Accelerated generation of human induced pluripotent stem cells with retroviral transduction and chemical inhibitors under physiological hypoxia.

Induced pluripotent stem (iPS) cells are generated from somatic cells by the forced expression of a defined set of pluripotency-associated transcription factors. Human iPS cells can be propagated indefinitely, while maintaining the capacity to differentiate into all cell types in the body except for extra-embryonic tissues. This technology not only represents a new way to use individual-specific stem cells for regenerative medicine but also constitutes a novel method to obtain large amounts of disease-specific cells for biomedical research. Despite their great potential, the long reprogramming process (up to 1month) remains one of the most significant challenges facing standard virus-mediated methodology. In this study, we report the accelerated generation of human iPS cells from adipose-derived stem (ADS) cells, using a new combination of chemical inhibitors under a setting of physiological hypoxia in conjunction with retroviral transduction of Oct4, Sox2, Klf4, and L-Myc. Under optimized conditions, we observed human embryonic stem (ES)-like cells as early as 6 days after the initial retroviral transduction. This was followed by the emergence of fully reprogrammed cells bearing Tra-1-81-positive and DsRed transgene-silencing properties on day 10. The resulting cell lines resembled human ES cells in many respects including proliferation rate, morphology, pluripotency-associated markers, global gene expression patterns, genome-wide DNA methylation states, and the ability to differentiate into all three of the germ layers, both in vitro and in vivo. Our method, when combined with chemical inhibitors under conditions of physiological hypoxia, offers a powerful tool for rapidly generating bona fide human iPS cells and facilitates the application of iPS cell technology to biomedical research.

Use of adipose tissue-derived stromal cells for prevention of esophageal stricture after circumferential EMR in a canine model.

BACKGROUND: EMR is an accepted treatment for early esophageal carcinoma. However, resection of a large mucosal area often causes postoperative esophageal stricture. OBJECTIVE: To investigate the efficacy of autologous adipose tissue-derived stromal cells (ADSCs) for prevention of stricture formation after EMR in dogs. DESIGN: Animal study. SETTING: University research center. INTERVENTION: Ten beagle dogs were randomized into a control group and an ADSCs-injected (ADSC) group. The ADSCs were isolated from autologous adipose tissue. Immediately after circumferential esophageal EMR, about 5 × 10(6) ADSCs suspended in 8 mL of phosphate-buffered saline solution were injected endoscopically into the residual submucosa of the ADSC group, whereas the control group received only 8 mL of phosphate-buffered saline solution. MAIN OUTCOME MEASUREMENTS: Dysphagia score, weight loss, rate of mucosal constriction, and histologic assessments. RESULTS: In the control and ADSC groups, the median dysphagia scores were 4 and 1 (P < .043), the mean degrees of mucosal constriction were 75.7% and 45.3% (P < .008), and the numbers of nascent microvessels in the submucosal layer were 7.4 and 16.2 per unit area (P = .007), respectively. Atrophy and fibrosis of the muscularis propria layer were observed in the control group. LIMITATIONS: Animal study, small sample size. CONCLUSION: Injection therapy with autologous ADSCs suppresses constriction of the esophageal mucosa and improves clinical symptoms after circumferential EMR in this canine model.

Observation of the esophagus, pharynx and lingual root by gastrointestinal endoscopy with a percutaneous retrograde approach.

AIM: To evaluate the efficacy of retrograde observation of the esophagus, pharynx, larynx and lingual root. METHODS: With the beagle dog under anesthesia, the anterior wall of the stomach was fixed on the abdominal wall in a similar way to percutaneous endoscopic gastrostomy. The gastrointestinal scope was inserted via a 12 mm laparoscopic port for subsequent retrograde observation from stomach to the oral cavity. RESULTS: With this technique, direct observation of gastric cardia was possible without restriction. The cervical esophagus was dilated well, also allowing clear observation of the hypopharyngo-esophageal junction. If the tongue was manually pulled out forward, observation of the lingual root was possible. CONCLUSION: This procedure is easy and effective for pre-treatment evaluation of the feasibility of endoscopic resection in cases of superficial carcinoma of head and neck.

Nucling recruits Apaf-1/pro-caspase-9 complex for the induction of stress-induced apoptosis.

Nucling is a novel protein isolated from murine embryonal carcinoma cells with an up-regulated expression during cardiac muscle differentiation. We show here that Nucling was up-regulated by proapoptotic stimuli and important for the induction of apoptosis after cytotoxic stress. We further demonstrated that overexpressed Nucling was able to induce apoptosis. In Nucling-deficient cells, the expression levels of Apaf-1 and cytochrome c, which are the major components of an apoptosis-promoting complex named apoptosome, were both down-regulated under cellular stress. A deficiency of Nucling also conferred resistance to apoptotic stress on the cell. After UV irradiation, Nucling was shown to reside in an Apaf-1/pro-caspase-9 complex, suggesting that Nucling might be a key molecule for the formation and maintenance of this complex. Nucling induced translocation of Apaf-1 to the nucleus, thereby distributing the Nucling/Apaf-1/pro-caspase-9 complex to the nuclear fraction. These findings suggest that Nucling recruits and transports the apoptosome complex during stress-induced apoptosis.