1 Department of Cell Biology, Medical School, 33014 University of Tampere, 2 Department of Obstetrics and Gynecology, Tampere University Hospital and 3 Department of Pathology, Centre for Laboratory Medicine, Tampere University Hospital, 33521Tampere, Finland
4 To whom correspondence should be addressed at: Department of Cell Biology, Medical School, FIN-33014 University of Tampere, Finland. Email: merja.blauer{at}uta.fi
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Abstract |
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Key words: endometrium/human/Matrigel/organoid/organotypic culture
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Introduction |
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Numerous cell culture models have been designed with an aim to experimentally address questions related to the various cellular and molecular factors underlying human endometrial function and dysfunction. As proliferative disorders (hyperplasia, cancer) of the human endometrium have been linked to hyperestrogenic states and to unopposed estrogen replacement therapy (Rose, 1996), methods to investigate steroid effects on epithelial proliferation in vitro are of particular clinical relevance. In view of the above it is evident that any culture model intended to mimic normal endometrial responses should include both the epithelial and stromal compartments of the tissue.
Explant cultures represent an in vitro model in which tissue integrity is well preserved. Such cultures readily lend themselves to biochemical and histochemical examination and have been used to study, for example, progestin effects (Illouz et al., 2003) and tamoxifen biotransformation (Sharma et al., 2003
) in human endometrial tissue. The separate contributions of epithelial and stromal cells cannot, however, be directly determined in this type of culture. Moreover, tissue explants are usually unable to maintain their histological structure intact but for a relatively short time in vitro. The assessment of proliferative responses by immunohistochemical means in endometrial explant culture has also proven problematic (Illouz et al., 2003
). In order to study stromalepithelial interactions in the human endometrium, more elaborate culture conditions recombining separated stromal and epithelial cells in different extracellular matrices have been employed (Bentin-Ley et al., 1994
; Arnold et al., 2001
; Pierro et al., 2001
). In these models, epithelial cells have been cultivated as monolayers on top of reconstituted basement membrane matrix (Matrigel) and stromal cells beneath them on plastic or embedded in collagen or Matrigel. Even in this type of organotypic model, in vivo-like proliferative responses to steroid hormones have been rarely documented (Pierro et al., 2001
).
Because of the paucity of in vitro models for the human endometrium able to simulate steroid hormonal responses, we set out to develop and validate culture conditions for this purpose. Here, we describe an organotypic model in which epithelial cells of the normal human endometrium are cultivated as glandular organoids within Matrigel matrix in co-culture with stromal cells seeded on plastic. We report in vivo-like effects of E2 and medroxyprogesterone acetate (MPA) on epithelial cell proliferation in this system. The concomitant expression and regulation of estrogen receptor (ER) and progesterone receptor (PR) is also evaluated.
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Materials and methods |
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Isolation of epithelial organoids and stromal cells
Endometrial glands and stromal cells were isolated by a modification of the method described by Laird et al. (1997). To remove blood and debris the tissue was first rinsed in culture medium consisting of phenol red-free DMEM/F12 (Gibco BRL) supplemented with 2% dextran-coated, charcoal-treated fetal calf serum (Gibco) and antibiotic/antimycotic agents as described above. The tissue was then minced finely with a sterile scalpel and after rinsing and removal of the medium by centrifugation at 50 g for 1 min, the tissue pieces were incubated in culture medium containing 0.1% collagenase A (Roche, Germany) for 1 h at 37 °C. To aid digestion, the tissue was gently pipetted several times during the incubation period. The digest was thereafter passed through a wire sieve to remove any undigested material and centrifuged at 250 g for 2 min. After resuspending the pellet in culture medium, glandular structures were separated from stromal cells by centrifugation at 50 g for 1 min. The red layer, mainly of vascular organoids on top of the white glandular sediment, was carefully removed by pipetting. The supernatant was collected and further centrifuged at 250 g for 2 min to pellet the stromal cells. The stromal pellet was freed of red blood cells by exposing it to 1 ml distilled water for not longer than 30 s, after which time 9 ml culture medium was added and the stromal cells were repelleted at 250 g. Further purification of the isolated glandular and stromal cells was done by gently pipetting the fractions, both suspended in 1 ml culture medium, onto 9 ml medium each and letting them sediment at unit gravity for 5 (epithelial glands) or 30 min (stromal cells). The procedure was repeated once or twice for organoids. Stromal cells were harvested from the top 9 ml and used for cell culture. The viability of the stromal cells thus obtained was between 80 and 95% as assessed by Trypan Blue exclusion. Only the sedimented epithelial glands were collected for organoid culture.
