Simple and Rapid Model to Generate Differentiated Endometrial Floating Organoids

In a mouse model of menstruation stromal cells respond to breakdown of the tissue by changing their identity to become epithelial cells that are incorporated into the luminal epithelium which is rapidly 'healed' without scarring.

Basal release of urokinase plasminogen activator, plasminogen activator inhibitor-1, and soluble plasminogen activator receptor from separated and cultured endometriotic and endometrial stromal and epithelial cells

To study the effects of stromal epithelial cell interactions on prostate cancer metastasis, we have used primary human prostatic stromal cells derived from malignant and non-malignant tissues and established epithelial cell lines from normal (PNT1a and PNT2-C2) and tumour (PC-3, DU145 and LNCaP) origins. The effects of stromal cells on epithelial cell growth were studied in direct and indirect (using culture inserts) co-culture and by exposure to stromal cell-conditioned medium (assessed by MTT assay). The influence of stromal cells on epithelial cell invasion was measured using matrigel invasion chambers and on epithelial cell motility using time lapse microscopy. Results indicated that epithelial cell line growth was similarly unaffected or inhibited by stromal cells derived from malignant (n = 8) or non-malignant tissue (n = 8). In contrast, PNT2-C2 and PC-3 cells were found to be the least and the most invasive and motile epithelia respectively. Stromal cultures enhanced the invasion of both epithelial cells, but no differences were observed between the use of malignant and non-malignant tissues. All stromal cultures modestly stimulated PNT2-C2 motility but displayed a greater stimulation of PC-3 cell motility, while stromal cells derived from malignant tissue stimulated PNT2-C2 and PC-3 cell motility more than stromal cultures from non-malignant tissues. © 2000 Cancer Research Campaign

Does mouse endometrial epithelial cells and stromal cells have a similar transient receptor potential (TRP)-channel expression profile and to that found in the human endometrium? Mouse endometrial epithelial and stromal cells have a distinct TRP channel expression profile analogous to what has been found in human endometrium, and hence suggests the mouse a good model to investigate the role of TRP channels in reproduction. An optimal intercellular communication between epithelial and stromal endometrial cells is crucial for successful reproduction. Members of the TRP family were recently described in the human endometrial stroma; however their functional expression in murine endometrium remains unspecified. Furthermore, epithelial and stromal cells have distinct functions in the reproductive process, implying the possibility for a different expression profile. However, knowledge about the functional expression pattern of TRP channels in either epithelial or stromal cells is not available. In this study, the expression pattern of TRP channels in the murine (C57BL/6 J strain) endometrium was investigated and compared to the human expression pattern. Therefore, expression was examined in uterine tissue isolated during the natural estrous cycle (n = 16) or during an induced menstrual cycle using the menstruating mouse model (n = 28). Next, the functional expression of TRP channels was assessed separately in endometrial epithelial and stromal cell populations. Quantitative RT-PCR was used to evaluate the relative mRNA expression of TRP channels in murine uterine tissue and cells. To further assess the functional expression in epithelial or stromal cells, primary endometrial cell cultures and Fura2-based calcium-microfluorimetry experiments were performed. The expression pattern of TRP channels during the natural estrous cycle or the induced menstrual cycle is analog to what has been shown in human samples. Furthermore, a very distinct expression pattern was observed in epithelial cells compared to stromal cells. Expression of TRPV4, TRPV6 and TRPM6 was significantly higher in epithelial cells whereas TRPV2, TRPC1/4 and TRPC6 were almost exclusively expressed in stromal cells. N/A. Although relevant mRNA levels are detected for TRPV6 and TRPM6, and TRPM4, lack of selective, available pharmacology restricted functional analysis of these ion channels. Successful reproduction, and more specifically embryo implantation, is a dynamic developmental process that integrates many signaling molecules into a precisely orchestrated program. Here, we describe the expression pattern of TRP channels in mouse endometrium that is similar to human tissue and their restricted functionality in either stromal cells or epithelial cells, suggesting a role in the epithelial-stromal crosstalk. These results will be very helpful to identify key players involved in the signaling cascades required for successful embryo implantation. In addition, these results illustrate that mouse endometrium is a valid representative for human endometrium to investigate TRP channels in the field of reproduction. The Research Foundation-Flanders (G.0856.13 N to J.V.); the Research Council of the Katholieke Universiteit Leuven (OT/13/113 to J.V. and PF-TRPLe to T.V.); the Planckaert-De Waele fund (to J.V.); Fonds Wetenschappelijk Onderzoek Belgium (to K.D.C. and A.H.). None of the authors have a conflict of interest.

