Christian Rimmbach

Programming and Isolation of Highly Pure Physiologically and Pharmacologically Functional Sinus-Nodal Bodies from Pluripotent Stem Cells

Summary Therapeutic approaches for “sick sinus syndrome” rely on electrical pacemakers, which lack hormone responsiveness and bear hazards such as infection and battery failure. These issues may be overcome via “biological pacemakers” derived from pluripotent stem cells (PSCs). Here, we show that forward programming of PSCs with the nodal cell inducer TBX3 plus an additional Myh6-promoter-based antibiotic selection leads to cardiomyocyte aggregates consisting of >80% physiologically and pharmacologically functional pacemaker cells. These induced sinoatrial bodies (iSABs) exhibited highly increased beating rates (300–400 bpm), coming close to those found in mouse hearts, and were able to robustly pace myocardium ex vivo. Our study introduces iSABs as highly pure, functional nodal tissue that is derived from PSCs and may be important for future cell therapies and drug testing in vitro.

TRAPLINE : a standardized and automated pipeline for RNA sequencing data analysis, evaluation and annotation

BackgroundTechnical advances in Next Generation Sequencing (NGS) provide a means to acquire deeper insights into cellular functions. The lack of standardized and automated methodologies poses a challenge for the analysis and interpretation of RNA sequencing data. We critically compare and evaluate state-of-the-art bioinformatics approaches and present a workflow that integrates the best performing data analysis, data evaluation and annotation methods in a Transparent, Reproducible and Automated PipeLINE (TRAPLINE) for RNA sequencing data processing (suitable for Illumina, SOLiD and Solexa).ResultsComparative transcriptomics analyses with TRAPLINE result in a set of differentially expressed genes, their corresponding protein-protein interactions, splice variants, promoter activity, predicted miRNA-target interactions and files for single nucleotide polymorphism (SNP) calling. The obtained results are combined into a single file for downstream analysis such as network construction. We demonstrate the value of the proposed pipeline by characterizing the transcriptome of our recently described stem cell derived antibiotic selected cardiac bodies (aCaBs).ConclusionTRAPLINE supports NGS-based research by providing a workflow that requires no bioinformatics skills, decreases the processing time of the analysis and works in the cloud. The pipeline is implemented in the biomedical research platform Galaxy and is freely accessible via www.sbi.uni-rostock.de/RNAseqTRAPLINE or the specific Galaxy manual page (https://usegalaxy.org/u/mwolfien/p/trapline---manual).

Enhanced stem cell migration mediated by VCAM-1/VLA-4 interaction improves cardiac function in virus-induced dilated cardiomyopathy

Endogenous circulation of bone marrow-derived cells (BMCs) was observed in patients with dilated cardiomyopathy (DCM) who showed cardiac upregulation of Vascular Cell Adhesion Protein-1 (VCAM-1). However, the underlying pathophysiology is currently unknown. Thus, we aimed to analyze circulation, migration and G-CSF-based mobilization of BMCs in a murine model of virus-induced DCM. Mice with coxsackievirus B3 (CVB3) induced DCM and healthy controls were analyzed regarding their myocardial homing factors by PCR. To determine cardiac VCAM-1 expression ELISA and immunohistochemistry were applied. Flow cytometry was performed to analyze BMCs. Cardiac diameters and function were evaluated by echocardiography before and 4xa0weeks after G-CSF treatment. In murine CVB3-induced DCM an increase of BMCs in peripheral blood and a decrease of BMCs in bone marrow was observed. We found an enhanced migration of Very Late Antigen-4 (VLA-4+) BMCs to the diseased heart overexpressing VCAM-1 and higher numbers of CD45−CD34−Sca-1+ and CD45−CD34−c-kit+ cells. Mobilization of BMCs by G-CSF boosted migration along the VCAM-1/VLA-4 axis and reduced apoptosis of cardiomyocytes. Significant improvement of cardiac function was detected by echocardiography in G-CSF-treated mice. Blocking VCAM-1 by a neutralizing antibody reduced the G-CSF-dependent effects on stem cell migration and cardiac function. This is the first study showing that in virus-induced DCM VCAM-1/VLA-4 interaction is crucial for recruitment of circulating BMCs leading to beneficial anti-apoptotic effects resulting in improved cardiac function after G-CSF-induced mobilization.

