The Experts below are selected from a list of 134544 Experts worldwide ranked by ideXlab platform
Thomas Eschenhagen - One of the best experts on this subject based on the ideXlab platform.
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Rat atrial engineered Heart Tissue: a new in vitro model to study atrial biology
Basic Research in Cardiology, 2018Co-Authors: Julia Krause, Thomas Eschenhagen, Marc D Lemoine, Alexandra Löser, Torsten Christ, Katharina Scherschel, Christian Meyer, Stefan Blankenberg, Tanja Zeller, Justus StenzigAbstract:Engineered Heart Tissue (EHT) from rat cells is a useful tool to study ventricular biology and cardiac drug safety. Since atrial and ventricular cells differ significantly, EHT and other 3D cell culture formats generated from ventricular cells have been of limited value to study atrial biology. To date, reliable in vitro models that reflect atrial physiology are lacking. Therefore, we established a novel EHT model using rat atrial cells (atrial EHT, aEHT) to assess atrial physiology, contractility and drug response. The Tissue constructs were characterized with regard to gene expression, histology, electrophysiology, and the response to atrial-specific drugs. We observed typical functional properties of atrial Tissue in our model such as more regular spontaneous beating with lower force, shorter action potential duration, and faster contraction and relaxation compared to ventricular EHT (vEHT). The expression of atrial-specific genes and proteins was high, whereas ventricle-specific transcripts were virtually absent. The atrial-selective drug carbachol had a strong negative inotropic and chronotropic effect on aEHT only. Taken together, the results demonstrate the feasibility of aEHT as a novel atrial 3D model and as a benchmark for Tissue engineering with human induced pluripotent stem cell-derived atrial-like cardiomyocytes. Atrial EHT faithfully recapitulates atrial physiology and shall be useful to study atrial molecular physiology in health and disease as well as drug response.
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human engineered Heart Tissue as a model system for drug testing
Advanced Drug Delivery Reviews, 2016Co-Authors: Alexandra Eder, Ingra Vollert, Arne Hansen, Thomas EschenhagenAbstract:Drug development is time- and cost-intensive and, despite extensive efforts, still hampered by the limited value of current preclinical test systems to predict side effects, including proarrhythmic and cardiotoxic effects in clinical practice. Part of the problem may be related to species-dependent differences in cardiomyocyte biology. Therefore, the event of readily available human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CM) has raised hopes that this human test bed could improve preclinical safety pharmacology as well as drug discovery approaches. However, hiPSC-CM are immature and exhibit peculiarities in terms of ion channel function, gene expression, structural organization and functional responses to drugs that limit their present usefulness. Current efforts are thus directed towards improving hiPSC-CM maturity and high-content readouts. Culturing hiPSC-CM as 3-dimensional engineered Heart Tissue (EHT) improves CM maturity and anisotropy and, in a 24-well format using silicone racks, enables automated, multiplexed high content readout of contractile function. This review summarizes the principal technology and focuses on advantages and disadvantages of this technology and its potential for preclinical drug screening.
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P778Physiological and pharmacological characterization of human engineered Heart Tissue
Cardiovascular Research, 2014Co-Authors: Ingra Vollert, Sebastian Schaaf, Christiane Neuber, David Letuffe-brenière, K. Breckwoldt, Aya Shibamiya, D Stimpel, Alexandra Eder, Thomas Eschenhagen, Arne HansenAbstract:Objective: Human induced pluripotent stem cell (iPS cell)-derived cardiomyocytes represent a valuable tool in cardiovascular research of tremendous potential. However, these cells are still immature and characterized by poor sarcomeric organization and cellular orientation in 2D cell culture. Therefore, the measurement of contractile force, the most important and best understood function of cardiomyocytes in vivo, is not established for these cells. This study describes the generation of three-dimensional, strip-format, force generating engineered Heart Tissues (EHTs) from human iPS cell-derived cardiomyocytes and presents a characterization of physiological and pharmacological parameters based on force development. Methods and results: Cardiomyocyte differentiation of human induced pluripotent cells was achieved by a growth factor-based three stage protocol. Strip-format EHTs were generated from dissociated cardiomyocytes in fibrin matrix between flexible silicone posts. Within two weeks after casting coherently beating human EHTs were formed and EHTs displayed a regular beating pattern for several weeks. Histological analysis revealed a high degree of sarcomeric organization and alignment of cardiomyocytes in EHTs. Functional analysis was performed by measuring force response to calcium concentration, pre-load, pacing frequency, beta-adrenergic and muscarinic agonists, modulators of sodium, calcium and potassium channels and revealed concentration-dependent effects. Comparison with native human Heart Tissue suggests an overall high level of similarity and minor differences. Conclusions: This study demonstrates feasibility to characterize human iPS cell-derived cardiomyocytes in EHTs by measuring contractile force. The analysis suggests high levels of similarity between EHTs and native human Heart Tissue. Human EHTs are a promising platform for automated toxicology screens in future drug development and for in vitro experiments on human cardiomyocytes in general.
