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.     

Monolayer culture of human endometrium: Methods of culture and identification of cell types

Summary Monolayer cultures can be established from human endometrial tissue after enzymatic dispersal into isolated glands or single cells. Three cell types that have distinct morphology by light and electron microscopy are observed in the resulting primary cultures. One cell type, an elongated spindle cell, is similar in appearance to fibroblasts derived from other tissues. A second cell type forms colonies of tightly cohesive cells, ranging in shape from oval to polygonal. These cells have typical organelles and junctional complexes characteristic of epithelial cells from the endometrium. The third cell type assumes a pavement-like appearance composed of polygonal cells when viewed by phase contrast microscopy, but lacks distinctive ultrastructural features of epithelial cells. These cells in culture resemble the endometrial stromal cell, the predominant cell type of the human endometrium in vivo. The epithelial cell does not survive subculturing but the other two cell types can be passaged through several generations and can be stored in liquid nitrogen and subsequently returned to culture. This work was supported by contract N01-CP75956 and grant R01-CA31733 from the National Cancer Institute. V. A. Varma is a recipient of an American Cancer Society fellowship; B. H. Dorman, a predoctoral fellowship from the Chemical Industry Institute of Toxicology; J. M. Siegfried, a training grant (CA09156) from the National Cancer Institute; and D. G. Kaufman, a Research Career Development Award (K04-CA-00431) from the National Cancer Institute.     

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.     

Morphologically stable epithelial vesicles cultured from normal human endometrium in defined media

Summary Epithelium from normal human endometrium was cultured as morphologically stable vesicular structures in a defined, hormone-supplemented PFMR-4 medium. The structures consisted of a single layer of polarized epithelial cells with the apical surface facing the external culture medium, and the basal surface resting on a well-defined basal lamina adjacent to the internal lumen. Vesicles were shown to (a) retain their viability for up to 3 mo. in culture, (b) to actively synthesize DNA after being cultured for over a month in a defined medium, and (c) to respond to steroid hormones. When embedded within a collagen gel, the vesicles reversed their epithelial polarity and formed branching, pseudoglandular structures. It was concluded that the three-dimensional shape of the epithelial vesicles had a critical role to play in their morphological stability nutient requirement, and hormone sensitivity.     

Normal human endometrium in cell culture

Summary The growth of short-term primary cultures of endometrial epithelium has been studied using Feulgen microspectrophotometry. A gradual increase in the number of polyploid nuclei up to 64C has been observed and is associated with a decline in the growth capacity of the cultures. The specific mechanism(s) of this polyploidization is not known.     

Variation in the electrical properties of cultured human proximal tubule cells

Summary Monolayers of human proximal tubule (HPT) cells, when grown on permeable supports and mounted in Ussing chambers, spontaneously display a transepithelial potential difference (PD), short-circuit current (Isc), and transepithelial specific resistance (R_T). These electrical parameters were used to determine the degree of heterogeneity among independent isolates of human proximal tubule cell cultures. Seventeen independent isolates of cells were assessed, totaling 260 individual determinations of spontaneous electrical properties. On average, these cell monolayers displayed an apicalnegative PD of 1.5 ± 0.1 mV, an Isc of 2.7 ± 0.2 μA/cm², and an R_T of 480 ± 19 ohms × cm². Each independent cell isolate, however, displayed electrical values within a narrow range, in some cases allowing isolates to be distinguished from one another. The individual isolates were also assessed for Na-coupled glucose transport, Na⁺,K⁺-ATPase activity, cAMP stimulation by parathyroid hormone (PTH), forskolin stimulation of Isc, and ouabain inhibition. With the exception of a strong correlation between Na⁺,K⁺-ATPase activity and Isc, these parameters, in contrast to electrical properties, were found to be consistent and did not reveal distinctions among the isolates. HPT cell cultures seem to consistently retain important features of proximal tubule differentiation while maintaining the variability, as demonstrated by electrical properties, that might be expected of cells isolated from a random population.     

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.     

Constitutive secretion of MMP9 by early-passage cultured human endothelial cells

Matrix metalloproteinase-9 (MMP9) plays an important role during angiogenesis. It is an inducible enzyme which is known to be secreted from human endothelial cells in response to phorbol myristate acetate (PMA), but thought not to be constitutively expressed. We examined the secretion of MMP9 by primary culture (P0), passage 1 (P1) and passage 2 (P2) human umbilical vein endothelial cells (HUVE). Whereas there was no detectable MMP9 in P2 cells under basal conditions, P0 HUVE secreted MMP9, as detected by zymography and ELISA. RT-PCR and cycloheximide inhibition studies confirmed that MMP was synthesized by P0 HUVE. MMP9 secretion was passage-dependent, decreasing rapidly as the cells were passaged in culture and was not detected at P2. The decrease was largely due to the population doubling of cells as they are cultured. This is the first report to show that cultured HUVE constitutively express MMP9 and that this secretion is restricted to very early-passage cells. These findings may be relevant to the angiogenic potential of human endothelial cells as they age.     

