Stromal–epithelial cell interactions and alteration of branching morphogenesis in macromastic mammary glands

True macromastia is a rare but disabling condition characterized by massive breast growth. The aetiology and pathogenic mechanisms for this disorder remain largely unexplored because of the lack of in vivo or in vitro models. Previous studies suggested that regulation of epithelial cell growth and development by oestrogen was dependent on paracrine growth factors from the stroma. In this study, a co‐culture model containing epithelial and stromal cells was used to investigate the interactions of these cells in macromastia. Epithelial cell proliferation and branching morphogenesis were measured to assess the effect of macromastic stromal cells on epithelial cells. We analysed the cytokines secreted by stromal cells and identified molecules that were critical for effects on epithelial cells. Our results indicated a significant increase in cell proliferation and branching morphogenesis of macromastic and non‐macromastic epithelial cells when co‐cultured with macromastic stromal cells or in conditioned medium from macromastic stromal cells. Hepatocyte growth factor (HGF) is a key factor in epithelial–stromal interactions of macromastia‐derived cell cultures. Blockade of HGF with neutralizing antibodies dramatically attenuated epithelial cell proliferation in conditioned medium from macromastic stromal cells. The epithelial–stromal cell co‐culture model demonstrated reliability for studying interactions of mammary stromal and epithelial cells in macromastia. In this model, HGF secreted by macromastic stromal cells was found to play an important role in modifying the behaviour of co‐cultured epithelial cells. This model allows further studies to investigate basic cellular and molecular mechanisms in tissue from patients with true breast hypertrophy.     

Normal human endometrial cells in culture: Characterization and immortalization of epithelial and stromal cells by SV 40 large T antigen

Summary— Thirty endometrial biopsies were cultured in order to separate stromal and epithelial cells. Using epidermal growth factor (EGF), cortisol, cholera toxin, insulin with 5% horse serum for epithelial cells or a medium with 20% fetal calf serum for stromal cells, we could specifically enrich endometrial culture in epithelial or stromal cells and culture them for 1 or 2 months. These cultures retained the phenotypic characteristics of epithelial (cytokeratins, mucin HMFG 1) and stromal (vimentin, smooth muscle actin, desmin) lineage by immunostaining analysis. Epithelial and stromal cultures from one individual were respectively immortalized by the SV 40 large T antigen. The immortalized cell lines kept the phenotype of the normal cells from which they derived.     

Estradiol-17β stimulates proliferation of uterine epithelial cells cultured with stromal cells but not cultured separately

Summary There is indirect evidence that the in vivo proliferative response of rodent uterine epithelium to estrogen requires interaction with the underlying stroma in pre- and post-pubescent animals. To examine this potential requirement directly, the proliferative response of epithelium to 17β-estradiol in the presence or absence of stroma was measured in vitro. Uterine epithelial and stromal cells were isolated separately from immature or adult mice, and were maintained as monocultures or cocultures in defined, serum-free medium with or without 8 × 10⁻⁹ M 17β-estradiol. Incorporation of bromodeoxyuridine into the DNA was determined by immunolabeling to assay proliferation in individual cells. Cell morphology and immunolabeling of cytokeratin were used to distinguish epithelial from stromal cells. Treatment of cocultures with 17β-estradiol for 24 h increased the proliferation of epithelial cells relative to controls approximately threefold, whereas, in monocultures of epithelial or stromal cells 17β-estradiol decreased the number of bromodeoxyuridine-incorporating cells by approximately half. Furthermore, cell contact between epithelial and stromal cells was important for the effects of 17β-estradiol on cells in cocultures. Approximately three quarters of the 17β-estradiol-induced proliferation of epithelial cells in cocultures was produced by epithelial cells within colonies that were also contacting stromal cells. These results are consistent with the hypothesis that stromal cells mediate the estrogenic proliferative response, and provide evidence that this mediation involves cell contact or stroma-mediated changes in the microenvironment immediately around the epithelial cell.     

