Multi-modal assessment of a cardiac stem cell therapy reveals distinct modulation of regional scar properties.

BACKGROUND: The initial idea of functional tissue replacement has shifted to the concept that injected cells positively modulate myocardial healing by a non-specific immune response of the transplanted cells within the target tissue. This alleged local modification of the scar requires assessment of regional properties of the left ventricular wall in addition to commonly applied measures of global morphological and functional parameters. Hence, we aimed at investigating the effect of cardiac cell therapy with cardiovascular progenitor cells, so-called cardiac induced cells, on both global and regional properties of the left ventricle by a multimodal imaging approach in a mouse model. METHODS: Myocardial infarction was induced in mice by ligation of the left anterior descending artery, the therapy group received an intramyocardial injection of 1 × 106 cardiac induced cells suspended in matrigel, the control group received matrigel only. [18F]FDG positron emission tomography imaging was performed after 17 days, to assess regional glucose metabolism. Three weeks after myocardial infarction, cardiac magnetic resonance imaging was performed for morphological and functional assessment of the left ventricle. Following these measurements, hearts were excised for histological examinations. RESULTS: Cell therapy had no significant effect on global morphological parameters. Similarly, there was no difference in scar size and capillary density between therapy and control group. However, there was a significant improvement in contractile function of the left ventricle - left ventricular ejection fraction, stroke volume and cardiac output. Regional analysis of the left ventricle identified changes of wall properties in the scar area as the putative mechanism. Cell therapy reduced the thinning of the scar and significantly improved its radial contractility. Furthermore, the metabolic defect, assessed by [18F]FDG, was significantly reduced by the cell therapy. CONCLUSION: Our data support the relevance of extending the assessment of global left ventricular parameters by a structured regional wall analysis for the evaluation of therapies targeting at modulation of healing myocardium. This approach will enable a deeper understanding of mechanisms underlying the effect of experimental regenerative therapies, thus paving the way for a successful translation into clinical application.

Cardiac cell therapies for the treatment of acute myocardial infarction in mice: systematic review and meta-analysis.

Backgound Aims: This meta-analysis aims at summarizing the whole body of research on cell therapies for acute myocardial infarction (MI) in the mouse model to bring forward ongoing research in this field of regenerative medicine. Despite rather modest effects in clinical trials, pre-clinical studies continue to report beneficial effects of cardiac cell therapies for cardiac repair following acute ischemic injury. Results: The authors' meta-analysis of data from 166 mouse studies comprising 257 experimental groups demonstrated a significant improvement in left ventricular ejection fraction of 10.21% after cell therapy compared with control animals. Subgroup analysis indicated that second-generation cell therapies such as cardiac progenitor cells and pluripotent stem cell derivatives had the highest therapeutic potential for minimizing myocardial damage post-MI. Conclusions: Whereas the vision of functional tissue replacement has been replaced by the concept of regional scar modulation in most of the investigated studies, rather basic methods for assessing cardiac function were most frequently used. Hence, future studies will highly benefit from integrating methods for assessment of regional wall properties to evolve a deeper understanding of how to modulate cardiac healing after acute MI.

Monitoring the maturation of the sarcomere network: a super-resolution microscopy-based approach.

The in vitro generation of human cardiomyocytes derived from induced pluripotent stem cells (iPSC) is of great importance for cardiac disease modeling, drug-testing applications and for regenerative medicine. Despite the development of various cultivation strategies, a sufficiently high degree of maturation is still a decisive limiting factor for the successful application of these cardiac cells. The maturation process includes, among others, the proper formation of sarcomere structures, mediating the contraction of cardiomyocytes. To precisely monitor the maturation of the contractile machinery, we have established an imaging-based strategy that allows quantitative evaluation of important parameters, defining the quality of the sarcomere network. iPSC-derived cardiomyocytes were subjected to different culture conditions to improve sarcomere formation, including prolonged cultivation time and micro patterned surfaces. Fluorescent images of α-actinin were acquired using super-resolution microscopy. Subsequently, we determined cell morphology, sarcomere density, filament alignment, z-Disc thickness and sarcomere length of iPSC-derived cardiomyocytes. Cells from adult and neonatal heart tissue served as control. Our image analysis revealed a profound effect on sarcomere content and filament orientation when iPSC-derived cardiomyocytes were cultured on structured, line-shaped surfaces. Similarly, prolonged cultivation time had a beneficial effect on the structural maturation, leading to a more adult-like phenotype. Automatic evaluation of the sarcomere filaments by machine learning validated our data. Moreover, we successfully transferred this approach to skeletal muscle cells, showing an improved sarcomere formation cells over different differentiation periods. Overall, our image-based workflow can be used as a straight-forward tool to quantitatively estimate the structural maturation of contractile cells. As such, it can support the establishment of novel differentiation protocols to enhance sarcomere formation and maturity.