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.     

Modulation of angiotensin-converting enzyme in cultured human vascular endothelial cells

Summary Previous work has suggested that not all immunoreactive angiotensin-converting enzyme (ACE) in tissues or cells is in a biologically active state. We have explored this possibility in cultured human umbilical vein endothelial cells (HUVEC), one of the most widely studied in vitro endothelial cell systems. Our approach included characterization of the effect of increasing passage number on ACE activity and expression of immunoreactive ACE at the single cell level, the subcellular compartmentalization of active ACE, and the effect of phorbol ester (PMA) treatment. We found that both ACE activity and expression of ACE antigen were downregulated by cultivation (30% of ACE-positive cells at seventh passage vs. 90% in primary culture). ACE downregulation is specific (number of CD31-positive cells did not change with cultivation) and correlated with downregulation of factor VIII-antigen. The percentage of ACE-positive cells in permeabilized HUVEC at third passage was almost twice that in nonpermeabilized HUVEC (90% vs. 50%), indicating that HUVEC contain intracellular immunoreactive ACE. ACE activity, however, was similar when measured in intact cells and in cell lysates. Moreover, diazonium salt of sulfanilic acid (DASA), a membrane-impermeable ACE inhibitor, inhibited ACE activity in intact cells and in cell lysates at the same extent, thus implying that intracellular ACE is inactive. PMA (100 nM) treatment increased the percentage of ACE-positive cells at third passage from 57 to 96%. ACE activity was increased 3-fold in cell and 1.5-fold in the culture medium of PMA-treated cells. Analysis of ACE activity in intact monolayers and cell lysates of control and PMA-treated cells revealed that all enzymatically active ACE in PMA-treated cells is localized on the plasma membrane and acts as an ectoenzyme. We conclude that expression of ACE by HUVEC is downregulated by repeated passage in culture but can be restored by PMA treatment. In addition, ACE expression is heterogeneous between neighboring cells, and total immunoreactive ACE protein associated with HUVEC includes an inactive pool of the enzyme.     

Biological characteristics of cultured cells derived from various types of human brain tumors

We placed in culture brain tumors from 45 cases (7 cases of astrocytoma, 2 from oligodendrogliomas, 2 glioblastomas, 2 ependymomas, 13 meningiomas, 6 pituitary adenomas, 5 neurinomas, a malignant lymphoma, a choroid plexus papilloma, and 6 metastatic tumors) and succeeded in making a primary culture from 33, and maintained 17 in vitro over a considerable period of time (greater than three months). In the early period of the primary cultures, the astrocytoma cells had cytoplasmic processes which contacted each other, the oligodendroglioma cells were small and spindle-shaped, the glioblastoma cells were neoplastic with pleopmorphic features and possessed cytoplasmic processes, the ependymoma cells formed a rosette-like cell arrangement, the meningioma cells were spindle- or round-shaped cells and characterized as forming psammoma bodies, the pituitary adenoma cells were round- or oval-shaped cells and produced growth hormone (GH), adenocorticoid tropic hormone (ACTH), prolactin, or other hypophyseal hormones, the choroid plexus papilloma cells were round-or polygonal and showed a papillary cell arrangement, the neurinoma cells were spindle- or fibrous-shaped cells, and the malignant lymphoma cells were round and formed cell aggregates floating in the culture medium.     

Outward-rectifying chloride channels in cultured adult and fetal human nasal epithelial cells

Summary The patch-clamp technique was used to characterize ion channels in the apical membranes of cultured human nasal epithelial cells, dissociated from fetal nasal mucosa and from adult nasal polyps. Outward-rectifying chloride channels were found in 4.3% of the cell-attached patches from fetal cells (n=258) and in 3.1% of the patches from adult cells (n=320). After exeision the number of patches containing active chloride channels increased threefold to 13% of the patches from the fetal cells and 10% from adult cells. The single-channel conductance at 0 mV in symmetrical 150mm NaCl solutions was 24.3 ±0.9 pS (n=28) and 26.0 ± 1.2 pS (n=30), respectively, in adult and fetal cells and showed outward rectification in the potential range from –80 to +80 mV. In fetal cells as well as in adult cells the channels were anion selective, and were almost impermeable for larger anions and monovalent cations. In cell-free patches the channels were Ca²⁺ independent. In most of the channels the open probability was voltage independent and high (±0.86); in 20% of the channels, however, the open probability increased with depolarization. In conclusion, fetal nasal epithelial cells contain chloride channels in their apical membranes with singlechannel properties and regulatory mechanisms similar to those found in cells from adults.     