Effect of mouse uterine stromal cells on epithelial cell transepithelial resistance (TER) and TNFalpha and TGFbeta release in culture

Recognizing that uterine stromal cells regulate several uterine epithelial cell function(s), the current study was undertaken to more fully define cell-cell communication in the uterus and to examine the role of uterine stromal cells in regulating epithelial cell monolayer integrity and cytokine release. Uterine epithelial and stromal cells from adult intact mice were isolated and cultured separately on cell culture inserts and/or in culture plates. Epithelial cells, which reach confluence as indicated by high transepithelial resistance (TER > 1000 ohms/well), preferentially release transforming growth factor-beta (TGFbeta) into the basolateral chamber ( approximately 70% > apical) and tumor necrosis factor-alpha (TNFalpha) into the apical compartment ( approximately 30% > basolateral). When epithelial cells on cell culture inserts were transferred to plates containing stromal cells, coculture for 24-48 h increased epithelial cell TER ( approximately 70% higher than control) and decreased TNFalpha release into both the apical and basolateral chambers ( approximately 30%-50%). In contrast, TGFbeta release was not affected by the presence of stromal cells. In other studies, the effects of stromal cells on epithelial cell TER and TNFalpha release persisted for 5-7 days following the removal of stromal cells and were also seen in coculture studies in which conditioned stromal media (CSM) was placed in the basolateral chamber. These studies indicate that uterine stromal cells produce a soluble factor(s) that regulates epithelial cell TER and release of TNFalpha without effecting TGFbeta release. These results suggest that uterine stromal cells communicate with epithelial cells via a soluble factor(s) to maintain uterine integrity and epithelial secretory function.     

Progesterone and estrogen receptor distribution in the endometrium of the mare

An immunoperoxidase staining technique was used to localize receptors for progesterone and estrogen in the uterus of the mare. Specific staining for receptors was limited to cell nuclei. During estrus, stromal cells tended to stain more intensely for both receptor types than myometrial cells or luminal and glandular epithelial cells. During diestrus, staining intensities in stromal and myometrial cells tended to decrease. Staining intensities of epithelial cells were not affected by the cycle stage. Early pregnancy did not markedly affect the staining intensities of pregnant mares compared with the nonpregnant mares on Day 14 of diestrus. In mares susceptible to endometritis from which samples were taken during diestrus, stromal and myometrial staining for estrogen receptors was more intense than in endometrium from genitally-normal mares.     

Two-dimensional protein profiles of cultured stromal and epithelial cells from hyperplastic human prostate

Studies were undertaken to compare and contrast the two-dimensional protein profiles of epithelial and stromal cells from hyperplastic human prostate to establish the protein composition of the two major cellular components of the prostate. Epithelial and stromal cells were isolated from human prostate obtained from patients undergoing open prostatectomy for benign prostatic hyperplasia (BPH). Proteins, isolated from the two cell populations and separated by two-dimensional (2D) electrophoresis, were analyzed by silver staining, fluorography of [35S]-methionine-labeled proteins, and immunoprotein blotting. Isolated prostatic epithelial cells, but not stromal cells, contained cytokeratin polypeptides 5, 6, 7, 8, 13, 14, 15, 16, 17, 18, and 19. Although vimentin could not be identified in silver stained 2D gels and fluorographs of cultured prostatic epithelial cells, a low level of immunoreactivity was noted following immunoblot analysis of epithelial cells proteins by the use of an anti-vimentin polyclonal. Vimentin was prominently expressed in cultured prostatic stromal cells and could be identified on silver stained 2D gels, fluorographs, and immunoblots of stroma-derived proteins. In addition, stromal marker proteins SM1, SM2, and SM3 were identified in 2D gels of stromal cells to distinguish them from epithelial cells. These studies demonstrate (1) the two-dimensional protein profile and cytokeratin polypeptide composition of cultured epithelial cells from hyperplastic human prostate and (2) the 2D protein profile of cultured prostatic stromal cells and identification of specific stromal marker proteins.     

Isolation and culture of glandular epithelial and stromal cells from the endometrium of mares.

