1 Department of Oncology, Johns Hopkins Medical Institutions, Baltimore, MD, 2 Departments of Laboratory Pathology and Medicine, University of North Carolina, Chapel Hill, NC, USA, 3 Department of Obstetrics and Gynecology, Helsinki University Central Hospital, Finland and 4 Department of Obstetrics and Gynecology, University of North Carolina, Chapel Hill, NC, USA
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Abstract |
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Key words: co-culture/endometrium/epithelium/glycodelin/stroma
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Introduction |
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In the female reproductive tract, stroma can promote epithelial development or reprogramme epithelial differentiation (Cooke et al., 1986; Bigsby and Cunha, 1986
; Donjacour and Cunha, 1991
). The endometrium is composed of mesodermal-derived glandular and luminal epithelia that are supported by a basement membrane and connective tissue stroma. Basement membrane likely plays a key role in promoting an epithelial phenotype (Classen-Linke et al., 1997
). In addition, stromal cells provide a regulatory role for growth and differentiation of the overlying epithelium (Cunha et al., 1985
). Mesenchymal regulation of epithelium is especially evident in the cycling endometrium, which undergoes monthly developmental changes in response to ovarian steroids. It now appears that steroid receptors in the stromal cells, but not the epithelium, may be required for the action of oestradiol and progesterone (Cooke et al., 1997
; Kurita et al., 1998
), demonstrating the paracrine role for the stromal cells in endometrial function.
The primary objective of the present work was to demonstrate that human stromal cells regulate normal, non-neoplastic human epithelial growth and differentiation in vitro. While this concept has been supported in animal models (Chung et al., 1992; Cunha and Young, 1992
; Cooke et al., 1997
), there have been few if any successful attempts demonstrating this regulatory role of stroma using in-vitro human cell model systems. The advantages of studying such interactions in vitro include: (i) the ability to manipulate cells in a defined environment and to retrieve and study specific cell types, (ii) to identify novel growth or inhibitory paracrine factors involved in regulation of growth, (iii) better to reproduce cell behaviour in situ and provide a physiological basis for future studies of molecular and genetic mechanisms of disease, and (iv) by using human cells the mechanisms of cellcell and cellmatrix interactions involved in human disease can be dissected, avoiding extrapolation of data from studies using other species. The use of a novel configuration of epithelial and stroma within appropriate extracellular matrices provides new insights into the paracrine role of stroma in directing epithelial cell function.
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Materials and methods |
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Isolation of primary endometrial epithelial and stromal cells
Endometrial tissue was rinsed in Hanks' balanced salt solution (HBSS) to remove blood and debris. Separation of glandular and stromal components was based on a modification of the work of Satyaswaroop and colleagues (Satyaswaroop et al., 1979). Following gentle centrifugation (600 g) the supernatant was removed and the tissue was placed on a 100 mm plastic tissue culture dish (Corning-Costar, Cambridge, MA, USA) under a sterile laminar flow hood. The tissue was minced with sterile scalpels into 1 mm2 fragments and then digested with collagenase (2 mg/ml, CLS-1, Worthington Biomedical, Freehold, NJ, USA) in isolation medium (as above) for 2.5 h at 37°C on a shaking rotor. The tissue digest was vigorously pipetted to break up any remaining tissue pieces and passed over a stacked sterile wire sieve assembly with number 100 wire cloth sieve (140 µm size, Newark Wire Co., Newark, NJ, USA), followed by a number 400 wire cloth sieve (37 µm) as shown in Figure 1
.
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Stromal cells were collected from the lower receptacle, pelleted by centrifugation and resuspended in 3 ml of isolation medium. Red blood cells were removed by carefully layering cell suspension over 3 ml of Ficoll-Paque (Pharmacia, Piscataway, NJ, USA) in a sterile 15 ml polycarbonate tube. The solution was centrifuged for 810 min at 400 g. The media/Ficoll interface layer containing the stromal cells was plated onto 100 mm plastic tissue culture dishes. Stromal cells were cultured for at least 3 days before being plated for co-culture experiments, as described below. Epithelial cells grew 35 days before co-culture, to insure no stromal cell contamination. Purity of epithelial and stromal components was assessed by morphological determination by light microscopy and reassessed by cytokeratin and vimentin staining for epithelial and stromal cells respectively. Each cell population was routinely over 98% pure as assessed by phase microscopy. In parallel studies with primary stromal and epithelial cultures both stromal and epithelial cells were found to respond to oestrogen by increased growth and [3H]-thymidine uptake, but these data are beyond the scope of the present paper.
