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Here, we describe a method for the isolation of intact cardiomyocytes from fresh or frozen human myocardium or fresh mouse hearts and the quantification of multinucleation, cardiomyocyte size, cell cycle activity, and total cardiomyocyte count per heart. We generalize this fixation-digestion method by isolating cells from a variety of mouse organs, including the liver, lung, and thymus.Human induced pluripotent stem cells (hiPSCs) are among the most promising tools for regenerative myocardial therapy and in vitro modeling of cardiac disease; however, their full potential cannot be met without robust methods for differentiating them into cardiac-lineage cells. Here, we present novel protocols for generating hiPSC-derived cardiomyocytes (CMs), endothelial cells (ECs), and smooth muscle cells (SMCs) and for assembling them into a patch of human cardiac muscle (hCMP). The differentiation protocols can be completed in just a few weeks and are substantially more efficient than conventional methods, while the hCMP fabrication procedure produces a patch of clinically relevant size and incorporates a simple method for maturing the engineered tissue via mechanical stimulation. We also describe how the patch can be evaluated in a large-animal (swine) model of myocardial injury.Engineered cardiac tissues hold tremendous promise for in vitro drug discovery, studies of heart development and disease, and therapeutic applications. Here, we describe a versatile "frame-hydrogel" methodology to generate engineered cardiac tissues with highly mature functional properties. This methodology has been successfully utilized with a variety of cell sources (neonatal rat ventricular myocytes, human and mouse pluripotent stem cell-derived cardiomyocytes) to generate tissues with diverse 3D geometries (patch, bundle, network) and levels of structural and functional anisotropy. Maturation of such engineered cardiac tissues is rapidly achieved without the need for exogenous electrical or mechanical stimulation or use of complex bioreactors, with tissues routinely reaching conduction velocities and specific forces of 25 cm/s and 20 mN/mm2, respectively, and forces per input cardiomyocyte of up to 12 nN. This method is reproducible and readily scalable to generate small tissues ideal for in vitro testing as well as tissues with large, clinically relevant dimensions.Direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs) holds great promise as a potential treatment for cardiovascular disease, many of which are associated with tremendous loss of functional cardiomyocytes and simultaneous formation of scar tissue. Burgeoning studies have shown that the introduction of three minimal transcriptional factors, Gata4, Mef2c, and Tbx5 (G/M/T), could convert murine fibroblasts into iCMs that closely resemble endogenous CMs both in vitro and in vivo. Recent studies on iCM cell fate determination have demonstrated that the removal of genetic and epigenetic barriers could facilitate iCM reprogramming. However, varied reprogramming efficiency among research groups hinders its further study and potential applicability. Here, we provide a newly updated and detailed protocol for in vitro generation and evaluation of functional iCMs from mouse embryonic fibroblasts and neonatal cardiac fibroblasts using retroviral polycistronic construct encoding optimal expression of G/M/T factors. We hope that this optimized protocol will lay the foundation for future mechanistic studies of murine iCMs and further improvement of iCM generation.The epicardium is a multipotent cell layer that is vital to myocardial development and regeneration. Epicardial cells contribute to cardiac fibroblast and smooth muscle populations of the heart and secrete paracrine factors that promote cardiomyocyte proliferation and angiogenesis. Despite a central role in cardiac biology, the mechanisms by which epicardial cells influence cardiac growth are largely unknown, and robust models of the epicardium are needed. Here, we review our protocol for differentiating induced pluripotent stem cells (iPSCs) into epicardial-like cells through temporal modulation of canonical Wnt signaling. iPSC-derived epicardial cells (iECs) resemble in vivo epicardial cells morphologically and display markers characteristic of the developing epicardium. We also review our protocol for differentiating iECs into fibroblasts and smooth muscle cells through treatment with bFGF and TGF-β1, respectively. iECs provide a platform for studying fundamental epicardial biology and can inform strategies for therapeutic heart regeneration.Failure to regenerate myocardium after injury is a major cause of mortality and morbidity in humans. selleck compound Direct differentiation of human induced pluripotent stem cells (iPSCs) into cardiomyocytes provides an invaluable resource to pursue cardiac regeneration based on cellular transplantation. Beyond the potential for clinical therapies, iPSC technology also enables the generation of cardiomyocytes to recapitulate patient-specific phenotypes, thus presenting a powerful in vitro cell-based model to understand disease pathology and guide precision medicine. Here, we describe protocols for reprogramming of human dermal fibroblasts and blood cells into iPSCs using the non-integrative Sendai virus system and for the monolayer differentiation of iPSCs to cardiomyocytes using chemically defined media.The heart is a complex organ consisting of a variety of different cardiomyocytes (ventricular vs. atrial, left vs. right ventricular, working vs. nodal) as well as other cell types, including endothelial cells and vascular smooth muscle cells. Pericytes, neurons, and immune cells are less abundant, yet still important. Whereas cardiomyocytes account for around 75% of the heart volume, 50-70% of