Sialic acid binding protein of human endometrium: Its regulation by steroids

In the present study, we have observed that sialic acid binding protein (SABP – a 54 kDa glycoprotein which was isolated from human endometrial scrapings taken at various stages of the menstrual cycle from normal cycling females and purified to apparent homogeneity and was earlier reported from this laboratory) was found in sufficiently detectable amount in the endometrium of normal cycling women whereas it was found in lesser amount in tissue from women who have recently entered the post-menopause stage. SABP was observed in both follicular and luteal phase of menstrual cycle which was found by western blot analysis. In the de-novo synthesis experiment, synthesis and secretion of SABP was found to be stimulated by estradiol (E₂) whereas progesterone (P₄) was found to have no significant stimulatory effect on it which was also confirmed by HEC cell culture. In the HEC cell culture, priming of cells with E₂ was found to influence the effect of P₄ on SABP when it was added 2 h after E₂ administration. This was observed by doing immunoprecipitation followed by SDS-PAGE and autoradiography. Hence this report clearly indicates that E₂ regulates the synthesis and secretion of 54 kDa SABP from human endometrium. How E₂ priming of endometrium influences the effect of P₄ on SABP has been discussed.     

Establishment and morphologic characterization of normal human endometrium in vitro

Summary Tissue culture offers a model system with which to study the endocrine-mediated growth, differentiation, and metabolic activities of the endometrium. We have established and continue to maintain monolayer cultures of normal human endometrial epithelial cells from each phase of the menstrual cycle. At present, eight proliferative, two secretory, and two menstrual phase cultures have been established. These have been passed at least three times. One proliferative phase culture has been growing for 18 mo, and passed 10 times. Colonies of epithelioid cells as well as single cells appear in the cultures within 2 to 8 h of initial culture and maintain this appearance throughout long-term growth. The cells are periodic acid Schiff positive for carbohydrates and positive for keratin, an immunochemical marker for epithelial tissues. Studies comparing the ultrastructure of the cultures with fresh endometrial tissue revealed morphologic features common to both, including prominent nucleoli, Golgi, mitochondria-rough endoplasmic reticulum complexes, and abundant glycogen. The cells are not tumorigenic in the nude mouse and do not form colonies on soft agarose, confirming the nonneoplastic identity of the cells. The opinions and assertions contained herein are those of the authors and are not to be construed as official or representing those of the Uniformed Services University of the Health Sciences, The Department of the Navy, or the Department of Defense. This project was supported by the following grants: C08509 from the Uniformed Services University and Clinical Investigation Study Protocol 82-06-1804, Naval Hospital, Bethesda. Presented at the Twenty-Second Annual Meeting of the Armed Forces District of the American College of Obstetricians and Gynecologists, 9–13 October 1983; Founders Award for the Best Paper on a Basic Scientific Subject.     

Serial culturing of human bronchial epithelial cells derived from biopsies

Summary In the present study we describe the establishment of serial cultures of human bronchial epithelial cells derived from biopsies obtained by fiberoptic bronchoscopy. The cell cultures were initiated from small amounts of material (2 mm forceps biopsies) using either explants or epithelial cell suspensions in combination with a feeder-layer technique. The rate of cell proliferation and the number of passages (up to 8 passages) achieved were similar, irrespective of whether the explants or dissociated cells were used. To modulate the extent of differentiation, the bronchial epithelial cells were cultured either under submerged, low calcium (0.06 mM) (proliferating), normal calcium (1.6 mM) (differentiation enhancing) conditions, or at the air-liquid interface. Characterization of the bronchial epithelial cell cultures was assessed on the basis of cell morphology, cytokeratin expression, and ciliary activity. The cells cultured under submerged conditions formed a multilayer consisting of maximally three layers of polygonal-shaped, small cuboidal cells, an appearance resembling the basal cells in vivo. In the air-exposed cultures, the formed multilayer consisted of three to six layers exhibiting squamous metaplasia. The cytokeratin profile in cultured bronchial epithelial cells was similar in submerged and air-exposed cultures and comparable with the profile found in vivo. In addition to cytokeratins, vimentin was co-expressed in a fraction of the subcultured cells. The ciliary activity was observed in primary culture, irrespective of whether the culture had been established from explants or from dissociated cells. This activity was lost upon subculturing and it was not regained by prolongation of the culture period. In contrast to submerged cultures and despite the squamous metaplasia appearance, the cells showed a reappearance of cilia when cultured at the air-liquid interface. Human bronchial epithelial cell cultures can be a representative model for controlling the mechanisms of regulation of bronchial epithelial cell function.     