Selection of a common multipotent cardiovascular stem cell using the 3.4-kb MesP1 promoter fragment

Common cardiovascular progenitor cells are characterized and induced by expression of the transcription factor MesP1. To characterize this population we used a 3.4-kb promoter fragment previously described by our group. This served to isolate MesP1-positive cells from differentiating ES stem cells via magnetic cell sorting based on a truncated CD4 surface marker. As this proximal promoter fragment omits a distal non-cardiovasculogenic enhancer region, we were able to achieve a synchronized fraction of highly enriched cardiovascular progenitors. These led to about 90xa0% of cells representing the three cardiovascular lineages: cardiomyocytes, endothelial cells and smooth muscle cells as evident from protein and mRNA analyses. In addition, electrophysiological and pharmacological parameters of the cardiomyocytic fraction show that almost all correspond to the multipotent early/intermediate cardiomyocyte subtype at day 18 of differentiation. Further differentiation of these cells was not impaired as evident from strong and synchronous beating at later stages. Our work contributes to the understanding of the earliest cardiovasculogenic events and may become an important prerequisite for cell therapy, tissue engineering and pharmacological testing in the culture dish using pluripotent stem cell-derived as well as directly reprogrammed cardiovascular cell types. Likewise, these cells provide an ideal source for large-scale transcriptome and proteome analyses.

Generation of murine cardiac pacemaker cell aggregates based on ES-cell-programming in combination with Myh6-promoter-selection.

Treatment of the sick sinus syndrome is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, they lack hormone responsiveness and the overall procedure is cost-intensive. Biological pacemakers generated from PSCs may become an alternative, yet the typical content of pacemaker cells in Embryoid Bodies (EBs) is extremely low. The described protocol combines forward programming of murine PSCs via the sinus node inducer TBX3 with Myh6-promoter based antibiotic selection. This yields cardiomyocyte aggregates consistent of >80% physiologically functional pacemaker cells. These induced-sinoatrial-bodies (iSABs) are spontaneously contracting at yet unreached frequencies (400-500 bpm) corresponding to nodal cells isolated from mouse hearts and are able to pace murine myocardium ex vivo. Using the described protocol highly pure sinus nodal single cells can be generated which e.g. can be used for in vitro drug testing. Furthermore, the iSABs generated according to this protocol may become a crucial step towards heart tissue engineering.

Non-viral magnetic engineering of endothelial cells with microRNA and plasmid-DNA—An optimized targeting approach

Genetic modulation of angiogenesis is a powerful tool for the treatment of multiple disorders. Here, we describe a strategy to produce modified endothelial cells, which can be efficiently magnetically guided. First, we defined optimal transfection conditions with both plasmid and microRNA, using a polyethyleneimine/magnetic nanoparticle-based vector (PEI/MNP), previously designed in our group. Further, two approaches were assessed in vitro: direct vector guidance and magnetic targeting of transfected cells. Due to its higher efficiency, including simulated dynamic conditions, production of miR/PEI/MNP-modified magnetically responsive cells was selected for further detailed investigation. In particular, we have studied internalization of transfection complexes, functional capacities and intercellular communication of engineered cells and delivery of therapeutic miR. Moreover, we demonstrated that 104 miRNA/PEI/MNP-modified magnetically responsive cells loaded with 0.37pg iron/cell are detectable with MRI. Taken together, our in vitro findings show that PEI/MNP is highly promising as a multifunctional tool for magnetically guided angiogenesis regulation.