The Effects of Hypoxic Preconditioned Murine Mesenchymal Stem Cells on Post-Infarct Arrhythmias in the Mouse Model.

Ventricular arrhythmias associated with myocardial infarction (MI) have a significant impact on mortality in patients following heart attack. Therefore, targeted reduction of arrhythmia represents a therapeutic approach for the prevention and treatment of severe events after infarction. Recent research transplanting mesenchymal stem cells (MSC) showed their potential in MI therapy. Our study aimed to investigate the effects of MSC injection on post-infarction arrhythmia. We used our murine double infarction model, which we previously established, to more closely mimic the clinical situation and intramyocardially injected hypoxic pre-conditioned murine MSC to the infarction border. Thereafter, various types of arrhythmias were recorded and analyzed. We observed a homogenous distribution of all types of arrhythmias after the first infarction, without any significant differences between the groups. Yet, MSC therapy after double infarction led to a highly significant reduction in simple and complex arrhythmias. Moreover, RNA-sequencing of samples from stem cell treated mice after re-infarction demonstrated a significant decline in most arrhythmias with reduced inflammatory pathways. Additionally, following stem-cell therapy we found numerous highly expressed genes to be either linked to lowering the risk of heart failure, cardiomyopathy or sudden cardiac death. Moreover, genes known to be associated with arrhythmogenesis and key mutations underlying arrhythmias were downregulated. In summary, our stem-cell therapy led to a reduction in cardiac arrhythmias after MI and showed a downregulation of already established inflammatory pathways. Furthermore, our study reveals gene regulation pathways that have a potentially direct influence on arrhythmogenesis after myocardial infarction.

Combined Gemcitabine and Immune-Checkpoint Inhibition Conquers Anti-PD-L1 Resistance in Low-Immunogenic Mismatch Repair-Deficient Tumors.

Tumors arising in the context of Lynch Syndrome or constitutional mismatch repair deficiency are hypermutated and have a good response towards immune-checkpoint inhibitors (ICIs), including α-PD-L1 antibodies. However, in most cases, resistance mechanisms evolve. To improve outcomes and prevent resistance development, combination approaches are warranted. Herein, we applied a combined regimen with an α-PD-L1 antibody and gemcitabine in a preclinical tumor model to activate endogenous antitumor immune responses. Mlh1-/- mice with established gastrointestinal tumors received the α-PD-L1 antibody (clone 6E11; 2.5 mg/kg bw, i.v., q2wx3) and gemcitabine (100 mg/kg bw, i.p., q4wx3) in mono- or combination therapy. Survival and tumor growth were recorded. Immunological changes in the blood were routinely examined via multi-color flow cytometry and complemented by ex vivo frameshift mutation analysis to identify alterations in Mlh1-/--tumor-associated target genes. The combined therapy of α-PD-L1 and gemcitabine prolonged median overall survival of Mlh1-/- mice from four weeks in the untreated control group to 12 weeks, accompanied by therapy-induced tumor growth inhibition, as measured by [18F]-FDG PET/CT. Plasma cytokine levels of IL13, TNFα, and MIP1ß were increased and also higher than in mice receiving either monotherapy. Circulating splenic and intratumoral myeloid-derived suppressor cells (MDSCs), as well as M2 macrophages, were markedly reduced. Besides, residual tumor specimens from combi-treated mice had increased numbers of infiltrating cytotoxic T-cells. Frameshift mutations in APC, Tmem60, and Casc3 were no longer detectable upon treatment, likely because of the successful eradication of single mutated cell clones. By contrast, novel mutations appeared. Collectively, we herein confirm the safe application of combined chemo-immunotherapy by long-term tumor growth control to prevent the development of resistance mechanisms.

