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Characterization of a Novel Telomerase-Immortalized Human

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Characterization of a Novel Telomerase-Immortalized Human Reproductive Biology and Endocrinology BioMed Central Research Open Access Characterization of a novel telomerase-immortalized human endometrial stromal cell line, St-T1b Annemarie Samalecos1, Katja Reimann1, Stefanie Wittmann1, Heinrich M Schulte1, Jan J Brosens2, Ana-Maria Bamberger3 and Birgit Gellersen*1 Address: 1Endokrinologikum Hamburg, 20251 Hamburg, Germany, 2Institute of Reproductive and Developmental Biology, Imperial College London, Hammersmith Hospital, London W12 ONN, UK and 3Section on Endocrinology and Ageing, University Clinic Hamburg-Eppendorf, 20251 Hamburg, Germany Email: Annemarie Samalecos - [email protected]; Katja Reimann - [email protected]; Stefanie Wittmann - [email protected]; Heinrich M Schulte - [email protected]; Jan J Brosens - [email protected]; Ana-Maria Bamberger - [email protected]; Birgit Gellersen* - [email protected] * Corresponding author Published: 20 July 2009 Received: 23 April 2009 Accepted: 20 July 2009 Reproductive Biology and Endocrinology 2009, 7:76 doi:10.1186/1477-7827-7-76 This article is available from: http://www.rbej.com/content/7/1/76 © 2009 Samalecos et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Coordinated differentiation of the endometrial compartments in the second half of the menstrual cycle is a prerequisite for the establishment of pregnancy. Endometrial stromal cells (ESC) decidualize under the influence of ovarian progesterone to accommodate implantation of the blastocyst and support establishment of the placenta. Studies into the mechanisms of decidualization are often hampered by the lack of primary ESC. Here we describe a novel immortalized human ESC line. Methods: Primary ESC were immortalized by the transduction of telomerase. The resultant cell line, termed St-T1b, was characterized for its morphological and biochemical properties by immunocytochemistry, RT-PCR and immunoblotting. Its progestational response was tested using progesterone and medroxyprogesterone acetate with and without 8-Br-cAMP, an established inducer of decidualization in vitro. Results: St-T1b were positive for the fibroblast markers vimentin and CD90 and negative for the epithelial marker cytokeratin-7. They acquired a decidual phenotype indistinguishable from primary ESC in response to cAMP stimulation. The decidual response was characterized by transcriptional activation of marker genes, such as PRL, IGFBP1, and FOXO1, and enhanced protein levels of the tumor suppressor p53 and the metastasis suppressor KAI1 (CD82). Progestins alone had no effect on St-T1b cells, but medroxyprogesterone acetate greatly enhanced the cAMP-stimulated expression of IGFBP-1 after 3 and 7 days. Progesterone, albeit more weakly, also augmented the cAMP-induced IGFBP-1 production but only after 7 days of treatment. The cell line remained stable in continuous culture for more than 150 passages. Conclusion: St-T1b express the appropriate phenotypic ESC markers and their decidual response closely mimics that of primary cultures. Decidualization is efficiently induced by cAMP analog and enhanced by medroxyprogesterone acetate, and, to a lesser extent, by natural progesterone. St-T1b cells therefore serve as a useful model for primary ESC. Page 1 of 11 (page number not for citation purposes) Reproductive Biology and Endocrinology 2009, 7:76 http://www.rbej.com/content/7/1/76 Background In the present study, we characterize a new telomerase- Decidualization is a differentiation process of endome- immortalized ESC line, termed St-T1b. The ability of these trial stromal cells (ESC) that, in humans, is initiated in the cells to undergo decidualization and their stability in secretory phase of the menstrual cycle independently of a long-term culture are investigated to validate the cell line blastocyst signal. The transformation of elongated stromal as a model for primary ESC. fibroblasts into secretory epithelioid-like decidual cells is first apparent approximately 10 days after the postovula- Methods tory surge in circulating progesterone (P4) levels in the Cell culture vicinity of terminal spiral arteries and then spreads Primary cultures of human ESC were prepared from ano- throughout the endometrial compartment through auto- nymized uterine biopsy samples obtained from premeno- crine and paracrine signals. Typical secretory products of pausal women (age 35–50) at the time of hysterectomy decidualized cells are IGFBP-1 and prolactin (PRL). for benign gynecological disorders. Purified ESC were pre- Induction of IGFBP1 and