Glandular epithelial and stromal cells were isolated from the endometrium of mares by collagenase digestion and were incubated on plastic for 7-9 days until the cells formed confluent monolayers. The cells differed in morphology: epithelial cells appeared polyhedral and stromal cells were spindle like. The monolayers were incubated in the presence and absence of oxytocin. Medium was removed from wells after 2, 8 and 24 h of incubation. Concentrations of prostaglandin F (PGF) in the medium increased significantly during this time. Glandular epithelial cells produced significantly more PGF than did stromal cells. Both types of cell responded significantly to oxytocin stimulation by increased secretion of PGF; the response of glandular epithelial cells tended to be greater than that of stromal cells. Secretion of PGF by cultured cells was not affected by cycle stage or pregnancy.     

Human mammary cell survival following ionizing radiation

We have developed and evaluated methods of culturing defined stromal and epithelial populations of normal human breast cells. These cell populations were used to generate radiation dose/survival curves. The epithelial cell population required specific hormones, growth factors, and conditioned media, as well as fibroblast feeder layers for clonal growth. Stromal cells grew well in a less complex medium. The stromal and parenchymal cell populations of the normal human breast were characterized by light and electron microscopy, immunohistochemical human fibronectin staining, gamma glutamyltranspeptidase histochemical staining, and cell sizing. Survival curves were generated using cells from four donors. The average D0 for epithelial cells was 122 cGy, with an average n value of 2.4. The average D0 and n values for stromal cells were 114 cGy and 2.0. The survival of human breast epithelial cells is compared to that of the cells of the rat mammary gland. The D0 values of both species are essentially the same, while the n value for human epithelial cells is lower. This difference in the n value may be a species specific response to radiation, or may merely reflect a difference in the two assay systems used to generate the survival curves.     

Steroid receptors in canine endometrial cells can be regulated by estrogen and progesterone under in vitro conditions

The expression of estrogen (ER) and progesterone receptors (PR) in the endometrium is regulated by steroid hormones. An increase in plasma estrogen leads to upregulation of the number of both steroid receptors, whereas a decrease in both receptors population is due to high concentration of plasma progesterone. To study the exact effect of different concentrations of beta-estradiol and progesterone on canine epithelial and stromal endometrial cells an in vitro model from dog uterus was developed and kept for 20 days. Material was obtained from healthy dogs, undergoing ovariohysterectomy. Endometrial epithelial and stromal cells were gained after collagenase treatment, followed by filtration steps. Electron microscopy and immunolabeling were used to study cell morphology and differentiation. Immunocytochemistry was used to determine proliferation rate (Ki-67), ER and PR status on Days 3, 8, 10, 13, and 20. Mitotic activity of both cells was stimulated with different concentrations of steroids and revealed high values until cells reached confluency. ER and PR expression in confluent layer from epithelial and stromal cells was upregulated with beta-estradiol. In addition progesterone significant downregulated both receptors population in stromal cells, whereas the reduction was less pronounced in epithelial cells. Results showed that our in vitro system is a useful tool to study the influence of beta-estradiol and progesterone on cell proliferation rate, ER and PR expression. The primary cell culture model helps to avoid experiments on living animals.     

Expression and action of hepatocyte growth factor in bovine endometrial stromal and epithelial cells in vitro.

Hepatocyte growth factor (HGF) is a pleiotropic growth factor that acts on various epithelial cells. The objectives of this study were to determine whether HGF altered the proliferation and prostaglandin (PG) secretion of bovine endometrial stromal and epithelial cells in vitro. We also observed HGF and HGF receptor (c-met) mRNA expression in cultured bovine endometrial stromal and epithelial cells by RT-PCR. Stromal and epithelial cells obtained from cows in early stage of the estrous cycle (days 2-5) were cultured in DMEM/Ham's F-12 supplemented with 10% calf serum. The cells were exposed to HGF (0-10 ng/ml) for 2, 4, or 6 days. HGF significantly increased the total DNA in epithelial (P < 0.05), but not stromal cells. In another experiment, when the cells reached confluence, the culture medium was replaced with fresh medium with 0.1% BSA containing HGF 0-100 ng/ml and the cells were cultured for 24 hr. The HGF stimulated PGF2alpha secretion in epithelial, but not stromal cells. RT-PCR revealed that mRNA of HGF is expressed only in stromal cells, and that c-met mRNA is expressed in both stromal and epithelial cells. These results suggest that HGF plays roles in the proliferation and the regulation of secretory function of bovine endometrial epithelial cells in a paracrine fashion.     

