ABSTRACT
We studied the effects of a dual-vector DYSF gene delivery system based on adeno-associated virus serotype 9 capsids on pathological manifestations of dysferlinopathy in skeletal muscles of Bla/J mice lacking DYSF expression. The mice received intravenous injection of 3×1013 genomic copies of the virus containing the dual-vector system. M. gastrocnemius, m. psoas major, m. vastus lateralis, and m. gluteus superficialis were isolated for histological examination in 3, 6, and 12 weeks after treatment. Healthy wild-type (C57BL/6) mice served as positive control and were sacrificed 3 weeks after injection of 150 µl of 0.9% NaCl into the caudal vein. To detect dysferlin in muscle cryosections, immunohistochemical analysis with diagnostic antibodies was performed; paraffin sections were stained with hematoxylin and eosin for morphometric analysis. After administration of gene-therapeutic constructs, muscle fibers with membrane or cytoplasmic dysferlin location were detected in all examined muscles. The proportion of necrotic muscle fibers decreased, the number of muscle fibers with central location of the nucleus increased, and the mean cross-section area of the muscle fibers decreased.
Subject(s)
Muscle, Skeletal , Muscular Dystrophies, Limb-Girdle , Mice , Animals , Dysferlin/genetics , Dysferlin/metabolism , Mice, Inbred C57BL , Muscle, Skeletal/metabolism , Muscular Dystrophies, Limb-Girdle/genetics , Muscular Dystrophies, Limb-Girdle/therapy , Muscular Dystrophies, Limb-Girdle/metabolism , Muscle Fibers, Skeletal/metabolism , Gene Transfer TechniquesABSTRACT
Extracellular vesicles derived from mesenchymal stem cells (MSCs) represent a novel approach for regenerative and immunosuppressive therapy. Recently, cytochalasin B-induced microvesicles (CIMVs) were shown to be effective drug delivery mediators. However, little is known about their immunological properties. We propose that the immunophenotype and molecular composition of these vesicles could contribute to the therapeutic efficacy of CIMVs. To address this issue, CIMVs were generated from murine MSC (CIMVs-MSCs) and their cytokine content and surface marker expression determined. For the first time, we show that CIMVs-MSCs retain parental MSCs phenotype (Sca-1+, CD49e+, CD44+, CD45-). Also, CIMVs-MSCs contained a cytokine repertoire reflective of the parental MSCs, including IL-1ß, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12(p40), IL-13, IL-17, CCL2, CCL3, CCL4, CCL5, CCL11, G-CSF, GM-CSF and TNF-α. Next, we evaluated the immune-modulating properties of CIMVs-MSCs in vivo using standard preclinical tests. MSCs and CIMVs-MSCs reduced serum levels of anti-sheep red blood cell antibody and have limited effects on neutrophil and peritoneal macrophage activity. We compared the immunomodulatory effect of MSCs, CIMVs and EVs. We observed no immunosuppression in mice pretreated with natural EVs, whereas MSCs and CIMVs-MSCs suppressed antibody production in vivo. Additionally, we have investigated the biodistribution of CIMVs-MSCs in vivo and demonstrated that CIMVs-MSCs localized in liver, lung, brain, heart, spleen and kidneys 48 h after intravenous injection and can be detected 14 days after subcutaneous and intramuscular injection. Collectively our data demonstrates immunomodulatory efficacy of CIMVs and supports their further preclinical testing as an effective therapeutic delivery modality.
Subject(s)
Cell-Derived Microparticles/immunology , Cytochalasin B/pharmacology , Cytokines/immunology , Extracellular Vesicles/immunology , Immunosuppressive Agents/pharmacology , Macrophages, Peritoneal/immunology , Mesenchymal Stem Cells/immunology , Animals , Cell-Derived Microparticles/drug effects , Cells, Cultured , Extracellular Vesicles/drug effects , Macrophages, Peritoneal/cytology , Macrophages, Peritoneal/drug effects , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/drug effects , MiceABSTRACT
At present a wide variety of methods have been proposed to treat eye disorders, drug therapies are most commonly used. It should be noted that effective treatment modalities especially for degeneration of the retina and optic nerve are lacking. In the last few years stem cell transplantation has been proposed as an alternative method. The opportunities that stem cells provide within clinical use are almost unlimited. These cells are presently applied to treat various traumatic and degenerative disorders due to their unique biologic properties. Stem cells have high proliferative capabilities and are a self-maintained population of cells capable of differentiating into different cell types. Thus, they are represent a very primary stage of a cell lineage. Their ability to differentiate into different pathways provides animals with great plasticity in the renewal of somatic cells in postnatal ontogenesis. Pre-clinical and clinical ophthalmology studies where mesenchymal stem cells are applied and various methods of their administration are discussed herein. In addition the safety and efficacy of using bone marrow- and adipose tissue-derived mesenchymal stem cells have been discussed.
