Ultrastructural characterization of two new human endometrial carcinoma cell lines and normal human endometrial epithelial cells cultured on extracellular matrix

Summary Two new lines of human endometrial carcinoma (HEC) cells, one from an adenocarcinoma and one from a highly metastatic serous papillary carcinoma, were established in culture. Structural and morphologic properties of these cells at early passage were compared with those of cultured normal human endometrial epithelial (NHEE) cells. For these studies, cells were grown on a conventional plastic surface or on an extracellular matrix substrate (Matrigel), and examined by transmission electron microscopy and immunofluorescent light microscopy. The HEC cells appeared morphologically similar on plastic and Matrigel, whereas the NHEE cells showed significantly greater epithelial morphologic differentiation on Matrigel than on plastic. On extracellular matrix, the morphologic differences observed between HEC cells and NHEE cells were primarily of an architectural nature, which may be in part explained by differences between NHEE and HEC cells in the arrangement of actin microfilaments and cytokeratin intermediate filaments. Furthermore, HEC cells displayed extensive networks of vimentin intermediate filaments, which were absent from the NHEE cells. These observations support the hypothesis that architectural deregulation is a prominent feature of endometrial carcinoma, and that cytoskeletal alterations may uncouple HEC cell ultrastructural morphology from the influence of extracellular matrix. This research was supported by research grants CA31733, CA45727, and ES07017, from the National Institutes of Health, Bethesda, MD. G. P. S. is a Jefferson Pilot Fellow in Academic Medicine. A preliminary account of this work was presented at the 1988 U.S.-Canadian Academy of Pathology Annual Meeting (Lab. Inves. 58:12a, 1988).     

Thymic epithelial progenitor cells and thymus regeneration： an update

The thymus provides the essential microenvironment for T-cell development and maturation. Thymic epithelial cells （TECs）, which are composed of thymic cortical epithelial cells （cTECs） and thymic medullary epithelial cells （mTECs）, have been well documented to be critical for these tightly regulated processes. It has long been controversial whether the common progenitor cells of TECs could give rise to both cTECs and mTECs. Great progress has been made to characterize the common TEC progenitor cells in recent years. We herein discuss the sole origin paradigm with regard to TEC differentiation as well as these progenitor cells in thymus regeneration.     

Bovine trophoblastic cell vesicle attachment to polarized endometrial epithelial cells in vitro

Summary Interactions between bovine trophoblastic cell vesicles and bovine endometrial epithelial cells were investigated by light and electron microscopy and lectin histochemistry in a cell culture model of early blastocyst attachment. Primary lines of bovine endometrial epithelial cells were polarized by subculturing on substrata and maintaining cultures at the air-medium interface. Trophoblastic cell vesicles were obtained from elongated Day 14 blastocysts. In co-cultures, trophoblastic cell vesicles adhered to endometrial epithelial cells through microvillus interdigitation and formation of primitive membrane junctional complexes. After 3 d in co-culture, a multilayered cellular plaque formed at the trophoblastic cell-endometrial epithelial cell interface. The type of cells contributing to this local proliferative response could not be identified specifically as trophoblastic or endometrial cells, and areas of membrane fusion between cells were noted. Ultrastructural features of vesicle adhesion in cultures were similar to features of conceptus attachment in vivo. Lectins bound to apical membranes of trophoblastic cells and endometrial epithelial cells in all locations except contact sites between vesicles and endometrial cells. These findings suggest that local cellular proliferation and membrane fusion between trophoblastic and endometrial epithelial cells may be early events in conceptus implantation in the cow and these events can be reproduced in culture. This work was supported by a grant from U.S. Department of Agriculture Animal Health and Disease Program, Washington, DC.     

Tissue culture of human epithelial cells from benign colonic tumors

Summary Human colonic epithelial cells from three classes of benign tumors have been reproducibly cultured free of fibroblasts for 8 wk using a supplemented Medium 199 (M 199S). The cultured colonic cells were identified as epithelial by the presence of junctional complexes (tight junctions, gap junctions, and desmosomes), a brush border on the apical surface, keratin fibrils, and by both a close-packed columnar or cuboidal morphology and the capability to transport water and ions to form hemicysts. Colony formation was initiated by groups of epithelial cells, not by single cells, and was inhibited by cocultivation with either lethally irradiated 3T3 cells or human diploid fibroblasts. Enhancement of epithelial colony formation was observed following culture on nonadherent, “floating” substrates compared with substrates attached directly to the bottom of the culture dish. Replication of epithelial cells in M 199S from the class of benign colonic tumors least prone to malignancy, the tubular, was significantly enhanced by epidermal growth factor (EGF). In contrast, EGF did not stimulate the growth of cells in M 199S from the other classes of benign tumors, the villotubular and the villous, which exhibit more malignant potential. These data imply that premalignant colonic epithelial cells lose responsiveness to growth modulation by EGF as they progress toward frank carcinoma. This study was supported by NCI Contract N01-CP43366 to M. L. and NCI Grant 1-R26-CA 28822 to E. F.     