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human engineered Heart Tissue as a versatile tool in basic research and preclinical toxicology
PLOS ONE, 2011Co-Authors: Sebastian Schaaf, Wolfram-hubertus Zimmermann, Aya Shibamiya, Alexandra Eder, Thomas Eschenhagen, Marc N Hirt, Marco Mewe, Andrea Stohr, L Conradi, Arne HansenAbstract:Human embryonic stem cell (hESC) progenies hold great promise as surrogates for human primary cells, particularly if the latter are not available as in the case of cardiomyocytes. However, high content experimental platforms are lacking that allow the function of hESC-derived cardiomyocytes to be studied under relatively physiological and standardized conditions. Here we describe a simple and robust protocol for the generation of fibrin-based human engineered Heart Tissue (hEHT) in a 24-well format using an unselected population of differentiated human embryonic stem cells containing 30–40% α-actinin-positive cardiac myocytes. Human EHTs started to show coherent contractions 5–10 days after casting, reached regular (mean 0.5 Hz) and strong (mean 100 µN) contractions for up to 8 weeks. They displayed a dense network of longitudinally oriented, interconnected and cross-striated cardiomyocytes. Spontaneous hEHT contractions were analyzed by automated video-optical recording and showed chronotropic responses to calcium and the β-adrenergic agonist isoprenaline. The proarrhythmic compounds E-4031, quinidine, procainamide, cisapride, and sertindole exerted robust, concentration-dependent and reversible decreases in relaxation velocity and irregular beating at concentrations that recapitulate findings in hERG channel assays. In conclusion this study establishes hEHT as a simple in vitro model for Heart research.
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development of a drug screening platform based on engineered Heart Tissue
Circulation Research, 2010Co-Authors: Arne Hansen, Sebastian Schaaf, Alexandra Eder, Marlene Bonstrup, Marianne Flato, Marco Mewe, Bulent Aksehirlioglu, Alexander Schworer, June Uebeler, Thomas EschenhagenAbstract:Rationale: Tissue engineering may provide advanced in vitro models for drug testing and, in combination with recent induced pluripotent stem cell technology, disease modeling, but available techniques are unsuitable for higher throughput. Objective: Here, we present a new miniaturized and automated method based on engineered Heart Tissue (EHT). Methods and Results: Neonatal rat Heart cells are mixed with fibrinogen/Matrigel plus thrombin and pipetted into rectangular casting molds in which two flexible silicone posts are positioned from above. Contractile activity is monitored video-optically by a camera and evaluated by a custom-made software program. Fibrin-based mini-EHTs (FBMEs) (150 μL, 600 000 cells) were transferred from molds to a standard 24-well plate two hours after casting. Over time FBMEs condensed from a 12×3×3 mm gel to a muscle strip of 8 mm length and, depending on conditions, 0.2 to 1.3 mm diameter. After 8 to 10 days, FBMEs started to rhythmically deflect the posts. Post properties and the extent of post deflection allowed calculation of rate, force (0.1 to 0.3 mN), and kinetics which was validated in organ baths experiments. FBMEs exhibited a well-developed, longitudinally aligned actinin-positive cardiac muscle network and lectin-positive vascular structures interspersed homogeneously throughout the construct. Analysis of a large series of FBME (n=192) revealed high yield and reproducibility and stability for weeks. Chromanol, quinidine, and erythromycin exerted concentration-dependent increases in relaxation time, doxorubicin decreases in contractile force. Conclusions: We developed a simple technique to construct large series of EHT and automatically evaluate contractile activity. The method shall be useful for drug screening and disease modeling.
