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Characterization of a novel telomerase-immortalized human endometrial stromal cell line, St-T1b.

Samalecos A, Reimann K, Wittmann S, Schulte HM, Brosens JJ, Bamberger AM, Gellersen B - Reprod. Biol. Endocrinol. (2009)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Endokrinologikum Hamburg, 20251 Hamburg, Germany. samalecos@endokrinologikum.com

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.

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Phenotypic markers of St-T1b cells. St-T1b were grown to near confluency and subjected to immunocytochemical analysis using antibodies to vimentin, CD90 and cytokeratin-7 (CK7). The trophoblast-derived cell line AC-1M88 was stained alongside as a control. Control insets (Co) shows staining with omission of the primary antibody (original magnification, 100×).
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Figure 1: Phenotypic markers of St-T1b cells. St-T1b were grown to near confluency and subjected to immunocytochemical analysis using antibodies to vimentin, CD90 and cytokeratin-7 (CK7). The trophoblast-derived cell line AC-1M88 was stained alongside as a control. Control insets (Co) shows staining with omission of the primary antibody (original magnification, 100×).

Mentions: St-T1b cells were initially characterized approximately 30 passages after immortalization with hTERT. First, we examined cell type-specific antigen expression of these fibroblast-derived cells. Vimentin and CD90 were used as fibroblast markers and contrasted to cytokeratin-7 (CK7) as an epithelioid marker (Fig. 1). St-T1b cells were found to display the appropriate phenotype being vimentin+/CD90+/CK7-. The trophoblast-derived cell line AC-1M88 served as a positive control for CK7 staining and lack of vimentin and CD90 immunoreactivity.


Characterization of a novel telomerase-immortalized human endometrial stromal cell line, St-T1b.

Samalecos A, Reimann K, Wittmann S, Schulte HM, Brosens JJ, Bamberger AM, Gellersen B - Reprod. Biol. Endocrinol. (2009)

Phenotypic markers of St-T1b cells. St-T1b were grown to near confluency and subjected to immunocytochemical analysis using antibodies to vimentin, CD90 and cytokeratin-7 (CK7). The trophoblast-derived cell line AC-1M88 was stained alongside as a control. Control insets (Co) shows staining with omission of the primary antibody (original magnification, 100×).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2719639&req=5

Figure 1: Phenotypic markers of St-T1b cells. St-T1b were grown to near confluency and subjected to immunocytochemical analysis using antibodies to vimentin, CD90 and cytokeratin-7 (CK7). The trophoblast-derived cell line AC-1M88 was stained alongside as a control. Control insets (Co) shows staining with omission of the primary antibody (original magnification, 100×).
Mentions: St-T1b cells were initially characterized approximately 30 passages after immortalization with hTERT. First, we examined cell type-specific antigen expression of these fibroblast-derived cells. Vimentin and CD90 were used as fibroblast markers and contrasted to cytokeratin-7 (CK7) as an epithelioid marker (Fig. 1). St-T1b cells were found to display the appropriate phenotype being vimentin+/CD90+/CK7-. The trophoblast-derived cell line AC-1M88 served as a positive control for CK7 staining and lack of vimentin and CD90 immunoreactivity.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Endokrinologikum Hamburg, 20251 Hamburg, Germany. samalecos@endokrinologikum.com

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.

Show MeSH
Related in: MedlinePlus