Identifizierung und Charakterisierung der CD4+AT2R+ T Zell Subpopulation bei Mensch und Ratte

The study contributes to the understanding of CD4+AT2R+ cell function after myocardial infarction (MI). This potential novel T cell subset, which expresses FoxP3 and various cytokines improves cardiac function and reduces infarct size. Results indicate that the CD4+AT2R+ T cells might be included in therapeutic approaches for the treatment of cardiac diseases, due to its upregulation in the infarcted state. The application of this T cell subset may improve the healing process of compromised heart tissue and contribute to a better quality of life in affected patients

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

Magnetic Nanoparticle Based Nonviral MicroRNA Delivery into Freshly Isolated CD105+ hMSCs

Genetic modifications of bone marrow derived human mesenchymal stem cells (hMSCs) using microRNAs (miRs) may be used to improve their therapeutic potential and enable innovative strategies in tissue regeneration. However, most of the studies use cultured hMSCs, although these can lose their stem cell characteristics during expansion. Therefore, we aimed to develop a nonviral miR carrier based on polyethylenimine (PEI) bound to magnetic nanoparticles (MNPs) for efficient miR delivery in freshly isolated hMSCs. MNP based transfection is preferable for genetic modifications in vivo due to improved selectivity, safety of delivery, and reduced side effects. Thus, in this study different miR/PEI and miR/PEI/MNP complex formulations were tested in vitro for uptake efficiency and cytotoxicity with respect to the influence of an external magnetic field. Afterwards, optimized magnetic complexes were selected and compared to commercially available magnetic vectors (Magnetofectamine, CombiMag). We found that all tested transfection reagents had high miR uptake rates (yielded over 60%) and no significant cytotoxic effects. Our work may become crucial for virus-free introduction of therapeutic miRs as well as other nucleic acids in vivo. Moreover, in the field of targeted stem cell therapy nucleic acid delivery prior to transplantation may allowfor initial cell modulation in vitro

Defining Optimized Properties of Modified mRNA to Enhance Virus- and DNA- Independent Protein Expression in Adult Stem Cells and Fibroblasts

Background: By far, most strategies for cell reprogramming and gene therapy are based on the introduction of DNA after viral delivery. To avoid the high risks accompanying these goals, non-viral and DNA-free delivery methods for various cell types are required. Methods: Relying on an initially established PCR-based protocol for convenient template DNA production, we synthesized five differently modified EGFP mRNA (mmRNA) species, incorporating various degrees of 5-methylcytidine-5'-triphosphate (5mC) and pseudouridine-5'-triphosphate (Ψ). We then investigated their effect on i) protein expression efficiencies and ii) cell viability for human mesenchymal stem cells (hMSCs) and fibroblasts from different origins. Results: Our protocol allows highly efficient mmRNA production in vitro, enabling rapid and stable protein expression after cell transfection. However, our results also demonstrate that the terminally optimal modification needs to be defined in pilot experiments for each particular cell type. Transferring our approach to the conversion of fibroblasts into skeletal myoblasts using mmRNA encoding MyoD, we confirm the huge potential of mmRNA based protein expression for virus- and DNA-free reprogramming strategies. Conclusion: The achieved high protein expression levels combined with good cell viability not only in fibroblasts but also in hMSCs provides a promising option for mmRNA based modification of various cell types including slowly proliferating adult stem cells. Therefore, we are confident that our findings will substantially contribute to the improvement of efficient cell reprogramming and gene therapy approaches

PARP1 inhibitor olaparib (Lynparza) exerts synthetic lethal effect against ligase 4-deficient melanomas

Cancer including melanoma may be ''addicted" to double strand break (DSB) repair and targeting this process could sensitize them to the lethal effect of DNA damage. PARP1 exerts an important impact on DSB repair as it binds to both single- and double- strand breaks. PARP1 inhibitors might be highly effective drugs triggering synthetic lethality in patients whose tumors have germline or somatic defects in DNA repair genes. We hypothesized that PARP1-dependent synthetic lethality could be induced in melanoma cells displaying downregulation of DSB repair genes. We observed that PARP1 inhibitor olaparib sensitized melanomas with reduced expression of DNA ligase 4 (LIG4) to an alkylatimg agent dacarbazine (DTIC) treatment in vitro, while normal melanocytes remained intact. PARP1 inhibition caused accumulation of DSBs, which was associated with apoptosis in LIG4 deficient melanoma cells. Our hypothesis that olaparib is synthetic lethal with LIG4 deficiency in melanoma cells was supported by selective anti-tumor effects of olaparib used either alone or in combination with dacarbazine (DTIC) in LIG4 deficient, but not LIG4 proficient cells. In addition, olaparib combined with DTIC inhibited the growth of LIG4 deficient human melanoma xenografts. This work for the first time demonstrates the effectiveness of a combination of PARP1 inhibitor olaparib and alkylating agent DTIC for treating LIG4 deficient melanomas. In addition, analysis of the TCGA and transcriptome microarray databases revealed numerous individual melanoma samples potentially displaying specific defects in DSB repair pathways, which may predispose them to synthetic lethality triggered by PARP1 inhibitor combined with a cytotoxic drug