Potential mechanisms of microRNA mobility.

microRNAs (miRNAs) are important epigenetic modulators of gene expression that control cellular physiology as well as tissue homeostasis, and development. In addition to the temporal aspects of miRNA-mediated gene regulation, the intracellular localization of miRNA is crucial for its silencing activity. Recent studies indicated that miRNA is even translocated between cells via gap junctional cell-cell contacts, allowing spatiotemporal modulation of gene expression within multicellular systems. Although non coding RNA remains a focus of intense research, studies regarding the intra-and intercellular mobility of small RNAs are still largely missing. Emerging data from experimental and computational work suggest the involvement of transport mechanisms governing proper localization of miRNA in single cells and cellular syncytia. Based on these data, we discuss a model of miRNA translocation that could help to address the spatial aspects of miRNA function and the impact of miRNA molecules on the intercellular signaling network.

SNX27 regulates DRA activity and mediates its direct recycling by PDZ-interaction in early endosomes at the apical pole of Caco2 cells.

DRA (downregulated in adenoma, SLC26A3) and NHE3 (Na+/H+ exchanger 3, SLC9A3) together mediate intestinal electroneutral NaCl absorption. Both transporters contain PDZ (postsynaptic density 95, disc large, zonula occludens 1) binding motifs and interact with PDZ adaptor proteins regulating their activity and recycling. SNX27 (sorting nexin 27) contains a PDZ domain and is involved in the recycling of cargo proteins including NHE3. The interaction of SNX27 with DRA and its potential role for the activity and recycling of DRA have been evaluated in this study. SNX27 specifically interacts with DRA via its PDZ domain. The knockdown (KD) of SNX27 reduced DRA activity by 50% but was not accompanied by a decrease of DRA surface expression. This indicates that DRA is trafficked to specific functional domains in the plasma membrane in which DRA is particularly active. Consistently, the disruption of lipid raft integrity by methyl-ß-cyclodextrin has an inhibitory effect on DRA activity that was strongly reduced after SNX27 KD. In differentiated intestinal Caco2 cells, superresolution microscopy and a novel quantitative axial approach revealed that DRA and SNX27 colocalize in rab5-positive early endosomes at the apical pole. SNX27 regulates the activity of DRA in the apical plasma membrane through binding with its PDZ domain. This interaction occurs in rab5-positive early endosomes at the apical pole of differentiated intestinal Caco2 cells. SNX27 is involved in the direct recycling of DRA to the plasma membrane where it is inserted into lipid rafts facilitating increased activity.NEW & NOTEWORTHY SNX27 has a PDZ domain and is involved in the regulation and recycling of transmembrane proteins. The role of SNX27 on the activity and recycling of the intestinal Cl-/HCO3- exchanger DRA has not yet been studied. This study shows that SNX27 directly interacts with DRA in early endosomes at the apical pole of intestinal Caco2 cells and mediates its direct recycling to facilitate high activity in lipid rafts in the apical plasma membrane.

Quantitative Evaluation of the Sarcomere Network of Human hiPSC-Derived Cardiomyocytes Using Single-Molecule Localization Microscopy.

The maturation of iPSC-derived cardiomyocytes is still a critical point for their application in cardiovascular research as well as for their clinical use. Although multiple differentiation protocols have been established, researchers failed to generate fully mature cardiomyocytes in vitro possessing identical phenotype-related and functional properties as their native adult counterparts. Besides electrophysiological and metabolic changes, the establishment of a well structured sarcomere network is important for the development of a mature cardiac phenotype. Here, we present a super resolution-based approach to quantitatively evaluate the structural maturation of iPSC-derived cardiomyocytes. Fluorescence labelling of the α-actinin cytoskeleton and subsequent visualization by photoactivated localization microscopy allows the acquisition of highly resolved images for measuring sarcomere length and z-disc thickness. Our image analysis revealed that iPSC and neonatal cardiomyocyte share high similarity with respect to their sarcomere organization, however, contraction capacity was inferior in iPSC-derived cardiac cells, indicating an early maturation level. Moreover, we demonstrate that this imaging approach can be used as a tool to monitor cardiomyocyte integrity, helping to optimize iPSC differentiation as well as somatic cell direct-reprogramming strategies.

