Histochemical identification of cultured cells from human endometrium

Summary Histochemical techniques have been applied to the identification of cell types cultured from human endometrium. Previous work from this laboratory characterized two principtal cell types found in cultures of endometrium: a mature epithelial cell and another cell which was classified as the endometrial stromal cell based on light and electron microscopy. In this report we compare the histochemical staining of endometrial tissue in frozen sections to that of cultured cells. These results confirm the epithelial and stromal nature of the respective cell types. Several markers were found that could distinguish between cells of epithelial and stromal origin. The enzymes alkaline phosphatase, γ-glutamyltranspeptidase, peroxidase, and β-glucuronidase were localized in glandular and surface epithelia in frozen sections and in colonies of epithelial cells in culture. Stroma in frozen sections and cultured stromal cells contained leucine aminopeptidase and fibronectin. Epithelial sections and in culture could also be distinguished from cells of stromal origin by preferential binding of lotus and peanut lectin. Several other markers were found in both endometrial epithelium and stroma. J. M. S. was recipient of National Research Service Award CA09156 (National Cancer Institute); K. G. N. was recipient of National Research Service Award ES07017 (National Institute of Environmental Health Sciences); and D. G. K. was recipient of Research Career Development Award CA00431 from the National Cancer Institute, Bethesda, MD. Supported by Grant CA 31733 from the National Cancer Institute, Bethesda, MD.     

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.     

Characterization in primary monolayer culture of separated cell types from rabbit endometrium

Epithelial cells and stromal cells of the rabbit endometrium were separated by successive enzymic digestion of the uterine mucosa. Isolated cell types were obtained in high yield, with good viability, and were maintained in monolayer cultures for up to 2 weeks. Epithelial cells in monolayers appeared as polygonal cells, displayed contact inhibition, and showed the presence of microvilli on the cell surface, with many desmosomes. Stromal cells grew rapidly to confluence, displayed overgrowth, and had a fibroblastic appearance with an absence of junctional complexes between cells. Indirect immunofluorescence showed uteroglobin on the surface of epithelial but not of stromal cells, and only epithelial cells secreted uteroglobin into the medium. These results confirm the identity of the cells and provide biochemical evidence for the epithelial cellular origin of uteroglobin. The method allows the culture of separate endometrial cell types, which retain their morphology and differentiated function in vitro.     

A cloned rat thymic epithelial cell line established from serum-free selective culture

Summary A serum-free system has been developed for selective growth and long-term culture of rat thymic epithelial cells. The growth media is a modification of McKeehan's WAJC 404, plus insulin, cholera toxin, dexamethasone, and epidermal growth factor. Cultures have been continuously passaged and maintained for over 6 mo., and a cloned cell line, TEA3A1, has been established. These cells are epithelial, judging by morphology and ultrastructure, and are positive for A2B5 and thymosin α markers for thymic endocrine cells. This work was partly supported by grant PCM-834 0582 from the National Science Foundation, Washington, DC, and grant P01 CA 37589-2 from the National Cancer Institute, Bethesda, MD.     

Ornithine decarboxylase activity in human endometrium and endometrial cancer cells

