119 research outputs found

    Human Induced Pluripotent Stem Cells on Autologous Feeders

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    BACKGROUND: For therapeutic usage of induced Pluripotent Stem (iPS) cells, to accomplish xeno-free culture is critical. Previous reports have shown that human embryonic stem (ES) cells can be maintained in feeder-free condition. However, absence of feeder cells can be a hostile environment for pluripotent cells and often results in karyotype abnormalities. Instead of animal feeders, human fibroblasts can be used as feeder cells of human ES cells. However, one still has to be concerned about the existence of unidentified pathogens, such as viruses and prions in these non-autologous feeders. METHODOLOGY/PRINCIPAL FINDINGS: This report demonstrates that human induced Pluripotent Stem (iPS) cells can be established and maintained on isogenic parental feeder cells. We tested four independent human skin fibroblasts for the potential to maintain self-renewal of iPS cells. All the fibroblasts tested, as well as their conditioned medium, were capable of maintaining the undifferentiated state and normal karyotypes of iPS cells. Furthermore, human iPS cells can be generated on isogenic parental fibroblasts as feeders. These iPS cells carried on proliferation over 19 passages with undifferentiated morphologies. They expressed undifferentiated pluripotent cell markers, and could differentiate into all three germ layers via embryoid body and teratoma formation. CONCLUSIONS/SIGNIFICANCE: These results suggest that autologous fibroblasts can be not only a source for iPS cells but also be feeder layers. Our results provide a possibility to solve the dilemma by using isogenic fibroblasts as feeder layers of iPS cells. This is an important step toward the establishment of clinical grade iPS cells

    Expression dynamics of HAND1/2 in in vitro human cardiomyocyte differentiation

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    転写因子HAND1とHAND2の発現パターンと役割の解明 --ヒトiPS細胞から増殖能の高い心筋細胞を回収する--. 京都大学プレスリリース. 2021-07-27.Three genes determine heart cell growth. 京都大学プレスリリース. 2021-07-27.Hand1 and Hand2 are transcriptional factors, and knockout mice of these genes show left and right ventricular hypoplasia, respectively. However, their function and expression in human cardiogenesis are not well studied. To delineate their expressions and assess their functions in human cardiomyocytes (CMs) in vitro, we established two triple-reporter human induced pluripotent stem cell lines that express HAND1[mCherry], HAND2[EGFP] and either MYH6-driven iRFP670 or tagBFP constitutively and investigated their expression dynamics during cardiac differentiation. On day 5 of the differentiation, HAND1 expression marked cardiac progenitor cells. We profiled the CM subpopulations on day 20 with RNA sequencing and found that mCherry+ CMs showed higher proliferative ability than mCherry− CMs and identified a gene network of LEF1, HAND1, and HAND2 to regulate proliferation in CMs. Finally, we identified CD105 as a surface marker of highly proliferative CMs

    Short-term clinicopathological outcome of neoadjuvant chemohormonal therapy comprising complete androgen blockade, followed by treatment with docetaxel and estramustine phosphate before radical prostatectomy in Japanese patients with high-risk localized prostate cancer

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    <p>Abstract</p> <p>Background</p> <p>To assess the outcome of neoadjuvant chemohormonal therapy comprising complete androgen blockade followed by treatment with docetaxel and estramustine phosphate before radical prostatectomy in Japanese patients with a high risk of localized prostate cancer (PCa).</p> <p>Methods</p> <p>Complete androgen blockade followed by 6 cycles of docetaxel (30 mg/m<sup>2</sup>) with estramustine phosphate (560 mg) were given to 18 PCa patients before radical prostatectomy. Subsequently, the clinical and pathological outcomes were analyzed.</p> <p>Results</p> <p>No patients had severe adverse events during chemohormonal therapy, and hence they were treated with radical prostatectomy. Two patients (11.1%) achieved pathological complete response. Surgical margins were negative in all patients. At a median follow-up of 18 months, 14 patients (77.8%) were disease-free without PSA recurrence. All 4 patients with PSA recurrence had pathologic T3b or T4 disease and 3 of these 4 patients had pathologic N1 disease.</p> <p>Conclusion</p> <p>We found that neoadjuvant chemohormonal therapy with complete androgen blockade followed by treatment with docetaxel and estramustine phosphate before radical prostatectomy was safe, feasible, and associated with favorable pathological outcomes in patients with a high risk of localized PCa.</p

    Dynamic Changes in Ultrastructure of the Primary Cilium in Migrating Neuroblasts in the Postnatal Brain