The endometrium is the secretory lining of the uterus that undergoes dynamic changes throughout the menstrual cycle in preparation for implantation and a pregnancy. Recently, endometrial organoids (EO) were established to study the glandular epithelium. We have built upon this advance and developed a multi-cellular model containing both endometrial stromal and epithelial cells. We use porous collagen scaffolds produced with controlled lyophilization to direct cellular organization, integrating organoids with primary isolates of stromal cells. The internal pore structure of the scaffold was optimized for stromal cell culture in a systematic study, finding an optimal average pore size of 101 µm. EO seeded organize to form a luminal-like epithelial layer, on the surface of the scaffold. The cells polarize with their apical surface carrying microvilli and cilia that face the pore cavities and their basal surface attaching to the scaffold with the formation of extracellular matrix proteins. Both cell types are hormone responsive on the scaffold, with hormone stimulation resulting in epithelial differentiation and stromal decidualization.

The objective of the present study was to define the afferent arm of the mucosal immune system in the female reproductive tract. When uterine cells were incubated with ovalbumin-specific T cells and ovalbumin, antigen presentation by purified luminal epithelial cells and stromal cells was measured. Analysis of uterine cells from intact rats throughout the reproductive cycle indicated that antigen presentation is controlled by the female sex hormones. Antigen presentation by epithelial cells was high at diestrus (day 3), when estradiol levels are elevated, and low at estrus and diestrus (days 1 and 2). In contrast, antigen presentation by stromal cells was low at diestrus (day 3) and high at estrus and diestrus (days 1 and 2). Estradiol given to ovariectomized rats stimulated epithelial cell and inhibited stromal cell antigen presentation compared with saline controls. To determine whether uterine cells interact with other cells to facilitate antigen presentation, thymus dendritic-like cells were incubated with uterine epithelial cells. Our findings indicated that antigen presentation is enhanced synergistically, suggesting that epithelial cells can interact with stromal antigen-presenting cells. Antibody neutralization studies indicated that both epithelial and stromal cell antigen presentation is mediated through class II, intercellular adhesion molecule-1 and lymphocyte function-associated molecules-1 alpha and -1 beta, which function as accessory molecules. These studies demonstrate that uterine epithelial and stromal cells are responsive to antigenic challenge and that sex hormones play a central role in regulating antigen presentation by uterine cells.

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.

In vitro culture of endometrial stromal and gland cells as a model for endometriosis: The effect of peritoneal fluid on proliferation

Role of endocrine status and cell type in adhesion of human endometrial cells to the peritoneum in nude mice

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)

Objective To establish an efficient method for isolating primary human endometrium stromal and epithelial cells in vitro. Methods Health endometrial tissue of hysterectomy patient was obtained, and was cut into a small piece. Firstly, the human endometrial stromal cells (HUSC) and human endometrial epithelial cells (HUEC) were isolated by the twice enzymatic digestion, twice separation and different adherent time of the two types of cells. And then HUSC and HUEC were identified and the percentage of vimentin (Vim)-positive and cytokeratin (CK)-positive ones were determined by flow cytometry. After that, primary HUSC were stimulated by cyclic adenosine monophosphate (cAMP)+medroxyprogesterone acetate (MPA) for 4 d and 7 d. Results The HUSC purity was (97.0±2.5)% and the HUEC purity was (90.0±4.1)% determined by flow cytometry, indicating that we had highly purified cultures of HUSC and HUEC in vitro. After cAMP+MPA induction for 4 d and 7 d, the HUSC showed more obvious changes in cell morphology and prolactin (PRL) level. Conclusion We successfully established an efficient method of isolation and characterization of primary human endometrium stromal and epithelial cells in vitro. Key words: Human endometrium stromal cells; Human endometrium epithelial cells; Isolation method; Molecular markers; Decidualization

Oxytocin has been demonstrated in the reproductive tissues of male mammals but has not previously been localised in human prostate tissue. Oxytocin has been shown to increase prostatic muscle tone and prostatic growth. It has been found in high levels in the prostate of aging dogs with benign prostatic hyperplasia (BPH). This may be of significance in this species and in humans, who both suffer from BPH, an androgen dependent condition that develops with age. Dihydrotestosterone (DHT) is the active hormone in the prostate and is produced from testosterone by the enzyme 5α-reductase. This conversion has been shown to be augmented in the presence of oxytocin. The aim of this study was to investigate the presence of oxytocin and oxytocin receptor in normal human prostate cells and correlate findings with the presence of 5α-reductase. Normal human prostate stromal epithelial cells (Clonetics) were grown in either prostate stromal cell media or prostate epithelial media (Clonetics). Stromal cells were at passage six and epithelial cells at passage four. Cells were fixed in Bouins and stained with antibodies for oxytocin, oxytocin receptor and 5α-reductase I and II. Western blot analysis was performed on the same cell types for the presence of oxytocin receptor. The presence of oxytocin and oxytocin receptor was confirmed in both epithelial and stromal cells by immunocytochemistry. Oxytocin receptor was confirmed by Western blot with a band present at 66 kDa. 5α-reductase I and II were also localised in both cell types by immunocytochemistry. These results demonstrate for the first time the expression of oxytocin and oxytocin receptor in both stromal and epithelial cells from normal human prostate. The demonstration of oxytocin, oxytocin receptor and 5α-reductase I and II in the same cell types suggest that oxytocin may play a role in the regulation of DHT in the human prostate.