Nkx2.5 Based Ventricular Programming of Murine ESC-Derived Cardiomyocytes.

BACKGROUND/AIMS: The availability of truly maturated cardiomyocytic subtypes is a major prerequisite for cardiovascular cell replacement therapies. Pluripotent stem cells provide a suitable source for the development of new strategies to overcome enormous hurdles such as yield, purity and safety of in vitro generated cells. METHODS: To address these issues, we have refined existing forward programming protocols by combining forced exogenous overexpression of the early cardiovascular transcription factor Nkx2.5 with a αMHC-promoter-based antibiotic selection step. Additionally, we applied small molecules such as ascorbic acid to enhance cardiomyogenic differentiation efficiency. Subsequently, we evaluated the cell fate of the resulting cardiomyocytes on the mRNA as well as protein levels. The latter was performed using high-resolution confocal microscopy. Furthermore, we examined the response of the cells` beating activities to pharmacological substance administration. RESULTS: Our results reveal an apparent influence of Nkx2.5 on the cell fate of ESC-derived cardiomyocytes. Resulting single cells exhibit characteristics of early ventricular cardiomyocytes, such as sarcomeric marker expression, spontaneous beating frequency, and distinct L-type calcium channel occurrence. CONCLUSION: Therefore, we demonstrate cardiovascular subtype forward programming of ESCs using a combination of transcription factors along with small molecule administration. However, our findings also underline current assumptions, that a terminal maturation of PSC derived cardiomyocytes in vitro is still an unsolved problem which urgently needs to be addressed in the field.

Stem Cell Therapy in Heart Diseases - Cell Types, Mechanisms and Improvement Strategies.

A large number of clinical trials have shown stem cell therapy to be a promising therapeutic approach for the treatment of cardiovascular diseases. Since the first transplantation into human patients, several stem cell types have been applied in this field, including bone marrow derived stem cells, cardiac progenitors as well as embryonic stem cells and their derivatives. However, results obtained from clinical studies are inconsistent and stem cell-based improvement of heart performance and cardiac remodeling was found to be quite limited. In order to optimize stem cell efficiency, it is crucial to elucidate the underlying mechanisms mediating the beneficial effects of stem cell transplantation. Based on these mechanisms, researchers have developed different improvement strategies to boost the potency of stem cell repair and to generate the "next generation" of stem cell therapeutics. Moreover, since cardiovascular diseases are complex disorders including several disease patterns and pathologic mechanisms it may be difficult to provide a uniform therapeutic intervention for all subgroups of patients. Therefore, future strategies should aim at more personalized SC therapies in which individual disease parameters influence the selection of optimal cell type, dosage and delivery approach.

Vimentin-Induced Cardiac Mesenchymal Stem Cells Proliferate in the Acute Ischemic Myocardium.

In-depth knowledge of the mechanisms induced by early postischemic cardiac endogenous mesenchymal stem cells (MSCs) in the acutely ischemic heart could advance our understanding of cardiac regeneration. Herein, we aimed to identify, isolate, and initially characterize the origin, kinetics and fate of cardiac MSCs. This was facilitated by in vivo genetic cell fate mapping through green fluorescent protein (GFP) expression under the control of vimentin induction after acute myocardial infarction (MI). Following permanent ligation of the left anterior descending coronary artery in CreER+ mTom/mGFP+ mice, vimentin/GFP+ cells revealed ischemia-responsive activation, survival, and local enrichment inside the peri-infarction border zone. Fluorescence-activated cell sorting (FACS)-isolated vimentin/GFP+ cells could be strongly expanded in vitro with clonogenic precursor formation and revealed MSC-typical cell morphology. Flow-cytometric analyses demonstrated an increase in cardiac vimentin/GFP+ cells in the ischemic heart, from a 0.6% cardiac mononuclear cell (MNC) fraction at 24 h to 1.6% at 72 h following MI. Sca-1+CD45- cells within the vimentin/GFP+ subtype of this MNC fraction increased from 35.2% at 24 h to 74.6% at 72 h after MI. The cardiac postischemic vimentin/GFP+ MNC subtype showed multipotent adipogenic, chondrogenic, and osteogenic differentiation potential, which is distinctive for MSCs. In conclusion, we demonstrated a seemingly proliferative first response of vimentin- induced cardiac endogenous MSCs in the acutely ischemic heart. Genetically, GFP-targeted in vivo cell tracking, isolation, and in vitro expansion of this cardiac MSC subtype could help to clarify their reparative status in inflammation, fibrogenesis, cell turnover, tissue homeostasis, and myocardial regeneration.