Cell culture media
Stromal cells were maintained in medium consisting of a 1:1 mixture of M199:Ham's F12 media (Gibco Life Technologies) supplemented with 4% heat-inactivated FBS, ITS+ [containing insulin (0.62 µg/ml), transferrin (0.62 µg/ml), and selenium (0.62 ng/ml), bovine serum albumin (125 µg/ml) and linoleic acid (52.6 µg/ml) (Collaborative Biomedical Products)] plus 100 units penicillin, 0.1 mg streptomycin and 0.25 µg amphotericin B per ml (antibiotic/antimycotic solution, Sigma). FBS was heat inactivated by incubating in a 56°C water bath for 1 h prior to filter sterilization. Culture medium was routinely changed every 34 days. Charcoal-stripped serum was used to remove steroids from culture media.
The primary epithelial gland cells required little or no serum and were cultured in media consisting of M199 and F12 (1:1) with added Mitoplus® (2 ml/l) (Collaborative Biomedical Products), bovine pituitary extract (BPE; 2ml/l) (Collaborative Biomedical Products), ITS+ (concentrations as above), and antibiotic and antimycotic agents as described above.
For co-culture, stromal and epithelial cells were grown in media containing Phenol Red-free M199 and F12 media (1:1) (Gibco) with ITS+ [(insulin (3.1 µg/ml), transferrin (3.1 µg/ml), and selenium (3.1 ng/ml), bovine serum albumin (625 µg/ml) and linoleic acid (263 µg/ml)] plus 0.1 mmol/l phospho-ethanolamine (Sigma) and 1% charcoal-stripped FBS.
Co-culture of stromal and epithelial cells
For purposes of co-culture, cells were plated either on plastic or on Millicell® CM filter inserts (Millipore, Bedford, MA, USA) containing Matrigel®. Endometrial epithelium was cultured either alone or in co-culture with stromal cells. Prior to plating, BME was kept at 4°C and was added undiluted to pre-cooled MilliCell® CM filter inserts (0.4 µm, 12 mm diameter).
Stromal and epithelial cell co-cultures were prepared from freshly isolated primary epithelial and early passage stromal cells (passage 14). Frozen cells were not used as new primary cell lots were prepared for each co-culture. Co-cultures mostly consisted of stromal and epithelial cells from the same patient. Typically, stromal cells were plated at 105 cells per well of a 24 well plate and all conditions were performed in triplicate. Stromal cells were either grown within the Matrigel® on the filter insert or beneath the filter insert on the plastic culture dish. These culture conditions are depicted in Figure 2, comparing epithelial monoculture (a) to co-culture with stromal cells on plastic below the filter insert (b) or embedded in Matrigel® within the filter insert (c). When suspended within the BME, stromal cells were pooled, pelleted and resuspended in the total volume of undiluted Matrigel® at 4°C (liquid phase). Aliquots of 100 µl Matrigel® plus 105 stromal cells per well were added to the cold inserts, spread evenly over the inserts and allowed to gel at 37°C for 1 h. Parallel inserts were prepared for epithelial monocultures using the undiluted Matrigel® in the absence of added stromal cells. Culture medium was gently added over the Matrigel® coated inserts at 1 ml/well after 1 h at 37°C (gel phase).
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Cell proliferation assays
Isolated primary cells were plated at 2x105 cells per well in six well dishes. Co-cultures were prepared as previously described using filter inserts and Matrigel®. After 7 days in culture, cells were released from the Matrigel® by digesting with a 1:1 mixture of trypsin-EDTA (Gibco Life Technologies) and Matrisperse® (Collaborative Biomedical Products) on ice for 3060 min. Cell number was quantified using a haemocytometer and/or Coulter cell counter (Coulter Corp., Miami, FL, USA).
