Human vaginal epithelial multilayer tissue culture

Summary Fragments of normal human adult vagina, when explanted onto glass slides gave rise to outgrowing sheets of pure epithelium, which had microscopic morphological features in common with normal vaginal epithelium. Infrequent fibroblast contamination was observed. Proliferating epithelial cells formedmultilayers of stratified squamous epithelium and demonstrated a progressive decrease in proliferative activity after 14 days. Continuous lines of epithelial cells were not obtained. Even in the absence of estrogens, transmission electron microscopy revealed evidence of keratinization of the superficial cells of the multilayer. Scanning electron microscopy of the surface of mature epithelial cells in culture revealed ultrastructural features that closely resembled those present on the surface of exfoliated cells obtained by scraping the vagina in vivo. This in vitro tissue culture model of human vaginal epithelium may provide a simple method of studying factors that influence vaginal epithelium growth, maturation and function.     

分步消化法原代培养鼠阴道黏膜上皮细胞的研究

目的探讨大鼠阴道黏膜上皮细胞的体外培养和扩增技术,为构建组织工程化阴道动物模型提供种子细胞。方法取大鼠阴道全层组织,经Dispase酶和胰酶分步消化后,接种于无血清角化细胞培养液中连续培养,观察细胞形态、体外生长特性和超微结构,绘制生长曲线,免疫组化鉴定。结果原代细胞培养24-36 h后开始贴壁,7-10d约80%融合,呈铺路石样外观,可连续传5-6代;扫描电镜下细胞表面可见微绒毛嵴;角蛋白染色阳性,细胞纯度98%;第五代细胞为正常二倍体核型。结论该方法培养的阴道上皮细胞增殖状态良好,细胞纯度高,扩增迅速,可在较短时间内获得大量细胞用于组织工程学研究。     

Structure and function of intercellular junctions in human cervical and vaginal mucosal epithelia

The mucosal epithelium is a major portal for microbial invasion. Mucosal barrier integrity is maintained by the physical interactions of intercellular junctional molecules on opposing epithelial cells. The epithelial mucosa in the female reproductive tract provides the first line of defense against sexually transmitted pathogenic bacteria and viruses, but little is known concerning the structure and molecular composition of epithelial junctions at this site. In the present study, the distribution of tight, adherens, and desmosomal junctions were imaged in the human endocervix (columnar epithelium) and ectocervix (stratified squamous epithelium) by electron microscopy, and permeability was assessed by tracking the penetration of fluorescent immunoglobulin G (IgG). To further define the molecular structure of the intercellular junctions, select junctional molecules were localized in the endocervical, ectocervical, and vaginal epithelium by fluorescent immunohistology. The columnar epithelial cells of the endocervix were joined by tight junctions that excluded apically applied fluorescent IgG. In contrast, the most apical layers of the ectocervical stratified squamous epithelium did not contain classical cell-cell adhesions and were permeable to IgG. The suprabasal and basal epithelial layers in ectocervical and vaginal tissue contained the most robust adhesions; molecules characteristic of exclusionary junctions were detected three to four cellular layers below the luminal surface and extended to the basement membrane. These data indicate that the uppermost epithelial layers of the ectocervix and vagina constitute a unique microenvironment; their lack of tight junctions and permeability to large-molecular-weight immunological mediators suggest that this region is an important battlefront in host defense against microbial pathogens.     

Epithelial-mesenchymal interactions in uterus and vagina alter the expression of the cell surface proteoglycan, syndecan