Organoid culture
Ten organoid/stromal cell co-cultures and in three cases parallel organoid cultures without stromal cells were prepared from freshly isolated epithelial organoids and stromal cells (Figure 1). The co-cultures always consisted of organoids and stromal cells from the same individual.
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The cultures were incubated at 37 °C in a humidified atmosphere of 5% CO2 in air. After 2 days, the culture medium was renewed and after another 24 h the cultures were subjected to hormonal treatment. One insert of each set was removed and fixed in 4% paraformaldehyde to serve as the day 0 control. The other three were cultured for a further 7 days with 10 nmol/l 17-E2 (Sigma) or 10 nmol/l E2 together with 100 nmol/l MPA (Sigma); the day 7 control insert received a similar amount of 100% ethanol used to dissolve the steroids. The medium was renewed every 2 days and fresh steroids were added. Seven days after treatment with hormones, the inserts were collected, fixed in 4% paraformaldehyde overnight and thereafter immersed in 70% ethanol. Under a stereomicroscope, the insert membrane was partially detached from its housing with an injection needle and the fixed Matrigel bed was carefully detached from the membrane. The gel was cut with a razor blade into four pieces, which were then closed into an embedding cassette and automatically infiltrated with paraffin (Tissue-Tek V.I.P.; Miles Inc., USA). The gel pieces were thereafter manually aligned with their cut sides up in a base mould and embedded in paraffin.
Immunohistochemistry
Immunohistochemical analysis was used to characterize and validate the culture method. Staining was performed using the broad spectrum Histostain Plus kit from Zymed (South San Francisco, USA). Deparaffinated and rehydrated sections were first immersed for 10 min in methanol with 0.5% H2O2 to inactivate endogenous peroxidases. Subsequently, the sections were boiled in a microwave oven (1000 W) in 0.01 mol/l citrate buffer (pH 6.0) for 10 min followed by incubation in the same buffer for a further 20 min. After rinsing in Tris-buffered saline, blocking reagent was applied for 20 min at room temperature. Sections were then incubated with primary antibodies overnight at 4 °C. The antibodies were diluted in phosphate-buffered saline (PBS) containing 0.5% BSA and 0.01% Tween. The following monoclonal anti-human antibodies were applied at the dilutions indicated: anti-ER (1:100) and PR (1:300) (NovoCastra, UK), anti-cytokeratin 18 (1:500) and anti-vimentin (1:500) (Dako, Denmark), anti-E-cadherin (1:1000; Santa Cruz) and anti-Ki67 (1:800; Roche, Germany). A polyclonal rabbit anti-human von Willebrand factor was used (1:5000; Sigma). After extensive rinsing in PBS, biotinylated second antibody was applied for 20 min at room temperature. After rinsing, the sections were incubated with enzyme conjugate for 10 min at room temperature. After a further wash step, the sections were covered with 3,3 -diaminobenzidine chromogen for 10 min. The sections were counterstained with haematoxylin. Controls were performed by replacing primary antibodies with PBS or normal mouse (2.5 µg/ml) or rabbit (2 µg/ml) immunoglobulin G.
Ki67 labelling index and statistical analysis
The percentage of Ki67 expression (the number of stained cells divided by the total number of cells) was calculated in representative 4 mm sections. All intact organoids in each section were counted. Sections from different levels of each sample were analysed to attain 500 counted cells in
20 individual organoids per sample. The percentage of Ki-positive organoids (bearing at least one Ki-positive cell) was also calculated. The means±SEM were calculated for each treatment and statistical differences between means were assessed using the Wilcoxon signed rank test. Correlation was considered statistically significant if P < 0.05.
Assessment of stromal cell proliferation
In six cases, stromal cells from each hormonal treatment were trypsinized at day 7 and their number was counted using a Bürker chamber. Statistical analysis was performed as above.
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Results |
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Isolation and purification of the two cellular compartments from primary tissues was done by selective centrifugation and sedimentation. Epithelial organoids with a minimum of contaminating vascular fragments were obtained by careful removal of vascular elements from the top of the first organoid pellet and by reducing the time for organoid sedimentation to 5 min. Cellular purity of the epithelial compartment was assessed by immunohistochemical analysis. All organoids were shown to express the epithelial cell markers cytokeratin 18 and E-cadherin, with no detectable changes in staining intensity during cultivation (Figure 2). Occasional strands of endothelial cells positive for von Willebrand factor were found in some samples together with adhering fibroblasts (data not shown). As previously reported (Marshburn et al., 1992; Matthews et al., 1992
; Nisolle et al., 1995
; Classen-Linke et al., 1997
), anti-vimentin staining was inconstantly positive also in epithelial cells and was, therefore, used in parallell with anti-cytokeratin 18 to reveal contaminating fibroblasts. In this manner, only a few vimentin-positive but cytokeratin 18-negative spindle-shaped cells or bundles thereof were detected in each sample (data not shown).