DNA synthesis was determined using tritiated thymidine incorporation, as a measure of cell proliferation. Epithelial cells were plated in triplicate in monoculture or co-cultured, as described. After 4 or 7 days of growth, tritiated (3H) thymidine was added in culture media at a final concentration of 2.5 µCi/ml. After 24 h, the medium was removed. Cultures were rinsed twice with HBSS and trichloro-acetic acid (TCA; 5%) was added to each culture well at 4°C for 20 min, to fix cells to the plates. The plates were rinsed three times with 5% TCA to remove unincorporated radioactive material. The cells were solublized with 0.2 mol/l NaOH, for 30 min at 37°C, before neutralization with 75 mmol/l HCl. The cell solution was added to scintillation vials and counted for 10 s in a Packard Scintillation Counter (Packard, Meriden, CT, USA). Thymidine incorporation was standardized to total cell counts (CPM/cell) or to DNA concentration (fmol/mg DNA). DNA concentrations were determined by isolating DNA from parallel wells using DNAzol (Molecular Research Center, OH, USA). Isolated DNA was quantified by spectrophotometric absorbency at 260 nm.
Glycodelin immunofluorometric assay (IFMA)
To compare indices of differentiated function in mono- and co-cultures of human endometrial epithelium with or without stromal cells, we used the epithelial gene product glycodelin (PP14). Primary endometrial epithelial cells (obtained only from proliferative phase endometrium to avoid pre-existing glycodelin expression) were plated alone or in co-culture with stromal cells either on plastic or in Matrigel® as previously described. Supernatant media were collected on days 2, 5, 9, and 12 at the time of media change and stored at 80°C. Media were assayed for glycodelin using an immunofluorometric assay (Koistinen et al., 1996). The immunofluorometric assay used monoclonal antibodies to glycodelin for coating the microtitre wells and secondary labelling with europium III chelate, as previously described (Rittinen et al., 1989
). The sensitivity of the immunofluorometric assay is more than x25 greater than that of radioimmunoassay and allows detection and accurate quantification of glycodelin in samples undetectable by radioimmunoassay. Experimental samples of 25 µl of cell culture supernatants were added to microtitre plates precoated with antibodies to glycodelin. Plates were incubated overnight at room temperature. Samples were washed and the secondary antibody, europium III chelate, was added to each well and incubated at room temperature for 2 h. The wells were washed and a fluorescence enhancement solution containing 2-naphtoyltrifluoroacetone and tri-n-octylphosphine oxide was added. Fluorescence was measured using a fluorometer. Results are presented as means of triplicate determinations in units of ng glycodelin/µg DNA.
Statistical analysis
Statistical significance of data was tested using analysis of variance (ANOVA) with Scheffé's correction to compare data from multiple conditions. For comparison of two populations, a two-sample Student's t-test was used.
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Results |
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To investigate the contributions of extracellular matrix on epithelial glandstroma interactions, we initially analysed the growth characteristics of isolated stromal or epithelial cells in monoculture when cultured with Matrigel® as compared with culture on plastic. The effect of the BME substrate on stromal and epithelial cell proliferation after 5 days in culture is depicted in Figure 3. Endometrial epithelial cells displayed less proliferation on plastic surfaces compared to Matrigel® (P < 0.05). Conversely, stromal cells exhibited greater proliferation on plastic surfaces as compared to when they were cultured in contact with Matrigel® (P < 0.05). The stromal cells remain viable as assessed by Trypan Blue exclusion, yet do not proliferate when embedded in the Matrigel®.
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Since a distinction between cell types in co-culture was difficult using phase microscopy alone (Figure 5a), epithelial cells were labelled using anti-cytokeratin antibodies and Texas Red fluorochrome and stromal cells with vimentin antibodies and using fluorescein (FITC-green fluorochrome). The nuclei of all cells were stained blue with DAPI. As shown in Figure 5bf
, the dynamic nature of epithelial cellstromal cell interaction is mediated in part by the presence of this extracellular matrix. As shown in Figure 5b, c and d
, epithelial cells (shown in red) formed spherical structures surrounded by stromal cells (green staining) that had adopted a position around the perimeter when cultured on Matrigel®. In cross-section and at higher power this was more evident (Figure 5d
). The cytoplasmic projections from single stromal cells sometimes appeared to project towards specific epithelial cells (Figure 5e
). When endometrial epithelial and stromal cells were co-cultured on plastic, this higher order of organized association was not apparent (Figure 5g
).