The cell surface proteoglycan, syndecan, exhibits molecular and histological dimorphism in the mouse uterus and vagina. In the mature vagina, syndecan is localized at the surfaces of the basal and intermediate cells of the stratified epithelium and has a modal molecular mass of ca. 92 kDa. The uterus expresses a larger form of syndecan (ca. 110 kDa) which is detected at the basolateral surfaces of the simple columnar epithelial cells. We have investigated whether epithelial-mesenchymal interactions influence the expression of syndecan in these organs by analyzing tissue recombinants composed of mouse epithelium and rat mesenchyme or vice versa with monoclonal antibody 281-2, which recognizes mouse syndecan. In tissue recombinants composed of newborn mouse uterine epithelium and rat vaginal stroma, the uterine epithelium was induced to form a stratified vaginal epithelium which expressed syndecan in same the pattern and mass typical of vaginal epithelium. Likewise, rat uterine stroma induced newborn mouse vaginal epithelium to undergo uterine development, and this epithelium exhibited a uterine pattern of syndecan expression. Although stromal cells normally express little syndecan in most adult organs, analysis of recombinants composed of mouse stroma and rat epithelium revealed that both uterine and vaginal mouse stromata synthesized syndecan that was larger (ca. 170-190 kDa) than the epithelial syndecans. A quantitative increase in the amount of stromal syndecan was evident when stroma was grown in association with epithelium in comparison to stroma grown by itself. These data suggest that epithelial-mesenchymal interactions influence the amount, localization, and mass of both epithelial and stromal syndecan.     

Formation and barrier function of tight junctions in human ovarian surface epithelium

The normal ovarian surface epithelium (OSE) is a primitive epithelium made up by a single layer of mesothelial-type epithelial cells. When these cells get trapped in the ovarian stroma, expression of epithelial specific markers, such as E-cadherin, are induced. Most epithelial cells are also characterized by the ability to form tight junctions (TJ). Incomplete TJ have earlier been demonstrated in the OSE by electron microscopy studies. We have investigated expression and localization of the TJ proteins ZO-1, occludin, and claudin-1 in tissue biopsies from normal human ovaries and OSE in culture. The dynamics of TJ formation were studied in human OSE cultured on porous filters in culture inserts by measuring trans epithelial resistance (TER) including Ca(2+) switch experiments. Confluent OSE cells were also analyzed by electron microscopy. The results show that normal human OSE has expression of all three TJ proteins investigated. These proteins, ZO-1, occludin, and claudin-1, were localized to OSE cell borders both in ovarian biopsies and in cultured OSE. There was no difference in this regard between fertile and postmenopausal women. Cells in culture were polarized and presented junctional complexes seen by electron microscopy. In the Ca(2+) switch experiments, removing free Ca(2+) transiently, TER decreased significantly (P < 0.05) in the Ca(2+)-free group compared with nontreated OSE. TER was fully restored after 24 h. N-cadherin but not E-cadherin was expressed in the OSE and localized to the cell borders. We conclude that normal human OSE express and form functional TJ both in vivo and vitro. This report also describes a method to study the influence of ovarian-derived mediators on TJ in cultured OSE.     

Langerhans cells phagocytose vaginal epithelial cells undergoing apoptosis during the murine estrous cycle.

Langerhans' cells (LCs) have been studied extensively in the epidermis, where they function as antigen-presenting cells. LCs are also present in the stratified epithelia of the murine vagina and cervix, but their function at these sites is not known. Recent reports noted the association of LCs with vaginal epithelial cells undergoing apoptosis and suggested that LCs might be involved in phagocytosis of dead cells. The present study describes the ultrastructural details of this process. The results demonstrate that LCs in murine vaginal epithelium during late metestrus and early diestrus phagocytose apoptotic epithelial cells and may thereby contribute to the normal turnover of the vaginal epithelium during the estrous cycle.     

Electron microscopy of adhesive interactions between Gardnerella vaginalis and vaginal epithelial cells, McCoy cells and human red blood cells

Exfoliated vaginal epithelial cells with attached bacteria, termed 'clue cells', which were procured from a patient with non-specific vaginitis, were stained with ruthenium red and examined by transmission electron microscopy. The attached bacteria appeared to adhere by means of an outer fibrillar coat. An epithelial tissue culture cell line (McCoy) and human red blood cells to which strains of Gardnerella vaginalis attached were similarly examined. The adherence of G. vaginalis to the epithelial cell line appeared to be mediated by an outer fibrillar coat while adherence to red cells appeared to be mediated by fimbriae. Transmission electron microscopy was performed on the Gardnerella strains used. Thin sections of tissue-culture-adherent strains revealed a dense outer fibrillar coat whereas the surface of the haemagglutinating strains showed fine fimbriae. Negative staining of haemagglutinating strains demonstrated fimbriae on a minority of organisms.     

