Tissue repair of uterine cervix--cell-biological properties of normal uterine cervical epithelia of transformation zone in vitro]

The objective of this study is to culture the epithelia of the transformation zone of the uterine cervix for long term and evaluate their biological characteristics, such as morphology, growth behavior, alkaline phosphatase activity and heterotransplantability. The epithelia of transformation zone of 15 cases of myoma uteri were cut into 1 x 1 x 1 mm fragments and placed directly on the cover glass. The explants were cultured at 37 degrees C in 5% CO2 and 95% air. In vitro outgrowth of squamous cells (squamous cell outgrowth pattern) was observed in 44, that of columnar cells (columnar cell outgrowth pattern) was observed 49, a mixture of squamous and columnar cell outgrowth patterns was 52 out of 198 explants of transformation zone. The squamous cells were polygonal in shape and showed a pavement-like cell arrangement. The glandular cells grew in whorled fashion. Along the margins of the outgrowth of glandular cells, two types of cells were seen after 2 weeks of culture. One type contained secretory vacuoles of glandular cell, and the other type contained a large number of tonofilaments of squamous metaplastic cells. These phenomena suggested that biological characteristics of the cells in vivo can well be retained in vitro for a relative long term (about 6 weeks).     

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.     

Differentiation of mouse embryonic palatal epithelium in culture: selective cytokeratin expression distinguishes between oral, medial edge and nasal epithelial cells

During normal murine palatogenesis, regional specific differentiation of the epithelium results in three cell phenotypes: nasal (ciliated pseudostratified columnar cells), oral (stratified squamous cells) and medial edge (migratory, epithelio-mesenchymally transformed cells). We have developed a defined, serum-free, culture system which supports the growth and differentiation of isolated murine embryonic palatal epithelia in vitro. Using immunofluorescence microscopy, an established panel of antibodies was used to characterise the cytokeratin intermediate filament profile of palatal epithelial sheets at a precise developmental stage, following culture in serum-free medium with and without either transforming growth factor alpha (TGF alpha) or 10% donor calf serum (DCS). The morphologically discernable oral, medial edge and nasal phenotypes exhibited distinctive cytokeratin profiles, which remained consistent for all culture conditions, and which correlated with the known differentiation states of the epithelial types. The oral epithelia stained positively for cytokeratin 19 and cytokeratins characteristic of multilayered epithelia (1, 5, 14). Nasal epithelia stained similarly but in addition expressed the simple-epithelial cytokeratin pair, 8 and 18. Medial edge epithelia also expressed cytokeratins 1, 5 and 14 but with the exception of a few isolated cells there was no staining for cytokeratins 8 and 18. Cytokeratin 19 was absent specifically from the medial edge epithelial cells: this result may be related to the loss of cytokeratin expression observed during epithelial-mesenchymal transformations. By exhibiting a complexity of expression linked to differentiation state and independent of culture conditions, cytokeratins constitute useful markers of palatal epithelial differentiation in vitro as well as in vivo.     

Use of microgravity bioreactors for development of an in vitro rat salivary gland cell culture model

During development, salivary gland (SG) cells both secrete factors which modulate cellular behavior and express specific hormone receptors. Whether SG cell growth is modulated by an autocrine epidermal growth factor (EGF) receptor-mediated signal transduction pathway is not clearly understood. SG tissue is the synthesis site for functionally distinct products including growth factors, digestive enzymes, and homeostasis maintaining factors. Historically, SG cells have proven difficult to grow and may be only maintained as limited three-dimensional ductal-type structures in collagen gels or on reconstituted basement membrane gels. A novel approach to establishing primary rat SG cultures is use of microgravity bioreactors originally designed by NASA as low-shear culture systems for predicting cell growth and differentiation in the microgravity environment of space. These completely fluid-filled bioreactors, which are oriented horizontally and rotate, have proven advantageous for Earth-based culture of three-dimensional cell assemblies, tissue-like aggregates, and glandular structures. Use of microgravity bioreactors for establishing in vitro models to investigate steroid-mediated secretion of EGF by normal SG cells may also prove useful for the investigation of cancer and other salivary gland disorders. These microgravity bioreactors promise challenging opportunities for future applications in basic and applied cell research. © 1993 Wiley-Liss, Inc.     

CA 125 is an excretory product of human endometrial glands

The present investigation was undertaken to study the cellular localization and kinetics of synthesis of CA 125 in the endometrium. CA 125 was localized by immunohistochemistry to the infranuclear region of epithelial cells during the proliferative phase and to the apical luminal border during the secretory phase. In gestational endometrium, both the cytoplasm and the apical luminal border of epithelial cells were intensely positive. No staining was seen in endometrial stromal cells during the normal cycle or in decidualized endometria. Results obtained from in vitro cultures of separated glandular and stromal cells were similar to those obtained by immunohistochemistry. That is, epithelial cells released between 5 and 25 times more CA 125 into the culture medium than did stromal cells. The release of CA 125 was highest in epithelial and stromal cells obtained during the early secretory phase. CA 125 concentrations were markedly elevated in endometrial aspirations obtained during the secretory phase or in endometria with crumbling stroma compared to plasma levels. Plasma levels of CA 125 were slightly elevated during menses. These results suggest that CA 125 is an exocrine product of endometrial epithelial cells. Plasma levels of CA 125 may be of endometrial origin only when the membrane barriers, which normally prevent its entry into the circulation, are damaged.     