Fine needle aspiration cytologic features of mammary phyllodes tumors

OBJECTIVE: To evaluate specific diagnostic fine needle aspiration cytologic (FNAC) features of phyllodes tumor (PT), particularly in the differentiation from fibroadenoma (FA). STUDY DESIGN: Twenty-eight FNAC of PT were reviewed for smear cellularity, epithelial and stromal fragments, their size and atypia, epithelial/stromal area ratio, background single stromal cells (oval or columnar), multinucleated giant cells, and squamous and apocrine cells. Twenty-one FNAC of fibroadenoma were also assessed for comparison. RESULTS: PT was significantly larger than FA. Epithelial fragments were found in all cases, with atypia present in PT. Stromal fragments were present in half the cases; there was no difference in stromal size, but the epithelial/stromal area ratio was significantly lower in PT than FA. Single columnar stromal cells with recognizable cytoplasm and multinucleated stromal giant cells were seen in some PT but not in FA. CONCLUSION: Cytologic diagnosis of PT remains difficult, with significant overlap with FA. The presence of large size, low epithelial/stromal ratio, epithelial atypia, columnar stromal cells with visible cytoplasm and stromal giant cells favors a diagnosis of PT over FA.     

Regulation of prostaglandin secretion from epithelial and stromal cells of the bovine endometrium by interleukin-1 beta, interleukin-2, granulocyte-macrophage colony stimulating factor and tumor necrosis factor-alpha.

Bovine endometrium was obtained on day 16 of pregnancy (estrus = 0) and separated into epithelial and stromal cell populations. When confluent, the two cell populations were treated for 24 h with cytokines at 1, 10 and 100 ng/ml. Prostaglandin (PG) E2 was the major prostaglandin produced by both cell types. For control cultures, more PGE2 was secreted into medium by stromal cells than by epithelial cells, whereas secretion of PGF was similar for epithelial and stromal cells. Interleukin-1 beta had no effect on prostaglandin production by stromal cell cultures but increased epithelial production of PGE2 and, to a lesser extent, PGF. Conversely, granulocyte-macrophage colony stimulating factor had no effect on epithelial cells but reduced secretion of PGE2 and PGF from stromal cells. There were no effects of interleukin-2 or tumor necrosis factor-alpha on prostaglandin secretion. Results indicate that certain cytokines can regulate endometrial prostaglandin secretion in a cell type-restricted manner.     

Separation and culture of ovine endometrial epithelial and stromal cells: evidence of morphological and functional polarity.

Epithelial and stromal cells from endometria of ovariectomized estradiol-treated Corriedale ewes were separated and purified after collagenase digestion. The separation method utilized differences in the speed and ease of detachment of cultured epithelial and stromal cells attached to plastic in response to brief trypsin exposure. Cells were characterized according to morphological, growth, and histochemical criteria. Contamination of each cell type with the other was less than 1%. Separated cells were grown on plastic or on Matrigel-coated Millicell inserts with nitrocellulose membranes. Transmission and scanning electron microscope analyses demonstrated the existence of tight junctions and prominent microvilli in the epithelial cultures on inserts but not on plastic. Asymmetrical secretion of prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E (PGE) by epithelial cells provided further evidence of polarization. Epithelial cell secretion of PGF2 alpha was greater than that by stromal cells whereas PGE secretion by stromal cells was greater than that by epithelial cells. Epithelial secretion in the basal direction was approximately 4 and 3 times that of apical secretion for PGF2 alpha and PGE, respectively. The separation protocol provides pure populations of ovine endometrial epithelial and stromal cells and the cultured epithelial cells exhibit characteristics of in vivo morphology and polarized function.     

