Tissue engineers and stem cell biologists have made exciting progress toward creating simplified models of human heart muscles or aligned monolayers to help bridge a longstanding gap between ...experimental animals and clinical trials. However, no existing human in vitro systems provide the direct measures of cardiac performance as a pump. Here, we developed a next-generation in vitro biomimetic model of pumping human heart chamber, and demonstrated its capability for pharmaceutical testing. From human pluripotent stem cell (hPSC)-derived ventricular cardiomyocytes (hvCM) embedded in collagen-based extracellular matrix hydrogel, we engineered a three-dimensional (3D) electro-mechanically coupled, fluid-ejecting miniature human ventricle-like cardiac organoid chamber (hvCOC). Structural characterization showed organized sarcomeres with myofibrillar microstructures. Transcript and RNA-seq analyses revealed upregulation of key Ca2+-handling, ion channel, and cardiac-specific proteins in hvCOC compared to lower-order 2D and 3D cultures of the same constituent cells. Clinically-important, physiologically complex contractile parameters such as ejection fraction, developed pressure, and stroke work, as well as electrophysiological properties including action potential and conduction velocity were measured: hvCOC displayed key molecular and physiological characteristics of the native ventricle, and showed expected mechanical and electrophysiological responses to a range of pharmacological interventions (including positive and negative inotropes). We conclude that such “human-heart-in-a-jar” technology could facilitate the drug discovery process by providing human-specific preclinical data during early stage drug development.
Traditional drug discovery is an inefficient process. Human pluripotent stem cell‐derived cardiomyocytes can potentially fill the gap between animal and clinical studies, but conventional ...two‐dimensional cultures inadequately recapitulate the human cardiac phenotype. Here, we systematically examined the pharmacological responses of engineered human ventricular‐like cardiac tissue strips (hvCTS) and organoid chambers (hvCOC) to 25 cardioactive compounds covering various drug classes. While hvCTS effectively detected negative and null inotropic effects, the sensitivity to positive inotropes was modest. We further quantified the predictive capacity of hvCTS in a blinded screening, with accuracies for negative, positive, and null inotropic effects at 100%, 86%, and 80%, respectively. Interestingly, hvCOC, with a pro‐maturation milieu that yields physiologically complex parameters, displayed enhanced positive inotropy. Based on these results, we propose a two‐tiered screening system for avoiding false positives and negatives. Such an approach would facilitate drug discovery by leading to better overall success.
Cardiac experimental biology and translational research would benefit from an in vitro surrogate for human heart muscle. This study investigated structural and functional properties and ...interventional responses of human engineered cardiac tissues (hECTs) compared to human myocardium. Human embryonic stem cell‐derived cardiomyocytes (hESC‐CMs, >90% troponin‐positive) were mixed with collagen and cultured on force‐sensing elastomer devices. hECTs resembled trabecular muscle and beat spontaneously (1.18±0.48 Hz). Microstructural features and mRNA expression of cardiac‐specific genes (α‐MHC, SERCA2a, and ACTC1) were comparable to human myocardium. Optical mapping revealed cardiac refractoriness with loss of 1:1 capture above 3 Hz, and cycle length dependence of the action potential duration, recapitulating key features of cardiac electrophysiology. hECTs reconstituted the Frank‐Starling mechanism, generating an average maximum twitch stress of 660 μN/mm2 at Lmax, approaching values in newborn human myocardium. Dose‐response curves followed exponential pharmacodynamics models for calcium chloride (EC50 1.8 mM) and verapamil (IC50 0.61 μM); isoproterenol elicited a positive chronotropic but negligible inotropic response, suggesting sarcoplasmic reticulum immaturity. hECTs were amenable to gene transfer, demonstrated by successful transduction with Ad.GFP. Such 3‐D hECTs recapitulate an early developmental stage of human myocardium and promise to offer an alternative preclinical model for cardiology research.—Turnbull, I. C., Karakikes, I., Serrao, G. W., Backeris, P., Lee, J.‐J., Xie, C., Senyei, G., Gordon, R. E., Li, R. A., Akar, F. G., Hajjar, R. J., Hulot, J.‐S., Costa, K. D. Advancing functional engineered cardiac tissues toward a preclinical model of human myocardium. FASEB J. 28, 644–654 (2014). www.fasebj.org
Research in photobiology is currently limited by a lack of devices capable of delivering precise and tunable irradiation to cells in a high-throughput format. This limits researchers to using ...expensive commercially available or custom-built light sources which make it difficult to replicate, standardize, optimize, and scale experiments. Here we present an open-source Microplate Photoirradiation System (MPS) developed to enable high-throughput light experiments in standard 96 and 24-well microplates for a variety of applications in photobiology research. This open-source system features 96 independently controlled LEDs (4 LEDs per well in 24-well), Wi-Fi connected control and programmable graphical user interface (GUI) for control and programming, automated calibration GUI, and modular control and LED boards for maximum flexibility. A web-based GUI generates light program files containing irradiation parameters for groups of LEDs. These