Generation of canine induced pluripotent stem cells under feeder-free conditions using Sendai virus vector encoding six canine reprogramming factors.

Although it is in its early stages, canine induced pluripotent stem cells (ciPSCs) hold great potential for innovative translational research in regenerative medicine, developmental biology, drug screening, and disease modeling. However, almost all ciPSCs were generated from fibroblasts, and available canine cell sources for reprogramming are still limited. Furthermore, no report is available to generate ciPSCs under feeder-free conditions because of their low reprogramming efficiency. Here, we reanalyzed canine pluripotency-associated genes and designed canine LIN28A, NANOG, OCT3/4, SOX2, KLF4, and C-MYC encoding Sendai virus vector, called 159cf. and 162cf. We demonstrated that not only canine fibroblasts but also canine urine-derived cells, which can be isolated using a noninvasive and straightforward method, were successfully reprogrammed with or without feeder cells. ciPSCs existed in undifferentiated states, differentiating into the three germ layers in vitro and in vivo. We successfully generated ciPSCs under feeder-free conditions, which can promote studies in veterinary and consequently human regenerative medicines.

Time-course colony tracking analysis for evaluating induced pluripotent stem cell culture processes.

Increasing the yield and maintaining a high quality of induced pluripotent stem cells (iPSCs) is necessary for manufacturing iPSCs at the industrial scale. However, because iPSCs are delicate, it is important to evaluate their quality during processing. To examine the status of cultured iPSCs non-invasively, morphology-based iPSC colony evaluation may be an efficient technology for cellular status monitoring and analysis. In this study, we examined the effectiveness of time-course colony tracking analysis for evaluating the iPSC culture process. Particularly, we obtained detailed time-course data to evaluate the effect of the pipetting technique on cell dissociation before seeding. Although the pipetting process causes severe shear stress to cells, which affects their quality, these effects have not been quantitatively analyzed because of their complex and uncontrollable parameters. By analyzing the heterogeneity and time-course responses of individual colonies, our colony tracking analysis revealed a critically damaged population caused by pipetting stress which could not be detected in conventional bulk analysis. Moreover, by comprehensively analyzing colony tracking data, which links the time-course morphology and marker staining results with each colony, we found that colony morphology is only highly correlated with the undifferentiated marker in the final stage, with a lower correlation in the early stages. Thus, colony tracking analysis provides a way to quantify cellular morphological information when evaluating complex iPSC manufacturing processes.

[Largest outbreak of legionellosis associated with spa baths: comparison of diagnostic tests].

In July 2002, a large outbreak of legionellosis occurred in a bathhouse with spa facilities in Miyazaki Prefecture. Among the visitors, 295, including suspected cases had pneumonia and/or symptoms of fever, coughing, etc. Of these, 37% were hospitalized and 7 died. Clinical samples from 95 mainly inpatients were collected and microbiologically tested at our laboratory. Legionella pneumophila serogroup (SG) 1 was isolated from 3 of 24 in sputum culture, and none of the 3 had been treated effectively with antibiotics at sputa collection. L. pneumophila antigen in urine was detected by using enzyme immunoassay and/or immunochromatographic kits in 23 of 75 patients. Serum antibodies to L. pneumophila SG1 and Legionella dumoffii were detected in 5 each of 66 patients--9 cases including a case at mixed infection-by microplate agglutination test and/or indirect immunofluorescence assay. At our laboratory, 32 were diagnosed with legionellosis. In this outbreak, 14 were diagnosed at other laboratories, resulting in 46 confirmed cases. Urine antigen was detected more frequently by Binax NOW immunochromatographic assay than by Biotest EIA-31% versus 16% of cases tested. Both assays detected urine antigen only in samples collected within 4 weeks after onset. Antigen concentration in urine enhanced sensitivity-58% and 51%-and extended the period of antigen detection beyond 5 weeks. Both antibody titers to L. pneumophila SG1 and L. dumoffii in more than 90% of sera collected within 3 weeks after onset were < 1:16. The rate of serum antibody titer to > or = 1:128 within 3 weeks was 1.6%, during 4 to 6 weeks less than 10%, and after 7 weeks or more 8 to 25%. After an administrative report was published, L. pneumophila DNA in sputa was detected in 5 of 17 patients by nested PCR, resulting in extra 3 cases. Altogether, urinary antigen detection and PCR were more effective in laboratory diagnostic tests than culture and serology. Culture combined with molecular epidemiology is critical, however, for confirming the source of infection.

Influence of the hypogonadism (hgn) locus on female reproduction and ovarian development in an altered genetic background.

Background and Aims: The hypogonadic rat (hgn/hgn) shows male sterility controlled by a single recessive gene hgn. The hgn/hgn females detected by the presence of renal hypoplasia in the HGN inbred strain show a reduced fertility. Recently, the gene responsible for male hypogonadism was mapped on chromosome 10 by a linkage analysis using only male backcross progeny. Because backcross females could not be categorized as affected or normal because of variations in their renal sizes, we could not examine female fertility in the backcross progeny. In the present experiment, we examined whether the gene mapped on rat chromosome 10 has any influences on female reproduction and ovarian development. Methods: The assumptive hgn/hgn females were identified in the backcross progeny by microsatellite markers closely linked to the hgn locus. Postnatal body growth, the weights of reproductive organs, estrus cycle and ovarian histology were examined. In addition, backcross embryos were genotyped, and their body growth and ovarian development was examined. Results: The hgn/hgn females showed growth retardation, a short reproductive life and an abnormal ovarian histology as adults. Regarding embryonic development, hgn/hgn females showed body growth retardation and ovarian hypoplasia. Conclusion: The mutation of the hgn mapped on chromosome 10 causes not only male sterility but also female reduced fertility related to ovarian hypoplasia beyond the altered genetic background. (Reprod Med Biol 2006; 5: 227-234).

[The largest outbreak of legionellosis in Japan associated with spa baths: epidemic curve and environmental investigation].

In July 2002, a large outbreak of legionellosis occurred in a bathhouse with spa facilities in Miyazaki Prefecture. Two hundred-ninety-five patients (including suspected cases) that had pneumonia and/or symptoms of fever, cough and so forth were reported; 37% of them were hospitalized and seven people died. In environmental investigations, Legionella pneumophila serogroups (SGs) land 8, L. dumoffii, L. londiniensis, some other Legionella species and many kinds of amoeba were isolated from 55 samples of bathtub water, tank water, filters and so forth in the spa facilities. The dominant isolates from the bathtab waters belonged to L. londiniensis, L. dumoffii and L. pneumophila SG1, and their maximum concentrations were 1.5 x 10(6), 5.2 x 10(5) and 1.6 x 10(5) cfu/100 mL, respectively. L. pneumophila SG1 strains isolated from bathtub water, tank water, filters and sputa of patients showed a indistinguishable DNA fingerprint pattern by pulsed-field gel electrophoresis (PFGE), confirming that the source of infection was the spa water. Our study indicate that spas may be a significant health hazard if hygienic management fails.