Characterization and co-culture of novel nontransformed cell lines derived from rat endometrial epithelium and stroma

Summary Normal and neoplastic growth of epithelial cells depends on mutual interactions between epithelial and stromal cells. As a tool for the study of the underlying molecular mechanisms, we have developed temperature-sensitive, nontransformed cell lines derived from rat uterine epithelium and stroma by transfecting primary cultures with a temperature-sensitive mutant of the SV40 large T antigen. The epithelial and stromal cell lines obtained shared relevant morphological characteristics with the primary cells from which they were derived. Immunocytochemical analysis showed that the epithelial cell lines expressed the intermediate filament cytokeratin, whereas the stromal lines expressed the intermediate filament vimentin. Alkaline phosphatase activity was present in all cell lines examined. All cell lines were anchorage dependent and did not form foci. One epithelial cell line expressed oxytocin mRNA, a gene product recently shown to be highly expressed in vivo in the uterine epithelium at term. If grown on Matrigel, this cell line formed domelike structures, a further characteristic of its differentiated phenotype. In an attempt to reconstitute an endometrium in vitro, epithelial cells were seeded on top of a layer of stromal cells. Paraffin cross sections showed that this in vitro system consisted of a bilayer structure. Four to five cuboidal epithelial cells were typically anchored atop one stromal cell, forming an endometriumlike tissue. The present in vitro system should provide a useful model for further studies on endometrial functions and epithelial/stromal cell interactions at a molecular level.     

Effects of cell storage and passage on basal and oxytocin-regulated prostaglandin secretion by equine endometrial epithelial and stromal cells

Cell cultures are useful for determining the responses of specific cell types to various factors under controlled conditions and for obtaining a better understanding of in vivo physiologic processes. The aims of the present study were (i) to establish methodologies for isolation, culture and cryopreservation of equine endometrial epithelial and stromal cells; and (ii) to determine the effect of passage and cryopreservation on endometrial cell physiology, based on their basal and oxytocin (OT)-stimulated prostaglandin (PG) release. Epithelial and stromal cells were obtained by enzymatic digestion of equine endometrium collected from Days 2-5 of the estrous cycle (n = 16). Primary epithelial and stromal cells, as well as cryopreserved cells were stimulated with OT (10⁻⁷m) for 24 h. The concentrations of PGE₂ and PGF_2α in the culture medium were measured by enzyme-linked immunosorbent assay (EIA). Oxytocin increased PGE₂ and PGF_2α release by primary cultures of unfrozen epithelial cells until passage I (P < 0.01) and by the primary culture of unfrozen and cryopreserved/thawed stromal cells until passage IV (P < 0.01). Cryopreserved/thawed stromal cells cultured up to passage IV and unfrozen epithelial cells derived from passage I have physiological properties similar to those observed in primary culture and may be successfully used for in vitro studies of PG secretion.     

In vitro prostaglandin release from and platelet-activating factor accumulation in isolated endometrial cells from pregnant and pseudopregnant rabbits.

Prostaglandin (PG) release from and platelet-activating factor (PAF) accumulation by enzymatically isolated endometrial epithelial and stromal cells from Day 6 pregnant and Day 6 pseudopregnant rabbits were studied in vitro, using RIA for PG measurement and a platelet aggregation assay for PAF measurement. On the first day of culture in serum-free media, PGF release into the medium was significantly higher from epithelial cells from Day 6 of pregnancy than from stromal cells from Day 6 of pregnancy or pseudopregnancy. PGE release did not differ significantly among these cell types. The addition of indomethacin (10(-5) M) to similar cultures inhibited release of both PGs from both cell types, but to a much greater extent from stromal than from epithelial cells. Significant stimulation of PG release by A23187 was achieved under all conditions on the fifth day of culture; PGE release was significantly greater than PGF release from stromal cells from Day 6 of pregnancy and pseudopregnancy, and release of both PGs from stromal cells was significantly greater from Day 6 of pregnancy than from Day 6 of pseudopregnancy. PG release from similar cells, cultured in medium containing 10% calf serum, was highest on the first or second day of culture and then, especially for PGF, declined with continued culture. PGE release was significantly higher than PGF release from stromal cells on the third and fourth days of culture. The ratios of PGF/PGE release from epithelial cells were significantly higher than those from stromal cells over the 5-day culture period for both reproductive stages. These ratios indicate the differential release of PGE and PGF from rabbit endometrial cell subpopulations and indicate a preferential release of PGE from stromal and of PGF from epithelial cells. Under basal conditions, PAF was not detected in epithelial or stromal cells cultured for 2 or 4 days, or in the associated culture media. If PAF had been released into the medium, it would have rapidly metabolized. Short exposure to calcium ionophore A23187 (10(-5) M) was able to stimulate PAF accumulation in epithelial and stroma cells in serum-free media, probably via the remodeling pathway. PAF was not detected in the medium. Intracellular PAF accumulation after exposure to A23187 (10(-5) M) for 5 min was significantly greater on the second day of culture than on the fourth day in epithelial and stromal cells from Day 6 of pregnancy.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cell-type specificity of interleukins 1alpha and 1beta on prostaglandin and plasminogen activator production in bovine endometrial cells