Normal and benign human prostatic epithelium in culture. I. Isolation.

Isolation of normal human glandular epithelia and their growth and maintenance in vitro have been major problems. The primary objective of studies presented here was to isolate postpubertal, normal human, viable prostatic epithelium for in vitro cultivation. The long-term objective of these investigations was to develop an in vitro human cell model system for studies on prostatic carcinogenesis. A method for isolation of viable, normal and benign human prostatic epithelium, using collagenase for tissue dissociation, is described. Intact acini were isolated, which, on plating gave rise to vigorously growing monolayer cultures of epithelial cells. The purity of epithelial cultures partly depended upon the source of tissue. Specimens of normal prostate and those of benign tissue derived from open prostatectomies provided primarily pure epithelial cultures with occasional fibroblast colonies in some cultures, which could be removed. Cultures from some specimens of transurethral resection of the prostate (TURP) contained many fibroblast colonies due to incomplete separation of acini from the stroma. This resulted from incomplete digestion of denatured tissue caused by electrocauterization during surgery. Cultures established in this manner are being used to study the effects of hormones, vitamins and other growth regulators in order to establish growth requirements of these cells in vitro, which would facilitate their long-term maintenance.     

A new method for the simultaneous analysis of growth and death of immunophenotypically defined cells in culture

BACKGROUND: Internal standards have been used in flow cytometry methods to enumerate lymphoid subsets and hemopoietic progenitor cells ex vivo. However, the currently available methods cannot be readily applied to the analysis of cultured cells because of the frequent occurrence of cell death during in vitro assays. METHODS: This paper reports a new method for the enumeration of both viable and nonviable cells in culture. Cells were counted with the aid of an internal reference standard of microbeads, and live versus dead cell discrimination was performed using 7-amino-actinomycin D which allows the double staining of surface antigens. RESULTS: The method is more precise, accurate and sensitive than either conventional light microscopy-based or automated cell counting. Additionally, it may be used to accurately measure the number of apoptotic cells in a culture. RESULTS: Through the enumeration of surviving cells it is demonstrated that, when applied to the study of mitogen-activated T lymphocytes, current flow cytometry techniques (which do not use internal standards) for the study of the viability and apoptosis overestimate the fraction of viable cells and underestimate both the fraction of dead and apoptotic cells. CONCLUSIONS: The new method overcomes these limitations and is of use in the in vitro study of cell growth and apoptosis.     

Influence of retinoids and EGF on growth of embryonic mouse palatal epithelia in culture

Summary Retinoids and growth factors seem to be important for normal mammalian reproduction and development. High levels of retinoic acid are teratogenic and induce cleft palate in the mouse. Little is known concerning the mechanisms through which retinoids induce cleft palate. Palatal epithelia from CD-1 embryonic mice on Day 12 of gestation were isolated from the mesenchyme and cultured in serum-free media, with all-trans retinoic acid or 13-cis retinoic acid, with or without epidermal growth factor (EGF). The epithelia attached and grew, and the cells differentiated over a 72-h culture period. Binding of [¹²⁵I]EGF was observed in all cultures in a pattern that correlated with thymidine (TdR) uptake by the epithelia. EGF enhanced growth and [³H]TdR incorporation of the oral cells, but nasal cells generally did not proliferate. In this culture system, both retinoids suppressed [³H]TdR incorporation in a concentration-dependent manner for epithelia cultured with or without EGF. Medial cells are important to normal palatogenesis as they play a role in fusion of opposing shelves and subsequently many of these cells undergo programmed cell death. Death of medial cells in vitro is prevented by EGF and by the retinoids, either with or without EGF. This response occurs in the absence of a mesenchymal interaction, suggesting that the medial cell response to EGF and retinoids is not mediated by or dependent on the mesenchymal tissues. The survival of medial cells may be responsible for the failure of opposing shelves to fuse.     

