Wittlyng3251

Exosomes are membrane-bound nano-vehicles shed by most eukaryotic cells. Exosomes contain specific proteins and RNAs from parent cells, and they play key signaling roles in cellular development, modulation, and tissue regeneration. Attempts to isolate and modify exosomes to increase their targeting efficiency to specific tissue are still in their infancy. Here, we describe generation of exosomes from biopsy, isolation of exosomes by centrifugal ultrafiltration method, and approaches for manipulation of cardiac homing exosomes by chemical engineering for the treatment of myocardial infarction.Modified mRNA (modRNA) is a promising new gene therapy approach that has safely and effectively delivered genes into different tissues, including the heart. Current efforts to use DNA-based or viral gene therapy to induce cardiac regeneration postmyocardial infarction (MI) or in heart failure (HF) have encountered key challenges, e.g., genome integration and delayed and noncontrolled expression. By contrast, modRNA is a transient, safe, non-immunogenic, and controlled gene delivery method that is not integrated into the genome. For most therapeutic applications, especially in regenerative medicine, the ability to deliver genes to the heart transiently and with control is vital for achieving therapeutic effect. Additionally, modRNA synthesis is comparatively simple and inexpensive compared to other gene delivery methods (e.g., protein), though a simple, clear in vitro transcription (IVT) protocol for synthesizing modRNA is needed for it to be more widely used. Here, we describe a simple and improved step-by-step IVT protocol to synthesize modRNA for in vitro or in vivo applications.Recombinant adeno-associated virus (rAAV) has been widely used for gene therapy. see more AAV-mediated gene transfer leads to durable protein expression in non-proliferating targeted tissues, which enables long-term modulation of gene expression. Here we describe a rAAV production protocol based on PEI-mediated triple transfection of HEK293T cells, followed by purification by iodixanol density gradient ultracentrifugation. Viral yield varies, depending on the size of the viral genome, but, typically, a yield of 3E11 viral genome (vg) can be achieved using the described protocol. Our results showed that injection of rAAV9 significantly transduces cardiac cells, which supports rAAV9 being an effective tool for gene delivery in the heart in vivo.The quantification of cell cycle activity is a prerequisite to defining the dynamics and scope of organ development or regeneration. Multi-isotope imaging mass spectrometry (MIMS) merges stable isotope tracers with an imaging mass spectrometry platform called NanoSIMS, which can quantitatively measure the incorporation of stable isotope tracers with high precision in suborganelle domains. MIMS has been applied to quantify the dynamics of postnatal cardiogenesis and mammalian cardiomyocyte regeneration during aging or in response to injury. Here, we present an approach to the conduct of MIMS experiments, with an emphasis on the application to the field of cardiac regeneration; however, the approach is also applicable, with, at most, minor modifications to broader biological questions.The development of the CreER/LoxP system has enabled temporal control and cell type specificity of gene activation or repression. A common application of this system involves lineage tracing and examining the proliferative capacity of cells of interest through clonal analysis. Here, we describe a method of performing 2- and 3-dimensional clonal analysis of cardiomyocytes (CMs) using the Rainbow reporter mouse model. We outline the process of using the Cell Counter plug-in tool in ImageJ to quantify the number of clones as well as the number of cells within each clone. For 3-dimensional analysis, we describe the tissue clearing technique, CLARITY, in conjunction with light-sheet imaging to obtain digital slices of the whole heart that can be reconstructed and clone volumes quantified using the Imaris software.Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes have become critically important for the detailed study of cardiac development, disease modeling, and drug screening. However, directed differentiation of hiPSCs into cardiomyocytes often results in mixed populations of cardiomyocytes and other cell types, which may confound experiments that require pure populations of cardiomyocytes. Here, we detail the use of a CRISPR/Cas9 genome editing strategy to develop cardiomyocyte-specific reporters that allow for the isolation of hiPSC-derived cardiomyocytes and chamber-specific myocytes. Moreover, we describe a cardiac differentiation protocol to derive cardiomyocytes from hiPSCs, as well as a strategy to use fluorescence-activated cell sorting to isolate pure populations of fluorescently labeled cardiomyocytes for downstream applications.Due to its pronounced regenerative capacity, the zebrafish heart represents an advantageous model system for exploring the cellular and molecular mechanisms of cardiac regeneration. Upon injury, the epicardium, the outermost mesothelial tissue layer of vertebrate hearts, serves dual purposes in the regenerating heart as both a signaling center and a source for crucial cell types. Traditional in vivo genetic approaches to study heart regeneration can be time consuming and are not applicable to large-scale approaches and live surveillance of cellular behaviors. Here, we demonstrate ex vivo methods to culture, maintain, and study the regenerative responses of epicardial tissue in excised zebrafish hearts. Epicardial cell proliferation and migration are monitored in real time after uninjured or injured hearts are excised, washed, and cultured for up to 30 days. In addition to these techniques, we describe ex vivo genetic ablation of the epicardium, cell proliferation assays, partial ventricular explant culturing, and chemical screening.Procurement and characterization of intact human cells are essential for studies in regenerative medicine and translational medical research. The selection of the currently available approaches to isolate intact cells depends on the age of the hearts. To isolate cardiomyocytes from the fetal or neonatal myocardium, the myocardium can be minced into small tissue blocks followed by enzyme incubation. However, the fetal and neonatal cardiomyocytes are very soft and the morphology changes from long rod or spindle shape to spheres after isolation. Because of the dense packing of the cardiomyocytes and the strong cell-cell connection in adult myocardium, it is difficult to isolate the cardiomyocytes from adult myocardium by enzyme incubation only. A perfusion method is necessary to deliver the enzyme solution to the deep layers of the myocardium. However, intact hearts, which are very rare, are required for the perfusion method. Therefore, lacking methods to efficiently isolate cardiomyocytes from myocardium of various ages builds a barrier between basic research and clinical studies.