Interleukin (IL)-1alpha is a potent stimulator of prostaglandin production in bovine endometrium, and IL-1 affects plasminogen activator (PA) activity in several types of cells. In this study, we determined the effects of IL-1alpha and IL-1beta on production of the prostaglandins PGF(2alpha) and PGE(2) and on PA activity in cultured bovine endometrial epithelial and stromal cells. We also determined the effects of PGE(2) and PGF(2alpha) on PA activity in these cells. Finally, we used RT-PCR to examine the expression of IL-1alpha, IL-1beta, and IL-1 receptor type 1 (IL-1R) mRNA in cultured bovine endometrial cells. This analysis revealed that IL-1alpha mRNA was present only in the stromal cells, whereas IL-1beta and IL-1R mRNAs were present in both cell types. When cultured cells were exposed to IL-1alpha and IL-1beta at concentrations ranging from 0.006 to 3 nM for 24h, IL-1alpha and IL-1beta were found to dose-dependently stimulate PGE(2) and PGF(2alpha) production in stromal cells (P<0.05) but not in epithelial cells. On the other hand, exposure to IL-1alpha and IL-1beta dose-dependently increased PA activity in the epithelial cells, whereas neither stimulated PA production in the stromal cells. When cells were exposed to IL-1alpha and IL-1beta at concentrations ranging from 0.06 to 3 nM for 24h, the two IL-1s differed in their effects on both PGE(2) and PGF(2alpha) production in stromal cells and had significantly differed in their effects on PA activity in epithelial cells. Exposure to PGE(2) and PGF(2alpha) did not affect PA activity in either stromal or epithelial cells (P>0.05). Taken together, these results suggest the possibility that both IL-1alpha and IL-1beta are produced by the stromal cells, that IL-1beta is produced by the epithelial cells, and that IL-1alpha is a far more potent stimulator than IL-1beta of prostaglandin and PA production in cultured bovine endometrial epithelial and stromal cells.     

Exon switching and activation of stromal and embryonic fibroblast growth factor (FGF)-FGF receptor genes in prostate epithelial cells accompany stromal independence and malignancy.

Stroma and the heparin-binding fibroblast growth factor (FGF) family influence normal epithelial cell growth and differentiation in embryonic and adult tissues. The role of stromal cells and the expression of isoforms of the FGF ligand and receptor family were examined during malignant progression of epithelial cells from a differentiated, slowly growing, nonmalignant model rat prostate tumor. In syngeneic hosts, a mixture of stromal and epithelial cells resulted in nonmalignant tumors which were differentiated and slowly growing. In the absence of the stromal cells, epithelial cells progressed to malignant tumors which were independent of the stroma and undifferentiated. The independence of the malignant epithelial cells from stromal cells was accompanied by a switch from exclusive expression of exon IIIb to exclusive expression of exon IIIc in the FGF receptor 2 (FGF-R2) gene. The FGF-R2(IIIb) isoform displays high affinity for stromal cell-derived FGF-7, whereas the FGF-R2(IIIc) isoform does not recognize FGF-7 but has high affinity for the FGF-2 member of the FGF ligand family. The switch from expression of exclusively exon IIIb to exclusively exon IIIc in the resident FGF-R2 gene was followed by activation of the FGF-2 ligand gene, the normally stromal cell FGF-R1 gene, and embryonic FGF-3 and FGF-5 ligand genes in malignant epithelial cells. Multiple autocrine and potentially intracrine ligand-receptor loops resulting from these alterations within the FGF-FGF-R family may underlie the autonomy of malignant tumor cells.     