Wolfram-hubertus Zimmermann - One of the best experts on this subject based on the ideXlab platform.
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human engineered Heart Tissue as a versatile tool in basic research and preclinical toxicology
PLOS ONE, 2011Co-Authors: Sebastian Schaaf, Wolfram-hubertus Zimmermann, Aya Shibamiya, Alexandra Eder, Thomas Eschenhagen, Marc N Hirt, Marco Mewe, Andrea Stohr, L Conradi, Arne HansenAbstract:Human embryonic stem cell (hESC) progenies hold great promise as surrogates for human primary cells, particularly if the latter are not available as in the case of cardiomyocytes. However, high content experimental platforms are lacking that allow the function of hESC-derived cardiomyocytes to be studied under relatively physiological and standardized conditions. Here we describe a simple and robust protocol for the generation of fibrin-based human engineered Heart Tissue (hEHT) in a 24-well format using an unselected population of differentiated human embryonic stem cells containing 30–40% α-actinin-positive cardiac myocytes. Human EHTs started to show coherent contractions 5–10 days after casting, reached regular (mean 0.5 Hz) and strong (mean 100 µN) contractions for up to 8 weeks. They displayed a dense network of longitudinally oriented, interconnected and cross-striated cardiomyocytes. Spontaneous hEHT contractions were analyzed by automated video-optical recording and showed chronotropic responses to calcium and the β-adrenergic agonist isoprenaline. The proarrhythmic compounds E-4031, quinidine, procainamide, cisapride, and sertindole exerted robust, concentration-dependent and reversible decreases in relaxation velocity and irregular beating at concentrations that recapitulate findings in hERG channel assays. In conclusion this study establishes hEHT as a simple in vitro model for Heart research.
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optimizing engineered Heart Tissue for therapeutic applications as surrogate Heart muscle
Circulation, 2006Co-Authors: Hiroshi Naito, Ivan Melnychenko, Michael Didié, Thomas Eschenhagen, Karin Schneiderbanger, Pia Schubert, Stephan Rosenkranz, Wolfram-hubertus ZimmermannAbstract:Background— Cardiac Tissue engineering aims at providing Heart muscle for cardiac regeneration. Here, we hypothesized that engineered Heart Tissue (EHT) can be improved by using mixed Heart cell populations, culture in defined serum-free and Matrigel-free conditions, and fusion of single-unit EHTs to multi-unit Heart muscle surrogates. Methods and Results— EHTs were constructed from native and cardiac myocyte enriched Heart cell populations. The former demonstrated a superior contractile performance and developed vascular structures. Peptide growth factor-supplemented culture medium was developed to maintain contractile EHTs in a serum-free environment. Addition of triiodothyronine and insulin facilitated withdrawal of Matrigel from the EHT reconstitution mixture. Single-unit EHTs could be fused to form large multi-unit EHTs with variable geometries. Conclusions— Simulating a native Heart cell environment in EHTs leads to improved function and formation of primitive capillaries. The latter may constitute a preformed vascular bed in vitro and facilitate engraftment in vivo. Serum- and Matrigel-free culture conditions are expected to reduce immunogenicity of EHT. Fusion of single-unit EHT allows production of large Heart muscle constructs that may eventually serve as optimized Tissue grafts in cardiac regeneration in vivo.