parameters are then uploaded wirelessly, stored and used on the MPS to run photoirradiation experiments inside any incubator. A rapid and semi-quantitative porphyrin metabolism assay was also developed to validate the system in wild-type fibroblasts. Protoporphyrin IX (PpIX) fluorescence accumulation was induced by incubation with 5-aminolevulinic acid (ALA), a photosensitization method leveraged clinically to destroy malignant cell types in a process termed photodynamic therapy (PDT), and cells were irradiated with 405nm light with varying irradiance, duration and pulsation parameters. Immediately after light treatment with the MPS, subsequent photobleaching was measured in live, adherent cells in both 96-well and a 24-well microplates using a microplate reader. Results demonstrate the utility and reliability of the Microplate Photoirradiation System to irradiate cells with precise irradiance and timing parameters in order to measure PpIx photobleaching kinetics in live adherent cells and perform comparable experiments with both 24 and 96 well microplate formats. The high-throughput capability of the MPS enabled measurement of enough irradiance conditions in a single microplate to fit PpIX fluorescence to a bioexponential decay model of photobleaching, as well as reveal a dependency of photobleaching on duty-cycle-but not frequency-in a pulsed irradiance regimen.
OBJECTIVES/SPECIFIC AIMS: Innovation in healthcare is increasingly dependent on technology and teamwork, requiring effective collaboration between disciplines. Through an intensive team-based ...competition event, Mount Sinai Health Hackathon 2017, aimed to harness the power of multidisciplinary and transdisciplinary collaboration to foster innovation in the field of cancer. Participants were immersed in an intensive weekend working in teams to develop technology solutions to important problems affecting patients and care providers in the field of cancer. The learning objectives were to enable participants to: Identify cancer-related healthcare problems which lend themselves to technology-based solutions. Delineate key behaviors critical to multidisciplinary team success Identify optimal strategies for communicating in multidisciplinary teams. Engage and inspire participants to apply knowledge of technology to meaningfully impact clinical care and well-being. METHODS/STUDY POPULATION: The Mount Sinai Health Hackathon is an annual 48-hour team-based competition, using a format adapted from guidelines provided by MIT Hacking Medicine. The 2017 event gathered a total of 87 participants (120 registered), representing 17 organizations from as far away as California, with a diverse range of backgrounds in bioinformatics, software and hardware, product design, business, digital health and clinical practice. The overall participation model included: Phase 0: Health Hackathon 101 summer workshops; Phase 1: pre-Hackathon priming activities using online forums Trello and Slack; Phase 2: a 48-hour onsite hackathon to catalyze innovation through problem sharing, solution pitches, team formation and development of prototype solutions; Phase 3: competitive presentations to judges and prize awards; Phase 4: a suite of post-hackathon support to stimulate continued development of innovations. The event sponsored by ConduITS, was also co-sponsored by Persistent Systems, IBM Watson, Tisch Cancer Institute, Sinai AppLab, Sinai Biodesign and other ISMMS Institutes. Mentors circulated throughout the event to support the teams in the technical, clinical, and business development aspects of their solutions. In total, the 14 teams formed during the Hackathon, created innovations ranging from diagnostic devices, networking apps, artificial intelligence tools, and others. The top 3 teams were each awarded $2500 to support their projects’ future development. RESULTS/ANTICIPATED RESULTS: Qualitative and quantitative post-event survey data revealed the Hackathon experience fostered collaborative attitudes and a positive experience for participants, providing insight into the potential benefits of team science. In the post-event survey (n=24) 92% of participants reported that the experience increased their ability to solve problems and 96% made new professional or personal connections. In addition, 96% of respondents would attend future Hackathon events and 75% reported they were likely to continue working on their project after the Hackathon. Qualitative feedback from 1 participant reported it was: “a wonderful event that really highlighted how much interdisciplinary team science can achieve.” Along with intermediate support interactions, including the winning teams participating in a Shark Tank style event with pitches to external entrepreneurs and investors, all teams will be followed up in 6 months time to determine if participants continue to work on projects, file new patents, create new companies, or leverage the new connections made through the Health Hackathon experience. DISCUSSION/SIGNIFICANCE OF IMPACT: Our experience indicates that a Health Hackathon is a compelling and productive forum to bring together students, trainees, faculty, and other stakeholders to explore tech-based solutions to problems in cancer and other areas of biomedicine. It is a valuable tool to foster collaboration and transdisciplinary team science and education. Follow-up analysis will determine to what extent the Mount Sinai Health Hackathon is contributing to an ecosystem that encourages professionals and trainees in healthcare and in technology development to work together to address unmet needs in healthcare with innovative technology solutions.