Summary Ornithine decarboxylase (ODC) activities were significantly higher in proliferative endometrium during the estrogen-dominated follicular phase of the menstrual cycle than in secretory endometrium after the formation of the progesterone-secreting corpus luteum. The enzymatic activity was increased about fivefold by renewal of the medium during incubations of endometrial fragments or isolated endometrial glands. Endometrial adenocarcinoma cells (HEC-1, HEG-50), both in monolayers and suspension, also responded to medium renewal by increasing ODC activity about 10-fold after 4 h, with subsequent reduction to control levels after 7 h. These effects were blocked by actinomycin D and cycloheximide. Endometrial stromal cells exhibited highly variable ODC activities at different passages. Difluoromethylornithine (DFMO) and sodium molybdate had marked antiproliferative effects in HEC-50 cultures, reducing cell numbers to 10 to 20% of control values 11 d after plating and inhibiting ODC activity by approximately 80% on Day 7. The antiproliferative effect of DFMO, but not that of molybdate, was reversed by 10 μM putrescine, the product of ODC activity. In contrast to DFMO, molybdate had no effect on ODC activity of cell homogenates. Molybdate did not elicit antizyme formation in HEC-50 cells under conditions in which putrescine did. These results indicate that ODC activity, present in both epithelial and stromal cells, as shown analytically and also by autoradiography after labeling with [³H]DFMO, may be related to cell proliferation in vivo and that proliferation of human endometrial cancer cells in culture can be arrested by DFMO and by molybdate. This investigation was supported by PHS grant HD 07197, awarded by the National Institute of Child Health and Human Development and PHS grant CA 15648, awarded by the National Cancer Institute.     

Maintenance of adult hamster pancreas cells on fibroblastic cells

Summary The maintenance of primary cultures of adult hamster pancreatic cells on layers of irradiated C3H/10T1/2 cells was studied. Various types of pancreatic cells, acinar, islet and ductular cells could be identified in the cultures by light and electron microscopy. Morphologically the various pancreatic cells retained many differentiated characteristics of their respective in vivo cell types. Insulin production was maintained at near Day 1 levels for the 16 d in culture for which it was measured. Colonies of epithelial cells continued to grow during a 20 d culture period. It is believed that this procedure for maintaining functional and growing pancreas cells in culture may be a useful in vitro model for studying the initiation of pancreatic carcinogenesis. Supported by Grant R01 CA 20022 and Contract N01 CP33278 from the National Cancer Institute, National Institutes of Health, Bethesda, Maryland.     

Ultrastructure of human endometrial epithelium in monolayer culture with and without steroid hormones

Summary Colonies of cells of epithelioid appearance were identified in monolayer cultures grown up to 50 days from normal human endometrial cell suspensions obtained by a method designed to insure a maximum harvest of glandular cells. Groups of these cells were separated from stromal cells by means of cloning cylinders. Studies comparing the ultrastructure of cells of this type to fresh endometrial tissue revealed a number of similarities. The morphological characteristics common to both types of samples included junctional complexes, perinuclear microfilaments and microvilli with glycocalyx. Other common features were prominent nucleoli, well developed Golgi, rough endoplasmic reticulum and membranebound electron-dense bodies in the cytoplasm. A stripping technique applie to the fetal bovine serum used in the nutrient medium made it possible to initiate cultures in a steroidfree environment and to maintain them in the presence of the specified concentration of estradiol and/or progesterone. Isolation of epithelial cells of endometrium in monolayer culture may provide a useful model system in which to study the specific effects of steroid hormones on cellular function and differentiation. Supported by grants from the National Institutes of Health (CA 18678 and CA 07368).     

Growth and characterization of human skin epithelial cell cultures

Summary In 129 of 140 attempts, human skin cells were successfully cultured on the dermal collagen bed of sterile, dead pigskin. Diploid epithelial cells grew selectively on the collagen bed; fibroblasts grew on the glass surfaces of the culture dishes. The cultures could be subdivided physically up to six times at a 1:2 split ratio, but at least 24 to 48 cell generations were produced over the months the cells could be carried. Much of the cell multiplication resulted in maturation into distinct basal, squamous, granular, and keratinized cell layers. The cultured cells were considered epithelial because of their shape, possession of intercellular bridges, desmosomes and tonofibrils, and because they formed maturating epithelium in vitro and upon transplantation back to the original human donor. As the cells grew they digested the pigskin collagen, thus producing clear zones that could be used to monitor and quantitate cell growth. Multiplication of epithelial cells, rather than migration, was indicated by mitotic figures in colchicine-treated cultures and by DNA synthesis. Expert technical assistance was provided by Nancy Allen (cell culture); William Towler (electron microscopy); James Malone, Nona Scaife, and Joy M. Nicolet (cytogenetics); R. Thomas Campbell and Dorothy Sarver (photography); and V. L. Angerstein, Susan Ekker, and Arnater Yarbrough (histology). This work was supported by The United Fund Cancer Society of Summit County, the Greater Cleveland Associated Foundation (grant no. 3G3490X1), the National Institute of General Medical Services (grant no. 1 R01 GM 21929-01), and the Charles E. Merrill Trust.     

