3D Heart Tissue Research Articles

Comparing fabrication techniques for engineered cardiac tissue.

Tissue engineering can provide in vitro models for drug testing, disease modeling, and perhaps someday, tissue/organ replacements. For building 3D heart tissue, the alignment of cardiac cells or cardiomyocytes (CMs) is important in generating a synchronously contracting tissue. To that end, researchers have generated several fabrication methods for building heart tissue, but direct comparisons of pros and cons using the same cell source is lacking. Here, we derived cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) and compare the assembly of these cells using three fabrication methods: cardiospheres, muscle rings, and muscle strips. All three protocols successfully generated compacted tissue comprised of hiPSC-derived CMs stable for at least 2 weeks. The percentage of aligned cells was greatest in the muscle strip (55%) and the muscle ring (50%) compared with the relatively unaligned cardiospheres (35%). The iPSC-derived CMs within the muscle strip also exhibited the greatest elongation, with elongation factor at 2.0 compared with 1.5 for the muscle ring and 1.2 for the cardiospheres. This is the first direct comparison of various fabrication techniques using the same cell source.

454 A Human 3D Model of Duchenne Muscular Dystrophy Cardiomyopathy to Investigate Calcium Regulation and Mitochondrial Dysfunction

OBJECTIVES/GOALS: We will use control- and DMD-engineered heart tissues to better model and investigate DMD cardiomyopathy. We will primarily assess cardiac calcium handling, mitochondrial function, and mitochondrial calcium handling, as calcium regulation and mitochondrial function are known to be affected in DMD. METHODS/STUDY POPULATION: We will use patient-derived stem cells, differentiated into cardiomyocytes in bioprinted 3D heart tissue muscle chambers to better model DMD cardiomyopathy. We will look at calcium handling and general mitochondrial function, as well as mitochondrial calcium handling, using a novel multifunctional genetic probe I previously developed allowing for simultaneous observation of cytosolic and mitochondrial calcium in real time. Optical mapping will also be used for tissue-level analysis. We will establish the functional differences at baseline, and then progress heart failure in the tissues to see how the abnormalities seen in the DMD tissues may get worse. Finally, we will investigate the effects of early restoration of dystrophin function on the effects of DMD cardiomyopathy development. RESULTS/ANTICIPATED RESULTS: We anticipate that DMD tissues will show more irregular/abnormal calcium handling, as seen in 2D hiPSC-CMs, as well as disruptions to mitochondrial function and ultrastructural development, as well as a decreased synchronization between cytosolic and mitochondrial calcium dynamics. We anticipate that these abnormalities will be exacerbated as the disease state progresses, but at least partially ameliorated with the restoration of dystrophin function. DISCUSSION/SIGNIFICANCE: DMD is a fatal disease with no known cure. Patients develop heart failure in their teens and die in their 20s, so any new insight that may prolong life and improve quality of life for patients is drastically needed. This would be the most accurate preclinical model of DMD cardiomyopathy to date and would investigate yet-untapped aspects of the disease state.

Contractility analysis of human engineered 3D heart tissues by an automatic tracking technique using a standalone application.

The use of Engineered Heart Tissues (EHT) as in vitro model for disease modeling and drug screening has increased, as they provide important insight into the genetic mechanisms, cardiac toxicity or drug responses. Consequently, this has highlighted the need for a standardized, unbiased, robust and automatic way to analyze hallmark physiological features of EHTs. In this study we described and validated a standalone application to analyze physiological features of EHTs in an automatic, robust, and unbiased way, using low computational time. The standalone application “EHT Analysis” contains two analysis modes (automatic and manual) to analyzes the contractile properties and the contraction kinetics of EHTs from high speed bright field videos. As output data, the graphs of displacement, contraction force and contraction kinetics per file will be generated together with the raw data. Additionally, it also generates a summary file containing all the data from the analyzed files, which facilitates and speeds up the post analysis. From our study we highlight the importance of analyzing the axial stress which is the force per surface area (μN/mm2). This allows to have a readout overtime of tissue compaction, axial stress and leave the option to calculate at the end point of an experiment the physiological cross-section area (PSCA). We demonstrated the utility of this tool by analyzing contractile properties and compaction over time of EHTs made out of a double reporter human pluripotent stem cell (hPSC) line (NKX2.5EGFP/+-COUP-TFIImCherry/+) and different ratios of human adult cardiac fibroblasts (HCF). Our standalone application “EHT Analysis” can be applied for different studies where the physiological features of EHTs needs to be analyzed under the effect of a drug compound or in a disease model.