Scanning microscopy of the developing vagina in postnatal gerbils

Scanning electron microscopy of postnatally developing gerbil vagina (birth to maturity) shows that longitudinal folds form prior to transverse folds; the process of fold formation is initiated on the dorsal wall and proceeds ventrally. From days 1 to 7 postnatally, the vaginal epithelium is composed of either flat or bulging cells, depending on the vaginal region. The luminal cell surface is covered with uniform stubby microvilli and solitary cilia. Between days 9 and 20, the flat cells with distinct cell boundaries spread toward more proximal areas, leading to the formation of mixed patches of cells with flat or rounded apices. Individual elongated microvilli or tufts of forked microvilli may sprout from their surfaces. Solitary cilia gradually disappear. The transition from immature to mature vaginal epithelium starts around day 20, when individual cells recess below the level of neighboring cells. This process spreads throughout the vagina during the following days, reflecting local changes in the subsurface layers of the epithelium preparatory to exfoliation. Around day 40 the actual exfoliation of the luminal cell layer starts. By this time the surface characteristics of many of the desquamating cells have changed. In addition to microvilli, microridges are being formed. The process of exfoliation is finished by about day 60. The newly appearing cell layers now transform into typical cornified cells of the cycling vaginal epithelium.     

Adherence of Candida albicans to buccal and vaginal epithelial cells: ultrastructural observations

The adherence of Candida albicans to human buccal and vaginal epithelial cells was studied by transmission electron microscopy. Adherence to epithelial cells was confirmed by both a radiometric assay as well as direct microscopic examination of stained cell preparations. Ultramicroscopic preparations revealed that yeast cells were closely appressed to epithelial cell surfaces and were often partially enclosed within phagocyticlike invaginations of the epithelial cells. A murine model of vaginitis caused by C. albicans was also used to study adherence to epithelial cells and to follow the course of colonization. Ultramicroscopic preparations of murine vaginal tissue revealed that within 2 h postinfection, yeast cells could be seen adhering to epithelial cells. At 6 h postinfection, hyphae and yeast cells were not only found on the epithelial cell surface but also within the submucosal tissue. When observed on the epithelial cell surface, Candida cells were either attached to host cells, or when infected tissue was stained with ruthenium red, Candida cells were observed on the epithelial surface embedded within an electron-dense matrix. Fungal elements were abundant in the submucosa at 24 h postinfection and were still observed on the epithelial cell surface; all of this was accompanied by an inflammatory response.     

The role of fibroblast growth factors on the differentiation of vaginal epithelium of neonatal mice

The uterus and upper 3/5 of the vagina originate from the Müllerian duct; however, these organs show quite distinct characteristics in morphology and function. To investigate factors controlling vaginal epithelial cell differentiation from a single layer of pseudostratified epithelium to a multi-layered stratified epithelium with keratin, we focused on fibroblast growth factors (Fgfs). Transformation related protein 63 (Trp63) expression, a marker of stratified epithelium, increased in the Müllerian vaginal epithelial cells from days 0 to 5, and keratin 14 (Krt14) was expressed from day 5, suggesting that Trp63-negative vaginal epithelial cells can differentiate into Trp63-positive cells after birth. Fgf7 and Fgf10 were localized in the vaginal stroma but their receptor, Fgf receptor 2IIIb (Fgfr2IIIb), was localized in the vaginal epithelium. Both Fgf9 and its receptor, Fgfr2IIIc, were localized in the vaginal epithelium. Vaginae cultured with FGF10 or anti-FGF9 antibody showed stratified epithelium with an intense Krt14 expression; however, an inhibitor of phosphorylation of mitogen-activated protein kinase 1/3 (MAPK1/3) canceled the effect of FGF10 and anti-FGF9 antibody. Thus, Fgf10 stimulates the differentiation of pseudostratified epithelial cells into stratified cells via MAPK1/3 pathway, and Fgf9 inhibits this differentiation in the neonatal mouse vagina.     