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Engineered Heart Tissue grafts improve systolic and diastolic function in infarcted rat Hearts
Nature Medicine, 2006Co-Authors: Wolfram-hubertus Zimmermann, Ivan Melnychenko, G Wasmeier, Michael Didié, Hiroshi Naito, Uwe Nixdorff, Andreas Hess, Lubos Budinsky, Kay Brune, Bjela MichaelisAbstract:The concept of regenerating diseased myocardium by implantation of Tissue-engineered Heart muscle is intriguing, but convincing evidence is lacking that Heart Tissues can be generated at a size and with contractile properties that would lend considerable support to failing Hearts. Here we created large (thickness/diameter, 1-4 mm/15 mm), force-generating engineered Heart Tissue from neonatal rat Heart cells. Engineered Heart Tissue formed thick cardiac muscle layers when implanted on myocardial infarcts in immune-suppressed rats. When evaluated 28 d later, engineered Heart Tissue showed undelayed electrical coupling to the native myocardium without evidence of arrhythmia induction. Moreover, engineered Heart Tissue prevented further dilation, induced systolic wall thickening of infarcted myocardial segments and improved fractional area shortening of infarcted Hearts compared to controls (sham operation and noncontractile constructs). Thus, our study provides evidence that large contractile cardiac Tissue grafts can be constructed in vitro, can survive after implantation and can support contractile function of infarcted Hearts.
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engineered Heart Tissue for regeneration of diseased Hearts
Biomaterials, 2004Co-Authors: Wolfram-hubertus Zimmermann, Ivan Melnychenko, Thomas EschenhagenAbstract:Abstract Cardiac Tissue engineering aims at providing contractile Heart muscle constructs for replacement therapy in vivo. At present, most cardiac Tissue engineering attempts utilize Heart cells from embryonic chicken and neonatal rats and scaffold materials. Over the past years our group has developed a novel technique to engineer collagen/matrigel-based cardiac muscle constructs, which we termed engineered Heart Tissue (EHT). EHT display functional and morphological properties of differentiated Heart muscle and can be constructed in different shape and size from collagen type I, extracellular matrix proteins (Matrigel®), and Heart cells from neonatal rats and embryonic chicken. First implantation studies in syngeneic Fischer 344 rats provided evidence of EHT survival and integration in vivo. This review will focus on our experience in Tissue engineering of cardiac muscle. Mainly, EHT construction, matrix requirements, potential applications of different cell types including stem cells, and our first implantation experiences will be discussed. Despite many critical and unresolved questions, we believe that cardiac Tissue engineering in general has an interesting perspective for the replacement of malfunctioning myocardium and reconstruction of congenital malformations.
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cardiac grafting of engineered Heart Tissue in syngenic rats
Circulation, 2002Co-Authors: Wolfram-hubertus Zimmermann, Ivan Melnychenko, G Wasmeier, Michael Didié, Uwe Nixdorff, Andreas Hess, Oliver Boy, Winfried Neuhuber, Michael Weyand, Thomas EschenhagenAbstract:Background Cell grafting has emerged as a novel approach to treat Heart diseases refractory to conventional therapy. We hypothesize that survival and functional and electrical integration of grafts may be improved by engineering cardiac Tissue constructs in vitro before grafting. Methods and Results Engineered Heart Tissue (EHT) was reconstituted by mixing cardiac myocytes from neonatal Fischer 344 rats with liquid collagen type I, matrigel, and serum-containing culture medium. EHTs were designed in circular shape (inner/outer diameter: 8/10 mm; thickness: 1 mm) to fit around the circumference of Hearts from syngenic rats. After 12 days in culture and before implantation on uninjured Hearts, contractile function of EHT was measured under isometric conditions. Baseline twitch tension amounted to 0.34±0.03 mN (n=33) and was stimulated by Ca 2+ and isoprenaline to 200±12 and 185±10% of baseline values, respectively. Despite utilization of a syngenic model immunosuppression (mg/kg BW: azathioprine 2, cyclosporine A 5, methylprednisolone 2) was necessary for EHT survival in vivo. Echocardiography conducted 7, 14, and 28 days after implantation demonstrated no change in left ventricular function compared with pre-OP values (n=9). Fourteen days after implantation, EHTs were heavily vascularized and retained a well organized Heart muscle structure as indicated by immunolabeling of actinin, connexin 43, and cadherins. Ultrastructural analysis demonstrated that implanted EHTs surpassed the degree of differentiation reached before implantation. Contractile function of EHT grafts was preserved in vivo. Conclusions EHTs can be employed for Tissue grafting approaches and might serve as graft material to repair diseased myocardium.
