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Endocrinology Vol. 145, No. 5 2291-2296
Copyright © 2004 by The Endocrine Society

A Novel Immortalized Human Endometrial Stromal Cell Line with Normal Progestational Response

Graciela Krikun, Gil Mor, Ayesha Alvero, Seth Guller, Frederick Schatz, Eva Sapi, Mizanur Rahman, Rebeca Caze, Mazin Qumsiyeh and Charles J. Lockwood

Department of Obstetrics, Gynecology and Reproductive Science and Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06520-8063

Address all correspondence and requests for reprints to: Dr. Graciela Krikun, Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine, 333 Cedar Street, P.O. Box 208063, New Haven, Connecticut 06520-8063. E-mail: graciela.krikun{at}yale.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Obtaining primary human endometrial stromal cells (HESCs) for in vitro studies is limited by the scarcity of adequate human material and the inability to passage these cells in culture for long periods. Immortalization of these cells would greatly facilitate studies; however, the process of immortalization often results in abnormal karyotypes and aberrant functional characteristics. To meet this need, we have introduced telomerase into cultured HESCs to prevent the normal shortening of telomeres observed in adult somatic cells during mitosis. We have now developed and analyzed a newly immortalized HESC line that contains no clonal chromosomal structural or numerical abnormalities. In addition, when compared with the primary unpassaged parent cells, the new cell line displayed similar biochemical endpoints after treatment with ovarian steroids. Classical decidualization response to estradiol plus medroxyprogesterone acetate were seen in both morphologically, and progestin was seen to induce or regulate the expression of IGF binding protein-1, fibronectin, prolactin, tissue factor, plasminogen activator inhibitor-1, and Fas/Fas ligand. In summary, an immortalized HESC line has been developed that is karyotypically, morphologically, and phenotypically similar to the primary parent cells, and it is a powerful and consistent resource for in vitro work.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROGESTERONE STIMULATES THE estrogen-primed human endometrium to undergo decidualization, the process of growth and differentiation that transforms precursor stromal cells into decidual cells. Decidualized stromal cells are phenotypically different from their precursors. For example, decidualization enhances expression of prolactin (PRL), IGF binding protein 1 (IGFBP-1), tissue factor (TF), plasminogen activator inhibitor-1 (PAI-1), and components of basal lamina such as laminin, collagen IV, and fibronectin (FN) in cultured stromal cells (1, 2, 3, 4). Studies of human endometrial stromal cells (HESCs) are limited by the capacity to obtain samples to establish primary cultures. Previously, two endometrial stromal cell lines were created using simian virus 40 transformation. However, both these cell lines displayed marked karyotypic abnormalities (5). To overcome this limitation, we used a new approach to immortalization of cells using telomerase.

Telomeres are repetitive DNA sequences at the ends of chromosomes that can be maintained by telomerase. When adult somatic cells divide, their telomeres shorten by 10–200 bp per division, because the cells do not contain functional telomerase. This progressive shortening of the telomeres with each cell division has been proposed as the mitotic clock that regulates the loss of replicative potential. Telomerase is a multicomponent enzyme that comprises a template RNA plus an essential catalytic protein subunit [human telomerase reverse transcriptase (hTERT)] (6). The function of telomerase is to add TTAGGG repeats to telomeres by reverse-transcribing the RNA template, hence compensating for the loss of telomeric DNA associated with normal cell division (7). Although most normal human cells are telomerase negative (8), telomerase activity can be induced by transfecting cells with vectors expressing exogenous hTERT, and, for some types of cells, the expression of exogenous hTERT is sufficient for immortalization (9). Thus, we sought to examine whether such an approach could be employed to establish a new endometrial stromal cell line to facilitate in vitro studies.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
HESC isolation and cell culture
The isolation and culturing of primary HESCs was carried out as previously described (10). Briefly, after obtaining written informed consent and institutional approval from Yale New Haven Hospital (New Haven, CT), endometria from women of reproductive age were obtained from hysterectomies for benign conditions (e.g. myomas without abnormal uterine bleeding). The endometria were transported to a sterile laminar flow hood, and isolation of the stromal cells was conducted by digesting endometrial fragments with 0.25% type I collagenase (267 U/mg; lot no. 47P1428; Worthington, Lakewood, NJ) for 30 min in a shaking water bath at 37 C. The digestate was filtered through a 38-µm stainless steel sieve to remove the glands. Purification of the stromal cell-enriched fraction was accomplished with a Percoll gradient together with incubation for 30 min at 37 C in a standard humidified 95% air/5% CO2 incubator. During this period, the HESCs, but not the other cell types, attach to polystyrene tissue culture plastic.