Soluble factors derived from human amniotic epithelial cells suppress collagen production in human hepatic stellate cells

BackgroundIntravenous infusion of human amniotic epithelial cells (hAECs) has been shown to ameliorate hepatic fibrosis in murine models. Hepatic stellate cells (HSCs) are the principal collagen-secreting cells in the liver. The aim of this study was to investigate whether factors secreted by hAECs and present in hAEC-conditioned medium (CM) have anti-fibrotic effects on activated human HSCs.MethodsHuman AECs were isolated from the placenta and cultured. Human hepatic stellate cells were exposed to hAEC CM to determine potential anti-fibrotic effects.ResultsHSCs treated for 48 h with hAEC CM displayed a significant reduction in the expression of the myofibroblast markers α-smooth muscle actin and platelet-derived growth factor. Expression of the pro-fibrotic cytokine transforming growth factor-β1 (TGF-β1) and intracellular collagen were reduced by 45% and 46%, respectively. Human AEC CM induced HSC apoptosis in 11.8% of treated cells and reduced HSC proliferation. Soluble human leukocyte antigen–G1, a hAEC-derived factor, significantly decreased TGF-β1 and collagen production in activated HSCs, although the effect on collagen production was less than that of hAEC CM. The reduction in collagen and TGF-B1 could not be attributed to PGE2, relaxin, IL-10, TGF-B3, FasL or TRAIL.ConclusionsHuman AEC CM treatment suppresses markers of activation, proliferation and fibrosis in human HSCs as well as inducing apoptosis and reducing proliferation. Human AEC CM treatment may be effective in ameliorating liver fibrosis and warrants further study.     

Indication of selective growth of human endometrial epithelial cells on extracellular matrix

Summary The culturing of human endometrium in conventional plastic dishes and media is only partially successful, mainly because a growth of a heterogeneous population of cells is achieved. Naturally produced extracellular matrix closely resembles the subepithelial basement membrane and seems to affect both growth and differentiation of cells. These qualities of the extracellular matrix (ECM) were applied for obtaining endometrial epithelial cultures. Endometrial tissue specimens were plated after slicing on ECM-coated dishes and kept for up to 8 d. The growth of a confluent homogeneous tissue composed of polygonal epithelial-like cells was demonstrated. To further characterize these cells, cultures were examined by scanning electron microscopy and transmission electron microscopy. Scanning electron microscopy revealed flattened polygonal cells covered with microvilli, among which ciliated cells were observed. By transmission electron microscopy the cells were seen as a monolayer, with some cells overlapping, closely adherent to the matrix. Microvilli, as well as intracellular vacuoles and glycogen granules were observed. Cell type specific cytoskeletal markers were demonstrated by antibodies to intermediate filament proteins (keratin and epithelial membrane antigen). Taken together, the morphologic and immunohistochemical studies indicate that a selective growth of the epithelial component of endometrial tissue was obtained after plating unprocessed endometrial tissue fragments on ECM-coated culture dishes. This work was supported by PHS grant no. CA 30289 to J.V.     

