Many cells make life work-multicellularity in stem cell-based cardiac disease modelling

Cardiac disease causes 33% of deaths worldwide but our knowledge of disease progression is still very limited. In vitro models utilising and combining multiple, differentiated cell types have been used to recapitulate the range of myocardial microenvironments in an effort to delineate the mechanical, humoral, and electrical interactions that modulate the cardiac contractile function in health and the pathogenesis of human disease. However, due to limitations in isolating these cell types and changes in their structure and function in vitro, the field is now focused on the development and use of stem cell-derived cell types, most notably, human-induced pluripotent stem cell-derived CMs (hiPSC-CMs), in modelling the CM function in health and patient-specific diseases, allowing us to build on the findings from studies using animal and adult human CMs. It is becoming increasingly appreciated that communications between cardiomyocytes (CMs), the contractile cell of the heart, and the non-myocyte components of the heart not only regulate cardiac development and maintenance of health and adult CM functions, including the contractile state, but they also regulate remodelling in diseases, which may cause the chronic impairment of the contractile function of the myocardium, ultimately leading to heart failure. Within the myocardium, each CM is surrounded by an intricate network of cell types including endothelial cells, fibroblasts, vascular smooth muscle cells, sympathetic neurons, and resident macrophages, and the extracellular matrix (ECM), forming complex interactions, and models utilizing hiPSC-derived cell types offer a great opportunity to investigate these interactions further. In this review, we outline the historical and current state of disease modelling, focusing on the major milestones in the development of stem cell-derived cell types, and how this technology has contributed to our knowledge about the interactions between CMs and key non-myocyte components of the heart in health and disease, in particular, heart failure. Understanding where we stand in the field will be critical for stem cell-based applications, including the modelling of diseases that have complex multicellular dysfunctions

Rolling circle transcription-amplified hierarchically structured organic-inorganic hybrid RNA flowers for enzyme immobilization

Programmable nucleic acids have emerged as powerful building blocks for the bottom-up fabrication of two- or three-dimensional nano- and microsized constructs. Here we describe the construction of organic–inorganic hybrid RNA flowers (hRNFs) via rolling circle transcription (RCT), an enzyme-catalyzed nucleic acid amplification reaction. These hRNFs are highly adaptive structures with controlled sizes, specific nucleic acid sequences, and a highly porous nature. We demonstrated that hRNFs are applicable as potential biological platforms, where the hRNF scaffold can be engineered for versatile surface functionalization and the inorganic component (magnesium ions) can serve as an enzyme cofactor. For surface functionalization, we proposed robust and straightforward approaches including in situ synthesis of functional hRNFs and postfunctionalization of hRNFs that enable facile conjugation with various biomolecules and nanomaterials (i.e., proteins, enzymes, organic dyes, inorganic nanoparticles) using selective chemistries (i.e., avidin–biotin interaction, copper-free click reaction). In particular, we showed that hRNFs can serve as soft scaffolds for β-galactosidase immobilization and greatly enhance enzymatic activity and stability. Therefore, the proposed concepts and methodologies are not only fundamentally interesting when designing RNA scaffolds or RNA bionanomaterials assembled with enzymes but also have significant implications on their future utilization in biomedical applications ranging from enzyme cascades to biosensing and drug delivery

Extracellular vesicles from human cardiac fibroblasts modulate calcium cycling in human stem cell-derived cardiomyocytes

Cardiac fibroblasts regulate the development of the adult cardiomyocyte phenotype and cardiac remodeling in disease. We investigate the role that cardiac fibroblasts-secreted extracellular vesicles (EVs) have in the modulation of cardiomyocyte Ca2+ cycling–a fundamental mechanism in cardiomyocyte function universally altered during disease. EVs collected from cultured human cardiac ventricular fibroblasts were purified by centrifugation, ultrafiltration and size-exclusion chromatography. The presence of EVs and EV markers were identified by dot blot analysis and electron microscopy. Fibroblast-conditioned media contains liposomal particles with a characteristic EV phenotype. EV markers CD9, CD63 and CD81 were highly expressed in chromatography fractions that elute earlier (Fractions 1–15), with most soluble contaminating proteins in the later fractions collected (Fractions 16–30). Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were treated with fibroblast-secreted EVs and intracellular Ca2+ transients were analyzed. Fibroblast-secreted EVs abbreviate the Ca2+ transient time to peak and time to 50% decay versus serum-free controls. Thus, EVs from human cardiac fibroblasts represent a novel mediator of human fibroblast-cardiomyocyte interaction, increasing the efficiency of hiPSC-CM Ca2+ handling

