Kenichiro Donai

Bovine and porcine fibroblasts can be immortalized with intact karyotype by the expression of mutant cyclin dependent kinase 4, cyclin D, and telomerase

Cattle and pigs comprise the most economically important livestock. Despite their importance, cultured cells from these species, which are useful for physiological analyses, are quite limited in cell banks. One of the reasons for the limited number of cell lines is the difficulty in their establishment. To overcome limitations in cell-line establishment, we attempted to immortalize bovine and porcine fibroblasts by transduction of multiple cell cycle regulators (mutant cyclin dependent kinase 4, cyclin D and telomerase reverse transcriptase). The transduced cells continued to display a stable proliferation rate and did not show cellular senescence. Furthermore, cell cycle assays showed that induction of these exogenous genes enhanced turnover of the cell cycle, especially at the G1-S phase. Furthermore, our established cell lines maintained normal diploid karyotypes at 98-100%. Our study demonstrated that bypassing p16/Rb-mediated cell arrest and activation of telomerase activity enabled efficient establishment of immortalized bovine- and porcine-derived fibroblasts. The high efficiency of establishing cell lines suggests that the networks of cell cycle regulators, especially p16/Rb-associated cell cycle arrest, have been conserved during evolution of humans, cattle, and pigs.

Coffee consumption delays the hepatitis and suppresses the inflammation related gene expression in the Long-Evans Cinnamon rat

BACKGROUND AND AIMS Large-scale epidemiological studies have shown that drinking more than two cups of coffee per day reduces the risks of hepatitis and liver cancer. However, the heterogeneity of the human genome requires studies of experimental animal models with defined genetic backgrounds to evaluate the coffee effects on liver diseases. We evaluated the efficacy of coffee consumption with one of experimental animal models for human disease. METHOD We used the Long Evans Cinnamon (LEC) rat, which onsets severe hepatitis and high incidence of liver cancer, due to the accumulation of copper and iron in livers caused by the genetic mutation in Atp7B gene, and leading to the continuous oxidative stress. We determined the expression of inflammation related genes, and amounts of copper and iron in livers, and incidence of the pre-neoplastic foci in the liver tissue of LEC rats. RESULTS Coffee administration for 25 weeks delayed the occurrence of hepatitis by two weeks, significantly improved survival, reduced the expression of inflammatory cytokines, and reduced the incidence of small pre-neoplastic liver foci in LEC rats. There was no significant difference in the accumulation of copper and iron in livers, indicating that coffee administration does not affect to the metabolism of these metals. These findings indicate that drinking coffee potentially prevents hepatitis and liver carcinogenesis through its anti-inflammatory effects. CONCLUSION This study showed the efficacy of coffee in the prevention of hepatitis and liver carcinogenesis in the LEC model.

Efficient establishment of primary fibroblast cultures from the hawksbill sea turtle (Eretmochelys imbricata)

The hawksbill sea turtle (Eretmochelys imbricata) is a critically endangered species at a risk of extinction. Preservation of the genomic and cellular information of endangered animals is important for future genetic and biological studies. Here, we report the efficient establishment of primary fibroblast cultures from skin tissue of the hawksbill sea turtle. We succeeded in establishing 19 primary cultures from 20 hawksbill sea turtle individuals (a success rate of 95%). These cells exhibited a fibroblast-like morphology and grew optimally at a temperature of 26°C, but experienced a loss of viability when cultured at 37°C. Chromosomal analysis using the primary cells derived here revealed that hawksbill sea turtles have a 2n = 56 karyotype. Furthermore, we showed that our primary cell cultures are free of several fish-related viruses, and this finding is important for preservation purposes. To our knowledge, this report is the first to describe primary cell cultures established from normal tissues of the hawksbill sea turtle. The results will contribute to the preservation of biodiversity, especially for the sea turtles that are critically endangered owing to human activities.

Establishment of Cell Lines Derived From the Genus Macaca Through Controlled Expression of Cell Cycle Regulators

Nonhuman primates are useful animal models for the study of human diseases. However, the number of established cell lines from nonhuman primates is quite limited compared with the number established from other experimental animals. The establishment of nonhuman primate cell lines would allow drug testing on those cell lines before moving experiments into primates. In this study, we established nonhuman primate primary cell lines by introducing the genes for CDK4R24C, cyclin D1, and hTERT. These cell lines proliferated more rapidly than primary cells and bypassed cellular senescence. Karyotype analysis showed that the chromosome patterns were intact in the immortalized cell lines. Furthermore, we showed that the expression of introduced genes could be precisely controlled through the Tet‐Off system with the addition of doxycycline. The present study shows that introduction of the CDK4R24C, cyclin D1, and hTERT genes are effective methods of establishing nonhuman primate cell lines. J. Cell. Biochem. 116: 205–211, 2015.