Serum-free culture conditions for the growth of normal rat mammary epithelial cells in primary culture

Summary A monolayer culture system has recently been developed for the extended growth and serial passage of normal rat mammary epithelial (RME) cells. In this system the cells undergo greater than 20 population doublings when grown on type I collagen-coated tissue culture dishes in Ham's F12 medium supplemented with insulin, hydrocortisone, epidermal growth factor, prolactin, progesterone, cholera toxin, and 5% fetal bovine serum (FBS). The purpose of the present studies was to define additional growth factors that would allow equivalent RME cell proliferation in serum-free medium. Ethanolamine (EA) was effective at reducing the FBS requirements for RME cell proliferation and at its optimum concentration did so by greater than 20-fold. Even with optimum levels of EA there was essentially no cell proliferation in the absence of FBS. However, addition of bovine serum albumin (BSA) to the hormone, growth factor, and EA-supplemented medium resulted in substantial proliferation in the absence of serum, and the further addition of transferrin (T) potentiated this effect. Thus, in this culture system, replacement of FBS with EA, BSA, and T resulted in RME cell proliferation in primary culture which was equivalent to that obtained in the 5% FBS-containing medium. This work was supported by grant RR-05529 from the Division of Research Resources, National Institutes of Health, Bethesda, MD, and by Public Health Service grant CA40064-01 from the National Cancer Institute, Bethesda, MD.     

Normal human endometrium in cell culture

Summary Separation of human endometrium into its epithelial and stromal components has been achieved through collagenase digestion and has permitted a study of these two cell populations under specific experimental culture conditions. The stromal cell populations showed a progesterone response, were easily handled in culture, and displayed a limited in vitro life span typical of human diploid fibroblasts. In contrast, epithelium only survived in shortterm primary culture and showed no clear hormone response. High-density epithelial cultures remained viable for longer periods in culture. Comparisons between resurfacing endometrial epithelial cells in vivo and epithelial cells migrating from explants in vitro suggested that this initial epithelial migration in vitro was the counterpart of the repair response in vivo. We are much in debt to Dr. R. C. Hallowes (Department of Pathology, Imperial Cancer Research Fund) for his guidance and encouragement throughout the course of this work. We also gratefully acknowledge Dr. P. N. Riddle (Time-Lapse Cinematography Unit, Imperial Cancer Research Fund) for carrying out the time-lapse cinematography; Mrs. Lyn Rolph (Stereoscan Unit, Bedford College, University of London) for assisting with the SEM; and Mr. G. D. Leach for his competent help with the photography.     

Morphology and growth potential of stromal cell cultures derived from human endometrium

Summary Propagable cell cultures derived from human endometrial tissue were determined to contain cells predominantly of stromal cell origin based on their morphologic resemblance to endometrial stromal cells. These features included nexi, solitary cilia, and predecidual cytology. In addition to morphology the cell cultures retained a normal karyotype and responded to steroid hormones as evidenced by cellular aggregation. The stromal cells were evaluated for a variety of characteristics associated with transformed cells and seemed to be biologically normal without neoplastic phenotypes. Growth potential of the stromal cell cultures was also characterized in normal maintenance medium, in nutritionally depleted medium with reduced levels of calcium or serum, and in medium with increased levels of serum. The prolonged survival of the stromal cells in vitro coupled with the retention of in vivo characteristics and an absence of neoplastic phenotype provides a human cell system that is amenable to a variety of long-term experimental analyses. This work was supported by contract CP75956 and grant CA31733 from the National Cancer Institute. B. Hugh Dorman was the recipient of a predoctoral scholarship from the Chemical Industry Institute of Toxicology. Jill M. Siegfried was supported by National Research Service Award CA09156. David G. Kaufman is the recipient of a Research Career Development Award (CA00431) from the National Cancer Institute.     