Arne Hansen - One of the best experts on this subject based on the ideXlab platform.
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human engineered Heart Tissue as a model system for drug testing
Advanced Drug Delivery Reviews, 2016Co-Authors: Alexandra Eder, Ingra Vollert, Arne Hansen, Thomas EschenhagenAbstract:Drug development is time- and cost-intensive and, despite extensive efforts, still hampered by the limited value of current preclinical test systems to predict side effects, including proarrhythmic and cardiotoxic effects in clinical practice. Part of the problem may be related to species-dependent differences in cardiomyocyte biology. Therefore, the event of readily available human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CM) has raised hopes that this human test bed could improve preclinical safety pharmacology as well as drug discovery approaches. However, hiPSC-CM are immature and exhibit peculiarities in terms of ion channel function, gene expression, structural organization and functional responses to drugs that limit their present usefulness. Current efforts are thus directed towards improving hiPSC-CM maturity and high-content readouts. Culturing hiPSC-CM as 3-dimensional engineered Heart Tissue (EHT) improves CM maturity and anisotropy and, in a 24-well format using silicone racks, enables automated, multiplexed high content readout of contractile function. This review summarizes the principal technology and focuses on advantages and disadvantages of this technology and its potential for preclinical drug screening.
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P778Physiological and pharmacological characterization of human engineered Heart Tissue
Cardiovascular Research, 2014Co-Authors: Ingra Vollert, Sebastian Schaaf, Christiane Neuber, David Letuffe-brenière, K. Breckwoldt, Aya Shibamiya, D Stimpel, Alexandra Eder, Thomas Eschenhagen, Arne HansenAbstract:Objective: Human induced pluripotent stem cell (iPS cell)-derived cardiomyocytes represent a valuable tool in cardiovascular research of tremendous potential. However, these cells are still immature and characterized by poor sarcomeric organization and cellular orientation in 2D cell culture. Therefore, the measurement of contractile force, the most important and best understood function of cardiomyocytes in vivo, is not established for these cells. This study describes the generation of three-dimensional, strip-format, force generating engineered Heart Tissues (EHTs) from human iPS cell-derived cardiomyocytes and presents a characterization of physiological and pharmacological parameters based on force development. Methods and results: Cardiomyocyte differentiation of human induced pluripotent cells was achieved by a growth factor-based three stage protocol. Strip-format EHTs were generated from dissociated cardiomyocytes in fibrin matrix between flexible silicone posts. Within two weeks after casting coherently beating human EHTs were formed and EHTs displayed a regular beating pattern for several weeks. Histological analysis revealed a high degree of sarcomeric organization and alignment of cardiomyocytes in EHTs. Functional analysis was performed by measuring force response to calcium concentration, pre-load, pacing frequency, beta-adrenergic and muscarinic agonists, modulators of sodium, calcium and potassium channels and revealed concentration-dependent effects. Comparison with native human Heart Tissue suggests an overall high level of similarity and minor differences. Conclusions: This study demonstrates feasibility to characterize human iPS cell-derived cardiomyocytes in EHTs by measuring contractile force. The analysis suggests high levels of similarity between EHTs and native human Heart Tissue. Human EHTs are a promising platform for automated toxicology screens in future drug development and for in vitro experiments on human cardiomyocytes in general.
