preparation-vitro-characterization-magnetized-mir-modified

Preparation and <em>In Vitro</em> Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

analysis-gap-junction-dependent-transfer-mirna-with-3d-frap

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

protocol-for-microrna-transfer-into-adult-bone-marrow-derived

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

University of Rostock

Reference and Translation Center for Cardiac Stem Cell Therapy (RTC)

Rostock University Medical Center

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.

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

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.

Defining optimized properties of modified mRNA to enhance virus- and DNA- independent protein expression in adult stem cells and fibroblasts.

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 10(4) 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.

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

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.

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

Aim. CD133(+) stem cells bear huge potential for regenerative medicine. However, low retention in the injured tissue and massive cell death reduce beneficial effects. In order to address these issues, we intended to develop a nonviral system for appropriate cell engineering. Materials and Methods. Modification of human CD133(+) stem cells with magnetic polyplexes carrying microRNA was studied in terms of efficiency, safety, and targeting potential. Results. High microRNA uptake rates (~80-90%) were achieved without affecting CD133(+) stem cell properties. Modified cells can be magnetically guided. Conclusion. We developed a safe and efficient protocol for CD133(+) stem cell modification. Our work may become a basis to improve stem cell therapeutical effects as well as their monitoring with magnetic resonance imaging.

Magnet-Bead Based MicroRNA Delivery System to Modify CD133(+) Stem Cells.

Standardization of stem cell therapy requires application of appropriate methods to evaluate safety and efficacy, including long-term pharmacovigilance. To accomplish this objective, a long-term registry programme was installed.

Article Total : 3	Year
Publication title	2017
Publication title	2017
Publication title	2018

Article	Year
Magnetic Nanoparticle Based Nonviral MicroRNA Delivery into Freshly Isolated CD105(+) hMSCs. Stem cells international\| PubMed ID: 24799915	2014
Defining optimized properties of modified mRNA to enhance virus- and DNA- independent protein expression in adult stem cells and fibroblasts. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology\| PubMed ID: 25720503	2015
Non-viral magnetic engineering of endothelial cells with microRNA and plasmid-DNA-An optimized targeting approach. Nanomedicine : nanotechnology, biology, and medicine\| PubMed ID: 27389150	2016
Applying 3D-FRAP microscopy to analyse gap junction-dependent shuttling of small antisense RNAs between cardiomyocytes. Journal of molecular and cellular cardiology\| PubMed ID: 27480520	2016
Magnet-Bead Based MicroRNA Delivery System to Modify CD133(+) Stem Cells. Stem cells international\| PubMed ID: 27795713	2016
Stem cell registry programme for patients with ischemic cardiomyopathy undergoing coronary artery bypass grafting: what benefits does it derive? ESC heart failure\| PubMed ID: 28451446	2017

Natalia Voronina

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