Cardiac Mesenchymal Stem Cells Proliferate Early in the Ischemic Heart.

BACKGROUND/PURPOSE: Cardiac mesenchymal stem cells (MSCs) could stimulate cell-specific regenerative mechanisms after myocardial infarction (MI) depending on spatial origin, distribution, and niche regulation. We aimed at identifying and isolating tissue-specific cardiac MSCs that could contribute to regeneration. METHODS: Following permanent ligation of the left anterior descending coronary artery in rats (n = 16), early cardiac tissues and cardiac mononuclear cells (MNCs) were analyzed by immunohistology, confocal laser scanning microscopy, and flow cytometry, respectively. Early postischemic specific MSCs were purified by fluorescence-activated cell sorting, cultivated under standardized culture conditions, and tested for multipotent differentiation in functional identification kits. RESULTS: Cardiac MSC niches were detected intramyocardially in cell clusters after MI and characterized by positive expression for vimentin, CD29, CD44, CD90, CD105, PDGFRα, and DDR2. Following myocardial ischemia, proliferation was induced early and proliferation density was approximately 11% in intramyocardial MSC clusters of the peri-infarction border zone. Cluster sizes increased by 157 and 64% in the peri-infarction and noninfarcted areas of infarcted hearts compared with noninfarcted hearts 24 h following MI, respectively. Coincidentally, flow cytometry analyses illustrated postischemic moderate enrichments of CD45-CD44+ and CD45-DDR2+ cardiac MNCs. We enabled isolation of early postischemic culturable cardiac CD45-CD44+DDR2+ MSCs that demonstrated typical clonogenicity with colony-forming unit-fibroblast formation as well as adipogenic, chondrogenic, and osteogenic differentiation. CONCLUSIONS: MI triggered early proliferation in specific cardiac MSC niches that were organized in intramyocardial clusters. Following targeted isolation, early postischemic cardiac CD45-CD44+DDR2+ MSCs exhibited typical characteristics with multipotent differentiation capacity and clonogenic expansion.

Applying 3D-FRAP microscopy to analyse gap junction-dependent shuttling of small antisense RNAs between cardiomyocytes.

Small antisense RNAs like miRNA and siRNA are of crucial importance in cardiac physiology, pathology and, moreover, can be applied as therapeutic agents for the treatment of cardiovascular diseases. Identification of novel strategies for miRNA/siRNA therapy requires a comprehensive understanding of the underlying mechanisms. Emerging data suggest that small RNAs are transferred between cells via gap junctions and provoke gene regulatory effects in the recipient cell. To elucidate the role of miRNA/siRNA as signalling molecules, suitable tools are required that will allow the analysis of these small RNAs at the cellular level. In the present study, we applied 3 dimensional fluorescence recovery after photo bleaching microscopy (3D-FRAP) to visualise and quantify the gap junctional exchange of small RNAs between neonatal cardiomyocytes in real time. Cardiomyocytes were transfected with labelled miRNA and subjected to FRAP microscopy. Interestingly, we observed recovery rates of 21% already after 13min, indicating strong intercellular shuttling of miRNA, which was significantly reduced when connexin43 was knocked down. Flow cytometry analysis confirmed our FRAP results. Furthermore, using an EGFP/siRNA reporter construct we demonstrated that the intercellular transfer does not affect proper functioning of small RNAs, leading to marker gene silencing in the recipient cell. Our results show that 3D-FRAP microscopy is a straightforward, non-invasive live cell imaging technique to evaluate the GJ-dependent shuttling of small RNAs with high spatio-temporal resolution. Moreover, the data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation where small RNAs act as signalling molecules within the intercellular network.

Spatio-temporal model of endogenous ROS and raft-dependent WNT/beta-catenin signaling driving cell fate commitment in human neural progenitor cells.