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human engineered Heart Tissue as a versatile tool in basic research and preclinical toxicology
PLOS ONE, 2011Co-Authors: Sebastian Schaaf, Wolfram-hubertus Zimmermann, Aya Shibamiya, Alexandra Eder, Thomas Eschenhagen, Marc N Hirt, Marco Mewe, Andrea Stohr, L Conradi, Arne HansenAbstract:Human embryonic stem cell (hESC) progenies hold great promise as surrogates for human primary cells, particularly if the latter are not available as in the case of cardiomyocytes. However, high content experimental platforms are lacking that allow the function of hESC-derived cardiomyocytes to be studied under relatively physiological and standardized conditions. Here we describe a simple and robust protocol for the generation of fibrin-based human engineered Heart Tissue (hEHT) in a 24-well format using an unselected population of differentiated human embryonic stem cells containing 30–40% α-actinin-positive cardiac myocytes. Human EHTs started to show coherent contractions 5–10 days after casting, reached regular (mean 0.5 Hz) and strong (mean 100 µN) contractions for up to 8 weeks. They displayed a dense network of longitudinally oriented, interconnected and cross-striated cardiomyocytes. Spontaneous hEHT contractions were analyzed by automated video-optical recording and showed chronotropic responses to calcium and the β-adrenergic agonist isoprenaline. The proarrhythmic compounds E-4031, quinidine, procainamide, cisapride, and sertindole exerted robust, concentration-dependent and reversible decreases in relaxation velocity and irregular beating at concentrations that recapitulate findings in hERG channel assays. In conclusion this study establishes hEHT as a simple in vitro model for Heart research.
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development of a drug screening platform based on engineered Heart Tissue
Circulation Research, 2010Co-Authors: Arne Hansen, Sebastian Schaaf, Alexandra Eder, Marlene Bonstrup, Marianne Flato, Marco Mewe, Bulent Aksehirlioglu, Alexander Schworer, June Uebeler, Thomas EschenhagenAbstract:Rationale: Tissue engineering may provide advanced in vitro models for drug testing and, in combination with recent induced pluripotent stem cell technology, disease modeling, but available techniques are unsuitable for higher throughput. Objective: Here, we present a new miniaturized and automated method based on engineered Heart Tissue (EHT). Methods and Results: Neonatal rat Heart cells are mixed with fibrinogen/Matrigel plus thrombin and pipetted into rectangular casting molds in which two flexible silicone posts are positioned from above. Contractile activity is monitored video-optically by a camera and evaluated by a custom-made software program. Fibrin-based mini-EHTs (FBMEs) (150 μL, 600 000 cells) were transferred from molds to a standard 24-well plate two hours after casting. Over time FBMEs condensed from a 12×3×3 mm gel to a muscle strip of 8 mm length and, depending on conditions, 0.2 to 1.3 mm diameter. After 8 to 10 days, FBMEs started to rhythmically deflect the posts. Post properties and the extent of post deflection allowed calculation of rate, force (0.1 to 0.3 mN), and kinetics which was validated in organ baths experiments. FBMEs exhibited a well-developed, longitudinally aligned actinin-positive cardiac muscle network and lectin-positive vascular structures interspersed homogeneously throughout the construct. Analysis of a large series of FBME (n=192) revealed high yield and reproducibility and stability for weeks. Chromanol, quinidine, and erythromycin exerted concentration-dependent increases in relaxation time, doxorubicin decreases in contractile force. Conclusions: We developed a simple technique to construct large series of EHT and automatically evaluate contractile activity. The method shall be useful for drug screening and disease modeling.
Jorge Kalil - One of the best experts on this subject based on the ideXlab platform.
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t cell response in rheumatic fever crossreactivity between streptococcal m protein peptides and Heart Tissue proteins
Current Protein & Peptide Science, 2007Co-Authors: Luiza Guilherme, Sandra E Oshiro, Ana C Tanaka, Pablo Maria Alberto Pomerantzeff, Jorge KalilAbstract:Molecular mimicry between streptococcal and human proteins has been proposed as the triggering factor leading to autoimmunity in rheumatic fever (RF) and rheumatic Heart disease (RHD). In this review we focus on the studies on genetic susceptibility markers involved in the development of RF/RHD and molecular mimicry mediated by T cell responses of RHD patients against streptococcal antigens and human Tissue proteins. We identified several M protein epitopes recognized by peripheral T cells of RF/RHD patients and by Heart Tissue infiltrating T cell clones of severe RHD patients. The regions of the M protein preferentially recognized by human T cells were also recognized by murine T cells. By analyzing the T cell receptor (TCR) we observed that some Vβ families detected on the periphery were oligoclonal expanded in the Heart lesions. These results allowed us to confirm the major role of T cells in the development of RHD lesions.
