Patterned Arteriole-Scale Vessels Enhance Engraftment, Perfusion, and Vessel Branching Hierarchy of Engineered Human Myocardium for Heart Regeneration.

Heart regeneration after myocardial infarction (MI) using human stem cell-derived cardiomyocytes (CMs) is rapidly accelerating with large animal and human clinical trials. However, vascularization methods to support the engraftment, survival, and development of implanted CMs in the ischemic environment of the infarcted heart remain a key and timely challenge. To this end, we developed a dual remuscularization-revascularization therapy that is evaluated in a rat model of ischemia-reperfusion MI. This study details the differentiation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for engineering cardiac tissue containing patterned engineered vessels 400 µm in diameter. Vascularized engineered human myocardial tissues (vEHMs) are cultured in static conditions or perfused in vitro prior to implantation and evaluated after two weeks. Immunohistochemical staining indicates improved engraftment of hiPSC-CMs in in vitro-perfused vEHMs with greater expression of SMA+ vessels and evidence of inosculation. Three-dimensional vascular reconstructions reveal less tortuous and larger intra-implant vessels, as well as an improved branching hierarchy in in vitro-perfused vEHMs relative to non-perfused controls. Exploratory RNA sequencing of explanted vEHMs supports the hypothesis that co-revascularization impacts hiPSC-CM development in vivo. Our approach provides a strong foundation to enhance vEHM integration, develop hierarchical vascular perfusion, and maximize hiPSC-CM engraftment for future regenerative therapy.

One Billion hiPSC-Cardiomyocytes: Upscaling Engineered Cardiac Tissues to Create High Cell Density Therapies for Clinical Translation in Heart Regeneration.

Despite the overwhelming use of cellularized therapeutics in cardiac regenerative engineering, approaches to biomanufacture engineered cardiac tissues (ECTs) at clinical scale remain limited. This study aims to evaluate the impact of critical biomanufacturing decisions-namely cell dose, hydrogel composition, and size-on ECT formation and function-through the lens of clinical translation. ECTs were fabricated by mixing human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) and human cardiac fibroblasts into a collagen hydrogel to engineer meso-(3 × 9 mm), macro- (8 × 12 mm), and mega-ECTs (65 × 75 mm). Meso-ECTs exhibited a hiPSC-CM dose-dependent response in structure and mechanics, with high-density ECTs displaying reduced elastic modulus, collagen organization, prestrain development, and active stress generation. Scaling up, cell-dense macro-ECTs were able to follow point stimulation pacing without arrhythmogenesis. Finally, we successfully fabricated a mega-ECT at clinical scale containing 1 billion hiPSC-CMs for implantation in a swine model of chronic myocardial ischemia to demonstrate the technical feasibility of biomanufacturing, surgical implantation, and engraftment. Through this iterative process, we define the impact of manufacturing variables on ECT formation and function as well as identify challenges that must still be overcome to successfully accelerate ECT clinical translation.

Activation of the Hedgehog signaling pathway leads to fibrosis in aortic valves.

BACKGROUND: Fibrosis is a pathological wound healing process characterized by excessive extracellular matrix deposition, which interferes with normal organ function and contributes to ~ 45% of human mortality. Fibrosis develops in response to chronic injury in nearly all organs, but the a cascade of events leading to fibrosis remains unclear. While hedgehog (Hh) signaling activation has been associated with fibrosis in the lung, kidney, and skin, it is unknown whether hedgehog signaling activation is the cause or the consequence of fibrosis. We hypothesize that activation of hedgehog signaling is sufficient to drive fibrosis in mouse models. RESULTS: In this study, we provide direct evidence to show that activation of Hh signaling via expression of activated smoothened, SmoM2, is sufficient to induce fibrosis in the vasculature and aortic valves. We showed that activated SmoM2 -induced fibrosis is associated with abnormal function of aortic valves and heart. The relevance of this mouse model to human health is reflected in our findings that elevated GLI expression is detected in 6 out of 11 aortic valves from patients with fibrotic aortic valves. CONCLUSIONS: Our data show that activating hedgehog signaling is sufficient to drive fibrosis in mice, and this mouse model is relevant to human aortic valve stenosis.