Primary cultures of epithelial cells and long-term cultures of fibroblasts from the human umbilical cord: ultrastructural and immunocytochemical characterization

Repeated trypsinization of the full term human umbilical cord epithelium allows an homogeneous and exclusively epithelial primary culture, without fibroblastic growth. Transmission electron microscopy observations of desmosomes, cytokeratin intermediate filaments as revealed by indirect immunofluorescence and cultural aspects confirm the epithelial nature of this primary culture. Fibroblasts obtained by an explants culture method exhibit neither desmosomes nor cytokeratin intermediate filaments, which are epithelial markers. They yield characteristic long term cultures of fibroblastic aspect and growth.     

Mechanisms underlying the regulation of intracellular and luminal pH in vaginal epithelium

The vagina provides a characteristic low-Na⁺ and low-pH fluid microenvironment that is considered generally protective. Previous studies have shown that various types of epithelial cells harbor the capacity of intracellular pH (pHi) regulation. However, it remains elusive whether vaginal epithelium could actively regulate pHi by transporting acid–base ions. In this study, we verified that after transient exposure to NH₄Cl, the pHi values could rapidly recover from acidification via Na⁺-H⁺ exchanger (NHE), Na⁺-HCO₃⁻ cotransporter (NBC), and carbonic anhydrase in human vaginal epithelial cell line VK2/E6E7. Positive expression of the main acid–base transporters including NHE1-2, NBCe1-2, and NBCn1 mRNA was also detected in VK2/E6E7 cells. Moreover, the in vivo study further showed that interfering with the function of V-type H⁺-ATPase, NHE or NBC expressed in vagina impaired vaginal luminal pH homeostasis in rats. Taken together, our study reveals the property of pH regulation in vaginal epithelial cells, which might provide novel insights into the potential role of vaginal epithelium in the formation of the vaginal acidic microenvironment.     

Immunocytochemical localization in normal and transformed human cells in tissue culture using a monoclonal antibody to the src protein of the Harvey strain of murine sarcoma virus

Using a rat monoclonal antibody directed against the p21 src protein of the Harvey strain of Murine Sarcoma Virus (MSV), we have examined the reactivity of human cells in tissue culture using immunofluorescence and electron microscopy. Qualitative results indicated that untransformed mouse and human fibroblastic cells have undetectable amounts of p21; these levels were greatly increased after transformation with Harvey MSV. A group of human tumor cell lines adapted to tissue culture were examined and almost all of the epithelial tumor lines showed significant localization with this antibody. Notable exceptions were two melanoma cell lines which were negative for p21 by immunofluorescence. When normal human epithelial cells derived from esophageal or foreskin epithelium were examined, the antibody showed significant reactivity with subconfluent growing cells. After the normal cells were allowed to become quiescent, the reactivity with this antibody decreased. All of the localization seen by fluorescence was in a distribution consistent with the previously demonstrated location of p21 scr on the inner aspect of the plasma membrane. Electron microscope localization showed labeling for this antigen on the inner surface of the plasma membrane in both transformed mouse cells and in the human tumor cell lines MCF-7 and HTB-2 (RT4). These results suggest that the amounts of p21-like proteins detectable in human epithelial tumor cells do not necessarily reflect their malignant potential, but may be related to their epithelial nature. The loss of detectable localization at quiescence suggests that p21 levels decrease when these epithelial cells stop growing, and raises the possibility that an analog of p21 may be used by these human epithelial cells to regulate cell growth.     

Clomiphene citrate alters vaginal surface morphology in cycling rats.

