Mouse embryoid bodies (mEBs) were evaluated in detail on the basis of respiratory activity and high-throughput quantitative reverse transcription-PCR (RT-qPCR) analysis. The hanging drop culture method was applied to prepare various sizes of mEBs ranging from 100 to 250 μm in radius by causing the aggregation of embryonic cells. The respiratory activity of individual mEBs was noninvasively measured using scanning electrochemical microscopy in a cone-shaped microwell. For gene expression analysis, we used 48.48 Dynamic Array chips (Fluidigm) integrating microfluidic circuits, which allowed high-throughput qPCR analysis in parallel. The respiratory activity of the mEBs that were cultured for 1 to 6 days could predict the mRNA levels of undifferentiation and differentiation markers. However, the sizes of the mEBs could also predict the gene expression of the undifferentiation/differentiation markers because the radii of the mEBs increased by more than 2-fold after incubation in hanging drop culture for 6 days. Next, mEBs with identical sample sizes were evaluated for respiratory activity and gene expression. For mEBs cultured at 1500 cells per droplet for 3 days, the respiratory activity was negatively correlated with the mRNA levels of pluripotent markers such as Nanog and Sox2. Many differentiation markers were positively correlated with the respiratory activity. However, there was no significant difference in respiration activity between the beating and nonbeating samples on day 3. Finally, principal component analysis (PCA) confirmed the relationship between respiratory activity and the mRNA levels of undifferentiation/differentiation markers.

The cancer/testis antigen (CTA) genes were identified as human genes preferentially expressed in cancer cells and testis, but the contribution of CTAs to cancer and male germ cell development is unclear. In this study, we comprehensively examined mouse CTA functions and found that the majority of CTAs are involved in growth and/or survival of cancer cells. We focused on one mouse CTA gene, Tekt5, for its detailed functional analysis. Tekt5 knockdown (KD) in ovarian cancer cells caused G1 arrest and apoptosis, and p27kip1 was concomitantly upregulated. Tekt5 KD also resulted in decreased levels of acetylated α-tubulin and subsequent fragmentation of β-III-tubulin, upregulation of HDAC6 that deacetylates α-tubulin, and nuclear accumulation of SMAD3 that induces p27kip1 expression. Because depolymerization of tubulin is known to cause translocation of SMAD3 to the nucleus, these results together suggested that TEKT5 negatively regulates Hdac6 expression and consequently maintains cell cycle via stabilization of tubulin. We also found that the number of spermatids was significantly decreased and acetylated α-tubulin levels were decreased in vivo by KD of Tekt5 in testis. Because acetylated α-tubulin is required for sperm morphogenesis, these results suggest that TEKT5 is necessary for spermiogenesis via maintenance of acetylated α-tubulin levels.

Oxygen consumption rate (OCR) of mouse embryoid body (EB) was measured repeatedly in glucose-depleted medium or PBS solution. Non-invasive nature of the scanning electrochemical microscopy allowed sequential OCR measurement of the identical EB sample for more than 24 h. We found that the OCR value for the undifferentiated EB (cultured for 2 days) reached almost zero after 12 h exposure in PBS. On the contrary, the OCR value for the differentiated EB (cultured for 8 days) indicated 40 to 28% normal to the OCR at 0 h-incubation after 24 h exposure in PBS-based solution. It is suggested that differentiated cells indicated a stronger adaptation to glucose deprivation comparing to the undifferentiated cells.

The Ferritin heavy polypeptide-like 17 (Fthl17) gene is a member of the cancer/testis antigen gene family, and is preferentially expressed in cancer cells and in testis. Although DNA methylation has been linked to the regulation of human FTHL17 gene expression, detailed epigenetic regulation of its expression has not been investigated. To address this, we assessed the epigenetic regulation of murine Fthl17 gene expression in cancer cells and germ cells. Fthl17 was more highly expressed in testis, a murine lung cancer cell line, KLN205, and in germline stem cells (GSCs) than in normal lung tissues. Furthermore, the Fthl17 expression level in GSCs was significantly higher than in KLN205 cells. We performed bisulfite-sequencing and luciferase (luc) reporter assays to examine the role of DNA methylation of the Fthl17 promoter in the regulation of Fthl17 expression. In KLN205 cells, testis, and GSCs, the Fthl17 5’-upstream region was hypo-methylated compared with normal lung tissues. Luc reporter assays indicated that hypo-methylation of the -0.6 kb to 0 kb region upstream from the transcription start site (TSS) was involved in the up-regulation of Fthl17 expression in KLN205 cells and GSCs. Because the -0.6 kb to -0.3 kb or the -0.3 kb to 0 kb region were relatively more hypo-methylated in KLN205 cells and in GSCs, respectively, compared with other regions between -0.6 kb to 0 kb, those regions may contribute to Fthl17 up-regulation in each cell type. Following treatment with 5-Azacytidine, the -0.3 kb to 0 kb region became hypo-methylated, and Fthl17 expression was up-regulated in KLN205 cells to a level comparable to that in GSCs. Together, the results suggest that hypo-methylation of different but adjacent regions immediately upstream of the Fthl17 gene contribute to differential expression levels in lung cancer cells and GSCs, and hypo-methylation of the TSS-proximal region may be critical for high level expression.

The effective radius of a bulk-wave ultrasonic transducer as a circular piston source, fabricated on one end of a synthetic silica (SiO2) glass buffer rod, was evaluated for accurate velocity measurements of dispersive specimens over a wide frequency range. The effective radius was determined by comparing measured and calculated phase variations due to diffraction in an ultrasonic transmission line of the SiO2 buffer rod/water-couplant/SiO2 standard specimen, using radio-frequency (RF) tone burst ultrasonic waves. Fourteen devices with different device parameters were evaluated. The velocities of the nondispersive standard specimen (C-7940) were found to be 5934.10 +/- 0.35 m/s at 70 to 290 MHz, after diffraction correction using the nominal radius (0.75 mm) for an ultrasonic device with an operating center frequency of about 400 MHz. Corrected velocities were more accurately found to be 5934.15 +/- 0.03 m/s by using the effective radius (0.780 mm) for the diffraction correction. Bulk-wave ultrasonic devices calibrated by this experimental procedure enable conducting extremely accurate velocity dispersion measurements.