Canonical WNT/ß-catenin signaling is a central pathway in embryonic development, but it is also connected to a number of cancers and developmental disorders. Here we apply a combined in-vitro and in-silico approach to investigate the spatio-temporal regulation of WNT/ß-catenin signaling during the early neural differentiation process of human neural progenitors cells (hNPCs), which form a new prospect for replacement therapies in the context of neurodegenerative diseases. Experimental measurements indicate a second signal mechanism, in addition to canonical WNT signaling, being involved in the regulation of nuclear ß-catenin levels during the cell fate commitment phase of neural differentiation. We find that the biphasic activation of ß-catenin signaling observed experimentally can only be explained through a model that combines Reactive Oxygen Species (ROS) and raft dependent WNT/ß-catenin signaling. Accordingly after initiation of differentiation endogenous ROS activates DVL in a redox-dependent manner leading to a transient activation of down-stream ß-catenin signaling, followed by continuous auto/paracrine WNT signaling, which crucially depends on lipid rafts. Our simulation studies further illustrate the elaborate spatio-temporal regulation of DVL, which, depending on its concentration and localization, may either act as direct inducer of the transient ROS/ß-catenin signal or as amplifier during continuous auto-/parcrine WNT/ß-catenin signaling. In addition we provide the first stochastic computational model of WNT/ß-catenin signaling that combines membrane-related and intracellular processes, including lipid rafts/receptor dynamics as well as WNT- and ROS-dependent ß-catenin activation. The model's predictive ability is demonstrated under a wide range of varying conditions for in-vitro and in-silico reference data sets. Our in-silico approach is realized in a multi-level rule-based language, that facilitates the extension and modification of the model. Thus, our results provide both new insights and means to further our understanding of canonical WNT/ß-catenin signaling and the role of ROS as intracellular signaling mediator.

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.

Ca2+-mediated mitochondrial reactive oxygen species metabolism augments Wnt/ß-catenin pathway activation to facilitate cell differentiation.

Emerging evidence suggests that reactive oxygen species (ROS) can stimulate the Wnt/ß-catenin pathway in a number of cellular processes. However, potential sources of endogenous ROS have not been thoroughly explored. Here, we show that growth factor depletion in human neural progenitor cells induces ROS production in mitochondria. Elevated ROS levels augment activation of Wnt/ß-catenin signaling that regulates neural differentiation. We find that growth factor depletion stimulates the release of Ca(2+) from the endoplasmic reticulum stores. Ca(2+) subsequently accumulates in the mitochondria and triggers ROS production. The inhibition of mitochondrial Ca(2+) uptake with simultaneous growth factor depletion prevents the rise in ROS metabolism. Moreover, low ROS levels block the dissociation of the Wnt effector Dishevelled from nucleoredoxin. Attenuation of the response amplitudes of pathway effectors delays the onset of the Wnt/ß-catenin pathway activation and results in markedly impaired neuronal differentiation. Our findings reveal Ca(2+)-mediated ROS metabolic cues that fine-tune the efficiency of cell differentiation by modulating the extent of the Wnt/ß-catenin signaling output.

Neuronal differentiation requires a biphasic modulation of gap junctional intercellular communication caused by dynamic changes of connexin43 expression.

It was suggested that gap junctional intercellular communication (GJIC) and connexin (Cx) proteins play a crucial role in cell proliferation and differentiation. However, the mechanisms of cell coupling in regulating cell fate during embryonic development are poorly understood. To study the role of GJIC in proliferation and differentiation, we used a human neural progenitor cell line derived from the ventral mesencephalon. Fluorescence recovery after photobleaching (FRAP) showed that dye coupling was extensive in proliferating cells but diminished after the induction of differentiation, as indicated by a 2.5-fold increase of the half-time of fluorescence recovery. Notably, recovery half-time decreased strongly (five-fold) in the later stage of differentiation. Western blot analysis revealed a similar time-dependent expression profile of Cx43, acting as the main gap junction-forming protein. Interestingly, large amounts of cytoplasmic Cx43 were retained mainly in the Golgi network during proliferation but decreased when differentiation was induced. Furthermore, down-regulation of Cx43 by small interfering RNA reduced functional cell coupling, which in turn resulted in a 50% decrease of both the proliferation rate and neuronal differentiation. Our findings suggest a dual function of Cx43 and GJIC in the neural development of ReNcell VM197 human progenitor cells. GJIC accompanied by high Cx43 expression is necessary (1) to maintain cells in a proliferative state and (2) to complete neuronal differentiation, including the establishment of a neural network. However, uncoupling of cells is crucial in the early stage of differentiation during cell fate commitment.

