Combined use of magnetic microbeads for endothelial cell isolation and enhanced cell engraftment in myocardial repair.

Background: The regenerative potential of the heart after injury is limited. Therefore, cell replacement strategies have been developed. However, the engraftment of transplanted cells in the myocardium is very inefficient. In addition, the use of heterogeneous cell populations precludes the reproducibility of the outcome. Methods: To address both issues, in this proof of principle study, we applied magnetic microbeads for combined isolation of eGFP+ embryonic cardiac endothelial cells (CECs) by antigen-specific magnet-associated cell sorting (MACS) and improved engraftment of these cells in myocardial infarction by magnetic fields. Results: MACS provided CECs of high purity decorated with magnetic microbeads. In vitro experiments revealed that the angiogenic potential of microbead-labeled CECs was preserved and the magnetic moment of the cells was strong enough for site-specific positioning by a magnetic field. After myocardial infarction in mice, intramyocardial CEC injection in the presence of a magnet resulted in a strong improvement of cell engraftment and eGFP+ vascular network formation in the hearts. Hemodynamic and morphometric analysis demonstrated augmented heart function and reduced infarct size only when a magnetic field was applied. Conclusion: Thus, the combined use of magnetic microbeads for cell isolation and enhanced cell engraftment in the presence of a magnetic field is a powerful approach to improve cell transplantation strategies in the heart.

Overexpression of Cx43 in cells of the myocardial scar: Correction of post-infarct arrhythmias through heterotypic cell-cell coupling.

Ventricular tachycardia (VT) is the most common and potentially lethal complication following myocardial infarction (MI). Biological correction of the conduction inhomogeneity that underlies re-entry could be a major advance in infarction therapy. As minimal increases in conduction of infarcted tissue markedly influence VT susceptibility, we reasoned that enhanced propagation of the electrical signal between non-excitable cells within a resolving infarct might comprise a simple means to decrease post-infarction arrhythmia risk. We therefore tested lentivirus-mediated delivery of the gap-junction protein Connexin 43 (Cx43) into acute myocardial lesions. Cx43 was expressed in (myo)fibroblasts and CD45+ cells within the scar and provided prominent and long lasting arrhythmia protection in vivo. Optical mapping of Cx43 injected hearts revealed enhanced conduction velocity within the scar, indicating Cx43-mediated electrical coupling between myocytes and (myo)fibroblasts. Thus, Cx43 gene therapy, by direct in vivo transduction of non-cardiomyocytes, comprises a simple and clinically applicable biological therapy that markedly reduces post-infarction VT.

Improved heart repair upon myocardial infarction: Combination of magnetic nanoparticles and tailored magnets strongly increases engraftment of myocytes.

Cell replacement in the heart is considered a promising strategy for the treatment of post-infarct heart failure. Direct intramyocardial injection of cells proved to be the most effective application route, however, engraftment rates are very low (<5%) strongly hampering its efficacy. Herein we combine magnetic nanoparticle (MNP) loading of EGFP labeled embryonic cardiomyocytes (eCM) and embryonic stem cell-derived cardiomyocytes (ES-CM) with application of custom designed magnets to enhance their short and long-term engraftment. To optimize cellular MNP uptake and magnetic force within the infarct area, first numerical simulations and experiments were performed in vitro. All tested cell types could be loaded efficiently with SOMag5-MNP (200 pg/cell) without toxic side effects. Application of a 1.3 T magnet at 5 mm distance from the heart for 10 min enhanced engraftment of both eCM and ES-CM by approximately 7 fold at 2 weeks and 3.4 fold (eCM) at 8 weeks after treatment respectively and also strongly improved left ventricular function at all time points. As underlying mechanisms we found that application of the magnetic field prevented the initial dramatic loss of cells via the injection channel. In addition, grafted eCM displayed higher proliferation and lower apoptosis rates. Electron microscopy revealed better differentiation of engrafted eCM, formation of cell to cell contacts and more physiological matrix formation in magnet-treated grafts. These results were corroborated by gene expression data. Thus, combination of MNP-loaded cells and magnet-application strongly increases long-term engraftment of cells addressing a major shortcoming of cardiomyoplasty.

Transgenic systems for unequivocal identification of cardiac myocyte nuclei and analysis of cardiomyocyte cell cycle status.