体外诱导人羊膜上皮细胞分化为胰岛素分泌细胞的研究

目的:通过体外诱导分化实验,探讨人羊膜上皮细胞(hAECs)向胰岛素分泌细胞(ISCs)分化的能力。方法:采用胰蛋白酶消化法从人羊膜组织分离提取hAECs,用流式细胞仪和免疫细胞化学法进行鉴定。取第3代hAECs在含尼克酰胺和N2补充物的无血清培养基中诱导培养,分别于诱导不同时间采用免疫细胞化学法检测胰岛素和β2微球蛋白的表达,采用放射免疫法检测上清液中胰岛素含量,采用RT-PCR检测胰岛素mRNA和胰十二指肠同源异型盒因子-1(PDX-1)mRNA的表达。结果:①hAECs高表达CD29、CD73、CD166和CK19;②hAECs诱导组第7、142、1天胰岛素阳性细胞百分率分别为74.00%±1.73%、75.33%±1.15%和75.67%±0.58%,而对照组未见胰岛素阳性细胞;③hAECs诱导组第7、14、21天培养物上清液中胰岛素含量分别达(328.47±3.22)μIU/ml、(332.26±1.22)μIU/ml和(329.68±2.57)μIU/ml,均显著高于对照组(P均<0.01);④hAECs诱导前后均有PDX-1 mRNA和β2微球蛋白表达,胰岛素mRNA表达仅见于诱导组。结论:hAECs能分化为ISCs,在Ⅰ型糖尿病细胞移植治疗方面具有潜在应用前景。     

In vitro deregulation of markers characteristic of human prostate epithelial cells

We have screened primary cultures of human prostate for the expression of markers reported to be characteristic of specific cell lineages in vivo, in order to ascertain whether human prostate cells in vitro maintain and reflect their in vivo differentiated phenotypes and to evaluate the homogeneity of the populations of cells that can be derived from this tissue. Using single and dual stain immunofluorescent microscopy to analyse very early organoid and subsequently derived monolayer stage cultures, we have observed that expression of markers characteristic of human prostate epithelial cells in vivo is deregulated within 48h, indicating that dissociation of human prostate tissue and cultivation of prostate epithelial cells in culture can result in promiscuous expression of cell type specific markers of prostate epithelial cells. These observations have important implications for studies of cell lineage and differentiation of prostate cells in vitro.     

Transformed human bronchial epithelial cells (beas-2b) alter the growth and morphology of normal human bronchial epithelial cells in vitro

Normal human bronchial epithelial cells (BE) and adenovirus-12 SV40 hybrid virus transformed, non-tumorigenic human bronchial epithelial cells (BEAS-2B) were cultured for 7 days in a serum free hormone supplemented medium. BE cells after 3 days in culture were exposed to conditioned medium (CM_t) from confluent BEAS-2B cells. By day 7, CM_t-treated BE cells exhibited a lower colony forming efficiency (CFE), fewer cells per colony, and a reduced mitotic index (MI) and BrdU (bromodeoxyuridine) labeling index. CM_t also enhanced the expression of a terminally differentiated squamous phenotype in BE cells. Cell free lysates from BEAS-2B cells (CFL_t) had effects similar to CM_t on the MI and morphology of BE cells. In contrast, CM_t and CFL_t did not inhibit the growth, or alter the morphology of BEAS-2B cells. Conditioned medium from BE cells (CM_n) did not reduce the growth of BEAS-2B cells, and had little effect on the morphology of BE cells. In co-culturesAbbreviations BE normal bronchial epithelial cells - BEAS-2B adenovirus-12 SV40 hybrid virus transformed bronchial epithelial cells - CM_n conditioned medium from BE cells - CM_t conditioned medium from BEAS-2B cells - CF_n cell free lysate from BE cells - CFL_t cell free lysate from BEAS-2B cells - BrdU bromodeoxyuridine - KGM keratinocyte growth medium - TGF- transforming growth factor type - NCI-LHC National Cancer Institute-Laboratory of Human Carcinogenesis Contribution No. 2801 from the Pathobiology Laboratory, University of Maryland.     

Rat lens epithelial cells in vitro

Summary Lens epithelial cells from rats aged 5 days were grown in long-term cultures. These cells age, differentiate and transform spontaneously. Morphological observations indicate five different stages (A-E). The epithelial character is lost after the first two passages. Elongated cells appearing afterwards are considered as cells that have started differentiation to fiberlike cells. Big flattened cells are considered as senescent cells that have lost their proliferative capacity. Data from population kinetics also reflect these five stages. Chromosome analysis shows that three of the five stages are no longer diploid. Two alternative modes of spontaneous transformation are possible. The proliferative capacity of rat lens epithelial cells is higher than that of rat embryonic fibroblast systems. The investigations were supported by the Deutsche Forschungsgemeinschaft (Biology of Aging, Grants Ri 285/2 and Ri 285/3).     

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.     

