Biocompatibility assessment of sub-5 nm silica-coated superparamagnetic iron oxide nanoparticles in human stem cells and in mice for potential application in nanomedicine.

Ultrasmall superparamagnetic iron oxide nanoparticles with a size <5 nm are emerging nanomaterials for their excellent biocompatibility, chemical stability, and tunable surface modifications. The applications explored include dual-modal or multi-modal imaging, drug delivery, theranostics and, more recently, magnetic resonance angiography. Good biocompatibility and biosafety are regarded as the preliminary requirements for their biomedical applications and further exploration in this field is still required. We previously synthesized and characterized ultrafine (average core size of 3 nm) silica-coated superparamagnetic iron oxide fluorescent nanoparticles, named sub-5 SIO-Fl, uniform in size, shape, chemical properties and composition. The cellular uptake and in vitro biocompatibility of the as-synthesized nanoparticles were demonstrated in a human colon cancer cellular model. Here, we investigated the biocompatibility of sub-5 SIO-Fl nanoparticles in human Amniotic Mesenchymal Stromal/Stem Cells (hAMSCs). Kinetic analysis of cellular uptake showed a quick nanoparticle internalization in the first hour, increasing over time and after long exposure (48 h), the uptake rate gradually slowed down. We demonstrated that after internalization, sub-5 SIO-Fl nanoparticles neither affect hAMSC growth, viability, morphology, cytoskeletal organization, cell cycle progression, immunophenotype, and the expression of pro-angiogenic and immunoregulatory paracrine factors nor the osteogenic and myogenic differentiation markers. Furthermore, sub-5 SIO-Fl nanoparticles were intravenously injected into mice to investigate the in vivo biodistribution and toxicity profile for a time period of 7 weeks. Our findings showed an immediate transient accumulation of nanoparticles in the kidney, followed by the liver and lungs, where iron contents increased over a 7-week period. Histopathology, hematology, serum pro-inflammatory response, body weight and mortality studies demonstrated a short- and long-term biocompatibility and biosafety profile with no apparent acute and chronic toxicity caused by these nanoparticles in mice. Overall, these results suggest the feasibility of using sub-5 SIO-Fl nanoparticles as a promising agent for stem cell magnetic targeting as well as for diagnostic and therapeutic applications in oncology.

Complete Assessment of Multilineage Differentiation Potential of Human Skeletal Muscle-Derived Mesenchymal Stem/Stromal Cells.

The minimal criteria for mesenchymal stem/stromal cell (MSC) identification set by the International Society for Cellular Therapy include plastic adherence, presence and absence of a set of surface antigens and in vitro multilineage differentiation. This differentiation is assessed through stimulation of MSCs with defined combination and concentration of growth factors towards specific lineages and histological confirmation of the presence of differentiated cells. Here we provide protocols for multilineage differentiation, namely, osteogenesis, adipogenesis, chondrogenesis and myogenesis. We also provide their respective histological analyses.

A novel in vitro model for the assessment of postnatal myonuclear accretion.

BACKGROUND: Due to the post-mitotic nature of myonuclei, postnatal myogenesis is essential for skeletal muscle growth, repair, and regeneration. This process is facilitated by satellite cells through proliferation, differentiation, and subsequent fusion with a pre-existing muscle fiber (i.e., myonuclear accretion). Current knowledge of myogenesis is primarily based on the in vitro formation of syncytia from myoblasts, which represents aspects of developmental myogenesis, but may incompletely portray postnatal myogenesis. Therefore, we aimed to develop an in vitro model that better reflects postnatal myogenesis, to study the cell intrinsic and extrinsic processes and signaling involved in the regulation of postnatal myogenesis. METHODS: Proliferating C2C12 myoblasts were trypsinized and co-cultured for 3 days with 5 days differentiated C2C12 myotubes. Postnatal myonuclear accretion was visually assessed by live cell time-lapse imaging and cell tracing by cell labeling with Vybrant® DiD and DiO. Furthermore, a Cre/LoxP-based cell system was developed to semi-quantitatively assess in vitro postnatal myonuclear accretion by the conditional expression of luciferase upon myoblast-myotube fusion. Luciferase activity was assessed luminometrically and corrected for total protein content. RESULTS: Live cell time-lapse imaging, staining-based cell tracing, and recombination-dependent luciferase activity, showed the occurrence of postnatal myonuclear accretion in vitro. Treatment of co-cultures with the myogenic factor IGF-I (p < 0.001) and the cytokines IL-13 (p < 0.05) and IL-4 (p < 0.001) increased postnatal myonuclear accretion, while the myogenic inhibitors cytochalasin D (p < 0.001), myostatin (p < 0.05), and TNFα (p < 0.001) decreased postnatal myonuclear accretion. Furthermore, postnatal myonuclear accretion was increased upon recovery from electrical pulse stimulation-induced fiber damage (p < 0.001) and LY29004-induced atrophy (p < 0.001). Moreover, cell type-specific siRNA-mediated knockdown of myomaker in myoblasts (p < 0.001), but not in myotubes, decreased postnatal myonuclear accretion. CONCLUSIONS: We developed a physiologically relevant, sensitive, high-throughput cell system for semi-quantitative assessment of in vitro postnatal myonuclear accretion, which can be used to mimic physiological myogenesis triggers, and can distinguish the cell type-specific roles of signals and responses in the regulation of postnatal myogenesis. As such, this method is suitable for both basal and translational research on the regulation of postnatal myogenesis, and will improve our understanding of muscle pathologies that result from impaired satellite cell number or function.