Cycling virgin female rats were treated with clomiphene citrate (CC) during dioestrus of the reproductive cycle. Animals were sacrificed 2 days after the initial injection and their vaginal tissue was examined by scanning electron microscopy. Animals treated with one dose of CC had an epithelium consistent with a prolonged dioestrus. Treatment with CC for 2 days induced changes in the epithelium that had no resemblance to any normal hormonally controlled event in the vagina. It was found that CC had effects consistent with progesterone alone as well as effects unique to this superovulatory drug.     

Characterization of Cell Surface Lectin-binding Patterns of Human Airway Epithelium

Glycosylated structures on the cell surface have a role in cell adhesion, migration, and proliferation. Repair of the airway epithelium after injury requires each of these processes, but the normal cell surface glycosylation of non-mucin producing airway epithelial cells is unknown. We examined cell surface glycosylation in human airway epithelial cells in tissue sections and in human airway epithelial cell lines in culture. Thirty-eight lectin probes were used to determine specific carbohydrate residues by lectin-histochemistry. Galactose or galactosamine-specific lectins labeled basal epithelial cells, lectins specific for several different carbohydrate structures bound columnar epithelial cells, and fucose-specific lectins labeled all airway epithelial cells. The epithelial cell lines 1HAEo– and 16HBE14o– bound lectins that were specific to basal epithelial cells. Flow cytometry of these cell lines with selected lectins demonstrated that lectin binding was to cell surface carbohydrates, and revealed possible hidden tissue antigens on dispersed cultured cells. We demonstrate specific lectin-binding patterns on the surface of normal human airway epithelial cells. The expression of specific carbohydrate residues may be useful to type epithelial cells and as a tool to examine cell events involved in epithelial repair.     

Epithelial heterogeneity in the murine thymus: a cell surface glycoprotein expressed by subcapsular and medullary epithelium

A monoclonal antibody (MAb), G8.8, was raised against glycoconjugates isolated from a cloned line of murine medullary thymic epithelial cells. Flow cytometric analysis of the reactivity of this MAb with cultured thymic epithelium demonstrated that the ligand was expressed on the cell surface. Immunohistochemical examination of normal murine thymus revealed labeling of cells in the subcapsular and medullary areas. Immunoelectron microscopy revealed surface labeling restricted to cells possessing ultrastructural features of epithelium (desmosomes, tonofilaments, and cytoplasmic cysts). During thymic ontogeny, G8.8+ cells predominated in fetal development at the earliest time point examined (Day 14 of gestation). There was an expansion of the cortical epithelial component so that by Day 18 cortical and medullary compartments could be clearly distinguished. Immunoprecipitation of radioiodinated thymic stroma with MAb G8.8 detected a molecule with an apparent Mr of approximately 38 KD under non-reducing conditions. When reduced, the apparent Mr was slightly increased (42 KD). This MAb also exhibited reactivity with gut and epidermal epithelium and some tubular epithelium in the kidney, but did not react with epithelial parenchymal cells of the liver.     

Scanning Electron Microscopy of Cell-Surface Changes in Methylnitrosurea (MNU)-Treated Rat Bladders in vivo and in vitro

Scanning electron microscopy has been used (1) to characterize epithelial cells of bladders from normal rats and from rats treated with a single initiating but non-carcinogenic dose of 2 mg methylnitrosurea (MNU), 24 h and 6 weeks after treatment; and (2) to compare morphological aspects of epithelial differentiation in organ culture of bladder explants taken from untreated and MNU-treated rats at these time intervals.
There are marked differences in vivo between the surface organization of normal urothelium and urothelium undergoing reversible hyperplasia following MNU treatment. Maturation of the normal rat bladder epithelium in vivo is shown to be related to a series of well-defined cell-surface changes readily identified by SEM. By contrast the maturation response is perturbed in the hyperplastic epithelium; the cells lose their ability to differentiate normally and form instead an excess of stubby globular microvilli which project from the cell surface.
In organ culture, maturation of normal bladder epithelium (both in re-epithelialized areas of the explant and in areas of epithelial outgrowth over cellulose acetate substrates) can be also related to a series of cell surface changes showing close similarities to those in vivo. However, epithelial maturation remains defective in organ cultures of bladders from MNU-treated animals. The closely parallel behaviour of the bladder epithelium in vivo and in vitro in both normal and treated tissues underlines the potential value of the bladder organ culture system for studying the comparative biology of hyperplastic development produced by a single initiating dose of MNU and suggests it will be useful with which to study carcinogenesis following multiple doses of MNU.     