Establishment of a reporter system to monitor silencing status in induced pluripotent stem cell lines

Induced pluripotent stem (iPS) cells have proven to be an effective technology in regenerative medicine; however, the low efficiency of reprogramming is a major obstacle to the successful generation of iPS cell lines. One of the most important characteristics of a high-quality iPS cell line is the inactivation of transgenes driven by a retrovirus-derived long terminal repeat promoter. In this study, we established a novel marker system containing three kinds of proteins: secreted-type luciferase (MetLuc), copepod Pontellina plumata green fluorescent protein (copGFP), and an antibiotic-resistant gene product (Neo(r)). The introduction of MetLuc-copGFP-Neo(r) in mouse embryonic fibroblasts (MEFs) allowed us to monitor the reporter expression changes as an indicator of the state of silencing during reprogramming. Transformation of iPS cells induced a remarkable reduction in reporter activity, indicating that the retroviral silencing was detected successfully. Our system enables us to monitor the silencing status of transgenes and to efficiently select iPS cell lines that can be used for further applications.

Induced Pluripotent Stem Cells With Six Reprogramming Factors From Prairie Vole, Which Is an Animal Model for Social Behaviors.

Prairie voles show strong pair bonding with their mating partners, and they demonstrate parental behavior toward their infants, indicating that the prairie vole is a unique animal model for analysis of molecular mechanisms of social behavior. Until a recent study, the signaling pathway of oxytocin was thought to be critical for the social behavior of prairie voles, but neuron-specific functional research may be necessary to identify the molecular mechanisms of social behavior. Prairie vole pluripotent stem cells of high quality are essential to elucidate the molecular mechanisms of social behaviors. Generation of high-quality induced pluripotent stem cells (iPSCs) would help to establish a genetically modified prairie vole, including knockout and knock-in models, based on the pluripotency of iPSCs. Thus, we attempted to establish high-quality prairie vole-derived iPSCs (pv-iPSCs) in this study. We constructed a polycistronic reprogramming vector, which included six reprograming factors (Oct3/4, Sox2, Klf4, c-myc, Lin28, and Nanog). Furthermore, we evaluated the effect of six reprogramming factors, which included Oct3/4 with the transactivation domain (TAD) of MyoD. Implantation of the pv-iPSCs into immunodeficient mice caused a teratoma with three germ layers. Furthermore, the established pv-iPSCs tested positive for stem cell markers, including alkaline phosphatase activity (ALP), stage-specific embryonic antigen (SSEA)-1, and dependence on leukemia inhibitory factor (LIF). Our data indicate that our newly established pv-iPSCs may be a useful tool for genetic analysis of social behavior.

Establishment of an immortalized cell line derived from the prairie vole via lentivirus-mediated transduction of mutant cyclin-dependent kinase 4, cyclin D, and telomerase reverse transcriptase

The prairie vole (Microtus ochrogaster) shows social behaviors such as monogamy and parenting of infants with pair bonding. These social behaviors are specific to the prairie vole and have not been observed in other types of voles, such as mountain voles. Although the prairie vole has several unique characteristics, an in vitro cell culture system has not been established for this species. Furthermore, establishment of cultured cells derived from the prairie vole may be beneficial based on the three Rs (i.e., Replacement, Reduction, and Refinement) concept. Therefore, in this study, we attempted to establish an immortalized cell line derived from the prairie vole. Our previous research has shown that transduction with mutant forms of cyclin-dependent kinase 4 (CDK4), cyclin D, and telomerase reverse transcriptase (TERT) could efficiently immortalize cells from multiple species, including humans, cattle, pigs, and monkeys. Here, we introduced these three genes into prairie vole-derived muscle fibroblasts. The expression of mutant CDK4 and cyclin D proteins was confirmed by western blotting, and telomerase activity was detected in immortalized vole muscle-derived fibroblasts (VMF-K4DT cells or VMFs) by stretch PCR. Population doubling analysis showed that the introduction of mutant CDK4, cyclin D, and TERT extended the lifespan of VMFs. To the best of our knowledge, this is the first report describing the establishment of an immortalized cell line derived from the prairie vole through the expression of mutant CDK4, cyclin D, and human TERT.