Properties of epithelial cells cultured from human carcinomas and nonmalignant tissues

Human epithelial cell cultures were examined for expression of plasminogen activator and fibronectin matrix. All of the cells examined showed ultrastructural evidence suggesting their epithelial origin, including microvilli and specialized junctions. The nonmalignant cells were also negative for endothelial cell markers (ie, they lacked factor VIII antigen, a nonthrombogenic surface and Weibel-Palade bodies). The nonmalignant lines all produced large amounts of plasminogen activator, whereas the tumor-derived lines showed a gradation of activities, ranging from lines having as much activity as the nonmalignant lines to lines having little or no activity above background. For both normal and malignant cells, addition of dexamethesone only slightly decreased the levels of plasminogen activator. By immunofluorescence microscopy, normal bladder and fetal intestine epithelial cells showed fibronectin in a globular and fibrillar matrix. In contrast, normal mammary epithelial cells had a much diminished amount of fibronectin with a punctate distribution.     

Rat lens epithelial cells in vitro

Summary Serially subcultured rat lens epithelial cells grow in different stages, which can be classified according to morphology, chromosome numbers and population kinetics. A lensspecific γ-crystallin appears in the diploid stage, when elongated cell types are observed. One of the β-crystallin bands (pH 5.7) disappears during aging in higher passage numbers of the diploid stage B. A weak band in the β-crystallin region (pH 6.4), which is present in all stages, becomes very intensive in aneuploid cells of stage D, which exhibit a fibroblast-like morphology. The work was supported by Deutsche Forschungsgemeinschaft, Grant Ri 285/3.     

Role of biotin-containing membranes and nuclear distribution in differentiating human endometrial cells

Human Ishikawa endometrial cells form domes when confluent monolayers are stimulated with fresh fetal bovine serum. Extensive structural and biochemical changes have been detected during the approximately 30 h differentiation period. The earliest detectable change involves the formation of multinucleated structures and the appearance of “granules” that stain for biotin within those structures. Nuclei become associated with each other and are ultimately enclosed within a biotin-containing membrane. Aggregated membrane-sheathed nuclei and the cells containing them begin to elevate from the dish as biotin staining becomes apparent in apical membranes. The elevated structures are called predomes and consist of one or more very large cells containing the sheathed nuclei. Apical membranes of these unusual cells extend far out into the medium in structures that resemble endometrial pinopods. A lumen under the elevated cells fills with transcytosed fluid. As differentiation proceeds, highly concentrated chromatin material that was flattened against apical and lateral membranes of the predome cells begins to disperse. Small mononuclear cells evolve from larger predome cells. Apical membranes of predome and dome cells continue to stain for biotin. Gel electrophoresis of SDS-solubilized biotin-containing membranes, followed by Western blot analysis using avidin-linked peroxidase, resulted in three stained bands with molecular weights similar to those of the mitochondrial carboxylases: propionyl carboxylase, methylmalonyl carboxylase, and pyruvate carboxylase. J. Cell. Biochem. 71:400–415, 1998. © 1998 Wiley-Liss, Inc.     

人胎儿鼻咽上皮细胞的背景氯电流

采用膜片钳和图像分析技术,研究人胎儿鼻咽上皮细胞背景电流的特性及其与容积激活性氯电流的关系。在等张溶液中,可记录到一背景电流,该电流呈微弱的外向整流性,无明显时间依赖性失活,其翻转电位为(?0.73±1.7)mV(n=21),接近氯离子平衡电位(?0.9mV)。细胞外高张刺激(440mOsmol/L)明显抑制此电流(59.6±7.1)%,而低张刺激(160mOsmol/L)则诱发细胞产生容积激活性氯电流。氯通道阻断剂tamoxifen和5-硝基-2-(3-苯丙胺基)苯甲酸[5-nitro-2-(3-phenylpropylamino)benzoicacid,NPPB]显著地抑制背景电流并使细胞基础容积增大。上述结果表明,人胎儿鼻咽上皮细胞的背景Cl?电流是背景电流的重要成分,此Cl?电流与容积激活性氯电流及细胞基础容积调节有关。     

Human endometrial carcinoma cells release factors which inhibit the growth of normal epithelial cells in culture

