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Whole body vibration (WBV) is a very widespread mechanical stimulus used in physical therapy, rehabilitation and fitness centres. It has been demonstrated that vibration induces improvements in muscular strength and performance and increases bone density. We investigated the effects of low-amplitude, high frequency vibration (HFV) at the cellular and tissue levels in muscle. We developed a system to produce vibrations adapted to test several parameters in vitro and in vivo. For in vivo experiments, we used newborn CD1 wild-type mice, for in vitro experiments, we isolated satellite cells from 6-day-old CD1 mice, while for proliferation studies, we used murine cell lines. Animals and cells were treated with high frequency vibration at 30 Hz. We analyzed the effects of mechanical stimulation on muscle hypertrophy/atrophy pathways, fusion enhancement of myoblast cells and modifications in the proliferation rate of cells. Results demonstrated that mechanical vibration strongly down-regulates atrophy genes both in vivo and in vitro. The in vitro experiments indicated that mechanical stimulation promotes fusion of satellite cells treated directly in culture compared to controls. Finally, proliferation experiments indicated that stimulated cells had a decreased growth rate compared to controls. We concluded that vibration treatment at 30 Hz is effective in suppressing the atrophy pathway both in vivo and in vitro and enhances fusion of satellite muscle cells.

article i nfo Several studies have demonstrated that tissue culture conditions influence the differentiation of human adipose-derived stem cells (hASCs). Recently, studies performed on SAOS-2 and bone marrow stromal cells (BMSCs) have shown the effectiveness of high frequency vibration treatment on cell differentiation to osteoblasts. The aim of this study was to evaluate the effects of low amplitude, high frequency vibrations on the differentiation of hASCs toward bone tissue. In view of this goal, hASCs were cultured in proliferative or osteogenic media and stimulated daily at 30 Hz for 45 min for 28 days. The state of calcification of the extracellular matrix was determined using the alizarin assay, while the expression of extracellular matrix and associated mRNA was determined by ELISA assays and quantitative RT-PCR (qRT-PCR). The results showed the osteogenic effect of high frequency vibration treatment in the early stages of hASC differentiation (after 14 and 21 days). On the contrary, no additional significant differences were observed after 28 days cell culture. Transmission Electron Microscopy (TEM) images performed on 21 day samples showed evidence of structured collagen fibers in the treated samples. All together, these results demonstrate the effectiveness of high frequency vibration treatment on hASC differentiation toward osteoblasts.

Production and isolation of forebrain interneuron progenitors are essential for understanding cortical development and developing cell-based therapies for developmental and neurodegenerative disorders. We demonstrate production of a population of putative calretinin-positive bipolar interneurons that express markers consistent with caudal ganglionic eminence identities. Using serum-free embryoid bodies (SFEBs) generated from human inducible pluripotent stem cells (iPSCs), we demonstrate that these interneuron progenitors exhibit morphological, immunocytochemical, and electrophysiological hallmarks of developing cortical interneurons. Finally, we develop a fluorescence-activated cell-sorting strategy to isolate interneuron progenitors from SFEBs to allow development of a purified population of these cells. Identification of this critical neuronal cell type within iPSC-derived SFEBs is an important and novel step in describing cortical development in this iPSC preparation.

In order to verify whether differentiation of adult stem cells toward bone tissue is promoted by high-frequency vibration (HFV), bone marrow stromal cells (BMSCs) were mechanically stimulated with HFV (30 Hz) for 45 minutes a day for 21 or 40 days. Cells were seeded in osteogenic medium, which enhances differentiation towards bone tissue. The effects of the mechanical treatment on differentiation were measured by Alizarin Red test, (q) real-time PCR, and protein content of the extracellular matrix. In addition, we analyzed the proliferation rate and apoptosis of BMSC subjected to mechanical stimulation. A strong increase in all parameters characterizing differentiation was observed. Deposition of calcium was almost double in the treated samples; the expression of genes involved in later differentiation was significantly increased and protein content was higher for all osteogenic proteins. Lastly, proliferation results indicated that stimulated BMSCs have a decreased growth rate in comparison with controls, but both treated and untreated cells do not enter the apoptosis process. These findings could reduce the gap between research and clinical application for bone substitutes derived from patient cells by improving the differentiation protocol for autologous cells and a further implant of the bone graft into the patient.