Development of matured hiPSCs-derived 3D cardiac tissue using ERR gamma agonist and mechanical stress and application for Hypertrophic Cardiomyopathy (HCM) model

Abstract Introduction Tissue engineering using human induced pluripotent stem cells-derived cardiomyocytes (hiPSCs-CMs) is one of the potential tools to replicate human heart in vitro. Although there are many publications on 3 dimensional (3D) heart tissues (1), these tissues show fetal like phenotypes. For that reason, several maturation methods such as electrical stimulation and mechanical stress have been investigated (2, 3). However, these methods have been inadequate in differentiating fetal like phenotype tissue from adult tissues. Previously, we identified a novel compound, T112, which induced hiPSCs-CMs maturation from approximately 9,000 compounds using Troponin I1-EmGFP and Troponin I3-mCherry double reporter hiPSCs-CMs. This compound enhanced morphological and metabolic maturation of hiPSCs-CMs via estrogen-rerated receptor gamma activation Purpose We hypothesized that our novel compound, T112, in combination with mechanical stress could result in further maturation of 3D heart tissue. Therefore, our specific aim is to develop a novel maturation method applicable to genetic disease model of HCM using 3D heart tissue combined with T112. Methods We constructed 3D heart tissue mixed with fibroblast and double reporter hiPSCs-CMs by the hydrogel methods using Flex cell system®. We added T112 with or without mechanical stretching to 3D tissue from 7 to 15 days after 3D heart tissue was constructed. Then we measured maturation related phenotype such as sarcomere gene expression, mitochondrial DNA content and cell size. Results Similar to hiPSCs-CM, the addition of T112 to the constructed 3D heart tissue significantly increased TNNI3 mRNA compared to that of DMSO. Furthermore, T112 treated 3D heart tissue showed increased cell size and oblong shape. Next, in order to promote more maturation of 3D heart tissue, we performed mechanical stretching with the addition of T112. The combination of T112 with mechanical stretching showed higher expression of mCherry, a reporter protein for TNNI3 expression, and higher isotropy of sarcomere alignment in 3D heart tissue than that with the static condition. Furthermore, 3D heart tissue in the treatment of T112 with or without mechanical stretching showed higher mitochondrial DNA content compared to the respective DMSO controls. Interestingly, we applied this combination method to hiPSCs carrying MYH7 R719Q mutation which is known to cause hypertrophic cardiomyopathy, and the 3D heart tissue composed of cardiomyocytes derived from mutant iPSCs demonstrated increased sarcomere disarray compared to isogenic wild-type 3D heart tissue. Conclusion These results suggest that the combination of T112 and mechanical stretching promotes metabolic and structural maturation of 3D heart tissue and would be useful for creating a HCM disease model. Funding Acknowledgement Type of funding source: Private company. Main funding source(s): T-CiRA project, Takeda Pharmaceutical Company Limited

Slicing and Culturing Pig Hearts under Physiological Conditions.