Action potential metrics and automated data analysis pipeline for cardiotoxicity testing using optically mapped hiPSC-derived 3D cardiac microtissues.

Recent advances in human induced pluripotent stem cell (hiPSC)-derived cardiac microtissues provide a unique opportunity for cardiotoxic assessment of pharmaceutical and environmental compounds. Here, we developed a series of automated data processing algorithms to assess changes in action potential (AP) properties for cardiotoxicity testing in 3D engineered cardiac microtissues generated from hiPSC-derived cardiomyocytes (hiPSC-CMs). Purified hiPSC-CMs were mixed with 5-25% human cardiac fibroblasts (hCFs) under scaffold-free conditions and allowed to self-assemble into 3D spherical microtissues in 35-microwell agarose gels. Optical mapping was performed to quantify electrophysiological changes. To increase throughput, AP traces from 4x4 cardiac microtissues were simultaneously acquired with a voltage sensitive dye and a CMOS camera. Individual microtissues showing APs were identified using automated thresholding after Fourier transforming traces. An asymmetric least squares method was used to correct non-uniform background and baseline drift, and the fluorescence was normalized (ΔF/F0). Bilateral filtering was applied to preserve the sharpness of the AP upstroke. AP shape changes under selective ion channel block were characterized using AP metrics including stimulation delay, rise time of AP upstroke, APD30, APD50, APD80, APDmxr (maximum rate change of repolarization), and AP triangulation (APDtri = APDmxr-APD50). We also characterized changes in AP metrics under various ion channel block conditions with multi-class logistic regression and feature extraction using principal component analysis of human AP computer simulations. Simulation results were validated experimentally with selective pharmacological ion channel blockers. In conclusion, this simple and robust automated data analysis pipeline for evaluating key AP metrics provides an excellent in vitro cardiotoxicity testing platform for a wide range of environmental and pharmaceutical compounds.

Human Atrial Cardiac Microtissues for Chamber-Specific Arrhythmic Risk Assessment.

INTRODUCTION: Although atrial fibrillation is the most prevalent disorder of electrical conduction, the mechanisms behind atrial arrhythmias remain elusive. To address this challenge, we developed a robust in vitro model of 3D atrial microtissue from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and evaluated chamber-specific chemical responses experimentally and computationally. METHODS: We differentiated atrial and ventricular cardiomyocytes (aCMs/vCMs) from GCaMP6f-expressing hiPSCs and assessed spontaneous AP activity using fluorescence imaging. Self-assembling 3D microtissues were formed with lactate purified CMs and 5% human cardiac fibroblasts and electrically stimulated for one week before high resolution action potential (AP) optical mapping. AP responses to the atrial-specific potassium repolarizing current I Kur-blocker 4-Aminopyridine (4-AP) and funny current I f-blocker Ivabradine were characterized within their therapeutic window. Finally, we expanded upon a published hiPSC-CM computational model by incorporating the atrial-specific I Kur current, modifying ion channel conductances to match the AP waveforms of our microtissues, and employing the updated model to reinforce our experimental findings. RESULTS: High purity CMs (> 75% cTnT+) demonstrated subtype specification by MLC2v expression. Spontaneous beating rates significantly decreased following 3D microtissue formation, with atrial microtissues characterized by their faster spontaneous beating rate, slower AP rise time, and shorter AP duration (APD) compared to ventricular microtissues. We measured atrial-specific responses, including dose-dependent APD prolongation with 4-AP treatment and dose-dependent reduction in spontaneous activity post-Ivabradine treatment. CONCLUSION: The presented in vitro platform for screening atrial-specific responses is both robust and sensitive, with high throughput, enabling studies focused at elucidating the mechanisms underlying atrial arrhythmias. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-021-00703-x.

Architected fibrous scaffolds for engineering anisotropic tissues.