the cells in the heart are non-myocytes. This complexity of the heart underlines the difficulties in interpreting data obtained in vivo. In the field of cardiac regeneration, it remains unclear whether it is possible to induce a significant number of cardiomyocytes to proliferate and whether the often-observed improvement in cardiac function after experimental therapies is due to the induction of cardiomyocyte proliferation. Therefore, the reductionist approach inherent to cultures of isolated cells continues to be of great importance, even though it is important to study heart disease in vivo due to interactions of the different cell types. Cultured cardiomyocytes allow for easy manipulation of cell behavior (e.g., cell division) and its analysis (e.g., live-cell imaging). In addition, isolated cells in culture are a valuable tool for pharmacological and toxicological studies. This chapter offers a practical guide to isolate and culture primary neonatal and adult rat cardiomyocytes and a detailed protocol for live-cell imaging of embryonic and neonatal cardiomyocytes.Small animal models are indispensable for cardiac regeneration research. Studies in mouse and rat models have provided important insights into the etiology and mechanisms of cardiovascular diseases and accelerated the development of therapeutic strategies. It is vitally important to be able to evaluate the therapeutic efficacy and have reliable surrogate markers for therapeutic development for cardiac regeneration research. Magnetic resonance imaging (MRI), a versatile and noninvasive imaging modality with excellent penetration depth, tissue coverage, and soft-tissue contrast, is becoming a more important tool in both clinical settings and research arenas. Cardiac MRI (CMR) is versatile, noninvasive, and capable of measuring many different aspects of cardiac functions, and, thus, is ideally suited to evaluate therapeutic efficacy for cardiac regeneration. CMR applications include assessment of cardiac anatomy, regional wall motion, myocardial perfusion, myocardial viability, cardiac function assessment, assessment of myocardial infarction, and myocardial injury. Myocardial infarction models in mice are commonly used model systems for cardiac regeneration research. In this chapter, we discuss various CMR applications to evaluate cardiac functions and inflammation after myocardial infarction.Adult zebrafish possess an elevated cardiac regenerative capacity as compared with adult mammals. In the past two decades, zebrafish have provided a key model system for studying the cellular and molecular mechanisms of innate heart regeneration. The ease of genetic manipulation in zebrafish has enabled the establishment of a genetic ablation injury model in which over 60% of cardiomyocytes can be depleted, eliciting signs of heart failure. After this severe injury, adult zebrafish efficiently regenerate lost cardiomyocytes and reverse heart failure. In this chapter, we describe the methods for inducing genetic cardiomyocyte ablation in adult zebrafish, assessing cardiomyocyte proliferation, and histologically analyzing regeneration after injury.The zebrafish (Danio rerio) possesses a spectacular capacity for cardiac regeneration. Zebrafish have been used in cardiac regeneration research for nearly two decades, contributing to the identification of signals and cellular mechanisms as potential targets for human heart repair. Investigations into cardiac regeneration in zebrafish have been facilitated by multiple methods of inducing cardiac tissue damage. Among the established methods, cardiac resection injury is a relatively simple, yet robust approach traditionally used to induce cardiac tissue damage in a reproducible manner. Here, we describe a detailed protocol to perform a cardiac resection injury in adult zebrafish and discuss potential complications for researchers who are new to this technique.Zebrafish have the capacity to regenerate most of its organs upon injury, including the heart. Due to its amenability for genetic manipulation, the zebrafish is an excellent model organism to study the cellular and molecular mechanisms promoting heart regeneration. Several cardiac injury models have been developed in zebrafish, including ventricular resection, genetic ablation, and ventricular cryoinjury. This chapter provides a detailed protocol of zebrafish ventricular cryoinjury and highlights factors and critical steps to be considered when performing this method.Myocardial infarction is a major clinical challenge for interventional, pharmacological, and potential molecular treatment of the ischemic insult. A large animal model with clinic-derived instrumentation allows for detailed imitation of interventional catheterization routines and application routes, whereas similar anatomy and heart proportions raise the possibility to precisely evaluate the efficacy of application modes, e.g., antegrade or retrograde intracoronary application of locally acting pharmaceutical agents, viruses, and cells. Here, we describe the techniques of left ventricular catheterization and induction of ischemia and reperfusion, as well as hemodynamic monitoring and regional application of therapeutic agents in pigs.Left ventricular catheterization in mice allows for in-depth assessment of myocardial function in healthy and diseased animals with the advent of pressure volume loop recordings greatly enhancing the technique. While a powerful tool, proper execution of the procedure is paramount to ensure reproducibility and reliability of the results obtained. Here, we describe the technique of left ventricular catheterization using the Scisense conductance catheter system by Transonic; however, the basic method applies to all murine catheter systems. We furthermore indicate possible pitfalls during the procedure and how to avoid them.