Mammalian γ2 AMPK regulates intrinsic heart rate

AMPK is a conserved serine/threonine kinase whose activity maintains cellular energy homeostasis. Eukaryotic AMPK exists as αβγ complexes, whose regulatory γ subunit confers energy sensor function by binding adenine nucleotides. Humans bearing activating mutations in the γ2 subunit exhibit a phenotype including unexplained slowing of heart rate (bradycardia). Here, we show that γ2 AMPK activation downregulates fundamental sinoatrial cell pacemaker mechanisms to lower heart rate, including sarcolemmal hyperpolarization-activated current (If) and ryanodine receptor-derived diastolic local subsarcolemmal Ca2+ release. In contrast, loss of γ2 AMPK induces a reciprocal phenotype of increased heart rate, and prevents the adaptive intrinsic bradycardia of endurance training. Our results reveal that in mammals, for which heart rate is a key determinant of cardiac energy demand, AMPK functions in an organ-specific manner to maintain cardiac energy homeostasis and determines cardiac physiological adaptation to exercise by modulating intrinsic sinoatrial cell behavior.AMPK regulates cellular energy balance using its γ subunit as an energy sensor of cellular AMP and ADP to ATP ratios. Here, the authors show that γ2 AMPK activation lowers heart rate by reducing the activity of pacemaker cells, whereas loss of γ2 AMPK increases heart rate and prevents the adaptive bradycardia of endurance training in mice.

(Re-)programming of subtype specific cardiomyocytes

Adult cardiomyocytes (CMs) possess a highly restricted intrinsic regenerative potential - a major barrier to the effective treatment of a range of chronic degenerative cardiac disorders characterized by cellular loss and/or irreversible dysfunction and which underlies the majority of deaths in developed countries. Both stem cell programming and direct cell reprogramming hold promise as novel, potentially curative approaches to address this therapeutic challenge. The advent of induced pluripotent stem cells (iPSCs) has introduced a second pluripotent stem cell source besides embryonic stem cells (ESCs), enabling even autologous cardiomyocyte production. In addition, the recent achievement of directly reprogramming somatic cells into cardiomyocytes is likely to become of great importance. In either case, different clinical scenarios will require the generation of highly pure, specific cardiac cellular-subtypes. In this review, we discuss these themes as related to the cardiovascular stem cell and programming field, including a focus on the emergent topic of pacemaker cell generation for the development of biological pacemakers and in vitro drug testing.

Smell of cells: Volatile profiling of stem- and non-stem cell proliferation

Bacterial and cell cultures are known to emit a large number of volatile organic compounds (VOCs). Conventional biochemical methods are often destructive, time-consuming and expensive. In contrast, VOC analysis of headspace over cultures may offer a non-destructive alternative for the monitoring of cell proliferation and metabolism. VOC profiles from cultures of murine pluripotent stem cells and fibroblasts were assessed every 24 h for 3 days. Pure cell media were measured as parallel controls. VOC analysis was highly standardized with respect to time of measurement and phases of cell growth. Cultures were grown in custom-made inert boxes. In order to determine the effects of fresh media supply on VOC emissions, both cell types were cultured with and without daily media exchange. VOCs from headspace were preconcentrated by means of needle trap micro-extraction and analysed by gas chromatography-mass spectrometry (GC-MS). Murine pluripotent stem cells emitted increasing concentrations of thiirane and methyl-methoxy-hydroxy-methyl-amine (MMHA). Substance concentration correlated with cell numbers. Murine fibroblasts did not emit thiirane or MMHA. Concentrations of aldehydes, especially benzaldehyde, were lower in both cell cultures than in pure media samples. Daily media exchange resulted in higher cell numbers, but had no major effects on VOC concentrations emitted from the cells. Investigation and monitoring of volatile substances such as thiirane and MMHA may enhance the understanding of stem cell properties and lead to a destruction-free characterization of pluripotent stem cells.

Increased numbers of bone marrow-derived cells in parathyroid adenoma.

In primary hyperparathyroidism (PHPT), the increased levels of parathyroid hormone (PTH) result in mobilisation of bone‐marrow‐derived cells (BMCs) into peripheral blood. However, the fate of these cells is still unknown.