Mimicking the native three-dimensional microenvironment is of crucial importance when biofabricating a new healthcare material. One aspect of the native tissue that is often omitted when designing a suitable scaffold is its anisotropy. Not only is matching native mechanical properties important when designing implantable scaffolds or healthcare materials, but matching physiological structure is also important as many cell populations respond differently to fiber orientation. Therefore, novel aligned electrospun scaffolds with varying fiber angles and spacing of bundles were created and mechanically characterized. Through controlling the angle between the fibers in each layer of the scaffold, a range of different physiological anisotropic mechanical properties were achieved that encompasses values found in native tissues. Extrapolation of this mechanical data allowed for any native tissue's anisotropic Young's modulus to be mimicked by electrospinning fibers at a particular angle. These electrospun scaffolds were then incorporated with cell-laden hydrogels to create hybrid structures that contain the benefits of both scaffolding techniques with the ability to encapsulate cells in the hydrogel. To conclude, this study develops a novel bundled fiber scaffold that was architected to yield anisotropic properties matching native tissues.

Rapid custom prototyping of soft poroelastic biosensor for simultaneous epicardial recording and imaging.

The growing need for the implementation of stretchable biosensors in the body has driven rapid prototyping schemes through the direct ink writing of multidimensional functional architectures. Recent approaches employ biocompatible inks that are dispensable through an automated nozzle injection system. However, their application in medical practices remains challenged in reliable recording due to their viscoelastic nature that yields mechanical and electrical hysteresis under periodic large strains. Herein, we report sponge-like poroelastic silicone composites adaptable for high-precision direct writing of custom-designed stretchable biosensors, which are soft and insensitive to strains. Their unique structural properties yield a robust coupling to living tissues, enabling high-fidelity recording of spatiotemporal electrophysiological activity and real-time ultrasound imaging for visual feedback. In vivo evaluations of custom-fit biosensors in a murine acute myocardial infarction model demonstrate a potential clinical utility in the simultaneous intraoperative recording and imaging on the epicardium, which may guide definitive surgical treatments.

A predictive in vitro risk assessment platform for pro-arrhythmic toxicity using human 3D cardiac microtissues.

Cardiotoxicity of pharmaceutical drugs, industrial chemicals, and environmental toxicants can be severe, even life threatening, which necessitates a thorough evaluation of the human response to chemical compounds. Predicting risks for arrhythmia and sudden cardiac death accurately is critical for defining safety profiles. Currently available approaches have limitations including a focus on single select ion channels, the use of non-human species in vitro and in vivo, and limited direct physiological translation. We have advanced the robustness and reproducibility of in vitro platforms for assessing pro-arrhythmic cardiotoxicity using human induced pluripotent stem cell-derived cardiomyocytes and human cardiac fibroblasts in 3-dimensional microtissues. Using automated algorithms and statistical analyses of eight comprehensive evaluation metrics of cardiac action potentials, we demonstrate that tissue-engineered human cardiac microtissues respond appropriately to physiological stimuli and effectively differentiate between high-risk and low-risk compounds exhibiting blockade of the hERG channel (E4031 and ranolazine, respectively). Further, we show that the environmental endocrine disrupting chemical bisphenol-A (BPA) causes acute and sensitive disruption of human action potentials in the nanomolar range. Thus, this novel human 3D in vitro pro-arrhythmic risk assessment platform addresses critical needs in cardiotoxicity testing for both environmental and pharmaceutical compounds and can be leveraged to establish safe human exposure levels.

Octanoate is differentially metabolized in liver and muscle and fails to rescue cardiomyopathy in CPT2 deficiency.