Functional microvascularization of human myocardium in vitro

In this study, we report static and perfused models of human myocardial-microvascular interaction. In static culture, we observe distinct regulation of electrophysiology of human induced pluripotent stem cell derived-cardiomyocytes (hiPSC-CMs) in co-culture with human cardiac microvascular endothelial cells (hCMVECs) and human left ventricular fibroblasts (hLVFBs), including modification of beating rate, action potential, calcium handling, and pro-arrhythmic substrate. Within a heart-on-a-chip model, we subject this three-dimensional (3D) co-culture to microfluidic perfusion and vasculogenic growth factors to induce spontaneous assembly of perfusable myocardial microvasculature. Live imaging of red blood cells within myocardial microvasculature reveals pulsatile flow generated by beating hiPSC-CMs. This study therefore demonstrates a functionally vascularized in vitro model of human myocardium with widespread potential applications in basic and translational research

Raman spectroscopy imaging reveals interplay between atherosclerosis and medial calcification in human aorta

Medial calcification in the human aorta accumulates during aging and is known to be aggravated in several diseases. Atherosclerosis, another major cause of cardiovascular calcification, shares some common aggravators. However, the mechanisms of cardiovascular calcification remain poorly understood. To elucidate the relationship between medial aortic calcification and atherosclerosis, we characterized the cross-sectional distributions of the predominant minerals in aortic tissue, apatite and whitlockite, and the associated extracellular matrix. We also compared the cellular changes between atherosclerotic and nonatherosclerotic human aortic tissues. This was achieved through the development of Raman spectroscopy imaging methods that adapted algorithms to distinguish between the major biomolecules present within these tissues. We present a relationship between apatite, cholesterol, and triglyceride in atherosclerosis, with the relative amount of all molecules concurrently increased in the atherosclerotic plaque. Further, the increase in apatite was disproportionately large in relation to whitlockite in the aortic media directly underlying a plaque, indicating that apatite is more pathologically significant in atherosclerosis-aggravated medial calcification. We also discovered a reduction of β-carotene in the whole aortic intima, including a plaque in atherosclerotic aortic tissues compared to nonatherosclerotic tissues. This unprecedented biomolecular characterization of the aortic tissue furthers our understanding of pathological and physiological cardiovascular calcification events in humans

Research data supporting "Ultrasound-triggered enzymatic gelation"

Research data supporting the publication: V. Nele et al., Ultrasound-triggered enzymatic gelation, 2020, DOI: 10.1002/adma.201905914Research data supporting the publication: V. Nele et al., Ultrasound-triggered enzymatic gelation, 2020, DOI: 10.1002/adma.201905914

Ultrasound-triggered enzymatic gelation

Hydrogels are formed using various triggers, including light irradiation, pH adjustment, heating, cooling or chemical addition. In this report, a new method for forming hydrogels is introduced: ultrasound-triggered enzymatic gelation. Specifically, ultrasound is used as a stimulus to liberate liposomal calcium ions, which then trigger the enzymatic activity of transglutaminase. The activated enzyme catalyzes the formation of fibrinogen hydrogels through covalent intermolecular crosslinking. The catalysis and gelation processes are monitored in real time and both the enzyme kinetics and final hydrogel properties are controlled by varying the initial ultrasound exposure time. This technology is extended to microbubble-liposome conjugates, which exhibit a stronger response to the applied acoustic field and are also used for ultrasound-triggered enzymatic hydrogelation. To the best of our knowledge, these results are the first instance in which ultrasound has been used as a trigger for either enzyme catalysis or enzymatic hydrogelation. This approach is highly versatile and Peer reviewed version of the manuscript published in final form at Advanced Materials (2020) 2 could be readily applied to different ion-dependent enzymes or gelation systems. Moreover, this work paves the way for the use of ultrasound as a remote trigger for in vivo hydrogelation