HESCs were grown to confluence in a phenol red-free 1:1 vol/vol mix of DMEM (Life Technologies, Inc., Grand Island, NY) and Ham’s F-12 (Flow Laboratories, Rockville, MD) with 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml fungizone that was supplemented with 10% charcoal-stripped calf serum.

Immortalization protocol
Immortalization of primary HESCs derived from the midsecretory phase was achieved by transfection of telomerase (hTERT) using a retroviral system using pA317 hTERT-expressing cell line. The actively proliferating monolayer of stromal cells was infected with supernatant derived from the cell line pA317-hTERT (Geron Corp., Menlo Park, CA), which expresses the hTERT and puromycin resistance genes. Infection was performed for 8 h in the presence of 10 µg/ml Polybrene (Sigma-Aldrich, St. Louis, MO). Forty-eight hours after transfection, 800 ng of puromycin (Sigma-Aldrich) was added per milliliter of media to select the hTERT-expressing clones. Cells were then serially propagated.

Karyotyping
HESCs were harvested when 50–70% confluent with actively dividing cells present. Colcemid was added to a final concentration of 0.04 mg/ml at least 2 h before harvesting. Cells were trypsinized and harvested to produce G-banded metaphase preparations as described (11). Karotype analysis and documentation were performed by a computerized acquisition and analysis system (Applied Imaging, Santa Clara, CA). A minimum of 20 cells were analyzed from each immortalized and parent primary cell culture for chromosomal clonal structural or numerical abnormalities.

Telomerase detection
Telomerase activity was assayed using the TRAPeze ELISA Detection Kit (Chemicon International, Inc., Temecula, CA) per the manufacturer’s instructions, and telomerase was allowed to add telomeric repeats onto a 3' end of a biotinylated substrate oligonucleotide for 30 min. The products were then amplified using PCR with a biotinylated primer and dinitrophenol-labeled deoxycytidine triphosphate. The resulting tagged PCR products were immobilized onto streptavidin-coated microtiter plates via biotin-streptavidin interaction, and then detected by antidinitrophenol antibody conjugated to horseradish peroxidase. The amount of product was determined by means of the horseradish peroxidase activity using the substrate 3,3',5,5'-tetramethylbenzidine and subsequent color development. Absorbance readings were determined at 450 and 595 nM and telomerase activity was measured using the equation Abs450 – Abs595. As positive control for the assay, lysate from a telomerase-positive cell pellet and TSR8 template provided in the kit were used. For negative control, the lysis buffer was used. In addition, cell lysates were heat-inactivated for 10 min at 85 C and also used as negative controls.

Steroid effects
Immortalized and nonimmortalized HESCs were incubated with 0.1% ethanol (vehicle control) or 10–8 M estradiol (E2) or 10–7 M medroxyprogesterone acetate (MPA) (Sigma-Aldrich) or E2 + MPA. After 8 d, cells were lysed with 10 mM Tris (pH 7.4), 1 mM sodium orthovanadate, and 1% SDS for protein analysis or Tri-Reagent (Sigma-Aldrich) for RNA analysis. Medium supernatants and cell lysates were stored at –70 C until used.

ELISAs
IGFBP-1.
Concentrations of IGFBP-1 in cultured HESCs after various treatments were determined from the supernatant using an IGFBP-1 ELISA from Alpha Diagnostic International (San Antonio, TX). Absorbance was measured at 414 nM and compared with a standard curve derived from pure IGFBP-1 included in the ELISA kit.

FN.
Concentrations of FN from cultured HESCs after various treatments were determined from the supernatant using an FN ELISA from American Diagnostica (Greenwich, CT). Absorbance was measured at 450 nM and compared with a standard curve of FN included in the ELISA kit.

PRL.
Concentrations of PRL from cultured HESCs after various treatments were determined from the supernatant using a Human Prolactin ELISA Kit from Alpha Diagnostic International. Absorbance was measured at 414 nM and compared with a standard curve derived from pure PRL included in the ELISA kit.

PAI-1.
Concentrations of PAI-1 from cultured HESCs after various treatments were determined from the supernatant using a PAI-1 ELISA from American Diagnostica. Absorbance was measured at 450 nM and compared with a standard curve derived from PAI-1 included in the ELISA kit.