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Cell proliferation in cultured organoids
The Ki67 nuclear antigen was used as a marker to immunohistochemically determine the fraction of proliferating epithelial cells in each sample (Figures 3 and 5ad). After 3 days in culture, at the time of hormone supplementation (day 0), the Ki67 index in epithelial organoids was 6.2±2.4 (mean±SEM), the control values after 10 days of culture (day 7 of hormone treatment) being somewhat lower, with a mean value of 4.6±0.8. There was individual variation in the percentage of proliferating cells in cultured organoids. The percentages in untreated controls (at day 7) varied between 1.8 and 9.3 with no apparent correlation with menstrual cycle phase.
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In three samples, the hormonal effects were compared in parallel organoid cultures with or without stromal cells (Figure 4). Whereas in co-cultures E2 was shown to increase and MPA to decrease the Ki67 index, in the absence of stromal cells, both hormonal treatments resulted in similar low Ki67 indices, which were below the control value. Due to the limited number of cases, the differences did not reach statistical significance.
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Regulation of ER and PR in cultured epithelial organoids
Immunohistochemical analysis was used to study the regulation of ER and PR by E2 and MPA in cultured endometrial organoids. Apart from minor differences in staining intensity in some samples, ER expression resembled the in vivo situation throughout the experiment with no apparent regulation with either hormonal treatment (Figure 5fh). PR expression, on the other hand, declined rapidly compared with that in non-cultured tissue, being markedly reduced or undetectable after the first 3 days of culture. After 10 days of culture, PR expression was no longer detected in any of the untreated controls (Figure 5j). PR expression was markedly increased under E2 treatment in all samples studied (Figure 5k). MPA counteracted the PR-inducing effect of E2, the PR expression being undetectable in organoids under simultaneous treatment with E2 and MPA (Figure 5l). The expression and regulation of ER and PR was similar in the presence and absence of stromal cells (data not shown).
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Discussion |
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Contacts with the extracellular matrix and with neighbouring cells are essential for epithelial cells to display differentiated morphology and function in vivo and in vitro. The present model was designed to mimic the in vivo epithelial organization by providing an extracellular matrix for epithelial organoids to grow in glandular arrangements with (or without) the inclusion of a separate compartment of stromal cells. Reconstituted basement membrane extract (Matrigel) has been widely used to grow epithelial cells in vitro and was chosen also here to serve as the supportive matrix for the organoids. Due to its protein composition, including the two main components of natural basement membranes, laminin and type IV collagen, Matrigel is able to promote epithelial cell attachment and differentiation in vitro. In previous studies, monolayers of endometrial epithelial cells on Matrigel have been shown to become polarized (Schatz et al., 1990; Bentin-Ley et al., 1994
; Classen-Linke et al., 1997
) and to form glandular structures within the gel (Rinehart et al., 1988
; White et al., 1990
). On bare cell culture plastic, on the other hand, primary endometrial epithelial cells tend to lose their epithelial phenotype and grow in whorl-like patterns with a loss of lateral junctions (Schatz et al., 1990
; Arnold et al., 2001
).
Instead of growing dissociated epithelial cells on Matrigel surface as in previous organotypic models (Bentin-Ley et al., 1994; Arnold et al., 2001
; Pierro et al., 2001
), we embedded fragments of epithelium directly inside the matrix (Hopfer et al., 1994
), an approach previously successfully used to grow human mammary gland epithelial organoids in vivo (Parmar et al., 2002
). With intact cellcell contacts, most organoids readily adapted to the culture environment and glandular structures could be detected within 24 h. The organoids maintained their glandular morphology and epithelial phenotype throughout the experimental period as indicated by their in vivo-like expression of cytokeratin 18 and E-cadherin, whose immunohistochemical expression has been shown to decrease with dedifferentiation in many tissues, including the human endometrium (Pötter et al., 1999
). Besides structural integrity, the organoids retained their ability to respond to hormonal cues under the optimized culture conditions. No substantial differences in hormonal responses between organoids from different phases of the menstrual cycle were detected. It appears that when brought into standardized culture conditions with a 3 day hormone-free period, the cells retain their inherent capacity to respond to steroid hormones regardless of their original hormonal milieu in vivo. Previously, comparable changes in the proliferation of epithelial cells isolated from proliferative and secretory epithelium in response to stromal cells have been described (Arnold et al., 2001
).