The regulation of prostaglandin E2 and prostaglandin F2 alpha in primary cultures of epithelial and stromal cells of bovine endometrium was investigated using two physiological agents, oxytocin and platelet-activating factor, and the protein kinase C activator, 4 beta-phorbol 12-myristate 13-acetate. At the basal level, epithelial cells produced more prostaglandin F2 alpha than did stromal cells (P < or = 0.0001) and stromal cells produced more prostaglandin E2 than did epithelial cells (P < or = 0.0001). In the presence of oxytocin, production of prostaglandin E2 and F2 alpha increased in a dose-dependent manner, only in epithelial cells. Stromal cells did not respond to oxytocin, suggesting that the oxytocin response is cell type specific, acting preferentially on the cell type in which prostaglandin F2 alpha is the major prostaglandin produced. Platelet-activating factor increased prostaglandin E2 and prostaglandin F2 alpha production in epithelial cells at 1 and 10 pmol l-1 (PGE2: 10 pmol l-1, P < or = 0.01; PGF2 alpha: 1 pmol l-1, P < or = 0.02). Stromal cells also responded to platelet-activating factor, but only at high concentrations (PGE2: 0.1 mumol l-1, P < or = 0.001; PGF2 alpha: 0.1 mumol l-1, P < or = 0.0001). These results further demonstrate the differences in epithelial and stromal cells; epithelial cells are more sensitive to platelet-activating factor than are stromal cells. Phorbol 12-myristate 13-acetate increased prostaglandin production in a dose-dependent manner in both epithelial and stromal cells, indicating that protein kinase C activation can increase prostaglandin production in these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Endometrial stromal and epithelial cell function is typically studied in vitro using standard two-dimensional monocultures, but these cultures fail to reflect the complex three-dimensional (3D) architecture of tissue. A 3D model of bovine endometrium that reflects the architectural arrangement of in vivo tissue would beneficially assist the study of tissue function. An electrospun polyglycolide (PGA) scaffold was selected to grow a 3D model of primary bovine endometrial epithelial and stromal cells, that reflects the architecture of the endometrium for the study of pathophysiology. Electrospun scaffolds were seeded with stromal and epithelial cells, and growth was assessed using histological techniques. Prostaglandin E2 and prostaglandin F2α responsiveness of endometrial scaffold constructs was tested using oxytocin plus arachidonic acid (OT + AA) or lipopolysaccharide (LPS). Stromal and epithelial cells growing on the electrospun scaffold had an architectural arrangement that mimicked whole tissue, deposited fibronectin, had appropriate expression of vimentin and cytokeratin and were responsive to OT + AA and LPS, as measured by prostaglandin accumulation. In conclusion, a functional 3D model of stromal and epithelial cells was developed using a PGA electrospun scaffold which may be used to study endometrial pathophysiology.

Objective(s):Endometriosis is a complex gynecologic disease with unknown etiology. Noscapine has been introduced as a cancer cell suppressor. Endometriosis was considered as a cancer like disorder, The aim of present study was to investigate noscapine apoptotic effect on human endometriotic epithelial and stromal cells in vitro.Materials and Methods:In this in vitro study, endometrial biopsies from endometriosis patients (n=9) were prepared and digested by an enzymatic method (collagenase I, 2 mg/ml). Stromal and epithelial cells were separated by sequential filtration through a cell strainer and ficoll layering. The cells of each sample were divided into five groups: control (0), 10, 25, 50 and 100 micromole/liter (µM) concentration of noscapine and were cultured for three different periods of times; 24, 48 and 72 hr. Cell viability was assessed by colorimetric assay. Nitric oxide (NO) concentration was measured by Griess reagent. Cell death was analyzed by Acridine Orange (AO)–Ethidium Bromide (EB) double staining and Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay. Data were analyzed by one-way ANOVA.Results:Viability of endometrial epithelial and stromal cells significantly decreased in 10, 25, 50 and 100 µM noscapine concentration in 24, 48, 72 hr (P<0.05) and apoptotic index increased in 25, 50 and 100 µM noscapine concentrations in 48 hr significantly (P<0.05). Concentrations of NO didn’t show a significant decrease.Conclusion:Noscapine increased endometriotic epithelial and stromal cell death and can be suggested as a treatment for endometriosis.