Long-chain fatty acid oxidation is frequently impaired in primary and systemic metabolic diseases affecting the heart; thus, therapeutically increasing reliance on normally minor energetic substrates, such as ketones and medium-chain fatty acids, could benefit cardiac health. However, the molecular fundamentals of this therapy are not fully known. Here, we explored the ability of octanoate, an eight-carbon medium-chain fatty acid known as an unregulated mitochondrial energetic substrate, to ameliorate cardiac hypertrophy in long-chain fatty acid oxidation-deficient hearts because of carnitine palmitoyltransferase 2 deletion (Cpt2M-/-). CPT2 converts acylcarnitines to acyl-CoAs in the mitochondrial matrix for oxidative bioenergetic metabolism. In Cpt2M-/- mice, high octanoate-ketogenic diet failed to alleviate myocardial hypertrophy, dysfunction, and acylcarnitine accumulation suggesting that this alternative substrate is not sufficiently compensatory for energy provision. Aligning this outcome, we identified a major metabolic distinction between muscles and liver, wherein heart and skeletal muscle mitochondria were unable to oxidize free octanoate, but liver was able to oxidize free octanoate. Liver mitochondria, but not heart or muscle, highly expressed medium-chain acyl-CoA synthetases, potentially enabling octanoate activation for oxidation and circumventing acylcarnitine shuttling. Conversely, octanoylcarnitine was oxidized by liver, skeletal muscle, and heart, with rates in heart 4-fold greater than liver and, in muscles, was not dependent upon CPT2. Together, these data suggest that dietary octanoate cannot rescue CPT2-deficient cardiac disease. These data also suggest the existence of tissue-specific mechanisms for octanoate oxidative metabolism, with liver being independent of free carnitine availability, whereas cardiac and skeletal muscles depend on carnitine but not on CPT2.

Use of an Integrated Low-Flow Anesthetic Vaporizer, Ventilator, and Physiological Monitoring System for Rodents.

Low-flow digital vaporizers commonly utilize a syringe pump to directly administer volatile anesthetics into a stream of carrier gas. Per animal welfare recommendations, animals are warmed and monitored during procedures requiring anesthesia. Common anesthesia and physiological monitoring equipment include gas tanks, anesthetic vaporizers and stands, warming controllers and pads, mechanical ventilators, and pulse oximeters. A computer is also necessary for data collection and to run equipment software. In smaller spaces or when performing field work, it can be challenging to configure all this equipment in limited space. The goal of this protocol is to demonstrate best practices for use of a low-flow digital vaporizer using both compressed oxygen and room air, along with an integrated mechanical ventilator, pulse oximeter, and far infrared warming as an all-inclusive anesthesia and physiological monitoring suite ideal for rodents.

Dynamic transition of the blood-brain barrier in the development of non-small cell lung cancer brain metastases.

Invasion of the brain by non-small cell lung cancer (NSCLC) results in a shift of the blood-brain barrier (BBB) to the insufficiently characterized blood-tumor barrier (BTB). Effective drug delivery through the BTB is one of the greatest therapeutic obstacles in treating brain metastases. Using an experimental model, we defined key changes within the BTB and the BBB in the brain around the tumor (BAT) region over time. Brain-seeking NSCLC cells were delivered into the circulation of athymic-nude mice via intracardiac injection and developing brain metastases were evaluated over six-weeks. Components of the BBB and BTB were analyzed using immunofluorescence microscopy and compared using a mixed model of regression. Our results demonstrate a dynamic time-dependent BTB phenotype. Capillaries of the BAT and BTB were dilated with increased CD31 expression compared to controls. Expression of collagen IV, a pan-basement membrane component, was significantly decreased in the BTB compared to the BBB. There was also a significant increase in the desmin-positive pericyte subpopulation in the BTB compared to the BBB. The most striking changes were identified in astrocyte water channels with a 12.18-fold (p < 0.001) decrease in aquaporin-4 in the BTB; the BAT was unchanged. Analysis of NSCLC brain metastases from patient samples similarly demonstrated dilated capillaries and loss of both collagen IV and aquaporin-4. These data provide a comprehensive analysis of the BTB in NSCLC brain metastasis. Astrocytic endfeet, pericytes, and the basement membrane are potential therapeutic targets to improve efficacy of chemotherapeutic delivery into NSCLC brain metastases.

Three-dimensional myocardial strain correlates with murine left ventricular remodelling severity post-infarction.