Although endometrial epithelial cells respond to follicular E2 by a marked increase in cell proliferation in vivo, their estrogen responsiveness has been difficult to reproduce in vitro. The results from monocultures of uterine epithelial cells, both rodent and human, imply that E2 may not have a direct mitogenic effect on these cells (Alkhalaf et al., 1991; Uchima et al., 1991
; Marshburn et al., 1992
; Classen-Linke et al., 1997
, 1998). That E2-induced epithelial mitogenesis in the female reproductive tract is rather an indirect process mediated by stromal ER
has been proposed by Cooke et al. (1997)
on the basis of experiments on a rodent model in vivo. A paracrine mode of action for progestins in uterine tissue has also been suggested (Kurita et al., 1998
). Comparison of the three mono- and co-cultures in the present study appears to be in agreement with the above findings. The limited number of replicates and lack of statistical significance do not, however, allow any definite conclusions to be made as to the significance of the stromal compartment to the proliferative effects observed in epithelial organoids. Moreover, the presence of steroid receptors in the stromal compartment, a prerequisite for stromal-mediated effects, was not investigated in the present study. In co-cultures, E2 doubled the number of Ki67-positive cells relative to controls. A somewhat smaller but statistically significant effect was observed when the relative number of Ki67-positive organoids was calculated, providing a convenient means to assess proliferative hormone effects in the present model. MPA was shown not only to counteract the effect of E2, but also to reduce cell proliferation to half of that in controls. As for E2 effects, our findings are in agreement with those of Pierro et al. (2001)
who used co-cultures of epithelial cell monolayers on Matrigel-coated inserts and stromal cells on plastic to study estrogen responsiveness in normal human endometrium. Conversely, in the three organoid monocultures analysed in the present study, E2 both alone and together with MPA decreased the Ki67 index by approximately half. A similar phenomenon has previously been reported in rodent epithelial monocultures (Astrahantseff and Morris, 1994
), whereas in the human model of Pierro et al. (2001)
no marked change compared to controls was detected. The difference between the two human models may reflect differences in their respective culture environments but, because of the limitations associated with the present monoculture data, the significance of these observations remains unclear.
The effect of stromal-produced factors on epithelial cell proliferation has also been shown in cultures without hormonal stimuli. Stromal cell-conditioned medium has been reported to increase the replication of human endometrial epithelial cells in monolayer cultures (Akoum et al., 1996). Also Pierro et al. (2001)
showed an enhanced epithelial proliferation in control co-cultures as compared with parallel epithelial monocultures and the same trend was seen, albeit less reproducibly, in the present study. In the study of Arnold et al. (2001)
, on the other hand, stromal cells were reported to inhibit epithelial cell growth especially when embedded in Matrigel in contact co-culture. Thus, it appears likely that proteins of the basement membrane may modulate the regulatory capacity of adhering stromal cells. Considering the potential impact of Matrigel on stromal cell function in our culture conditions, adequate cellular purity of the epithelial compartment is important to accomplish.
The expression of ER and PR in the human endometrial epithelium changes characteristically during the normal endometrial cycle, being maximal in the proliferative phase and diminishing in the mid-secretory phase (Snijders et al., 1992; Noe et al., 1999
; Vienonen et al., 2004
). In agreement with the known effects of estrogen and progestins on PR expression, E2 was found to induce and MPA to down-regulate PR expression in the present organoid culture. Previously, hormonal regulation of PR has been reported in human primary endometrial epithelial cells cultured on top of Matrigel (Classen-Linke et al., 1997
, 1998) and in explant cultures of the human proliferative endometrium (Illouz et al., 2003
). In both these models, MPA was shown to decrease not only PR but also ER expression. Also, different types of progestins have been shown to have different in vitro effects. (Illouz et al., 2003
). With regard to ER
, only minor changes in the staining intensity of positive cells could be detected in our study after steroid treatment. The high level of ER
detected in epithelial organoids without hormonal stimuli resembles their constitutive expression in the post-menopausal uterus (Snijders et al., 1992
; Noe et al., 1999
).
In conclusion, most of our current knowledge concerning the effects of steroids on the human endometrium derives from clinical data, and in vitro methods allowing experimentation on human endometrial tissue have been limited. The organotypic model presented here provides an in vitro setting in which to study steroid hormone effects on the normal human endometrium both in terms of cell proliferation and gene expression. The model facilitates the manipulation of the culture environment and may help to elucidate the molecular basis of the effects of steroids and other biological agents in human endometrial cells, thereby obviating the need of extrapolation from animal models. The culture system may have potential uses in preclinical screening of hormones for therapeutic use.
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Acknowledgements |
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References |
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Submitted on August 18, 2004; accepted on December 7, 2004.
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