Alexandra Eder - One of the best experts on this subject based on the ideXlab platform.
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human engineered Heart Tissue as a model system for drug testing
Advanced Drug Delivery Reviews, 2016Co-Authors: Alexandra Eder, Ingra Vollert, Arne Hansen, Thomas EschenhagenAbstract:Drug development is time- and cost-intensive and, despite extensive efforts, still hampered by the limited value of current preclinical test systems to predict side effects, including proarrhythmic and cardiotoxic effects in clinical practice. Part of the problem may be related to species-dependent differences in cardiomyocyte biology. Therefore, the event of readily available human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CM) has raised hopes that this human test bed could improve preclinical safety pharmacology as well as drug discovery approaches. However, hiPSC-CM are immature and exhibit peculiarities in terms of ion channel function, gene expression, structural organization and functional responses to drugs that limit their present usefulness. Current efforts are thus directed towards improving hiPSC-CM maturity and high-content readouts. Culturing hiPSC-CM as 3-dimensional engineered Heart Tissue (EHT) improves CM maturity and anisotropy and, in a 24-well format using silicone racks, enables automated, multiplexed high content readout of contractile function. This review summarizes the principal technology and focuses on advantages and disadvantages of this technology and its potential for preclinical drug screening.
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Human Engineered Heart Tissue: Analysis of Contractile Force
Stem Cell Reports, 2016Co-Authors: Ingra Mannhardt, Sebastian Schaaf, Christiane Neuber, David Letuffe-brenière, K. Breckwoldt, Alexandra Eder, Herbert Schulz, Anika Benzin, Tessa Werner, Thomas SchulzeAbstract:Analyzing contractile force, the most important and best understood function of cardiomyocytes in vivo is not established in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). This study describes the generation of 3D, strip-format, force-generating engineered Heart Tissues (EHT) from hiPSC-CM and their physiological and pharmacological properties. CM were differentiated from hiPSC by a growth factor-based three-stage protocol. EHTs were generated and analyzed histologically and functionally. HiPSC-CM in EHTs showed well-developed sarcomeric organization and alignment, and frequent mitochondria. Systematic contractility analysis (26 concentration-response curves) reveals that EHTs replicated canonical response to physiological and pharmacological regulators of inotropy, membrane- and calcium-clock mediators of pacemaking, modulators of ion-channel currents, and proarrhythmic compounds with unprecedented precision. The analysis demonstrates a high degree of similarity between hiPSC-CM in EHT format and native human Heart Tissue, indicating that human EHTs are useful for preclinical drug testing and disease modeling.
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P778Physiological and pharmacological characterization of human engineered Heart Tissue
Cardiovascular Research, 2014Co-Authors: Ingra Vollert, Sebastian Schaaf, Christiane Neuber, David Letuffe-brenière, K. Breckwoldt, Aya Shibamiya, D Stimpel, Alexandra Eder, Thomas Eschenhagen, Arne HansenAbstract:Objective: Human induced pluripotent stem cell (iPS cell)-derived cardiomyocytes represent a valuable tool in cardiovascular research of tremendous potential. However, these cells are still immature and characterized by poor sarcomeric organization and cellular orientation in 2D cell culture. Therefore, the measurement of contractile force, the most important and best understood function of cardiomyocytes in vivo, is not established for these cells. This study describes the generation of three-dimensional, strip-format, force generating engineered Heart Tissues (EHTs) from human iPS cell-derived cardiomyocytes and presents a characterization of physiological and pharmacological parameters based on force development. Methods and results: Cardiomyocyte differentiation of human induced pluripotent cells was achieved by a growth factor-based three stage protocol. Strip-format EHTs were generated from dissociated cardiomyocytes in fibrin matrix between flexible silicone posts. Within two weeks after casting coherently beating human EHTs were formed and EHTs displayed a regular beating pattern for several weeks. Histological analysis revealed a high degree of sarcomeric organization and alignment of cardiomyocytes in EHTs. Functional analysis was performed by measuring force response to calcium concentration, pre-load, pacing frequency, beta-adrenergic and muscarinic agonists, modulators of sodium, calcium and potassium channels and revealed concentration-dependent effects. Comparison with native human Heart Tissue suggests an overall high level of similarity and minor differences. Conclusions: This study demonstrates feasibility to characterize human iPS cell-derived cardiomyocytes in EHTs by measuring contractile force. The analysis suggests high levels of similarity between EHTs and native human Heart Tissue. Human EHTs are a promising platform for automated toxicology screens in future drug development and for in vitro experiments on human cardiomyocytes in general.