Many novel drugs fail in clinical studies due to cardiotoxic side effects as the currently available in vitro assays and in vivo animal models poorly predict human cardiac liabilities, posing a multi-billion-dollar burden on the pharmaceutical industry. Hence, there is a worldwide unmet medical need for better approaches to identify drug cardiotoxicity before undertaking costly and time consuming 'first in man' trials. Currently, only immature cardiac cells (human induced pluripotent stem cell-derived cardiomyocytes [hiPSC-CMs]) are used to test therapeutic efficiency and drug toxicity as they are the only human cardiac cells that can be cultured for prolonged periods required to test drug efficacy and toxicity. However, a single cell type cannot replicate the phenotype of the complex 3D heart tissue which is formed of multiple cell types. Importantly, the effect of drugs needs to be tested on adult cardiomyocytes, which have different characteristics and toxicity responses compared to immature hiPSC-CMs. Culturing human heart slices is a promising model of intact human myocardium. This technology provides access to a complete multicellular system that mimics the human heart tissue and reflects the physiological or pathological conditions of the human myocardium. Recently, through optimization of the culture media components and the culture conditions to include continuous electrical stimulation at 1.2 Hz and intermittent oxygenation of the culture medium, we developed a new culture system setup that preserves viability and functionality of human and pig heart slices for 6 days in culture. In the current protocol, we are detailing the method for slicing and culturing pig heart as an example. The same protocol is used to culture slices from human, dog, sheep, or cat hearts. This culture system has the potential to become a powerful predictive human in situ model for acute cardiotoxicity testing that closes the gap between preclinical and clinical testing results.

Cardiomyocyte maturation: advances in knowledge and implications for regenerative medicine.

Our knowledge of pluripotent stem cell (PSC) biology has advanced to the point where we now can generate most cells of the human body in the laboratory. PSC-derived cardiomyocytes can be generated routinely with high yield and purity for disease research and drug development, and these cells are now gradually entering the clinical research phase for the testing of heart regeneration therapies. However, a major hurdle for their applications is the immature state of these cardiomyocytes. In this Review, we describe the structural and functional properties of cardiomyocytes and present the current approaches to mature PSC-derived cardiomyocytes. To date, the greatest success in maturation of PSC-derived cardiomyocytes has been with transplantation into the heart in animal models and the engineering of 3D heart tissues withelectromechanical conditioning. In conventional 2D cell culture, biophysical stimuli such as mechanical loading, electrical stimulation and nanotopology cues all induce substantial maturation, particularly of the contractile cytoskeleton. Metabolism has emerged as a potent means to control maturation with unexpected effects on electrical and mechanical function. Different interventions induce distinct facets of maturation, suggesting that activating multiple signalling networks might lead to increased maturation. Despite considerable progress, we are still far from being able to generate PSC-derived cardiomyocytes with adult-like phenotypes in vitro. Future progress will come from identifying the developmental drivers of maturation and leveraging them to create more mature cardiomyocytes for research and regenerative medicine.

Pulsatile Cardiac Tissue Grafts Using a Novel Three-Dimensional Cell Sheet Manipulation Technique Functionally Integrates With the Host Heart, In Vivo

We devised a method of fabricating easily transplantable scaffoldless 3D heart tissue, made with a novel cell-sheet (CS) technology from cultured cardiomyocytes using a fibrin polymer coated dish. In the present study, we tested in vivo electrical communication which is essential for improving heart function between the host heart and the grafted CS. The epicardial surface of the ventricle of an anesthetized open-chest nude rat was ablated by applying a heated metal. Bilayered CS was obtained from neonatal rat primary culture. CS was transplanted onto the injured myocardial surface (sMI) (sMI+sheet group). The rats were allowed to recover for 1 to 4 weeks, to stabilize the grafts. Action potentials (APs) from the excised perfused heart were monitored by the fluorescence signal of di-4ANEPPS with a high speed charge-coupled device camera. The APs were observed under epicardial pacing of the host heart or the CS grafts. The pacing threshold of the current output was measured in the sMI+sheet group and in the nongrafted sMI group at the center of the sMI and in the normal zone (Nz). Bidirectional AP propagation between the sMI and Nz was observed in the sMI+sheet group (n=14), but was blocked at the marginal area of the sMI in the sMI group (n=9). The ratio of the pacing threshold (sMI/Nz) was significantly lower in the sMI+sheet than in the sMI group (3.0+/-0.7, 19.0+/-6.1 respectively P<0.05). There were neither spontaneous nor pacing-induced arrhythmias in these two groups. Bidirectional smooth AP propagation between the host heart and the grafted CS was observed. This finding suggested functional integration of this CS graft with the host heart without serious arrhythmia.