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Discussion |
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Attempts to demonstrate stromal regulation of epithelial cell function have been explored in other tissues with the development of various cell culture models. In collagen gels, fibroblasts induced epithelial tubular morphogenesis in MDCK kidney cells (Montesano et al., 1991). Media conditioned by stromal cells was shown to inhibit proliferation of prostate epithelial cells in vitro (Kooistra et al., 1995
). Stromal induction of prostatic epithelial differentiated function has also been studied by Chung et al. (Chung et al., 1992
) in an in-vitroin-vivo model and in three-dimensional organoid cultures (Zhau et al., 1997
). Co-culture models have also been developed for keratinocytes (Konig and Bruckner-Tderman, 1991
; Fusenig, 1992
) and for mammary cells in which stromal cells were found to regulate epithelial function through interaction with extracellular matrix constituents (Haslam, 1986
; Haslam and Counterman, 1991
). Few in-vitro studies using human endometrial cells have been successful at demonstrating such differences in epithelial proliferation or regulation of differentiated cell function that are dependent on the presence of stromal cells. As revealed in the current study, contact with basement membrane extract (Matrigel®) provides a key modification to published methods which successfully demonstrates in-vitro regulation of endometrial epithelial growth and differentiation that is mediated by endometrial stroma.
The human endometrium is a very relevant tissue for investigating the influence of the tissue micro-environment on epithelial growth and differentiation. In the adult, the endometrium undergoes cyclic developmental changes that can be studied in the context of endocrine, paracrine and extracellular influences. It is a tissue that is readily accessible and many of the regulated genes have now been identified (Bell and Drife, 1989; Seppälä et al., 1992
). Methods to separate and culture endometrial stromal and epithelial elements have now been well established, and the purity and morphology have been described (Kirk et al., 1978
; Satyaswaroop et al., 1979
; Varma et al., 1982
; Osteen et al., 1989
; Classen-Linke et al., 1997
). The importance of the extracellular matrix for epithelial polarization has previously been demonstrated for endometrial cells (Schatz et al., 1990
) as well as for re-establishment of three-dimensional morphology (Kaufman et al., 1980
; Rinehart et al., 1988
; Bentin-Ley et al., 1994
; Classen-Linke et al., 1997
). Epithelial cells in culture express endometrial specific secretory products (Wegner and Carson, 1992
) and can respond to the ovarian steroid hormones (Astrahantseff and Morris, 1994
; Osteen et al., 1994
; Classen-Linke et al., 1997
). With the exception of work by Osteen demonstrating stromal regulation of epithelial metalloproteases, mediated by tumour growth factor (TGF)-ß (Osteen et al., 1994
), stromal regulation of epithelial function had not been reproduced in cell co-culture for human endometrium.
The configuration of cell co-culture used in this study is the first that we are aware of to place stromal cells in direct contact with basement membrane material. Our data suggest that optimal configuration for stromal growth inhibitory effect in co-culture was when stromal cells were not only in the presence of BME but also in direct proximity to the epithelial cells. Diffusible factors created by the stromal cells in Matrigel® were not sufficient for inhibition. Whereas much of the extracellular matrix of the interstitial stroma is primarily comprised of fibronectin (Fazleabas et al., 1997), this study offers the hypothesis of a potential unique regulatory role for the periglandular subpopulation of stromal cells which are in contact with the basement membrane.
BME or Matrigel® has been used in studies of epithelial cells in vitro for several tissues, including the endometrium (Rinehart et al., 1988; Kliman and Feinberg, 1990
; Mahfoudi et al., 1991
; Streuli et al., 1991
; Bentin-Ley et al., 1994
; Baatout, 1997
; Classen-Linke et al., 1997
). This material is derived from the Engelbreth-Holm-Swarm (EHS) tumour cell line (Kleinman et al., 1986
). This reconstituted basement membrane extract contains laminin (60%), type IV collagen (30%), heparan sulphate proteoglycan (8%) entactin or nidogen (1%) and resembles the basal lamina at the electron microscopic level when polymerized. Endometrial epithelial cells attach to the basement membrane proteins and become polarized through specific interactions with cell adhesion molecules (Davis and Camarillo, 1995
; Strunck and Vollmer, 1996
; Fukushima et al., 1998
). In a model using fetal mouse mammary cells, just the presence of Matrigel® was enough to restore hormone-dependent gene expression (Streuli et al., 1991
; Streuli and Bissell, 1991
), but Matrigel® alone does not appear to be sufficient for full differentiation of adult human endometrial epithelial cells, as demonstrated by our study.