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human engineered Heart Tissue as a versatile tool in basic research and preclinical toxicology
PLOS ONE, 2011Co-Authors: Sebastian Schaaf, Wolfram-hubertus Zimmermann, Aya Shibamiya, Alexandra Eder, Thomas Eschenhagen, Marc N Hirt, Marco Mewe, Andrea Stohr, L Conradi, Arne HansenAbstract:Human embryonic stem cell (hESC) progenies hold great promise as surrogates for human primary cells, particularly if the latter are not available as in the case of cardiomyocytes. However, high content experimental platforms are lacking that allow the function of hESC-derived cardiomyocytes to be studied under relatively physiological and standardized conditions. Here we describe a simple and robust protocol for the generation of fibrin-based human engineered Heart Tissue (hEHT) in a 24-well format using an unselected population of differentiated human embryonic stem cells containing 30–40% α-actinin-positive cardiac myocytes. Human EHTs started to show coherent contractions 5–10 days after casting, reached regular (mean 0.5 Hz) and strong (mean 100 µN) contractions for up to 8 weeks. They displayed a dense network of longitudinally oriented, interconnected and cross-striated cardiomyocytes. Spontaneous hEHT contractions were analyzed by automated video-optical recording and showed chronotropic responses to calcium and the β-adrenergic agonist isoprenaline. The proarrhythmic compounds E-4031, quinidine, procainamide, cisapride, and sertindole exerted robust, concentration-dependent and reversible decreases in relaxation velocity and irregular beating at concentrations that recapitulate findings in hERG channel assays. In conclusion this study establishes hEHT as a simple in vitro model for Heart research.
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development of a drug screening platform based on engineered Heart Tissue
Circulation Research, 2010Co-Authors: Arne Hansen, Sebastian Schaaf, Alexandra Eder, Marlene Bonstrup, Marianne Flato, Marco Mewe, Bulent Aksehirlioglu, Alexander Schworer, June Uebeler, Thomas EschenhagenAbstract:Rationale: Tissue engineering may provide advanced in vitro models for drug testing and, in combination with recent induced pluripotent stem cell technology, disease modeling, but available techniques are unsuitable for higher throughput. Objective: Here, we present a new miniaturized and automated method based on engineered Heart Tissue (EHT). Methods and Results: Neonatal rat Heart cells are mixed with fibrinogen/Matrigel plus thrombin and pipetted into rectangular casting molds in which two flexible silicone posts are positioned from above. Contractile activity is monitored video-optically by a camera and evaluated by a custom-made software program. Fibrin-based mini-EHTs (FBMEs) (150 μL, 600 000 cells) were transferred from molds to a standard 24-well plate two hours after casting. Over time FBMEs condensed from a 12×3×3 mm gel to a muscle strip of 8 mm length and, depending on conditions, 0.2 to 1.3 mm diameter. After 8 to 10 days, FBMEs started to rhythmically deflect the posts. Post properties and the extent of post deflection allowed calculation of rate, force (0.1 to 0.3 mN), and kinetics which was validated in organ baths experiments. FBMEs exhibited a well-developed, longitudinally aligned actinin-positive cardiac muscle network and lectin-positive vascular structures interspersed homogeneously throughout the construct. Analysis of a large series of FBME (n=192) revealed high yield and reproducibility and stability for weeks. Chromanol, quinidine, and erythromycin exerted concentration-dependent increases in relaxation time, doxorubicin decreases in contractile force. Conclusions: We developed a simple technique to construct large series of EHT and automatically evaluate contractile activity. The method shall be useful for drug screening and disease modeling.