decidua-specific PRL gene tran- pared as previously described [15] and maintained in ESC scription serve as molecular markers of decidualization medium: phenolred-free Dulbecco's modified Eagle [1-3]. Furthermore, expression of the forkhead transcrip- medium (DMEM)/Ham's F12 with 10% steroid-depleted tion factor FOXO1 is induced with decidualization, both dialyzed fetal bovine serum (FCS) (PAA Laboratories, in vivo and in vitro [4], and levels of the tumor suppressor Cölbe, Germany), 100 U/ml penicillin, 100 g/ml strep- protein p53 and the tetraspanin KAI1 (CD82), a cell sur- tomycin, and 0.2% Primocin (Invivogen, San Diego, CA), face protein that functions as a metastasis suppressor in and supplemented with insulin (1 g/ml) and 17-estra- tumor cells, are increased posttranscriptionally in decidu- diol (E2; 1 nM) (Sigma-Aldrich, Deisenhofen, Germany). alized cells [5-7]. Decidualization was performed in minimal medium 1 (MM1; ESC medium without insulin or E2) in the pres- Investigations into the mechanisms of decidualization in ence of 0.5 mM 8-Br-cAMP (Biolog, Bremen, Germany). humans are largely restricted to cell culture experimenta- The local ethics committee approved this study and tion. Yet such studies are hampered by difficulties in patient consent was obtained before tissue collection. obtaining tissues for the preparation of primary decidual cell cultures. Consequently, attempts have been made to Immortalization of primary ESC was performed in collab- immortalize human endometrial stromal cells by onco- oration with Gordon Peters (Cancer Research UK London genic transformation. However, SV40-transformed cells Research Institute, London, UK). ESC were isolated from display genomic instability [8] and, with time in culture, a biopsy taken during the proliferative phase from a 30 lose the crucial capability of undergoing decidual differ- year old patient with tubal disease awaiting IVF treatment. entiation in response to the appropriate stimuli (our own Briefly, the receptor for ecotropic retroviruses was first sta- unpublished observations on the St-2 cell line described bly introduced into the primary ESC using neomycin in [9]). Recently, the use of human telomerase reverse selection, then the cells were infected with a retrovirus transcriptase (hTERT) as the immortalizing agent for ESCs encoding hTERT and selected for hygromycin resistance. has yielded more promising results [10,11]. The pBABE retrovirus system was used in which the cDNA of interest is driven by the Moloney murine leukemia Normal primary cells undergo senescence because linear virus promoter in the viral long terminal repeat and the chromosomes shorten progressively with every round of marker is driven by the SV40 promoter [16]. St-T1b cells replication unless the ends of the chromosomes, the tel- were maintained and decidualized in the same media as omers, are maintained by telomerase, a reverse tran- described for ESC, but in the absence of Primocin. Stimu- scriptase that adds telomeric repeats de novo after each lations with P4, MPA, dexamethasone (all from Sigma- cell division. While human tumor cells often have aber- Aldrich) and R5020 (Promegestone; PerkinElmer, rantly high telomerase levels, most differentiated adult Rodgau, Germany) were performed in MM1. With the cells display silencing of telomerase activity, leading to exception of the long-term observation, all experiments progressive telomere shortening and thus a limited were performed repeatedly between passage numbers 30 lifespan [12]. The re-introduction of telomerase into pri- and 53, and representative experiments are shown. mary cells can bestow them with extension of replicative lifespan or even immortalization without the characteris- The trophoblastic hybridoma cell line AC-1M88, a fusion tics of neoplastic transformation such as anchorage-inde- of primary extravillous trophoblast from term placenta pendent growth and lack of contact inhibition [13,14]. with a mutant of the JEG-3 choriocarcinoma cell line, was Telomerase-immortalized ESC lines have been shown to kindly provided by H.G. Frank and P. Kaufmann (Univer- undergo decidualization in response to extended treat- sity Hospital Aachen, Germany) [17,18], and was main- ment with 17-estradiol (E2) and medroxyprogesterone tained in DMEM/Ham's F12 with 10% complete FCS, 100 acetate (MPA), or upon sequential stimulation with epi- U/ml penicillin, and 100 g/ml streptomycin. dermal growth factor (EGF), E2 and MPA [10,11]. Page 2 of 11 (page number not for citation purposes) Table 1: Oligonucleotide Primers and Amplification Conditions for RT-PCR Page11 3 of Amplified Product Forward/Reverse Primer Amplification Conditions
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