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.     

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.     

Biological characteristics and viral susceptibility of a stable dog kidney cell line

Summary A cell line derived in 1956 from normal dog kidney is described. The cells are epithelial, contact-inhibited, and can be maintained in the same culture vessels for periods of more than 2.5 yr. Karyologically, the cells are hypodiploid with a modal number of 72 as opposed to the diploid number of 78. The karyotype indicates male origin of the cells and clonal derivation of extant cultures due to the presence of two marker chormosomes in all metaphases observed. At the 159th passage the dog kidney (DK) cells did not produce tumors in athymic rats. At least 13 viruses of various types produced transmissible cytopathogenic effects in the DK cells, including all of the human influenza viruses investigated. The project was supported in part by an Institutional Research and Development grant from SRI International and Contract Y01 CP8-0500, National Cancer Institute, National Institutes of Health, Bethesda, MD.     

Histochemical identification of cultured cells from human endometrium

Histochemical techniques have been applied to the identification of cell types cultured from human endometrium. Previous work from this laboratory characterized two principal cell types found in cultures of endometrium: a mature epithelial cell and another cell which was classified as the endometrial stromal cell based on light and electron microscopy. In this report we compare the histochemical staining of endometrial tissue in frozen sections to that of cultured cells. These results confirm the epithelial and stromal nature of the respective cell types. Several markers were found that could distinguish between cells of epithelial and stromal origin. The enzymes alkaline phosphatase, gamma-glutamyltranspeptidase, peroxidase, and beta-glucuronidase were localized in glandular and surface epithelia in frozen sections and in colonies of epithelial cells in culture. Stroma in frozen sections and cultured stromal cells contained leucine aminopeptidase and fibronectin. Epithelia in sections and in culture could also be distinguished from cells of stromal origin by preferential binding of lotus and peanut lectin. Several other markers were found in both endometrial epithelium and stroma.     

Long-term culture of epithelial cells from the normal rat colon

Summary Serial passage cultures of colonic epithelial cells from young rats have been maintained for more than 6 months in Eagle's minimum essential medium buffered with HEPES (25 mM) and supplemented with 2.5% fetal bovine serum, 0.5 μg/ml insulin, 5.0 μg/ml transferrin, and antibiotics. The cells proliferated in this medium with a population doubling time of approximately 53 h. The cells retained differentiated morphology as evidenced by secretory activity and the presence of secretory granules, microvilli, tonofilaments, and desmosomal junctions. Further cells at the fourth passage had normal karyotypes with 42 chromosomes and exhibited anchorage dependent growth. High concentrations of fetal bovine serum (10 to 15%) exerted toxic effects on the colonic epithelial cell cultures. Supported by National Cancer Institute Contract N01-CP-75914.     

Culture of adult rat lung cells: Benzo(a)pyrene metabolism and mutagenesis

Summary A method is described for obtaining and culturing large numbers of lung cells from normal adult male rats. The lungs were perfused in situ to remove blood cells and then perfused via the trachea with a trypsin-collagenase solution to initiate tissue digestion. The tissue was further digested in the enzyme solution and approximately 2×10⁸ viable lung cells were obtained per animal. Primary cultures contained a mixed cell population. Through eight subcultures about 70% of the cell population possessed an epithelial-like morphology, whereas the remaining 30% was fibroblast-like. Three clones of epithelial-like cells were isolated at the fourth subculture. The mass culture lung cells and the epithelial-like clone that was studied retained a normal karyotype and did not grow in soft agar. Both the mass culture cells and the epithelial clone metabolized the lung carcinogen benzo(a)pyrene (BP) to water-soluble products. Furthermore, the mass culture lung cells metabolized BP to intermediate(s) which mutated Chinese hamster V79 cells from ouabain sensitivity to ouabain resistance. These lung cell cultures have potential use in cell transformation, mutation and carcinogen metabolism studies. Visiting scientist from Hungary. This research was supported by Grant 5 R01 CA20022 and Public Health Service Contract N01 CP33278 from the Division of Cancer Cause and Prevention, National Cancer Institute, National Institutes of Health.     