Western blots
TF.
Western blot analysis and densitometry for TF was conducted on extracted pellets from primary or immortalized HESCs that had been treated with E2 or E2 + MPA as previously described (10). The mouse monoclonal antibody was a kind gift from Yale Nemerson (Mount Sinai Medical Center, New York, NY).

Fas/Fas ligand (FasL).
The expression of Fas/FasL was determined using Western blot analysis as previously described (12 .)

Power Blot
Power Blot analysis simultaneously detects several proteins by a modified Western blot technique. We employed this technique to compare 40 different proteins from control and progestin-treated primary or immortalized HESCs using the services of BD Transduction Laboratories (Lexington, KY) as follows: a 4–15% gradient sodium dodecyl sulfate-polyacrylamide gel was prepared and transferred to an Immobilon-P membrane (Millipore, Billerica, MA) for 2 h at 200 mAmp using a wet electrophoretic transfer apparatus. After transfer, the membrane was dried and rewet in methanol. The membrane was blocked for 1 h with blocking buffer. The membrane was clamped with a Western blotting manifold that isolates 40 channels across the membrane. In each channel, a complex antibody cocktail was added. Table 1Go lists the antibodies studied in these experiments plus internal controls that were run in lanes 20 and 41.


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  		TABLE 1. List of antibodies detected in the Power Blot

 
The membrane was allowed to hybridize for 1 h at 37 C, washed, and hybridized for 30 additional min at 37 C with secondary goat antimouse conjugated to Alexa680 fluorescent dye (Molecular Probes, Eugene, OR). The membrane was washed, dried, and scanned using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE).

Real-time quantitative RT-PCR
Primary or immortalized HESCs were treated with vehicle control, E2, MPA or E2 + MPA, and RNA was extracted as described above. Real- time quantitative RT-PCR was then conducted as follows. Reverse transcriptase was initially carried out with avian myeloblastosis virus reverse transcriptase (Invitrogen, San Diego, CA). A quantitative standard curve was then created using a range of 500 pg to 250 ng of cDNA. The curve was created with the Roche Light Cycler (Roche, Indianapolis, IN) by monitoring the increasing fluorescence of PCR products during amplification. Once the standard curve was established, quantitation of our unknowns was determined with the Roche Light Cycler and adjusted to the quantitative expression of ß-actin from these same samples. Melting curve analysis was conducted to determine the specificity of the amplified products and to ensure the absence of primer-dimer formation. All products obtained yielded the correct melting temperature. The primers described in Table 2Go were synthesized and gel-purified at the Yale DNA Synthesis Laboratory, Critical Technologies.


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  		TABLE 2. Primers used

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Immortalization: telomerase activity, karyotype, and morphology
As described, puromycin was used as the selection agent for telomerase-immortalized cells. Although immortalized cells grew well and could be consistently passaged, the nonimmortalized parent cells died within 72 h after puromycin addition. To ensure the continued presence of telomerase, cells passaged seven and nine times were assayed for telomerase-activity as described in Materials and Methods. Figure 1Go shows that immortalized HESCs and positive controls displayed similar levels of hTERT expression. The samples were tested in triplicate for each cell line. By contrast, primary parent cells or heat-inactivated cells displayed no fold induction or background levels of hTERT expression. The very small fold induction in telomerase activity after heat inactivation of immortalized cells at passage 7 is likely due to some resistance of the telomerase after treatment at 85 C for a short period of time.



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  		FIG. 1. Telomerase activity. The activity of telomerase is reported as fold induction compared with negative control (neg. cont.) and was measured using the equation Abs450 – Abs595. Positive controls (pos. cont.) consisted of a telomerase-positive cell pellet or TSR8 template provided by the manufacturer. Negative control (neg. cont.) consisted of buffer. Parent and immortalized (immort) cells from passage 7 and 9 (p7 & p9) were assayed before and after heat inactivation (heat inact.) as described in Materials and Methods.

 
To investigate the effect of immortalization on cell karyotypes, cytogenetic analysis was performed using standard G-banding analysis. This revealed no clonal structural or numerical abnormalities at the 500- to 550-band level in either parent or immortalized cell lines after examining a minimum of 20 well-banded metaphases per cell line (data not shown).