Human epithelial cells cultured from urine: Growth properties and keratin staining

Summary Epithelial cells can be cultured from the urine of newborn infants, providing a simple, noninvasive biopsy method. We established such cultures by standard techniques from 44% of uncontaminated, specimens obtained from newborn infants up to 1 week of age. There was an average of three colonies per milliliter of urine. Many cultures accomplished 15 to 25 population doublings in as many as five subcultures and yielded total potential culture sizes of 10⁴ to 6×10⁸ cells. Plating efficiency was high at each passage. The cultures displayed two morphologically distinct epithelial cell types. Immunofluorescent staining of keratin fibers in most of these cells further, identified them as epithelial. This work was supported by NIH grants, CA16754 (J. S. F., J. W. L.) and EY02472, AM25140, AM21358, and a Research Career Development Award (EY00125) to T.-T. S.     

Characterization of mouse fetal lung cells cultured on a pigskin substrate

Summary Lung organ bits taken from full-term mice were explanted on the dermal surface of sterile, dead pigskin. The cells migrated onto the pigskin dermis and proliferated to form an organoid culture consisting of ductular structures separated by a matrix of epithelial cells. Cells within the ductular structures were ciliated, produced mucin, and exhibited the activities of nonspecific esterase and gamma-glutamyl transferase; therefore they were considered to be derived from bronchial epithelium. Cells forming the matrix possessed the activities of nonspecific esterase and alkaline phosphatase and contained lamellar structures typical of surfactant-producing pneumocyte Type II cells; therefore they were considered to be derived from alveolar precursor cells. This research was supported by Grant-in-Aid 1203 M from the Council for Tobacco Research, awarded to Aaron E. Freeman.     

Acute cytotoxicity testing with cultured human lung and dermal cells

Summary An extensive in vitro study with cultured cells was conducted to test the basal cytotoxicity theory. This theory suggests that most chemical injury, at least in vitro, is a manifestation of one or more insults to the basic cellular structures and functions common to mammalian cells. This accounts for the similarity of results in multilaboratory studies. Human fetal lung fibroblasts (HFL1), and human skin fibroblasts (WS1, Detroit551) were studied in culture to evaluate their potential to screen for cytotoxicity. Confluent monolayers were incubated in the absence or presence of increasing concentrations of test chemicals for 24 h, and the MTT assay was used to assess toxicity. Inhibitory concentrations were extrapolated from concentration-effect curves after linear regression analysis. Twenty-nine chemicals were tested with each cell line and the cytotoxicity data compared to rodent and human lethal concentrations. The data suggest that the experimental IC₅₀ values are as accurate predictors of human toxicity as equivalent toxic blood concentrations derived from rodent LD₅₀s. In addition, lung and skin fibroblasts revealed no significant differences among the three cell lines. The results support the conclusion that finite cell lines of human origin have the potential for screening chemicals for human toxicity. In combination with previously published reports, the data suggest that a basal cytotoxic phenomenon may explain the similarity of results among different human cell lines.     

Cytoskeletal markers and specific protein production in cells cultured from human first and third trimester placentae

Summary In primary, short-term cultures derived from first and third trimester placentae, 60 to 90 and 70 to 95%, respectively, of the total cell population positively stain for cytokeratin intermediate filaments, typical of epithelial, i.e. trophoblastic cells. The rest of the cells express only vimentin intermediate filaments and thus are of mesenchymal origin. Only the cytokeratin-positive cells express human chorionic gonadotropin (hCG), whereas both the epithelial and the mesenchymal cells stain positively for pregnancy-specific beta-1-glycoprtein (SP₁). Cytokeratin-negative and vimentin-positive cell overgrowth is observed in cultures derived from first and early third trimester placentae. The cells constituting the monolayer thus formed are of fetal origin as evidenced by the expression of Y-body in over 80% of them. The cultured cells synthesize and secrete hCG and SP₁. The activity of these trophoblast-specific functions is inversely proportional to the gestational age of the placenta. Production of specific proteins and expression of intermediate filaments are proposed as criteria for defining the nature and origin of placental cells in primary, short-term cultures.