In the development process for drugs used to treat skeletal muscle, cell‐based contractile force assays have been considered as a useful in vitro test. Immortalized human myogenic cells are promising as cell sources for reproducible and well‐characterized in vitro models. In this study, it is investigated whether immortalized human myogenic cells, Hu5/KD3, have suitable contractile ability and the potential to be used as cell sources for contractile force assays. Muscle tissues are fabricated using Hu5/KD3 cells on the microfabricated devices used to measure contractile force. The tissues generate a tetanic force of ≈30 µN in response to the electrical pulse stimulation (EPS). Gene expression analysis of the myosin heavy chain (MYH) isoform indicates that the tissues mostly consisted of muscle fibers expressing MYH7 or/and MYH8. The addition of dexamethasone or lovastatin decreases the contractile force of the tissues, indicating that the tissues have the potential to evaluate drug candidates designed to treat muscle atrophy or statin‐induced myopathy. It is also demonstrated that the contractile force of tissues increased when EPS is applied as an artificial exercise. These results indicate that the Hu5/KD3 tissues can be employed for contractile force assays and would be useful for in vitro human skeletal muscle models.
Human pluripotent stem cells, including human induced pluripotent stem cells (hiPSCs), are promising materials for regenerative medicine and cell transplantation therapy. However, tumorigenic potential of residual undifferentiated stem cells hampers their use in these therapies. Therefore, it is important to develop methods that selectively eliminate undifferentiated stem cells from a population of differentiated cells before their transplantation. In the present study, we investigated whether plasma-activated medium (PAM) selectively eliminated undifferentiated hiPSCs by inducing external oxidative stress. PAM was prepared by irradiating cell culture medium with non-thermal atmospheric pressure plasma. We observed that PAM selectively and efficiently killed undifferentiated hiPSCs cocultured with normal human dermal fibroblasts (NHDFs), which were used as differentiated cells. We also observed that undifferentiated hiPSCs were more sensitive to PAM than hiPSC-derived differentiated cells. Gene expression analysis suggested that lower expression of oxidative stress-related genes, including those encoding enzymes involved in hydrogen peroxide (H 2 O 2 ) degradation, in undifferentiated hiPSCs was one of the mechanisms underlying PAM-induced selective cell death. PAM killed undifferentiated hiPSCs more efficiently than a medium containing the same concentration of H 2 O 2 as that in PAM, suggesting that H 2 O 2 and various reactive oxygen/nitrogen species in PAM selectively eliminated undifferentiated hiPSCs. Thus, our results indicate that PAM has a great potential to eliminate tumorigenic hiPSCs from a population of differentiated cells and that it may be a very useful tool in regenerative medicine and cell transplantation therapy.
Engineered three-dimensional models of neuromuscular tissues are promising for use in mimicking their disorder states in vitro. Although several models have been developed, it is still challenging to mimic the...
Human pluripotent stem cells, including human induced pluripotent stem cells (hiPSCs), serve as highly valuable sources for both cell-based therapies and basic research, owing to their abilities to self-renew and differentiate into any cell type of the human body. However, tumorigenic risks of residual undifferentiated stem cells limit the clinical application of hiPSCs, necessitating methods to eliminate undifferentiated hiPSCs from differentiated cells. Here, we found that undifferentiated hiPSCs were more sensitive to the treatment with a medium supplemented with high concentration of L-alanine than human fibroblasts (hFBs), human skeletal muscle cells (hSkMCs), hiPSC-derived cardiomyocytes (iCMs) or hiPSC-derived fibroblast-like cells (iFLCs), which were used as differentiated cells. Undifferentiated hiPSCs co-cultured with differentiated cells were selectively eliminated following treatment. In addition, we found that the medium supplemented with high concentration of D-alanine or β-alanine also induced cell death of hiPSCs and the treatment at 4 °C didn’t induce cell death of hiPSCs. The cell death induced would be associated partly with high osmotic pressure of the medium supplemented with L-alanine. As L-alanine is a component of proteins in human body and popular ingredient of cell culture media, treatment with high concentration of L-alanine may be useful for eliminating tumorigenic residual hiPSCs for stem cell-based therapies.
Skeletal muscle atrophy is characterized by decreases in protein content, myofiber diameter, and contractile force generation. As muscle atrophy worsens the quality of life, the development of anti‐atrophic substances is desirable. In this study, we aimed to demonstrate a screening process for anti‐atrophic peptides using photo‐cleavable peptide array technology and human contractile atrophic muscle models. We developed a 96‐well system and established a screening process with less variability. Dexamethasone‐induced human atrophic tissue was constructed in the system. Eight peptides were selected from the literature and used for the screening of peptides for preventing the decrease of the contractile forces of tissues. The peptide QIGFIW, which showed preventive activity, was selected as the seed sequence. As a result of amino acid substitution, we obtained QIGFIQ as a peptide with higher anti‐atrophic activity. These results indicate that the combinatorial use of the photo‐cleavable peptide array technology and 96‐well screening system could comprise a powerful approach to obtaining anti‐atrophic peptides, and suggest that the 96‐well screening system and atrophic model represent a practical and powerful tool for the development of drugs/functional food ingredients.
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