The effect of hormone treatment on morphological characteristics of the cultures was studied in immortalized vs. nonimmortalized HESCs. As can be seen in Fig. 2Go, no significant gross differences are observed in the morphology of the control-treated primary cultured HESCs compared with the control-treated immortalized HESCs. Furthermore, treatment of the primary or the immortalized cells with E2 showed essentially no morphological changes when compared with vehicle control treatment in both control and immortalized cells whereas treatment with E2 + MPA for 8 d induced an identical pattern of decidualized morphology in both the primary and immortalized HESCs (2, 3, 13). Hence, immortalized HESCs retain the ability to undergo morphological decidualization when exposed to a progestational milieu.



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  		FIG. 2. Progestational decidualization. Primary (top panel) or immortalized HESCs (bottom panel) were treated with vehicle control, 10–8 M E2 or E2 + 10–7 M MPA for 8 d to assess the effects on decidualization.

 
Power Blot analysis
Of the 40 proteins chosen to be studied by Power Blot analysis (Table 1Go), only two proteins, the phosphorylated form of p38 and connexin-43, demonstrated more than 2-fold changes in expression by parent vs. immortalized HESCs after treatment with vehicle control (Table 3Go). After treatment with E2 + MPA, the expression pattern of parent vs. immortalized cells was again similar with only four proteins demonstrating more than a 2-fold difference including the phosphorylated form of p38, connexin-43, phospho-Stat-3, and laminin-b. No CD 45 band was expressed by any of the cultured cells under any conditions demonstrating no white cell contamination in our preparations.


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  		TABLE 3. Differential protein expression for immortalized vs. parent HESCs treated with vehicle control or E2 + MPA

 
Hormone effects on primary and immortalized HESCs
Consistent with observations from previous studies on primary HESCs (1, 2, 3, 13, 14), Fig. 3Go demonstrates that classic progestational responses were seen in both primary and immortalized HESC cultures for the decidualization endpoints (PRL, IGFBP-1, and FN). Treatment with E2 + MPA compared with E2 alone resulted in significantly higher PRL expression in primary cells, whereas such treatment resulted in significantly higher FN expression in the immortalized cells.



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  		FIG. 3. Progestational induction of decidualization endpoints. Fold induction by E2 + MPA vs. E2 alone on PRL, IGFBP-1, and FN protein expression by primary and immortalized HESCs was assessed as described in Materials and Methods. (n = 4; *, P < 0.03 for PRL and P < 0.02 for FN in primary vs. immortalized cells as assessed by the Mann Whitney test).

 
Figure 4Go shows that the hemostatic endpoints TF and PAI-1 are also induced in both primary and immortalized HESCs treated with E2 + MPA compared with E2 alone. In this case, the induction of TF was similar in the immortalized cells compared with the primary cells whereas the induction of PAI-1 was significantly higher in the primary cells.



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  		FIG. 4. Progestational induction of hemostatic endpoints. Fold induction by E2 + MPA vs. E2 alone on TF and PAI-1 protein expression by primary and immortalized HESCs was assessed as described in Materials and Methods (n = 4; *, P < 0.02 for PAI-1 in primary vs. immortalized cells as assessed by the Mann Whitney test).

 
Finally, E2 + MPA decreased FAS and increased FasL expression in both primary and immortalized cells compared with E2 alone (data not shown).

To study the effect of immortalization at the mRNA levels, real-time quantitative RT-PCR was conducted on some select endpoints, namely IGFBP-1, TF, and PAI-1. Figure 5Go demonstrates that, as was observed at the protein level, treatment of primary or immortalized HESCs with progestin induced IGFBP-1 mRNA expression by 25- and 125-fold, respectively. In addition, treatment with progestin induced both TF and PAI-1 mRNA expression by about 20-fold in both parent and immortalized cells.



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  		FIG. 5. Progestational induction on mRNA expression of IGFBP-1, TF, and PAI-1. The fold induction by E2 + MPA vs. E2 alone on IGFBP-1, TF, and PAI-1 mRNA expression by primary and immortalized HESCs was detected by real-time quantitative RT-PCR as described in Materials and Methods (n = 3).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Decidualized endometrium plays a key role in implantation and pregnancy serving to restrain trophoblast invasion and ensuring adequate hemostasis (4, 15). Conversely, menstruation only occurs in species with spontaneous decidualizations suggesting that the decidua also regulates this unique cyclical bleeding phenomenon (15, 16, 17). Indeed, absent or deficient decidualization is associated with the potentially catastrophic hemorrhage seen in patients with ectopic pregnancy and placenta accreta. The presence of ectopic HESCs contributes to the pathophyisology of endometriosis. Abnormal endometrial bleeding is a major cause for emergent gynecological visits, hysterectomy, and discontinuation of long-term progestin-only contraceptives (18).

