Abstract
Bone marrow derived human mesenchymal stem cells (hMSCs) show promising potential in regeneration of defective tissue. Recently, gene silencing strategies using microRNAs (miR) emerged with the aim to expand the therapeutic potential of hMSCs. However, researchers are still searching for effective miR delivery methods for clinical applications. Therefore, we aimed to develop a technique to efficiently deliver miR into hMSCs with the help of a magnetic non-viral vector based on cationic polymer polyethylenimine (PEI) bound to iron oxide magnetic nanoparticles (MNP). We tested different magnetic complex compositions and determined uptake efficiency and cytotoxicity by flow cytometry. Additionally, we monitored the release, processing and functionality of delivered miR-335 with confocal laser scanning microscopy, real-time PCR and live cell imaging, respectively. On this basis, we established parameters for construction of magnetic non-viral vectors with optimized uptake efficiency (~75%) and moderate cytotoxicity in hMSCs. Furthermore, we observed a better transfection performance of magnetic complexes compared to PEI complexes 72 h after transfection. We conclude that MNP-mediated transfection provides a long term effect beneficial for successful genetic modification of stem cells. Hence, our findings may become of great importance for future in vivo applications.
Highlights
Bone marrow derived human mesenchymal stem cells show great therapeutic potential in treatment of cardiovascular diseases
We have recently shown for plasmid based complexes, that transfection efficiency was enhanced by conjugation of PEI complexes to magnetic nanoparticles (MNP) even without application of magnetic field, as magnetic polyplexes provided a faster release of DNA into the cytosol compared to PEI polyplexes
In this work we investigated the intracellular processing of precursor miR-335 to a mature strand as well as efficient knockdown of known target genes comparing the performance of PEI - mediated transfection and MNP-mediated transfection
Summary
Bone marrow derived human mesenchymal stem cells (hMSCs) show great therapeutic potential in treatment of cardiovascular diseases. The protective function of hMSCs can be explained by secretion of antiapoptotic, angiogenic [1,2] and matrix-mediating factors [3]. Due to their multipotency, hMSCs are able to differentiate into endothelial like cells leading to an improved cardiac function [4]. Research was concentrated on viral vectors, e.g., retroviruses and adenoviruses, as they provide high transduction efficiencies and long term gene expression. This approach has many disadvantages, such as toxicity, immunogenicity, mutagenicity, low genetic material load and high costs [10]. A focus has been placed on alternative non-viral delivery approaches
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