neutrophil recruitment induced by HMGB1 required the presence of receptor for advanced glycation end products (RAGE) on neutrophils but not on endothelial cells. In vitro , HMGB1 enhanced the interaction between Mac‐1 and RAGE. Consistently, HMGB1 activated Mac‐1 as well as Mac‐1‐mediated adhesive and migratory functions of neutrophils in a RAGE‐dependent manner. Moreover, HMGB1‐induced activation of nuclear factor‐κB in neutrophils required both Mac‐1 and RAGE. Together, a novel HMGB1‐dependent pathway for inflammatory cell recruitment and activation that requires the functional interplay between Mac‐1 and RAGE is described here.

Cardiomyocytes and endothelial cells in the heart are in close proximity and in constant dialogue. Endothelium regulates the size of the heart, supplies oxygen to the myocardium and secretes factors that support cardiomyocyte function. Robust and predictive cardiac disease models that faithfully recapitulate native human physiology in vitro would therefore ideally incorporate this cardiomyocyte-endothelium crosstalk. Here, we generated and characterized human cardiac microtissues in vitro that integrate both cell types in complex 3D structures. We established conditions for simultaneous differentiation of cardiomyocytes and endothelial cells from human pluripotent stem cells following initial cardiac mesoderm induction. The endothelial cells expressed cardiac markers also present in primary cardiac microvasculature suggesting cardiac endothelium identity. These cell populations were further enriched based on surface markers expression, then recombined allowing development of beating 3D structures termed cardiac microtissues. This in vitro model was robustly reproducable in both embryonic and induced pluripotent stem cells. It thus represents an advanced human stem cell-based platform for cardiovascular disease modelling and testing of relevant drugs.

Human neutrophil-specific CD177 (NB1 and PRV-1) has been reported to be up-regulated in a number of inflammatory settings, including bacterial infection and granulocyte-colony-stimulating factor application. Little is known about its function. By flow cytometry and immunoprecipitation studies, we identified platelet endothelial cell adhesion molecule-1 (PECAM-1) as a binding partner of CD177. Real-time protein-protein analysis using surface plasmon resonance confirmed a cation-dependent, specific interaction between CD177 and the heterophilic domains of PECAM-1. Monoclonal antibodies against CD177 and against PECAM-1 domain 6 inhibited adhesion of U937 cells stably expressing CD177 to immobilized PECAM-1. Transendothelial migration of human neutrophils was also inhibited by these antibodies. Our findings provide direct evidence that neutrophil-specific CD177 is a heterophilic binding partner of PECAM-1. This interaction may constitute a new pathway that participates in neutrophil transmigration.