Although studies of the cellular mechanisms involved in these pathologies are ideally carried out on primary cultured HESCs, the limited availability of tissue, difficulties in culturing, and limited life span of these cells makes these studies difficult to undertake and reproduce. The immortalization of HESCs would permit establishment of continuous cell lines facilitating study of their cell growth, differentiation, and function. Normal human cells are stringently destined to cellular aging and very rarely become immortalized. In vivo, when human cells become immortalized, they generally become neoplastic. It is believed that the p53 cascade plays an important role in the immortalization of human cells (19).

Previously, other groups conducted immortalization of primary HESCs by employing the simian virus 40, but these cell lines displayed numerous karyotypic abnormalities (5). To avoid this problem, the present study developed an immortalized HESC line that is karyotipically normal by transfection with hTERT. Indeed, Kyo et al. (20) have recently published work describing their ability to immortalize glandular epithelial cells that are karyotipically normal by Rb/p16/p53 inactivation and telomerase activation.

The replication of chromosomes in somatic cells is associated with the loss of chromosomal terminal nucleic acid sequences (telomeres) during each replication cycle. However, in germ cells and in most tumors the cellular enzyme telomerase counteracts the replication-dependent loss of telomere sequences (21). In addition, transfection of cells with expression vectors containing hTERT maintains telomere length and effectively gives normal cells an unlimited life span in culture (22, 23). Indeed, a recent study by Condon et al. (6) successfully used this technique to immortalize human myometrial cells that retained markers of differentiation like those observed in primary cultured cells.

The current study demonstrates that a newly immortalized HESC line displayed the morphological pattern and biochemical endpoints of decidualization including IGFBP-1, FN, PRL, TF, PAI-1, and FasL after treatment with E2 + MPA. These changes paralleled those observed with primary cultured HESCs.

Immortalized HESC lines, which are karyotypically and phenotypically similar to the primary parent cells and display similar phenotypic characteristics, have not previously been developed and are critical for in vitro work. We have developed an immortalized HESC line, which is karyotypically normal and responds to hormone stimulation similar to cultured primary stromal cells, and these should prove to be an invaluable tool for consistent in vitro work.


   Footnotes
 
This work was supported in part by grants from the National Institutes of Health: RO1 HD33937-06 (to C.J.L.) and RO1 HL70004-01A1 (to C.J.L.).

Abbreviations: E2, Estradiol; FN, fibronectin; FasL, Fas ligand; HESC, human endometrial stromal cell; hTERT, human telomerase reverse transcriptase; IGFBP-1, IGF binding protein 1; MPA, medroxyprogesterone acetate; PAI-1, plasminogen activator inhibitor-1; PRL, prolactin; TF, tissue factor.

Received November 26, 2003.