Autocrine and paracrine interactions between cells are important homeostatic mediators in normal tissues. Alterations to growth factor signalling pathways are likely to play a role in multistep carcinogenesis. In this study normal human endometrial epithelial cells (NHEC) after 3 days in culture were treated with serum-free medium conditioned for 24 h by log phase or confluent cultures of established RL95-2, HEC1A, or AN3CA endometrial carcinoma (EC) cell lines. By day 4, NHEC treated with either log phase or confluent conditioned medium (CM) showed a significant decrease (50–90% of control) in [³H]thymidine ([³H]TdR) incorporation. DNA synthesis was inhibited more by confluent than by log phase CM. By day 7, NHEC treated with CM exhibited fewer colonies per culture, fewer cells per colony, and an increased percentage of single cells. Several growth-regulatory gene products found in the nucleus or at the cell membrane have been shown to be expressed differently in normal and transformed cells. We selected the p53 and c-Ha-ras p21 proteins to further investigate the mechanism of alteration of proliferation in cells treated with carcinoma CM. Thus, by day 7, the percentage of NHEC with nuclear localization of wild type p53 (wt p53) was elevated by treatment with CM. In contrast, CM-treated EC cells continued to proliferate, and showed a decrease in the percentage of cells expressing nuclear wt p53 and an increase in the cytoplasmic expression of c-Ha-ras p21. Our studies show that EC cell lines release factors which inhibit the proliferation of NHEC, thus favoring the proliferation of EC cells.Abbreviations CM conditioned medium - EC endometrial adenocarcinoma - NHEC normal human endometrial epithelial cells     

Cholera toxin stimulation of human mammary epithelial cells in culture

Summary Addition of cholera toxin to human mammary epithelial cultures derived from reduction mammoplasties and primary carcinomas greatly stimulated cell growth and increased the number of times the cells could be successfully subcultured. Other agents known to increase intracellular cAMP levels were also growth stimulatory. The increased growth potential conferred by cholera toxin enhances the usefulness of this cell culture system. This work was supported by USPHS Grant CA-24844 from the National Cancer Institute and Grant CD-61B from the American Cancer Society.     

Notch ligand-dependent gene expression in human endometrial stromal cells

The purpose of this study is to investigate the expression patterns and role of Notch signaling in human endometrial cells. Notch receptors, Notch 1-3 were expressed in both endometrial epithelial and stromal cells. Notch ligands, Jag1 and Dll4 and Notch target genes, Hes1 and Hey1 were predominantly expressed in endometrial epithelial cells and scarce in stromal cells. Increased de novo synthesis of Dll4 or Jag1 in stromal cells by retroviral delivery significantly induced Hes1 and Hey1. Evaluations of global gene expression by microarrays revealed that more than 400 genes in stromal cells were significantly regulated by Jag1. Gene annotation-based functional analysis classified these genes into biological processes of cell adhesion, cell structure and motility, cell communication, cell cycle, and angiogenesis. This study provides evidence that Notch ligands control the Notch gene activities and may enhance development of human endometrium.     

In vitro optimization of the Gallus domesticus oviduct epithelial cells culture

The aim of this experiment was to establish an efficient method for isolation and further culture in vitro of the normal chicken oviduct epithelial cells (COEC) for cell-based research models. Different factors were tested to optimize COEC primary culture for repeatable results: the origin of isolated cells (oviduct Infundibulum or Magnum section); the oviduct tissue dissociation procedure (mechanical scrapping or mincing), tissue digestion times (15, 30 and 45 min), the culture plates coating (colagene I, polystyrene surface or 3T3 feeder layer), the growth media (classic DMEM/Ham's F12 and defined serum-free medium, Lonza Switzerland), incubation temperature (37 °C vs 41°C) and different cell seeding numbers: 0.2M, 0.5M and 1.0M cells/well. The COEC isolated by mincing the Infundibular neck and digestion of tissue for 30 min formed cell aggregates of bright colour and gave proliferating colonies of epithelial-like character which was the best result obtained from all applied procedures in our studies. The fibroblast-like cells considered as contaminants occurred only sporadically up to day 7 of culture. Seeding about 1M cells in 1 mL of serum-free medium onto 12-well dishes gave the optimal growth of colonies resulting in 5 to 7 confluent culture wells from a single oviduct sample. Feeder layer and collagen I did not improve adhesion of the COEC to the culture vessel. Adoption of 37 °C and 41 °C did not reveal apparent differences to the condition of cultured COEC. Cell differentiation and proliferation potential depends on number and replicative capacity of isolated progenitors. The progenitors are responsible for holoclones formation and good culture growth. The percentage of colonies developed from the cells isolated from Infundibulum was greater than that of other samples in our studies. We conclude that the model of COEC primary cultures from different segments of oviduct, in particular infundibulum, should be incorporated to the range of avian cells research as this work generates questions about undocumented sources of oviduct progenitor cells.