Primary fibroblast cultures and karyotype analysis for the olive ridley sea turtle (Lepidochelys olivacea)

The number of sea turtles in the wild is decreasing because of human activities, such as fishery bycatch (Peckham et al. 2007), oil spills, and marine pollution (Witherington 2001). Furthermore, illegal hunting for their oil, meat, and shells are still continuing (Koch et al. 2013). Hence, protection measures are being implemented in various countries to conserve the endangered species of sea turtles. In the past few decades, a major concern regarding sea turtles in the wild has been hybridization. Interspecific hybridization has been reported in several areas of the world (Karl et al. 1995; Barber et al. 2003; Lara-Ruiz et al. 2006). The original sea turtle species, such as hawksbill, loggerhead, and olive ridley, had established as independent species for more than millions of years despite overlaps among their habitats (Bowen et al. 1993). However, recent reports on intensive hybridization among these species clearly indicate that these unique original species are at a high risk of extinction in the near future. Acquiring information about the basic genetic background of these species, such as karyotype, is necessary to understand the cause of this intensive hybridization. In this study, we established a primary culture from olive ridley sea turtles and determined the karyotype of the primary cells. Since 1976, the San Diego Zoo started the preservation of biological specimens derived from critically endangered species in order to facilitate the use of these specimens as research materials for the following generations; this project was named “Frozen Zoo.” The significance of this project was internationally recognized, and similar projects, such as the Frozen Ark project in the UK, are underway. The establishment of cell cultures from critically endangered animals might contribute to these cryopreservation projects by increasing the stability of cryopreserved materials and facilitating efficient expansion of cultured cells. Since cultured cells have intact genetic information of the critically endangered animals, the cells have a potential to be used for the genetic analysis of future generations. The olive ridley sea turtles were maintained at the Port of Nagoya Public Aquarium. Small (3×3 mm) dermal tissue biopsy specimens were obtained from the flipper-like fin of two olive ridley sea turtles. The tissue biopsy specimens were immediately immersed in the cell culture medium. The biopsy process was supervised by a veterinary doctor of the Port of Nagoya Public Aquarium. For the primary culture, a six-well cell culture dish was coated with type I collagen. The detailed method for collagen coating has been described in our previous study (Fukuda et al. 2012). The cell culture was maintained at 26°C under 5% CO2 in a humidified chamber T. Fukuda (*) :M. Katayama :K. Donai : T. Uchida : E. Isogai Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi Aoba-ku, Sendai 981-8555, Japan e-mail: tomofukuda@bios.tohoku.ac.jp

Software development for estimating the concentration of radioactive cesium in the skeletal muscles of cattle from blood samples

The 2011 earthquake severely damaged the Fukushima Daiichi Nuclear Power Plant (FNPP), resulting in the release of large quantities of radioactive material into the environment. The deposition of these radionuclides in rice straw as livestock feed led to the circulation of contaminated beef in the market. Based on the safety concern of the consumers, a reliable method for estimating concentrations of radioactive cesium in muscle tissue is needed. In this study, we analyzed the concentrations of radioactive cesium in the blood and skeletal muscle of 88 cattle, and detected a linear correlation between them. We then developed software that can be used to estimate radioactive cesium concentrations in muscle tissue from blood samples. Distribution of this software to the livestock production field would allow us to easily identify high-risk cattle, which would be beyond the safety regulation, before shipping out to the market. This software is planned to be released as freeware. This software would contribute to food safety, and aid the recovery of the livestock industry from the damage creacted by the 2011 Tohoku earthquake and tsunami.

Leptospiral lipopolysaccharide stimulates the expression of toll-like receptor 2 and cytokines in pig fibroblasts

Pigs throughout the world are afflicted with leptospirosis, causing serious economic losses and potential hazards to human health. Although it has been known that leptospiral lipopolysaccharide (L-LPS) is involved in an immunological reaction between an antigen and a host cell, little is known about how the immune system of pigs can respond to L-LPS. Here, we stimulated pig fibroblasts by L-LPS and then quantitatively measured gene and protein expression levels of two toll-like receptors (TLRs), TLR2 and TLR4, by real-time PCR and Western blotting. As a result, expression of TLR2 was found to be significantly up-regulated within 24 h after L-LPS stimulation whereas induction of TLR4 expression was relatively weak. We also revealed that of myeloid differentiation primary response gene 88 (MyD88), interleukin 6 (IL-6) and IL-8 gene expressions were markedly up-regulated by L-LPS stimulation. These results may suggest that the pig cell can activate TLR2 rather than TLR4 by L-LPS stimulation, thereby inducing expression of cytokines.