Even though the mammalian heart has been investigated for many years, there are still uncertainties in the fields of cardiac cell biology and regeneration with regard to exact fractions of cardiomyocytes (CMs) at different developmental stages, their plasticity after cardiac lesion and also their basal turnover rate. A main shortcoming is the accurate identification of CM and the demonstration of CM division. Therefore, an in vivo model taking advantage of a live reporter-based identification of CM nuclei and their cell cycle status is needed. In this technical report, we describe the generation and characterization of embryonic stem cells and transgenic mice expressing a fusion protein of human histone 2B and the red fluorescence protein mCherry under control of the CM specific αMHC promoter. This fluorescence label allows unequivocal identification and quantitation of CM nuclei and nuclearity in isolated cells and native tissue slices. In ventricles of adults, we determined a fraction of <20 % CMs and binucleation of 77-90 %, while in atria a CM fraction of 30 % and a binucleation index of 14 % were found. We combined this transgenic system with the CAG-eGFP-anillin transgene, which identifies cell division and established a novel screening assay for cell cycle-modifying substances in isolated, postnatal CMs. Our transgenic live reporter-based system enables reliable identification of CM nuclei and determination of CM fractions and nuclearity in heart tissue. In combination with CAG-eGFP-anillin-mice, the cell cycle status of CMs can be monitored in detail enabling screening for proliferation-inducing substances in vitro and in vivo.

Systemic gene transfer enables optogenetic pacing of mouse hearts.

AIMS: Optogenetic pacing of the heart has been demonstrated in transgenic animals expressing channelrhodopsin-2 (ChR2). However, for the clinical use of optogenetics to treat cardiac arrhythmias, gene transfer to non-transgenic hearts is required. The aim of this study was to describe a reliable method for gene transfer of ChR2 into a sufficient percentage of cardiomyocytes to overcome the electrical sink of all the coupled non-expressing cardiomyocytes during optical pacing of the whole heart in vivo. METHODS AND RESULTS: Adeno-associated virus (AAV) with cardiac tropism for expression of ChR2 in fusion with mCherry was systemically injected into wild-type mouse hearts. Bright mCherry fluorescence was detected in the whole heart 4-10 weeks later. Single-cell dissociation revealed that on average 58% cardiomyocytes were mCherry-positive. These showed light-induced inward currents, action potentials, and contractions. Pulsed illumination of the left ventricle induced ventricular pacing in vivo in 74% of mice, and higher light intensities were required for reduced pulse duration or size of illumination. Non-responding hearts showed low AAV expression, and the threshold for optical pacing was estimated to be 35-40% ChR2-expressing cardiomyocytes. Optical pacing in vivo was stable over extended periods without negative effects on normal sinus rhythm and ECG parameters after termination of stimulation indicating sufficient cardiac output during pacing. CONCLUSIONS: Gene transfer generates sufficient ChR2 photocurrent for reliable optogenetic pacing in vivo and lays out the basis for future optogenetic pacemaker and pain-free defibrillation therapies.

Live monitoring of small vessels during development and disease using the flt-1 promoter element.

Vessel formation is of critical importance for organ function in the normal and diseased state. In particular, the labeling and quantitation of small vessels prove to be technically challenging using current approaches. We have, therefore, established a transgenic embryonic stem (ES) cell line and a transgenic mouse model where the vascular endothelial growth factor receptor VEGFR-1 (flt-1) promoter drives the expression of the live reporter eGFP. Fluorescence microscopy and immunostainings revealed endothelial-specific eGFP labeling of vascular networks. The expression pattern recapitulates that of the endogenous flt-1 gene, because small and large vessels are labeled by eGFP during embryonic development; after birth, the expression becomes more restricted to small vessels. We have explored this in the cardiovascular system more in detail and found that all small vessels and capillaries within the heart are strongly eGFP+. In addition, myocardial injuries have been induced in transgenic mice and prominent vascular remodeling, and an increase in endothelial cell area within the peri-infarct area could be observed underscoring the utility of this mouse model. Thus, the transgenic flt-1/eGFP models are powerful tools to investigate and quantify vascularization in vivo and to probe the effect of different compounds on vessel formation in vitro.

Local gene targeting and cell positioning using magnetic nanoparticles and magnetic tips: comparison of mathematical simulations with experiments.

PURPOSE: Magnetic nanoparticles (MNPs) and magnets can be used to enhance gene transfer or cell attachment but gene or cell delivery to confined areas has not been addressed. We therefore searched for an optimal method to simulate and perform local gene targeting and cell delivery in vitro. METHODS: Localized gene transfer or cell positioning was achieved using permanent magnets with newly designed soft iron tips and MNP/lentivirus complexes or MNP-loaded cells, respectively. Their distribution was simulated with a mathematical model calculating magnetic flux density gradients and particle trajectories. RESULTS: Soft iron tips generated strong confined magnetic fields and could be reliably used for local (~500 µm diameter) gene targeting and positioning of bone marrow cells or cardiomyocytes. The calculated distribution of MNP/lentivirus complexes and MNP-loaded cells concurred very well with the experimental results of local gene expression and cell attachment, respectively. CONCLUSION: MNP-based gene targeting and cell positioning can be reliably performed in vitro using magnetic soft iron tips, and computer simulations are effective methods to predict and optimize experimental results.