Unusual tubulin-clustering ability of specifically c7-modified colchicine analogues.

Highly cytotoxic C7-modified colchicine analogues, exemplified by tubuloclustin, promote microtubule disassembly followed by the formation of very stable tubulin clusters, both in vitro and in cells. The proposed mechanism of action of tubuloclustin and its analogues, beyond that of colchicine, includes additional specific interactions with the α-tubulin subunit.

Sarcomeric network analysis of ex vivo cultivated human atrial appendage tissue using super-resolution microscopy.

Investigating native human cardiac tissue with preserved 3D macro- and microarchitecture is fundamental for clinical and basic research. Unfortunately, the low accessibility of the human myocardium continues to limit scientific progress. To overcome this issue, utilizing atrial appendages of the human heart may become highly beneficial. Atrial appendages are often removed during open-heart surgery and can be preserved ex vivo as living tissue with varying durability depending on the culture method. In this study, we prepared living thin myocardial slices from left atrial appendages that were cultured using an air-liquid interface system for overall 10 days. Metabolic activity of the cultured slices was assessed using a conventional methyl thiazolyl tetrazolium (MTT) assay. To monitor the structural integrity of cardiomyocytes within the tissue, we implemented our recently described super-resolution microscopy approach that allows both qualitative and quantitative in-depth evaluation of sarcomere network based on parameters such as overall sarcomere content, filament size and orientation. Additionally, expression of mRNAs coding for key structural and functional proteins was analyzed by real-time reverse transcription polymerase chain reaction (qRT-PCR). Our findings demonstrate highly significant disassembly of contractile apparatus represented by degradation of [Formula: see text]-actinin filaments detected after three days in culture, while metabolic activity was constantly rising and remained high for up to seven days. However, gene expression of crucial cardiac markers strongly decreased after the first day in culture indicating an early destructive response to ex vivo conditions. Therefore, we suggest static cultivation of living myocardial slices derived from left atrial appendage and prepared according to our protocol only for short-termed experiments (e.g. medicinal drug testing), while introduction of electro-mechanical stimulation protocols may offer the possibility for long-term integrity of such constructs.

CCR2 macrophage response determines the functional outcome following cardiomyocyte transplantation.

BACKGROUND: The immune response is a crucial factor for mediating the benefit of cardiac cell therapies. Our previous research showed that cardiomyocyte transplantation alters the cardiac immune response and, when combined with short-term pharmacological CCR2 inhibition, resulted in diminished functional benefit. However, the specific role of innate immune cells, especially CCR2 macrophages on the outcome of cardiomyocyte transplantation, is unclear. METHODS: We compared the cellular, molecular, and functional outcome following cardiomyocyte transplantation in wildtype and T cell- and B cell-deficient Rag2del mice. The cardiac inflammatory response was assessed using flow cytometry. Gene expression profile was assessed using single-cell and bulk RNA sequencing. Cardiac function and morphology were determined using magnetic resonance tomography and immunohistochemistry respectively. RESULTS: Compared to wildtype mice, Rag2del mice show an increased innate immune response at steady state and disparate macrophage response after MI. Subsequent single-cell analyses after MI showed differences in macrophage development and a lower prevalence of CCR2 expressing macrophages. Cardiomyocyte transplantation increased NK cells and monocytes, while reducing CCR2-MHC-IIlo macrophages. Consequently, it led to increased mRNA levels of genes involved in extracellular remodelling, poor graft survival, and no functional improvement. Using machine learning-based feature selection, Mfge8 and Ccl7 were identified as the primary targets underlying these effects in the heart. CONCLUSIONS: Our results demonstrate that the improved functional outcome following cardiomyocyte transplantation is dependent on a specific CCR2 macrophage response. This work highlights the need to study the role of the immune response for cardiomyocyte cell therapy for successful clinical translation.