What has not been appreciated is the effect basement membrane proteins may have on the cells that reside on the side opposite to the epithelial cells, namely the stroma. Since stromal cells are now suspected of having major roles in epithelial growth and differentiation in adult tissues (Cunha et al., 1985), we reasoned that stromal cells closest to the epithelial cells would be the most likely to be involved in emitting regulatory signals. Basement membrane itself, which has two sides, may be the stimulus required for a differentiation of stromal cells toward this paracrine role. We have previously demonstrated this phenomenon in situ by examining HBepidermal growth factor (EGF). Only the stromal cells adjacent to basement membrane expressed this growth factor, while stromal cells away from the basement membrane (and epithelial cells) displayed little if any HBEGF expression (B.A.Lessey et al., unpublished data). As witnessed by the significant morphological changes that occur in both cell types shown in the present study, it appears that Matrigel® may activate a collection of genes that alter cellular phenotype in vitro by direct and indirect effects.
To investigate further the paracrine effects of stromal cells on epithelial cell function, several gene products were considered as potential markers of differentiation. Glycodelin, also known as placental protein-14 (PP-14) or 2-PEG, is an epithelial secretory product present in the late luteal phase and in the decidualized endometrium during early pregnancy (Julkunen et al., 1986a
, b
; Seppälä et al., 1994
). This glycoprotein has an extensive carbohydrate content and may have contraceptive potential by inhibiting binding of human spermatozoa to the zona pellucida in vitro (Dell et al., 1995
; Oehninger et al., 1995
; Clark et al., 1996
). Glycodelin has also been demonstrated to exhibit immunosuppressive activity (Okamoto et al., 1991
; Dell et al., 1995
). While the exact function of glycodelin remains to be elucidated, its synthesis increases in endometrium around the time of implantation and early pregnancy, suggesting a role in the establishment of pregnancy (Klentzeris et al., 1994
). Glycodelin can be assayed readily using immunofluorometric techniques as described in the present study. As such, the use of this hormone-regulated endometrial gene product was ideal for examining the effect of stromal cells on epithelial differentiation in our co-culture model system.
The regulation of glycodelin expression has previously been used to assess differentiation in endometrial epithelial cells in vitro. Preliminary data by White et al. (White et al., 1990) demonstrated that a mixed culture of endometrial stromal and epithelial cells in Matrigel® expressed glycodelin (PP14) more than epithelial cells growing in a monolayer without Matrigel®. Mixed endometrial cell cultures grown on a plastic substrate were reported to exhibit glycodelin concentrations that rapidly declined in culture within the first week (Chatzaki et al., 1994
). It was noted that this decrease in glycodelin was associated with a parallel increase in epithelial cell senescence. With successful separation of epithelial and stromal cells in culture we have further demonstrated that the increase in glycodelin concentrations was greatly facilitated by co-culture with stromal cells in the presence of Matrigel®.
As with any model, this co-culture system has both advantages as well as limitations. While we would like to recreate conditions in vivo as much as possible, we have not included other endometrial cellular components such as endothelial cells, immunological cells or myometrium; all of these cells may play a role in the function of this complex tissue. It was, perhaps, fortuitous that inclusion of only isolated epithelial and stromal cells demonstrated the restoration of epithelial responsiveness. Stromal cells, epithelial cells and contact with basement membrane proteins may be the minimal requirements for in-vitro restoration of endometrial growth and differentiation.
For endometrium, this model will have significant impact for the investigation of implantation and the establishment of uterine receptivity as well as elucidating factors that favour the development of endometrial hyperplasia or cancer. Furthermore, the important relationships between cells, endocrine and paracrine responses, and the extracellular matrix can now be more logically dissected and analysed. The model proposed here, in which stromal cells encounter BME, illustrates the importance of the tissue micro-environment and its effect on cell function. This simple alteration to the traditional methods for endometrial cell culture yields striking new results and provides potentially new insights into important questions in cell biology.
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Acknowledgements |
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Notes |
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References |
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Submitted on October 6, 2000; accepted on February 2, 2001.