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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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Induction of KAI1 and p53 protein expression in decidualizing St-T1b cells. A) Total protein was harvested from St-T1b cells after 1, 3, or 7 d of treatment with 8-Br-cAMP and from untreated cells. Western blot analysis was performed for KAI1 and p53, and for GAPDH as a loading control. B) RNA was harvested from the same cultures as described in panel A and analyzed for KAI1, p53 and GAPDH transcript levels by RT-PCR.
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Figure 3: Induction of KAI1 and p53 protein expression in decidualizing St-T1b cells. A) Total protein was harvested from St-T1b cells after 1, 3, or 7 d of treatment with 8-Br-cAMP and from untreated cells. Western blot analysis was performed for KAI1 and p53, and for GAPDH as a loading control. B) RNA was harvested from the same cultures as described in panel A and analyzed for KAI1, p53 and GAPDH transcript levels by RT-PCR.

Mentions: St-T1b cells were treated with 8-Br-cAMP, a widely established stimulus for decidualization [1]. Morphological decidualization was evident by phase contrast microscopy and upon staining of the actin cytoskeleton, showing the same reorganization from elongated to polygonal shape in St-T1b cells as seen in primary ESC (Fig. 2A). St-T1b cells were also compared to primary ESC for their ability to express typical marker genes of decidualization, namely those coding for decidual PRL (dPRL) and IGFBP-1. Maximum induction of dPRL and IGFBP-1 transcripts was seen after 3 d of cAMP treatment, and induction was maintained for at least 7 d in both cell systems (Fig. 2B). Furthermore, treatment with 8-Br-cAMP over a 7-day time-course induced a gradual increase in both p53 and KAI1 proteins but not mRNA levels (Fig. 3). In summary, the cAMP responses of St-T1b cells closely reflected those of primary ESC.


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)

Induction of KAI1 and p53 protein expression in decidualizing St-T1b cells. A) Total protein was harvested from St-T1b cells after 1, 3, or 7 d of treatment with 8-Br-cAMP and from untreated cells. Western blot analysis was performed for KAI1 and p53, and for GAPDH as a loading control. B) RNA was harvested from the same cultures as described in panel A and analyzed for KAI1, p53 and GAPDH transcript levels by RT-PCR.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Induction of KAI1 and p53 protein expression in decidualizing St-T1b cells. A) Total protein was harvested from St-T1b cells after 1, 3, or 7 d of treatment with 8-Br-cAMP and from untreated cells. Western blot analysis was performed for KAI1 and p53, and for GAPDH as a loading control. B) RNA was harvested from the same cultures as described in panel A and analyzed for KAI1, p53 and GAPDH transcript levels by RT-PCR.
Mentions: St-T1b cells were treated with 8-Br-cAMP, a widely established stimulus for decidualization [1]. Morphological decidualization was evident by phase contrast microscopy and upon staining of the actin cytoskeleton, showing the same reorganization from elongated to polygonal shape in St-T1b cells as seen in primary ESC (Fig. 2A). St-T1b cells were also compared to primary ESC for their ability to express typical marker genes of decidualization, namely those coding for decidual PRL (dPRL) and IGFBP-1. Maximum induction of dPRL and IGFBP-1 transcripts was seen after 3 d of cAMP treatment, and induction was maintained for at least 7 d in both cell systems (Fig. 2B). Furthermore, treatment with 8-Br-cAMP over a 7-day time-course induced a gradual increase in both p53 and KAI1 proteins but not mRNA levels (Fig. 3). In summary, the cAMP responses of St-T1b cells closely reflected those of primary ESC.

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