Clonogenicity of human endometrial epithelial and stromal cells

The human endometrium regenerates from the lower basalis layer, a germinal compartment that persists after menstruation to give rise to the new upper functionalis layer. Because adult stem cells are present in tissues that undergo regeneration, we hypothesized that human endometrium contains small populations of epithelial and stromal stem cells responsible for cyclical regeneration of endometrial glands and stroma and that these cells would exhibit clonogenicity, a stem-cell property. The aims of this study were to determine 1) the clonogenic activity of human endometrial epithelial and stromal cells, 2) which growth factors support this clonogenic activity, and 3) determine the cellular phenotypes of the clones. Endometrial tissue was obtained from women undergoing hysterectomy. Purified single- cell suspensions of epithelial and stromal cells were cultured at cloning density (300-500/cm(2)) in serum medium or in serum- free medium supplemented with one of eight growth factors. Small numbers of epithelial (0.22%) and stromal cells (1.25%) initiated colonies in serum-containing medium. The majority of colonies were small, containing large, loosely arranged cells, and 37% of epithelial and 1 in 60 of stromal colonies were classified as large, comprising small, densely packed cells. In serum-free medium, transforming growth factor-alpha (TGF alpha), epidermal growth factor (EGF), platelet-derived growth factor-BB (PDGF-BB) strongly supported clonogenicity of epithelial cells, while leukemia-inhibitory factor (LIF), hepatocyte growth factor (HGF), stem-cell factor (SCF), insulin-like growth factor-I (IGF- I) were weakly supportive, and basic fibroblast growth factor (bFGF) was without effect. TGF alpha, EGF, PDGF-BB, and bFGF supported stromal cell clonogenicity, while HGF, SCF, LIF, and IGF- I were without effect. Small epithelial colonies expressed three epithelial markers but not stromal markers; however, large epithelial colonies showed little reactivity for all markers except alpha(6)-integrin. All stromal colonies contained fibroblasts, expressing stromal markers, and in some colonies, myofibroblasts were also identified. This analysis of human endometrium has demonstrated the presence of rare clonogenic epithelial and stromal cells with high proliferative potential, providing the first evidence for the existence of putative endometrial epithelial and stromal stem cells.     

Polarized uterine epithelial cells preferentially present antigen at the basolateral surface: role of stromal cells in regulating class II-mediated epithelial cell antigen presentation

To study Ag presentation in the female reproductive tract, DO11.10 TCR transgenic mice specific for the class II MHC-restricted OVA(323-339) peptide and non-transgenic BALB/c mice were used. We report here that freshly isolated uterine epithelial cells, uterine stromal, and vaginal APCs present OVA and OVA(323-339) peptide to naive- and memory T cells, which is reduced when cells are incubated with Abs to CD80 and 86. To determine whether polarized primary epithelial cells present Ags, uterine epithelial cells were cultured on cell inserts in either the upright or inverted position. After reaching confluence, as indicated by high transepithelial resistance (>2000 ohms/well), Ag presentation by epithelial cells incubated with memory T cells and OVA(323-339) peptide placed on the basolateral surface (inverted) was 2- to 3-fold greater than that seen with epithelial cells in contact with T cells and peptide on the apical surface (upright). In contrast, whereas freshly isolated epithelial cells process OVA, polarized epithelial cells did not. When epithelial cells grown upright on inserts were incubated with T cells and OVA(323-339) peptide, coculture with either hepatocyte growth factor or conditioned stromal medium increased epithelial cell Ag presentation (approximately 90% higher than controls). These studies indicate that uterine stromal cells produce a soluble factor(s) in addition to a hepatocyte growth factor, which regulates epithelial cell Ag presentation. Overall, these results demonstrate that polarized epithelial cells are able to present Ags and suggest that uterine stromal cells communicate with epithelial cells via a soluble factor(s) to regulate Ag presentation in the uterus.