Heart failure continues to be a common and deadly sequela of myocardial infarction (MI). Despite strong evidence suggesting the importance of myocardial mechanics in cardiac remodelling, many MI studies still rely on two-dimensional analyses to estimate global left ventricular (LV) function. Here, we integrated four-dimensional ultrasound with three-dimensional strain mapping to longitudinally characterize LV mechanics within and around infarcts in order to study the post-MI remodelling process. To induce infarcts with varying severities, we separated 15 mice into three equal-sized groups: (i) sham, (ii) 30 min ischaemia-reperfusion, and (iii) permanent ligation of the left coronary artery. Four-dimensional ultrasound from a high-frequency small animal system was used to monitor changes in LV geometry, function and strain over 28 days. We reconstructed three-dimensional myocardial strain maps and showed that strain profiles at the infarct border followed a sigmoidal behaviour. We also identified that mice with mild remodelling had significantly higher strains in the infarcted myocardium than those with severe injury. Finally, we developed a new approach to non-invasively estimate infarct size from strain maps, which correlated well with histological results. Taken together, the presented work provides a thorough approach to quantify regional strain, an important component when assessing post-MI remodelling.

Epithelial-mesenchymal Transition Phenotypes in Vertebral Metastases of Lung Cancer.

Vertebral metastases of non-small cell lung cancer (NSCLC) are frequently diagnosed in the metastatic setting and are commonly identified in the thoracic vertebrae in patients. Treatment of NSCLC bone metastases, which are often multiple, is palliative, and the median survival times are 3 to 6 months. We have characterized spontaneous vertebral metastases in a brain metastases model of NSCLC and correlated these findings with epithelial-mesenchymal transition (EMT). Brain metastases were established in athymic nude mice following intracardiac injection of brain-seeking adenocarcinoma NSCLC cells. Thirty-nine percent of mice (14/36) developed spontaneous vertebral metastases, spinal cord compression, and hind-limb paralysis. Vertebral metastases consisted of an adenocarcinoma phenotype with neoplastic epithelial cells arranged in cords or acini and a mesenchymal phenotype with spindloid neoplastic cells arranged in bundles and streams. Quantitative and qualitative immunohistochemical and immunofluorescence assays demonstrated an increase in vimentin expression compared to cytokeratin expression in vertebral metastases. A correlation with EMT was supported by an increase in CD44 in vertebral metastases and parenchymal metastases. These data demonstrate a translational lung cancer metastasis model with spontaneous vertebral metastasis. The mesenchymal and epithelial phenotype of these spontaneous metastases coupled with EMT provide a conduit to improve drug delivery and overall patient survival.

Strain Mapping From Four-Dimensional Ultrasound Reveals Complex Remodeling in Dissecting Murine Abdominal Aortic Aneurysms.

Current in vivo abdominal aortic aneurysm (AAA) imaging approaches tend to focus on maximum diameter but do not measure three-dimensional (3D) vascular deformation or strain. Complex vessel geometries, heterogeneous wall compositions, and surrounding structures can all influence aortic strain. Improved understanding of complex aortic kinematics has the potential to increase our ability to predict aneurysm expansion and eventual rupture. Here, we describe a method that combines four-dimensional (4D) ultrasound and direct deformation estimation to compute in vivo 3D Green-Lagrange strain in murine angiotensin II-induced suprarenal dissecting aortic aneurysms, a commonly used small animal model. We compared heterogeneous patterns of the maximum, first-component 3D Green-Lagrange strain with vessel composition from mice with varying AAA morphologies. Intramural thrombus and focal breakage in the medial elastin significantly reduced aortic strain. Interestingly, a dissection that was not detected with high-frequency ultrasound also experienced reduced strain, suggesting medial elastin breakage that was later confirmed via histology. These results suggest that in vivo measurements of 3D strain can provide improved insight into aneurysm disease progression. While further work is needed with both preclinical animal models and human imaging studies, this initial murine study indicates that vessel strain should be considered when developing an improved metric for predicting aneurysm growth and rupture.

Cardiac and respiratory-gated volumetric murine ultrasound.

