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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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Long-term maintenance of St-T1b cells. A) St-T1b cells, obtained at passage number 30, were kept in continuous culture for the indicated number of passages and tested for their ability to decidualize in response to 3 d of 8-Br-cAMP stimulus. Transcript levels of dPRL, IGFBP-1 and FOXO1 were determined by RT-PCR; GAPDH mRNA was amplified for normalization. B) St-T1b cells at passage number 44 or 114 were treated with the indicated combinations of 8-Br-cAMP, P4 (1 μM) and E2 (10 nM) for 7 d before Western blot analysis for FOXO1. The blot was stripped and reprobed with GAPDH antibody. C) St-T1b cells at passage number 114, untreated or treated with 8-Br-cAMP for 7 d, were subjected to immunocytochemistry with vimentin antibody (original magnification, 100×).
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Figure 4: Long-term maintenance of St-T1b cells. A) St-T1b cells, obtained at passage number 30, were kept in continuous culture for the indicated number of passages and tested for their ability to decidualize in response to 3 d of 8-Br-cAMP stimulus. Transcript levels of dPRL, IGFBP-1 and FOXO1 were determined by RT-PCR; GAPDH mRNA was amplified for normalization. B) St-T1b cells at passage number 44 or 114 were treated with the indicated combinations of 8-Br-cAMP, P4 (1 μM) and E2 (10 nM) for 7 d before Western blot analysis for FOXO1. The blot was stripped and reprobed with GAPDH antibody. C) St-T1b cells at passage number 114, untreated or treated with 8-Br-cAMP for 7 d, were subjected to immunocytochemistry with vimentin antibody (original magnification, 100×).

Mentions: We wondered if the cells retained their ability to decidualize in extended culture. Cells obtained at passage number 30 were therefore maintained in continuous culture for more than 113 additional passages. At intervals, a 3-day decidualization experiment was performed, measuring the relative induction of dPRL, IGFBP-1 and FOXO1 transcripts as the endpoints (Fig. 4A). The induction of dPRL mRNA was clearly detectable up to passage 82 and then began to weaken. IGFBP-1 transcripts were less robustly induced and faded around passage number 72. St-T1b displayed a cAMP-dependent induction of FOXO1 message levels throughout the entire study period with a decline at later passages. This was reflected by a decrease in cAMP-induced FOXO1 protein levels when comparing passage numbers 44 and 114. The response to cAMP was not altered by the addition of P4 or P4 plus E2 over 7 d (Fig. 4B). Morphological features of decidualization, and vimentin expression, were still maintained at passage 114 (Fig. 4C).


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)

Long-term maintenance of St-T1b cells. A) St-T1b cells, obtained at passage number 30, were kept in continuous culture for the indicated number of passages and tested for their ability to decidualize in response to 3 d of 8-Br-cAMP stimulus. Transcript levels of dPRL, IGFBP-1 and FOXO1 were determined by RT-PCR; GAPDH mRNA was amplified for normalization. B) St-T1b cells at passage number 44 or 114 were treated with the indicated combinations of 8-Br-cAMP, P4 (1 μM) and E2 (10 nM) for 7 d before Western blot analysis for FOXO1. The blot was stripped and reprobed with GAPDH antibody. C) St-T1b cells at passage number 114, untreated or treated with 8-Br-cAMP for 7 d, were subjected to immunocytochemistry with vimentin antibody (original magnification, 100×).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 4: Long-term maintenance of St-T1b cells. A) St-T1b cells, obtained at passage number 30, were kept in continuous culture for the indicated number of passages and tested for their ability to decidualize in response to 3 d of 8-Br-cAMP stimulus. Transcript levels of dPRL, IGFBP-1 and FOXO1 were determined by RT-PCR; GAPDH mRNA was amplified for normalization. B) St-T1b cells at passage number 44 or 114 were treated with the indicated combinations of 8-Br-cAMP, P4 (1 μM) and E2 (10 nM) for 7 d before Western blot analysis for FOXO1. The blot was stripped and reprobed with GAPDH antibody. C) St-T1b cells at passage number 114, untreated or treated with 8-Br-cAMP for 7 d, were subjected to immunocytochemistry with vimentin antibody (original magnification, 100×).
Mentions: We wondered if the cells retained their ability to decidualize in extended culture. Cells obtained at passage number 30 were therefore maintained in continuous culture for more than 113 additional passages. At intervals, a 3-day decidualization experiment was performed, measuring the relative induction of dPRL, IGFBP-1 and FOXO1 transcripts as the endpoints (Fig. 4A). The induction of dPRL mRNA was clearly detectable up to passage 82 and then began to weaken. IGFBP-1 transcripts were less robustly induced and faded around passage number 72. St-T1b displayed a cAMP-dependent induction of FOXO1 message levels throughout the entire study period with a decline at later passages. This was reflected by a decrease in cAMP-induced FOXO1 protein levels when comparing passage numbers 44 and 114. The response to cAMP was not altered by the addition of P4 or P4 plus E2 over 7 d (Fig. 4B). Morphological features of decidualization, and vimentin expression, were still maintained at passage 114 (Fig. 4C).

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