Given the difficulties inherent in maintaining human pluripotent stem cells (hPSCs) in a healthy state, hPSCs should be routinely characterized using several established standard criteria during expansion for research or therapeutic purposes. hPSC colony morphology is typically considered an important criterion, but it is not evaluated quantitatively. Thus, we designed an unbiased method to evaluate hPSC colony morphology. This method involves a combination of automated non-labelled live-cell imaging and the implementation of morphological colony analysis algorithms with multiple parameters. To validate the utility of the quantitative evaluation method, a parent cell line exhibiting typical embryonic stem cell (ESC)-like morphology and an aberrant hPSC subclone demonstrating unusual colony morphology were used as models. According to statistical colony classification based on morphological parameters, colonies containing readily discernible areas of differentiation constituted a major classification cluster and were distinguishable from typical ESC-like colonies; similar results were obtained via classification based on global gene expression profiles. Thus, the morphological features of hPSC colonies are closely associated with cellular characteristics. Our quantitative evaluation method provides a biological definition of ‘hPSC colony morphology’, permits the non-invasive monitoring of hPSC conditions and is particularly useful for detecting variations in hPSC heterogeneity.
Three human pancreatic cancer cell lines, designated as KP·IN, KP·2 and Kp·3 have been established in both tissue cultures and in nude mice. The Kp·IN and Kp·3 tumors were obtained from liver metastases of pancreatic tumors and the KP·2 tumor was of primary pancreatic origin. The patients' tumors from which KP·IN and Kp·2 were derived showed characteristics of adenocarcinoma, and the KP·3 tumor had adenosquamous carcinoma characteristics. Inoculations of samples from surgical specimens into athymic nude mice resulted in tumor formation, with the tumors histologically closely resembling the original neoplasms. Subcutaneous injections of the established cell lines also induced tumor formation and the tumors histologically resembled the original lesion in the cases of KP·2 and KP·3 tumors, but the KP·IN tumors in the mice were histologically different from the surgical specimen. The KP·IN, Kp·2 and Kp·3 cell lines have been cultured continuously in a medium Supplemented with 10% fetal calf serum for more than 20, 21 and 17 months, respectively. The Kp· 2 and KP·3 cell lines produced and released carbohydrate antigen 19·9 into the spent medium but the KP·IN cell line did not. KP·IN and KP·3, produced liver metastases after intrasplenic injection into nUde mice, whereas Kp·2 produced few liver colonies. Cell lines highly metastatic to the liver, Kp· INLs and KP·3Ls, were isolated from the liver colonies of KP·IN and KP·3, respectively. Carcinomas of the pancreas are one of the most frequent causes of cancer deaths in Japan, and the incidence is increasing. I) Biological models are necessary for the study of pancreatic cancer, in that little is known about the biology, chemotherapy, or metastasis of this neo· plasm. Thus, permanent cell lines and xenografts of human pancreatic cancers could provide information on the biological properties and therapeutic response of this tumor that could lead to effective approaches for treatment of this disease.to demonstrate metastatic activity in the liver from colon tumors has been carried out using intrasplenic (LS.)6 injections of cells. 15, 16) It is necessary to study an experimental model system to determine whether pancreatic tumor cells metastasize to the liver.
The purpose of this study was to investigate the properties of several antimicrobial agents found to be effective against Chlamydia trachomatis and to verify the eradication therapy schedule. The in vitro activities of two quinolones (sparfloxacin, ofloxacin), of three macrolides (azithromycin, erythromycin, clarithromycin) and of a tetracycline (doxycycline) against C. trachomatis were evaluated by several methods for the determination of the minimum inhibitory concentration (MIC) and minimal lethal concentration (MLC). MLC of azithromycin was only 2 times higher than that of MIC. On the other hand, MLCs of other antibiotics were 4–16 times higher than their respective MICs. When all antimicrobial agents were added to the infected culture at different times, we found that the quinolones even at a concentration of 64 μg/ml could not inhibit the formation of inclusion if they were added after 20 h from the start of infection. The corresponding period for macrolides and doxycycline was 24 h. When the antibiotics were removed at 8 h after the start of the infection, all antibiotics except azithromycin and clarithromycin were needed at a concentration much higher than their MLCs to inhibit the formation of inclusion. We consider macrolides, especially azithromycin, to be an excellent anti-C. trachomatis drug because of its lower MICs and MLCs values which were also closer together.
Aims-To examine the detection limit of the ligase chain reaction kit for Chlamydia trachomatis, to study the inhibitory eVect of phosphate on the ligase chain reaction, and to clarify the mechanism of inhibition. Methods-Three reference serovars of C trachomatis-D/UW-3/Cx, F/UW-6/Cx, and L2/434/Bu-were used to test the sensitivity of the chlamydia ligase chain reaction. Comparison was made of the inhibition by phosphate before and after DNA amplification. Phosphate in up to 2.4 mM concentration was added to specimens of C trachomatis serovar D (1 to 50 inclusion forming units (IFU)/reaction) before DNA amplification to examine the concentration dependency of phosphate inhibition of the ligase chain reaction. Results-The detection limits were 0.6 IFU/reaction for serovar D/UW-3/Cx and F/UW-6/Cx, and 0.4 IFU/reaction for L2/ 434/Bu. Phosphate inhibited the ligase chain reaction only when it was added before the amplification stage. The specimens containing chlamydia at 1 to 50 IFU/reaction were negative when the concentration of phosphate added at the prethermocycle stage was more than 1.2 mM. Conclusions-Ligase chain reaction analysis is a reliable method of diagnosing C trachomatis infection because of its high sensitivity. It would be clearly superior to the currently used methods if the problem of inhibitors could be eliminated. The mechanism of inhibition of the ligase chain reaction by phosphate was thought to be blockade of the amplification of the target DNA. The eYcacy of the ligase chain reaction could be inhibited by phosphate in the urine, so duplicate dilution analysis of some negative specimens should be useful. (J Clin Pathol 1998;51:306-308) Keywords: Chlamydia trachomatis; ligase chain reaction; phosphate inhibition Chlamydia trachomatis, an obligate intracellular bacterium, is the major cause of sexually transmitted disease in well developed countries. In men who are sexually active this organism causes about 50% of cases of non-gonococcal urethritis.1 Asymptomatic infection is characteristic of this pathogen. In women, asymptomatic cervicitis contributes to many sequelae. Furthermore, maternal chlamydial infections have a direct and harmful influence on the fetus and can lead to several diseases in infants. Therefore, rapid, accurate, reliable, non-invasive, and convenient tests to detect C trachomatis are required for clinical screening. A genetic detection kit for C trachomatis-plasmid based ligase chain reaction-has been developed (Abbott Laboratories, Chicago, Illinois, USA).2 The specificity of this kit was reported to be better than that achieved by the polymerase chain reaction (PCR) (Amplicor, Roche Diagnostic Systems, Branchburg, New Jersey, USA).Sensitive and specific identification of C trachomatis in clinical specimens is essential for the eVective control of chlamydial infection. However, as the sensitivity increases, so does the possibility of false negative results. One of the reasons for false negatives is the presence of inhibitors in the test sample. In the present s...
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