Current cardiovascular ultrasound mainly employs planar imaging techniques to assess function and physiology. These techniques rely on geometric assumptions, which are dependent on the imaging plane, susceptible to inter-observer variability, and may be inaccurate when studying complex diseases. Here, we developed a gated volumetric murine ultrasound technique to visualize cardiovascular motion with high spatiotemporal resolution and directly evaluate cardiovascular health. Cardiac and respiratory-gated cine loops, acquired at 1000 frames-per-second from sequential positions, were temporally registered to generate a four-dimensional (4D) dataset. We applied this technique to (1) evaluate left ventricular (LV) function from both healthy mice and mice with myocardial infarction and (2) characterize aortic wall strain of angiotensin II-induced dissecting abdominal aortic aneurysms in apolipoprotein E-deficient mice. Combined imaging and processing times for the 4D technique was approximately 2-4 times longer than conventional 2D approaches, but substantially more data is collected with 4D ultrasound and further optimization can be implemented to reduce imaging times. Direct volumetric measurements of 4D cardiac data aligned closely with those obtained from MRI, contrary to conventional methods, which were sensitive to transducer alignment, leading to overestimation or underestimation of estimated LV parameters in infarcted hearts. Green-Lagrange circumferential strain analysis revealed higher strain values proximal and distal to the aneurysm than within the aneurysmal region, consistent with published reports. By eliminating the need for geometrical assumptions, the presented 4D technique can be used to more accurately evaluate cardiac function and aortic pulsatility. Furthermore, this technique allows for the visualization of regional differences that may be overlooked with conventional 2D approaches.

High-Frequency 4-Dimensional Ultrasound (4DUS): A Reliable Method for Assessing Murine Cardiac Function.

In vivo imaging has provided a unique framework for studying pathological progression in various mouse models of cardiac disease. Although conventional short-axis motion-mode (SAX MM) ultrasound and cine magnetic resonance imaging (MRI) are two of the most prevalent strategies used for quantifying cardiac function, there are few notable limitations including imprecision, inaccuracy, and geometric assumptions with ultrasound, or large and costly systems with substantial infrastructure requirements with MRI. Here we present an automated 4-dimensional ultrasound (4DUS) technique that provides comparable information to cine MRI through spatiotemporally synced imaging of cardiac motion. Cardiac function metrics derived from SAX MM, cine MRI, and 4DUS data show close agreement between cine MRI and 4DUS but overestimations by SAX MM. The inclusion of a mouse model of cardiac hypertrophy further highlights the precision of 4DUS compared with that of SAX MM, with narrower groupings of cardiac metrics based on health status. Our findings suggest that murine 4DUS can be used as a reliable, accurate, and cost-effective technique for longitudinal studies of cardiac function and disease progression.

Imaging of small animal peripheral artery disease models: recent advancements and translational potential.

Peripheral artery disease (PAD) is a broad disorder encompassing multiple forms of arterial disease outside of the heart. As such, PAD development is a multifactorial process with a variety of manifestations. For example, aneurysms are pathological expansions of an artery that can lead to rupture, while ischemic atherosclerosis reduces blood flow, increasing the risk of claudication, poor wound healing, limb amputation, and stroke. Current PAD treatment is often ineffective or associated with serious risks, largely because these disorders are commonly undiagnosed or misdiagnosed. Active areas of research are focused on detecting and characterizing deleterious arterial changes at early stages using non-invasive imaging strategies, such as ultrasound, as well as emerging technologies like photoacoustic imaging. Earlier disease detection and characterization could improve interventional strategies, leading to better prognosis in PAD patients. While rodents are being used to investigate PAD pathophysiology, imaging of these animal models has been underutilized. This review focuses on structural and molecular information and disease progression revealed by recent imaging efforts of aortic, cerebral, and peripheral vascular disease models in mice, rats, and rabbits. Effective translation to humans involves better understanding of underlying PAD pathophysiology to develop novel therapeutics and apply non-invasive imaging techniques in the clinic.