The present study aimed at verifying “in vivo” and “in vitro” the effects of mechanical vibrations on muscle development and on differentiation of satellite cells, the “stem cells” of muscle tissue. We realized a bioreactor composed by an eccentric motor which produces a displacement of 11 mm at frequencies between 1 and 120 Hz on a plate connected to the motor. On the plate we fixed a cage used for animals and the dishes for satellite cells and linear acceleration provoked by the motor to samples was measured. We used 30 Hz as stimulating frequency and we treated newborn mice from their birth for the next four weeks one 1h/day and satellite cells 1 and 4 days 1h/day always at 30 Hz. Every week we collected from a control and a treated mouse tibialis anterior muscles and we performed Western Blot analysis and quantitative Real-Time PCR (qRT-PCR) to investigate proteins and genes involved in hypertrophy and atrophy pathways of skeletal muscle. On satellite cells we studied some genes involved in differentiation and fusion of primary myoblasts. Results demonstrate that mechanical vibration induces muscle hypertrophy within the first week of treatment and enhances terminal differentiation of myoblasts of treated satellite cells respect to control ones.

The aim of this work is to test the effects of a specific mechanical stimulation (Low Amplitude, high Frequency Vibrations) on the bone matrix formation of hBMSCs. Previous studies demonstrated that chemical culture conditions could influence the differentiation of hBMSCs toward bone: by plating the cells in appropriate osteogenic culture medium hBMSCs differentiate into osteoblasts [1-3].

The aim of the work was to understand the consequences of low-amplitude, high-frequency vibrations on proliferation and differentiation of SAOS-2 cells (sarcoma osteogenetic), an osteoblastic and tumorigenic cell line. We realized a bioreactor composed of an eccentric motor that produces a displacement of 11 mm at frequencies between 1 and 120 Hz on a plate connected to the motor. The cultures of SAOS-2 cells were fixed on the plate, and the linear acceleration provoked by the motor to the cultures was measured. We used 30 Hz as stimulating frequency after a preliminary test on the effect of different frequencies on differentiation of cells. Afterward, SAOS-2 cells were stimulated with 30 Hz for different durations, every day for 4 days. The expression of some genes involved in the differentiation process was analyzed first with a reverse transcriptase-polymerase chain reaction and afterward with a real-time polymerase chain reaction on the most expressed genes. Moreover, the proliferation of cells was evaluated. The results suggest a strong increase in the expression of the genes involved in tissue differentiation in the treated groups with respect to the controls. On the other hand, the proliferation seems to be slowed down, so probably the acceleration perceived by the mechanosensors of the cells changes the cellular cycle by blocking the duplication to early differentiate toward bone tissue.

Many protocols have been designed to differentiate human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs) into neurons. Despite the relevance of electrophysiological properties for proper neuronal function, little is known about the evolution over time of important neuronal electrophysiological parameters in iPSC-derived neurons. Yet, understanding the development of basic electrophysiological characteristics of iPSC-derived neurons is critical for evaluating their usefulness in basic and translational research. Therefore, we analyzed the basic electrophysiological parameters of forebrain neurons differentiated from human iPSCs, from day 31 to day 55 after the initiation of neuronal differentiation. We assayed the developmental progression of various properties, including resting membrane potential, action potential, sodium and potassium channel currents, somatic calcium transients and synaptic activity. During the maturation of iPSC-derived neurons, the resting membrane potential became more negative, the expression of voltage-gated sodium channels increased, the membrane became capable of generating action potentials following adequate depolarization and, at day 48-55, 50% of the cells were capable of firing action potentials in response to a prolonged depolarizing current step, of which 30% produced multiple action potentials. The percentage of cells exhibiting miniature excitatory post-synaptic currents increased over time with a significant increase in their frequency and amplitude. These changes were associated with an increase of Ca2+ transient frequency. Co-culturing iPSC-derived neurons with mouse glial cells enhanced the development of electrophysiological parameters as compared to pure iPSC-derived neuronal cultures. This study demonstrates the importance of properly evaluating the electrophysiological status of the newly generated neurons when using stem cell technology, as electrophysiological properties of iPSC-derived neurons mature over time.