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.

Progesterone-induced sphingosine kinase-1 expression in the rat uterus during pregnancy and signaling consequences

“…Consistent with the idea that LPA might play a role in the initiation of labor, there was also more than a twofold (P Ͻ 0.05) rise in LPA 1 receptor expression in the uterus on days 21 and 22. LPA might stimulate myometrial contractions, as indicated by its ability to increase myosin light-chain phosphorylation in cultured human myometrial cells (42). It is not known whether reproductive tissues contribute significantly to the production of LPA.…”

Immortalization of Human Fetal Hepatocyte by Ectopic Expression of Human Telomerase Reverse Transcriptase, Human Papilloma Virus (E7) and Simian Virus 40 Large T (SV40 T) Antigen Towards Bioartificial Liver Support

“…[16][17][18][19] But cell population without hTERT expression in even in SV 40 expressing will enter into crisis, hence immortalization is imcomplete. 20 It has been demonstrated that immortalization of primary rat hepatocytes by early region of T-Ag comprising large T and small t gene have led to tumorigenic phenotype. 21 It is important to use role of hTERT for complete functional immortalization.…”

<p>In vitro comparison of liposomal drug delivery systems targeting the oxytocin receptor: a potential novel treatment for obstetric complications</p>

“…hTERT-immortalized myometrial cells were used to evaluate specificity of OTR targeting, as they exhibit the phenotype and properties of highly differentiated smooth muscle cells, without the high variability of responses seen with primary myometrial cells. 37,38 Human myometrial cells express OTRs [39][40][41] and have been reported to acquire additional OTRs in culture, [42][43][44] with upregulation likely due to the presence of fetal bovine serum in the culture medium. 45 It should be noted that serum and supplement free media were used in the in vitro cellular association studies to determine binding and cellular uptake of the OTR-targeted liposomal platforms in a reductionist in vitro system, which would mimic the in vivo situation of when the liposomes reached the myometrium of the uterus.…”