Hyperthermia has been studied as a noninvasive cancer treatment. Cancer cells show stronger thermal cytotoxicity than normal cells, which is exploited in hyperthermia. However, the absence of methods evaluating the thermal cytotoxicity in cells prevents the development of hyperthermia. To investigate the thermal cytotoxicity, culture temperature should be regulated. We, thus, developed a culture system regulating culture temperature immediately and accurately by employing metallic culture vessels. Michigan Cancer Foundation-7 cells and normal human dermal fibroblasts were used for models of cancer and normal cells. The findings showed cancer cells showed stronger thermal cytotoxicity than normal cells, which is quantitatively different from previous reports. This difference might be due to regulated culture temperature. The thermal stimulus condition (43 °C/30 min) was, further, focused for assays. The mRNA expression involving apoptosis changed dramatically in cancer cells, indicating the strong apoptotic trend. In contrast, the mRNA expression of heat shock protein (HSP) of normal cells upon the thermal stimulus was stronger than cancer cells. Furthermore, exclusively in normal cells, HSP localization to nucleus was confirmed. These movement of HSP confer thermotolerance to cells, which is consistent with the different thermal cytotoxicity between cancer and normal cells. In summary, our developed system can be used to develop hyperthermia treatment.

Alteration of the nuclear Ca2+ transient is an early event in cardiac remodeling. Regulation of the nuclear Ca2+ transient is partly independent of the cytosolic Ca2+ transient in cardiomyocytes. One nuclear membrane protein, emerin, is encoded by EMD, and an EMD mutation causes Emery-Dreifuss muscular dystrophy (EDMD). It remains unclear whether emerin is involved in nuclear Ca2+ homeostasis. The aim of this study is to elucidate the role of emerin in rat cardiomyocytes by means of hypertrophic stimuli and in EDMD induced pluripotent stem (iPS) cell-derived cardiomyocytes in terms of nuclear structure and the Ca2+ transient. The cardiac hypertrophic stimuli increased the nuclear area, decreased nuclear invagination, and increased the half-decay time of the nuclear Ca2+ transient in cardiomyocytes. Emd knockdown cardiomyocytes showed similar properties after hypertrophic stimuli. The EDMD-iPS cell-derived cardiomyocytes showed increased nuclear area, decreased nuclear invagination, and increased half-decay time of the nuclear Ca2+ transient. An autopsied heart from a patient with EDMD also showed increased nuclear area and decreased nuclear invagination. These data suggest that Emerin plays a crucial role in nuclear structure and in the nuclear Ca2+ transient. Thus, emerin and the nuclear Ca2+ transient are possible therapeutic targets in heart failure and EDMD.

Accumulating evidence suggests that human pluripotent stem cell-derived cardiomyocytes can affect "heart regeneration", replacing injured cardiac scar tissue with concomitant electrical integration. However, electrically coupled graft cardiomyocytes were found to innately induce transient post-transplant ventricular tachycardia in recent large animal model transplantation studies. We hypothesised that these phenomena were derived from alterations in the grafted cardiomyocyte characteristics. In vitro experiments showed that human embryonic stem cell-derived cardiomyocytes (hESC-CMs) contain nodal-like cardiomyocytes that spontaneously contract faster than working-type cardiomyocytes. When transplanted into athymic rat hearts, proliferative capacity was lower for nodal-like than working-type cardiomyocytes with grafted cardiomyocytes eventually comprising only relatively matured ventricular cardiomyocytes. RNA-sequencing of engrafted hESC-CMs confirmed the increased expression of matured ventricular cardiomyocyte-related genes, and simultaneous decreased expression of nodal cardiomyocyte-related genes. Temporal engraftment of electrical excitable nodal-like cardiomyocytes may thus explain the transient incidence of post-transplant ventricular tachycardia, although further large animal model studies will be required to control post-transplant arrhythmia.

Proteinases that digest the extracellular matrix are usually used to harvest cells from culture vessels in a general culture process, which lowers the initial adhesion rate in regenerative medicine. Cell sheet engineering is one of the most important technologies in this field, especially for transplantation, because fabricated cell sheets have rich extracellular matrixes providing strong initial adhesion. Current cell sheet fabrication relies on temperature-responsive polymer-coated dishes. Cells are cultured on such specialized dishes and subjected to low temperature. Thus, we developed a simple but versatile cell sheet fabrication method using ubiquitous culture dishes/flasks without any coating or temperature modulation. Confluent mouse myoblasts (C2C12 cell line) were exposed to ultrasonic vibration from underneath and detached as cell sheets from entire culture surfaces. Because of the absence of low temperature, cell metabolism was statically increased compared with the conventional method. Furthermore, viability, morphology, protein expression, and mRNA expression were normal. These analyses indicated no side effects of ultrasonic vibration exposure. Therefore, this novel method may become the standard for cell sheet fabrication. Our method can be easily conducted following a general culture procedure with a typical dish/flask, making cell sheets more accessible to medical experts.

Clinical application of human induced pluripotent stem cells (hiPSCs) has been hampered by the lack of a practical, scalable culture system. Stacked culture plates (SCPs) have recently attracted attention. However, final cell yields depend on the efficiency of cell detachment, and inefficient cell recovery from SCPs presents a major challenge to their use. We have developed an effective detachment method using resonance vibrations (RVs) of substrates with sweeping driving frequency. By exciting RVs that have 1-3 antinodes with ultra-low-density enzyme spread on each substrate of SCPs, 87.8% of hiPSCs were successfully detached from a 5-layer SCP compared to 30.8% detached by the conventional enzymatic method. hiPSC viability was similar after either method. Moreover, hiPSCs detached by the RV method maintained their undifferentiated state. Additionally, hiPSCs after long-term culture (10 passages) kept excellent detachment efficiency, had the normal karyotypes, and maintained the undifferentiated state and pluripotency. These results indicated that the RV method has definite advantages over the conventional enzymatic method in the scalable culture of hiPSCs using SCPs.