Microliter scale synthesis of luciferase-encapsulated polymersomes as artificial organelles for optogenetic modulation of cardiomyocyte beating

Constructing artificial systems that effectively replace or supplement natural biological machinery within cells is one of the fundamental challenges underpinning bioengineering. At the sub-cellular scale, artificial organelles (AOs) have significant potential as long-acting biomedical implants, mimicking native organelles by conducting intracellularly compartmentalized enzymatic actions. The potency of these AOs can be heightened when judiciously combined with genetic engineering, producing highly tailorable biohybrid cellular systems. Here, the authors present a cost-effective, microliter scale (10 µL) polymersome (PSome) synthesis based on polymerization-induced self-assembly for the in situ encapsulation of Gaussia luciferase (GLuc), as a model luminescent enzyme. These GLuc-loaded PSomes present ideal features of AOs including enhanced enzymatic resistance to thermal, proteolytic, and intracellular stresses. To demonstrate their biomodulation potential, the intracellular luminescence of GLuc-loaded PSomes is coupled to optogenetically engineered cardiomyocytes, allowing modulation of cardiac beating frequency through treatment with coelenterazine (CTZ) as the substrate for GLuc. The long-term intracellular stability of the luminescent AOs allows this cardiostimulatory phenomenon to be reinitiated with fresh CTZ even after 7 days in culture. This synergistic combination of organelle-mimicking synthetic materials with genetic engineering is therefore envisioned as a highly universal strategy for the generation of new biohybrid cellular systems displaying unique triggerable properties

Multiplexing physical stimulation on single human induced pluripotent stem cell-derived cardiomyocytes for phenotype modulation

Traditional in vitro bioengineering approaches whereby only individual biophysical cues are manipulated at any one time are highly inefficient, falling short when recapitulating the complexity of the cardiac environment. Multiple biophysical cues are present in the native myocardial niche and are essential during development, as well as in maintenance of adult cardiomyocyte (CM) phenotype in both health and disease. This study establishes a novel biofabrication workflow to study and manipulate hiPSC-CMs and to understand how these cells respond to a multiplexed biophysical environment, namely microscopic topography (3D shape resembling that of adult CM) and substrate stiffness, at a single cell level. Silicon masters were fabricated and developed to generate pillars of the desired 3D shapes, which would be used to mould the designed microwell arrays into a hydrogel. Polyacrylamide was modified with the incorporation of acrylic acid to provide a carboxylic group conjugation site for adhesion motifs, without comprising its capacity to modulate the stiffness. In this manner, individual parameters can be finely tuned independently within the hydrogel: the dimension of 3D shaped microwell and its stiffness. The design allows the platform to isolate single hiPSC-CMs to study solely biophysical cues in an absence of cell-cell physical interaction. Under physiologic-like physical conditions (3D shape resembling that of adult CM and 9.83 kPa substrate stiffness), isolated single hiPSC-CMs exhibit increased Cx-43 density, cell Peer reviewed version of the manuscript published in final form at Biofabrication (2020). membrane stiffness and calcium transient amplitude; co-expression of the subpopulation-related MYL2- MYL7 proteins; while displaying higher anisotropism in comparison to pathologic-like conditions (flat surface and 112 kPa substrate stiffness). This demonstrates that supplying a physiological or pathological microenvironment to an isolated single hiPSC-CM in absence of any physical cell-to-cell communication in this biofabricated platform leads to a significantly different set of cellular features, thus presenting a differential phenotype. Importantly, this demonstrates the high plasticity of hiPSC-CMs even in isolation. The ability of multiple biophysical cues to significantly influence isolated single hiPSC-CM phenotype and functionality highlights the importance of fine-tuning such cues for specific applications. This has the potential to produce more fit-for purpose hiPSC-CMs. Further understanding of human cardiac development is enabled by the robust, versatile and reproducible biofabrication techniques applied here. We envision this system to be easily applied to other tissues and cell types where the influence of stiffness and cellular shape plays also an important role in its physiology. Keywords: microfabrication, cardiomyocyte, physical interaction, cell shape, calcium handling, human induced pluripotent stem cell