In this study, we used three-dimensional human engineered cardiac tissue technology to directly show that phospholamban (PLN) R14del mutation impairs cardiac contractility and to demonstrate ...restoration of contractile properties with targeted genetic correction of this inheritable form of dilated cardiomyopathy.
The aim of this study was to explore the role of abnormal coronary microvasculature morphology and hemodynamics in the development of congestive heart failure (CHF). CHF was induced in rats by aortic ...banding, followed by ischemia-reperfusion and later aortic debanding. Polymerized casts of coronary vasculature were imaged under a scanning electron microscope (SEM). Matrix Laboratory (MATLAB) software was used to calculate capillary structure index (CSI), a measure of structural alignment also called mean vector length (MVL), for 93 SEM images of coronary capillaries (CSI→1 perfect linearity; CSI→0 circular disarray). CSI was incorporated as a constant to represent tortuosity and nonlaminar flow in Poiseuille's equation to estimate the differences in capillary blood flow rate, velocity, and resistance for CHF vs.
The morphology of CHF capillaries is significantly disordered and tortuous compared with control (CSI: 0.35 ± 0.02 for 61 images from 7 CHF rats; 0.58 ± 0.02 for 32 images from 7 control rats; P < 0.01). Estimated capillary resistance in CHF is elevated by 173% relative to control, while blood flow rate and blood velocity are 56 and 43% slower than control. Capillary resistance increased 67% due to the significantly narrower capillary diameter in CHF, while it increased an additional 105% due to tortuosity. The significant structural abnormalities of CHF coronary capillaries may drastically stagnate hemodynamics in myocardium and increase resistance to blood flow. This could play a role in the development of CHF. NEW & NOTEWORTHY In the present study, coronary capillary tortuosity was measured by applying Matrix Laboratory software to scanning electron microscope images of capillaries in a rat model of congestive heart failure. Stagnant blood flow in coronary capillaries may play a role in the development of congestive heart failure. The application of computer modeling to histological and physiological data to characterize the hemodynamics of coronary microcirculation is a new area of study.
Abstract only In ischemic congestive heart failure (CHF), the heart is damaged and undergoes compensatory remodeling, a pathological process associated with harmful effects. The goal of this study is ...to explore the manifestation of CHF by examining the morphological changes occurring in the coronary microvasculature in CHF versus normal rat hearts. We tested the hypothesis that coronary capillaries in rats with CHF exhibit significantly more morphological disorder than those in control rats. Methods: CHF was induced by aortic banding, ischemia/reperfusion injury two months post-banding (left coronary artery ligation for 30 minutes) and aortic debanding one month post-injury. Resin polymer containing fluorescent dye was injected into coronary vasculature of excised hearts. Muscle tissue was digested using NaOH to reveal vascular casts that were sputter coated with gold for imaging under a Scanning Electron Microscope (SEM). A total of 93 SEM images from 14 rats (7 control, 7 CHF) were analyzed for structural alignment using an automated gradient detection algorithm and circular statistics implemented using MATLAB software; Mean Vector Length (MVL) was calculated for each image as a measure of capillary organization (0<MVL<0. MVL->1 perfect alignment, MVL->0 random disarray). Results: CHF capillaries exhibit significantly more structural disorder than control (MVL 0.35±0.02 for 61 CHF, 0.58±0.02 for 32 control. p<0.01). Conclusions: Coronary capillaries in CHF rats exhibit significant abnormal morphological disarray that may impair blood flow hemodynamics and material and oxygen exchange in myocytes. Such disordered capillary remodeling could have detrimental consequences for the progression and prognosis of heart failure.
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