Transient and Stable Overexpression of Extracellular Superoxide Dismutase Is Positively Associated with the Myogenic Function of Human Skeletal Muscle-Derived Stem/Progenitor Cells.

In the present study, the genetic modification of human skeletal muscle-derived stem/progenitor cells (SkMDS/PCs) was investigated to identify the optimal protocol for myogenic cell preparation for use in post-infarction heart therapy. We used two types of modifications: GFP-transfection (using electroporation) and SOD3 transduction (using a lentiviral vector). SkMDS/PCs were cultured under different in vitro conditions, including standard (21% oxygen) and hypoxic (3% oxygen), the latter of which corresponded to the prevailing conditions in the post-infarction heart. Transfection/transduction efficacy, skeletal myogenic cell marker expression (CD56), cellular senescence, and apoptosis, as well as the expression of antioxidant (SOD1, SOD2, and SOD3), anti-aging (SIRT1 and FOXO), anti-apoptotic (BCL2), and myogenic (MyoD and MyoG) genes, were evaluated. The percentage of GFP-positive SkMDS/PCs was determined as an indicator of the efficacy of transfection, which reached 55%, while transduction showed better efficiency, reaching approximately 85% as estimated by fluorescence microscopy. The CD56-positive SkMDS/PCs were present in approximately 77% of the tested cells after transient transfection and approximately 96% after transduction. Under standard in vitro culture conditions, the ability of the differentiated, transfected SkMDS/PCs to form myotubes was greater than that of the wild type (WT) cell population (p < 0.001), while the cells transduced with the SOD3 gene exhibited an increase in cell fusion under both standard (p < 0.05) and hypoxic conditions (p < 0.001). In transduced SkMDS/PCs, we observed a positive influence of SOD3 overexpression on cell ageing and apoptosis. We observed an increase in the percentage of young cells under standard (p < 0.05) and hypoxic (p < 0.001) in vitro culture conditions, with a notable decrease in the percentage of senescent and advanced senescent cells in the SOD3-overexpressing cell population detected compared to that observed for the untransduced muscle-derived cells. A lower percentage of apoptotic cells was observed for transduced SkMDS/PCs than that for WT cells under hypoxic in vitro culture conditions. In transiently transfected SkMDS/PCs, we observed significantly higher gene expression levels of SOD2 (almost 40-fold) (p < 0.001) and FOXO (p < 0.05) (approximately 3-fold) under both normoxic and hypoxic culture conditions and of BCL2 under hypoxia compared to those observed in untreated cells (WT). In addition, myogenic genes showed a significant increase in MyoD (almost 18-fold) expression under standard culture conditions (p < 0.0001) and decreased MyoG expression (approximately 2-fold) after transfection (p < 0.05) compared with that detected in the WT skeletal muscle-derived cell control. Taken together, these results demonstrate that SOD3-tranduced skeletal muscle-derived cells may have potential for use in the regenerative treatment of the post-infarction heart.

Potential use of superparamagnetic iron oxide nanoparticles for in vitro and in vivo bioimaging of human myoblasts.

Myocardial infarction (MI) is one of the most frequent causes of death in industrialized countries. Stem cells therapy seems to be very promising for regenerative medicine. Skeletal myoblasts transplantation into postinfarction scar has been shown to be effective in the failing heart but shows limitations such, e.g. cell retention and survival. We synthesized and investigated superparamagnetic iron oxide nanoparticles (SPIONs) as an agent for direct cell labeling, which can be used for stem cells imaging. High quality, monodisperse and biocompatible DMSA-coated SPIONs were obtained with thermal decomposition and subsequent ligand exchange reaction. SPIONs' presence within myoblasts was confirmed by Prussian Blue staining and inductively coupled plasma mass spectrometry (ICP-MS). SPIONs' influence on tested cells was studied by their proliferation, ageing, differentiation potential and ROS production. Cytotoxicity of obtained nanoparticles and myoblast associated apoptosis were also tested, as well as iron-related and coating-related genes expression. We examined SPIONs' impact on overexpression of two pro-angiogenic factors introduced via myoblast electroporation method. Proposed SPION-labeling was sufficient to visualize firefly luciferase-modified and SPION-labeled cells with magnetic resonance imaging (MRI) combined with bioluminescence imaging (BLI) in vivo. The obtained results demonstrated a limited SPIONs' influence on treated skeletal myoblasts, not interfering with basic cell functions.

HBcAg produced in transgenic tobacco triggers Th1 and Th2 response when intramuscularly delivered.

Hepatitis B core Antigen (HBcAg) assembled into Capsid-Like Particles (CLPs) is investigated as a therapeutic vaccine in treatment of chronic hepatitis B (CHB) and in diagnostic tests or as a carrier for various epitopes. While the expression of HBcAg has been thoroughly clarified in E. coli and yeast, it has also been investigated in other expression systems. Stably transformed tobacco expressed HBcAg at a level of 110-250µg/g fresh weight, therefore in view of its large leaf biomass it offers a production platform comparable with transient expression systems regarding the final yield of HBcAg. Several extraction and purification methods were tested and finally the antigen was purified up to 43% using sucrose density gradient centrifugation. The purified HBcAg retained its antigenicity, as confirmed by ELISA and western blot, while maintaining its CLP-structure as observed in TEM. In mice HBcAg intramuscularly delivered at 2×10µg triggered a significant response (serum anti-HBc titre around 150,000), being statistically equivalent to that induced by the reference antigen. Among anti-HBc IgG isotypes, IgG2a and then IgG1 were increasing during immune response. However IgG2b and IgG3 were also induced, especially in mice immunised with the plant-derived antigen. Analysis of the isotype profile indicates mainly Th1 polarisation, but completed with Th2 response. Obtained results indicate a considerable potential of plant-derived HBcAg as a therapeutic vaccine, since a mixed immune response with a stronger Th1 component is particularly required for treatment of CHB.