Immortalization and characterization of human myometrial cells from term-pregnant patients using a telomerase expression vector.
ABSTRACT An examination of cellular processes involved in myometrial function has been greatly assisted by the use of human myometrial cells in primary culture. However, these cells can be used only for several passages before they senesce, and responses to various agents change with time in culture. The use of transformed cells is limited, as they can be polynucleated and can lose or gain chromosomes. We have developed three telomerase-immortalized cell lines from term-pregnant human myometrium to eliminate variability between passage numbers and allow genetic manipulations of myometrial cells to fully characterize signal pathways. These cells have a normal karyotype and were verified to be uterine smooth muscle by immunocytochemical staining for smooth muscle cell-specific alpha-actin and high affinity oxytocin antagonist binding sites. The three cell lines and the cells in primary culture from which they were derived were examined by cDNA microarray analysis. Of >10 000 expressed genes, there were consistent changes in the expression of approximately 1% in the three immortalized cell lines. We were unable to detect any significant differences between primary and immortalized cells in signal pathways such as epidermal growth factor-stimulated epidermal growth factor receptor phosphorylation, insulin-stimulated Akt phosphorylation, oxytocin and lysophosphatidic acid-stimulated extracellular signal-regulated kinase 1 and 2 phosphorylation, myosin light chain phosphorylation, and interleukin-1 induction of IkappaBalpha degradation. The immortalized cells should be useful for a range of studies, including high throughput analyses of the effects of environmental agents on the human myometrium.
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ABSTRACT: Bats are the suspected natural reservoir hosts for a number of new and emerging zoonotic viruses including Nipah virus, Hendra virus, severe acute respiratory syndrome coronavirus and Ebola virus. Since the discovery of SARS-like coronaviruses in Chinese horseshoe bats, attempts to isolate a SL-CoV from bats have failed and attempts to isolate other bat-borne viruses in various mammalian cell lines have been similarly unsuccessful. New stable bat cell lines are needed to help with these investigations and as tools to assist in the study of bat immunology and virus-host interactions. Black flying foxes (Pteropus alecto) were captured from the wild and transported live to the laboratory for primary cell culture preparation using a variety of different methods and culture media. Primary cells were successfully cultured from 20 different organs. Cell immortalisation can occur spontaneously, however we used a retroviral system to immortalise cells via the transfer and stable production of the Simian virus 40 Large T antigen and the human telomerase reverse transcriptase protein. Initial infection experiments with both cloned and uncloned cell lines using Hendra and Nipah viruses demonstrated varying degrees of infection efficiency between the different cell lines, although it was possible to infect cells in all tissue types. The approaches developed and optimised in this study should be applicable to bats of other species. We are in the process of generating further cell lines from a number of different bat species using the methodology established in this study.PLoS ONE 01/2009; 4(12):e8266. · 4.09 Impact Factor