Contractile Activity of Myotubes Derived from Human Induced Pluripotent Stem Cells: A Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a genetic disorder that results from deficiency of the dystrophin protein. In recent years, DMD pathological models have been created using induced pluripotent stem (iPS) cells derived from DMD patients. In addition, gene therapy using CRISPR-Cas9 technology to repair the dystrophin gene has been proposed as a new treatment method for DMD. However, it is not known whether the contractile function of myotubes derived from gene-repaired iPS cells can be restored. We therefore investigated the maturation of myotubes in electrical pulse stimulation culture and examined the effect of gene repair by observing the contractile behaviour of myotubes. The contraction activity of myotubes derived from dystrophin-gene repaired iPS cells was improved by electrical pulse stimulation culture. The iPS cell method used in this study for evaluating muscle contractile activity is a useful technique for analysing the mechanism of hereditary muscular disease pathogenesis and for evaluating the efficacy of new drugs and gene therapy.

Miniaturized skeletal muscle tissue fabrication for measuring contractile activity.

The contractile function of skeletal muscle is essential for maintaining the vital activity of life. Muscular diseases such as muscular dystrophy severely compromise the quality of life of patients and ultimately lead to death. There is therefore an urgent need to develop therapeutic agents for these diseases. In a previous study, we showed that three-dimensional skeletal muscle tissues fabricated using the magnetic force-based tissue engineering technique exhibited contractile activity, and that drug effects could be evaluated based on the contractile activity of the skeletal muscle tissues. However, the reported method requires a large number of cells and the tissue preparation procedure is complex. It is therefore necessary to improve the tissue preparation method. In this study, a miniature device made of polydimethylsiloxane was used to simplify the production of contracting skeletal muscle tissues applicable to high-throughput screening. The effects of model drugs on the contractile force generation of skeletal muscle tissues prepared from mouse C2C12 myoblast and human induced pluripotent stem cells were evaluated using the miniature muscle device. The results indicated that the muscle device system could provide a useful tool for drug screening.

Magnetic heating of nanoparticles as a scalable cryopreservation technology for human induced pluripotent stem cells.

Scale-up of production is needed for industrial applications and clinical translation of human induced pluripotent stem cells (hiPSCs). However, in cryopreservation of hiPSCs, successful rewarming of vitrified cells can only be achieved by convective warming of small volumes (generally 0.2 mL). Here, we present a scalable nano-warming technology for hiPSC cryopreservation employing inductive heating of magnetic nanoparticles under an alternating magnetic field. The conventional method by water bath heating at 37 °C resulted in a decrease of cell viability owing to devitrification caused by slow warming of samples with large volumes (≥ 20 mL). Nano-warming showed uniform and rapid rewarming of vitrified samples and improved viability of hiPSCs in the 20-mL system. In addition to single cells, hiPSC aggregates prepared using a bioreactor-based approach were successfully cryopreserved by the nano-warming technique. These results demonstrate that nano-warming is a promising methodology for cryopreservation in mass production of hiPSCs.

Novel neuromuscular junction model in 2D and 3D myotubes co-cultured with induced pluripotent stem cell-derived motor neurons.

Motor neurons differentiated from induced pluripotent stem (iPS) cells have attracted attention for use in the construction of drug screening systems for neuronal diseases, such as amyotrophic lateral sclerosis. However, conventional drug screening systems using 2-dimensional (2D) cultures of iPS cell-derived motor neurons often evaluate the cell survival rate, morphological changes in the cells and/or gene expression analysis, and these parameters do not always reflect the actual functions of motor neurons, i.e., the induction of muscle contractions. In the present study, we developed a neuromuscular junction model comprising motor neurons and myotubes, which were differentiated from iPS cells and C2C12 myoblasts, respectively. Using this model, the contractile activity and force generation of the myotubes via the neuromuscular junction were successfully measured in both two- and three-dimensional (3D) cell culture systems. The results suggested that this neuromuscular junction model can be used to construct a drug candidate screening system for neuronal diseases.

Neural differentiation of mouse induced pluripotent stem cells using cadherin gene-engineered PA6 feeder cells.

Investigating neural differentiation of pluripotent stem cells, including induced pluripotent stem (iPS) cells, is of importance for studying early neural development and providing a potential source of cells for nerve regeneration. Stromal cell-derived inducing activity (SDIA) using PA6 stromal cells promotes neural differentiation of iPS cells. Thus, we hypothesized that cadherin gene-engineered PA6 feeder cells will enhance the performance of SDIA by facilitating cell-cell interactions. Consequently, we created cadherin gene-engineered PA6 cells. Efficiency of neural differentiation from mouse iPS cells on PA6 feeder cells overexpressing E-cadherin gene (46%) or N-cadherin gene (27%) was significantly higher compared with parental PA6 feeder cells (19%). In addition, efficiency of motor neuron differentiation from mouse iPS cells on cadherin-gene engineered feeder cells (E-cadherin, 7.4%; N-cadherin, 11%) was significantly higher compared with parental PA6 feeder cells (4.1%). Altogether, these results indicate that cadherin gene-engineered feeder cells are a potent tool for promoting neural differentiation of pluripotent stem cells.