Accepted for publication January 8, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Tseng L, Mazella J 2002 Endometrial cell specific gene activation during implantation and early pregnancy. Front Biosci 7:d1566–d1574
  2. Giudice LC, Irwin JC 1999 Roles of the insulinlike growth factor family in nonpregnant human endometrium and at the decidual: trophoblast interface. Semin Reprod Endocrinol 17:13–21[Medline]
  3. Salamonsen LA, Dimitriadis E, Jones RL, Nie G 2003 Complex regulation of decidualization: a role for cytokines and proteases—a review. Placenta 24(Suppl A):S76–S85
  4. Lockwood CJ, Krikun G, Schatz F 1999 The decidua regulates hemostasis in human endometrium. Semin Reprod Endocrinol 17:45–51[Medline]
  5. Rinehart CA, Laundon CH, Mayben JP, Lyn-Cook BD, Kaufman DG 1993 Conditional immortalization of human endometrial stromal cells with a temperature-sensitive simian virus 40. Carcinogenesis 14:993–999[Abstract/Free Full Text]
  6. Condon J, Yin S, Mayhew B, Word RA, Wright WE, Shay JW, Rainey WE 2002 Telomerase immortalization of human myometrial cells. Biol Reprod 7:506–514
  7. Toouli CD, Huschtscha LI, Neumann AA, Noble JR, Colgin LM, Hukku B, Reddel RR 2002 Comparison of human mammary epithelial cells immortalized by simian virus 40 T-antigen or by the telomerase catalytic subunit. Oncogene 21:128–139[CrossRef][Medline]
  8. Weinrich SL, Pruzan R, Ma L, Ouellette M, Tesmer VM, Holt SE, Bodnar AG, Lichtsteiner S, Kim NW, Trager JB, Taylor RD, Carlos R, Andrews WH, Wright WE, Shay JW, Harley CB, Morin GB 1997 Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT. Nat Genet 17:498–502[CrossRef][Medline]
  9. Harada H, Nakagawa H, Oyama K, Takaoka M, Andl CD, Jacobmeier B, von Werder A, Enders GH, Opitz OG, Rustgi AK 2003 Telomerase induces immortalization of human esophageal keratinocytes without p16INK4a inactivation. Mol Cancer Res 1:729–738[Abstract/Free Full Text]
  10. Lockwood CJ, Nemerson Y, Guller S, Krikun G, Alvarez M, Hausknecht V, Gurpide E, Schatz F 1993 Progestational regulation of human endometrial stromal cell tissue factor expression during decidualization. J Clin Endocrinol Metab 76:231–236[Abstract]
  11. Baker RJ, Qumsiyeh MB, Rautenbach IL 1988 Evidence for eight tandem and five centric fusions in the evolution of the karyotype of Aethomys namaquensis A. Smith (Rodentia: Muridae). Genetica 76:161–169[CrossRef][Medline]
  12. Song J, Rutherford T, Naftolin F, Brown S, Mor G 2002 Hormonal regulation of apoptosis and the Fas and Fas ligand system in human endometrial cells. Mol Hum Reprod 8:447–455[Abstract/Free Full Text]
  13. Irwin JC, Kirk D, Gwatkin RB, Navre M, Cannon P, Giudice LC 1996 Human endometrial matrix metalloproteinase-2, a putative menstrual proteinase. Hormonal regulation in cultured stromal cells and messenger RNA expression during the menstrual cycle. J Clin Invest 97:438–447[Medline]
  14. Lane B, Oxberry W, Mazella J, Tseng L 1994 Decidualization of human endometrial stromal cells in vitro: effects of progestin and relaxin on the ultrastructure and production of decidual secretory proteins. Hum Reprod 9:259–266[Abstract/Free Full Text]
  15. Lockwood CJ, Krikun G, Schatz F 2001 Decidual cell-expressed tissue factor maintains hemostasis in human endometrium. Ann NY Acad Sci 943:77–88[Medline]
  16. Bell SC 1983 Decidualization and associated cell types: implications for the role of the placental bed in the materno-fetal immunological relationship. J Reprod Immunol 5:185–194[CrossRef][Medline]
  17. Critchley HO, Kelly RW, Brenner RM, Baird DT 2001 The endocrinology of menstruation—a role for the immune system. Clin Endocrinol (Oxf) 55:701–710[CrossRef][Medline]
  18. d’Arcangues C 2000 Management of vaginal bleeding irregularities induced by progestin-only contraceptives. Human Reprod 15(Suppl 3):24–29
  19. Namba M, Mihara K, Fushimi K 1996 Immortalization of human cells and its mechanisms. Crit Rev Oncog 7:19–31[Medline]
  20. Kyo S, Nakamura M, Kiyono T, Maida Y, Kanaya T, Tanaka M, Yatabe N, Inoue M 2003 Successful immortalization of endometrial glandular cells with normal structural and functional characteristics. Am J Pathol 163:2259–2269[Abstract/Free Full Text]
  21. Stoppler H, Hartmann DP, Sherman L, Schlegel R 1997 The human papillomavirus type 16 E6 and E7 oncoproteins dissociate cellular telomerase activity from the maintenance of telomere length. J Biol Chem 272:13332–13337[Abstract/Free Full Text]
  22. Vaziri H, Squire JA, Pandita TK, Bradley G, Kuba RM, Zhang H, Gulyas S, Hill RP, Nolan GP, Benchimol S 1999 Analysis of genomic integrity and p53-dependent G1 checkpoint in telomerase-induced extended-life-span human fibroblasts. Mol Cell Biol 19:2373–2379[Abstract/Free Full Text]
  23. Dickson MA, Hahn WC, Ino Y, Ronfard V, Wu JY, Weinberg RA, Louis DN, Li FP, Rheinwald JG 2000 Human keratinocytes that express hTERT and also bypass a p16 (INK4a)-enforced mechanism that limits life span become immortal yet retain normal growth and differentiation characteristics. Mol Cell Biol 20:1436–1447[Abstract/Free Full Text]



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