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    New neurons, referred to as neuroblasts, are continuously generated in the ventricular-subventricular zone of the brain throughout an animal's life. These neuroblasts are characterized by their unique potential for proliferation, formation of chain-like cell aggregates, and long-distance and high-speed migration through the rostral migratory stream (RMS) toward the olfactory bulb (OB), where they decelerate and differentiate into mature interneurons. The dynamic changes of ultrastructural features in postnatal-born neuroblasts during migration are not yet fully understood. Here we report the presence of a primary cilium, and its ultrastructural morphology and spatiotemporal dynamics, in migrating neuroblasts in the postnatal RMS and OB. The primary cilium was observed in migrating neuroblasts in the postnatal RMS and OB in male and female mice and zebrafish, and a male rhesus monkey. Inhibition of intraflagellar transport molecules in migrating neuroblasts impaired their ciliogenesis and rostral migration toward the OB. Serial section transmission electron microscopy revealed that each migrating neuroblast possesses either a pair of centrioles or a basal body with an immature or mature primary cilium. Using immunohistochemistry, live imaging, and serial block-face scanning electron microscopy, we demonstrate that the localization and orientation of the primary cilium are altered depending on the mitotic state, saltatory migration, and deceleration of neuroblasts. Together, our results highlight a close mutual relationship between spatiotemporal regulation of the primary cilium and efficient chain migration of neuroblasts in the postnatal brain

    Second nationwide surveillance of bacterial pathogens in patients with acute uncomplicated cystitis conducted by Japanese Surveillance Committee from 2015 to 2016: antimicrobial susceptibility of Escherichia coli, Klebsiella pneumoniae, and Staphylococcus saprophyticus

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    The Japanese Surveillance Committee conducted a second nationwide surveillance of antimicrobial susceptibility patterns of uropathogens responsible for acute uncomplicated cystitis (AUC) in premenopausal patients aged 16–40 years old at 31 hospitals throughout Japan from March 2015 to February 2016. In this study, the susceptibility of causative bacteria (Escherichia coli, Klebsiella pneumoniae, Staphylococcus saprophyticus) for various antimicrobial agents was investigated by isolation and culturing of organisms obtained from urine samples. In total, 324 strains were isolated from 361 patients, including E. coli (n = 220, 67.9%), S. saprophyticus (n = 36, 11.1%), and K. pneumoniae (n = 7, 2.2%). The minimum inhibitory concentrations (MICs) of 20 antibacterial agents for these strains were determined according to the Clinical and Laboratory Standards Institute (CLSI) manual. At least 93% of the E. coli isolates showed susceptibility to fluoroquinolones and cephalosporins, whereas 100% of the S. saprophyticus isolates showed susceptibility to fluoroquinolones and aminoglycosides. The proportions of fluoroquinolone-resistant and extended-spectrum β-lactamase (ESBL)-producing E. coli strains were 6.4% (13/220) and 4.1% (9/220), respectively. The antimicrobial susceptibility of K. pneumoniae was retained during the surveillance period, while no multidrug-resistant strains were identified. In summary, antimicrobial susceptibility results of our second nationwide surveillance did not differ significantly from those of the first surveillance. Especially the numbers of fluoroquinolone-resistant and ESBL-producing E. coli strains were not increased in premenopausal patients with AUC in Japan

    Natural frequencies and vibration modes of laminated composite plates reinforced with arbitrary curvilinear fiber shape paths

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    The development of tow-placement technology has made it possible to control fiber tows individually and place fibers in curvilinear distinct paths in each layer of a laminated plate. This paper presents an analytical method for determining natural frequencies and vibration modes of laminated plates having such curvilinear reinforcing fibers. Spline functions are employed to represent arbitrarily shaped fibers, and Ritz solutions are used to derive frequency equations using series type shape functions. The strain energy is evaluated by numerical integration involving the fiber orientation angle, and is calculated using the derivative of the spline function in minute intervals. The results show that the natural frequencies obtained by the present method agree well with results from finite element analyses. The vibration mode shape contour plots of the plates are seen to reflect clear influences of the fiber shapes

    Vibration design of laminated fibrous composite plates with local anisotropy induced by short fibers and curvilinear fibers

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    The present paper studies an optimum design method for proposing new types of fiber-reinforced composite plates with locally anisotropic structure. A finite element program is developed to analyze vibration of such locally anisotropic plates and the fundamental frequency is taken as an object function to be maximized. First, for demonstrating the effectiveness of local anisotropy, the optimum distributions of short fibers are calculated without directional constraints using a simple genetic algorithm (GA), and the layerwise optimization (LO) concept is used to reduce the computation time in the finite element calculation. Secondly, optimum arrangements of continuous curvilinear fibers are obtained under the continuity constraints where fibers directions are considered as projections of contour lines of a cubic polynomial surface. Numerical results show that the local anisotropy successfully improves frequency property and the optimum directions of short fibers indicate physically reasonable orientations. Also, the plates with optimally shaped continuous fibers yield higher fundamental frequencies than the conventional plates with parallel fibers
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