Cell size in aging monolayer cultures

Summary Changes in the size of the area covered by individual cultured WI-38 cells as the cultures age have been studied by using a new microphotographic paper cutout technique. This method is nondestructive and nonintrusive and avoids a number of artifacts which can occur in the measurement of suspended cells. The measurements reveal that the decreased cell yield of late passage cultures-reflects not only the appearance of a subpopulation of larger cells but also the failure of the cells to utilize all the growth surface available to them. This work was supported in part by USPHS research grant AG-00378 and by a fellowship, AG-05019, from the National Institute on Aging.     

Human ovarian surface epithelium in primary culture

Summary The ovarian surface epithelium (OSE) represents a minute fraction of the cell mass of the ovary but gives rise to over 80% of human ovarian carcinomas. No experimental models for the study of human OSE exist. To characterize OSE cells in culture, explants of ovarian surface from normal ovary of premenopausal women were grown on plastic, glass, and collagen gel in 25% fetal bovine serum/Waymouth's medium 752/1. About 25% of explants produced epithelial outgrowths. Morphologically, these outgrowths resembled OSE in vivo and endothelial and mesothelial cells in culture, but they differed from cultured ovarian stromal, granulosa, and luteal cells. Only OSE among ovarian cell types were intensely keratin positive by immunofluorescence. Keratin also distinguished OSE cells from the keratin-negative endothelial cells. Most but not all OSE colonies tested showed 17β-hydroxysteroid dehydrogenase (HSD) activity, which was absent in peritoneal mesothelial cells. Colonies from most patients were limited to a few millimetres and became stationary within a few weeks. Changes that accompanied cessation of growth included senescence, increased keratin content, or the formation of multicellular papillary aggregates. With time, OSE cells tended to assume a fibroblast-like morphology but remained keratin positive and continued to resemble OSE by scanning electron microscopy (SEM). Subcultured OSE cells persisted in a stationary keratin-positive form for many weeks. Throughout this study, all pavementlike epithelial outgrowths that were contiguous with an explant stained for keratin; thus, such colonies can be assumed to be OSE. Conversely, fibroblast-shaped cells may represent OSE as indicated by keratin content and SEM appearance. The methods presented here permit culture of normal human OSE under conditions in which the cells exhibit morphologic plasticity, variable 17β-HSD activity, and presence of keratin. Supported by a grant and a research associateship to N. A. by the National Cancer Institute of Canada.     

Biological properties of human melanoma cells in culture

Summary Three human melanoma cell lines derived from one primary and two metastatic tumors from three different patients were characterized for growth properties usually associated with malignant transformation; these include cell morphology, growth rate, saturation density, growth in semisolid media, colony-forming ability on contact-inhibited monolayers of normal fibroblasts and epithelial cells, and tumorigenicity in immunosuppressed mice. Variations in expression of aberrant properties were evident among the lines. One of the metastatic lines satisfied all the parameters of malignancy tested and the other showed a number of these properties, whereas the primary essentially fulfilled only one. These results suggest that cultured melanoma cells reflect the clinical variability often observed among melanoma patients and the metastatic melanoma seems to display a higher degree of malignant transformation than the primary. THis work was supported in part by USPHS Grant No. 5 T01 AI00332-06 from the National Institutes of Health, Contract E73-2001-N01-CP-3-3237 from the Virus Cancer Program of the National Cancer Institute, and USPHS Grant No. 0H00714-02 from the National Institute for Occupational Safety and Health.