Ultrastructure of the nonhuman primate vaginal mucosa: epithelial changes during the menstrual cycle and pregnancy

Ultrastructural changes in the vaginal epithelium of the rhesus monkey during the menstrual cycle and pregnancy were studied by scanning and transmission electron microscopy. During the menstrual cycle, the epithelium was keratinized but varied in thickness. Cells of the basal and parabasal layers were polyhedral in shape but as they differentiated they accumulated glycogen and filaments. Cells in the intermediate layers had keratohyaline and membrane-coating granules. Cells in the superficial layers had a thickened cell envelope, abundant keratin filaments, electron-dense intercellular material, and focal tight junctions. The epithelial surface had numerous microridges and numerous adherent bacteria; bacteria were rare on desquamating cells. The epithelium remained keratinized for about the first month of gestation, then underwent "mucification." The cells contained abundant granules and Golgi apparatus. Concomitant with this transformation, bacteria were no longer adherent to the epithelial surface and the surface cells had microvilli instead of microridges. The epithelial changes during pregnancy were roughly associated with the changing pattern of steroid hormone secretion during gestation.     

Ultrastructural analysis of the pathogenesis of Neisseria gonorrhoeae endometrial infection

We have studied gonococcal infection in human endometrium organ culture and in human primary endometrial epithelial cells using various microscopic techniques including scanning electron microscopy, transmission electron microscopy, bright field light microscopy and laser scanning confocal microscopy. Here we describe the interactions between Neisseria gonorrhoeae and human endometrial luminal epithelial cells at the ultrastructural levels. N. gonorrhoeae attached to cilia but were not observed associated with the plasma membrane of ciliated epithelial cells or internalized into ciliated epithelial cells. N. gonorrhoeae could be found in intracellular vacuoles in secretory epithelial cells. N. gonorrhoeae have diverse interactions with endometrial epithelium. These include intimate association and colocalization with asialoglycoprotein receptor (ASGP-R) and CEACAM, lamellipodia and ruffle formation and colocalization with CR3, and microvillus engagement. These studies indicate that N. gonorrhoeae utilize multiple mechanisms to associate with endometrial epithelial cells and can associate with both ciliated and secretory cells. This diversity is consistent with a role of the endometrium as a transition zone between frequently asymptomatic cervical gonorrhoea and symptomatic pelvic inflammatory disease.     

An air-liquid interface promotes the differentiation of gastric surface mucous cells (GSM06) in culture

The gastric surface epithelium is situated at an air-liquid interface because the luminal surface of the alimentary tract is in continuity with the air phase. However, the effects of this microenvironment on the gastric epithelium remain unclear. The aim of this study was to clarify the effects of an air-liquid interface on gastric epithelial cell biology. Gastric surface mucous cells (GSM06) were cultured at an air-liquid interface. Cultured cells were examined by histology, histochemistry, and transmission electron microscopy. When the cells were cultured at an air-liquid interface, the surface cells on the collagen gel became tall columnar and secreted periodic acid-Shiff-positive substances at the apical surface. These cells indicated many mucous granules in the apical cytoplasm and organized the basal lamina at the contact side with the gel. In contrast, under immersed condition, the surface cells showed immature features. This is the first report of an air-liquid interface promoting the differentiation of gastric surface mucous cells in a reconstruction culture of the gastric surface epithelial layer, suggesting that an air-liquid interface may function as a crucial luminal factor to maintain the homeostasis of gastric mucosa.