Growth of fetal rat gastro-intestinal epithelial cells is region-specifically controlled by growth factors and substrata in primary culture

The mammalian gastro-intestinal tract can be divided into three parts: esophagus and forestomach, glandular stomach, and intestine. We have previously reported primary culture systems for duodenal and glandular stomach epithelial cells in which the cells express tissue-specific marker proteins. However, the effects of growth factors and substrata on cell growth have not been fully investigated. In this study a primary culture system was established for forestomach epithelial cells and the mechanism by which the growth of gastro-intestinal epithelial cells is controlled in primary culture was examined. Forestomach, glandular stomach and duodenal epithelial cells proliferated rapidly in culture, increasing their numbers about 30-, 20-and 10-fold, respectively, in the first 5 days. Scanning electron microscopy showed that these three types of epithelial cells exhibited region-specific morphologies in culture. Results on the effects of growth factors and substrata on the proliferation of the epithelial cells revealed that the culture conditions required to induce maximal epithelial growth differed. Forestomach and glandular stomach epithelial cells required similar combinations of growth factors to proliferate, and these were quite different from those required for duodenal epithelial cells. Glandular stomach and duodenal epithelial cells could proliferate in a serum-free condition while forestomach epithelial cells could not. Thus, glandular stomach epithelial cells exhibited intermediate characteristics between forestomach and duodenal epithelial cells regarding their growth factor requirement. Glandular stomach and duodenal epithelial cells could not proliferate on plastic without collagen substrata while forestomach epithelial cells could. Duodenal epithelial cells proliferated faster on collagen gels than on collagen films, and forestomach epithelial cells faster on collagen films than on collagen gels. Glandular stomach epithelial cells proliferated similarly on both substrata. Thus again, glandular stomach epithelial cells exhibited intermediate characteristics between forestomach and duodenal epithelial cells regarding their substratum dependency. We conclude that the growth of gastro-intestinal epithelial cells is affected by both growth factors and substrata, and that glandular stomach epithelial cells exhibit intermediate characteristics between forestomach and duodenal epithelial cells in responding to these factors. These results suggest that a head-to-tail gradient exists in the gastro-intestinal tract which controls the epithelial response to growth factors and substrata.     

Regulation of cell proliferation in a stratified culture system of epithelial cells from prostate tissue

Mechanisms controlling epithelial proliferation and differentiation in the prostate have been primarily investigated in mouse models. The regulation of proliferation and differentiation is poorly understood in human prostate epithelial cells. In vivo, the glandular prostate epithelium consists of a p63-positive proliferating basal cell layer and a post-mitotic p27-positive secretory cell layer. We have established an organized stratified culture system of human primary prostate epithelial cells to gain insight into mechanisms regulating proliferation and differentiation. In this system, expression of p63 is observed in the bottom layer. In addition, BrdU incorporation persists even though cells are confluent. In contrast, in the upper layer, p63 expression is greatly diminished, p27 is expressed, and the cells are growth arrested. Overexpression of cyclin D1 or knockdown of p27 does not increase proliferation. After inactivation of the nuclear phosphoprotein Rb, the cell layers remain organized and cell proliferation increases only in the bottom layer. Furthermore, the expression of p63 remains confined to the bottom layer after Rb inactivation. Altogether, this in vitro model recapitulates certain aspects of in vivo growth regulation and differentiation and suggests that the loss of Rb family proteins in human cells trigger hyperplasia but is not sufficient for transformation.This work was supported by the Departments of Pathology and Urology at Weill Medial College, by grants DAMD-17-02-1-0159, MEDC-GR-355, and P30 CA015704-30, and by grant RO1CA84069 to B.E.C.     

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.     

Dental papilla cells in culture. Comparison of morphology, growth and collagen synthesis with two other dental-related embryonic mesenchymal cell populations

The dental papilla is a mesenchymal cell condensation which plays an important regulatory role during tooth development. Dental papilla mesenchymes were enzymatically separated from the dental epithelia from tooth germs of 17-day-old mouse embryos and disaggregated for monolayer culture. These cells were compared with gingival mesenchyme overlying the same tooth germs and with undifferentiated jaw mesenchyme from mandibles of 11-day-old embryos. The dental papilla cells were large and flat with numerous cell processes, whereas the gingival cells resembled typical spindle-shaped fibroblasts and grew to a higher cell density. Although the two mesenchymes differ in their collagen contents in vivo, no differences were detected either in the amount or type of collagen synthesized in vitro. Type I and III collagens were found in the culture media and type V collagen in the cell layer of both cell populations. The mandibular mesenchymal cells of the younger embryos resembled the dental papilla cells in morphology and growth rate. This may reflect retention of undifferentiated embryonic characteristics in the dental papilla. The successful culture of dental papilla cells now enables subsequent studies on the cellular properties related to the unique morphogenetic capabilities of these cells.     

Self-renewing endodermal progenitor lines generated from human pluripotent stem cells

The use of human pluripotent stem cells for laboratory studies and cell-based therapies is hampered by their tumor-forming potential and limited ability to generate pure populations of differentiated cell types in vitro. To address these issues, we established endodermal progenitor (EP) cell lines from human embryonic and induced pluripotent stem cells. Optimized growth conditions were established that allow near unlimited (>10(16)) EP cell self-renewal in which they display a morphology and gene expression pattern characteristic of definitive endoderm. Upon manipulation of their culture conditions in vitro or transplantation into mice, clonally derived EP cells differentiate into numerous endodermal lineages, including monohormonal glucose-responsive pancreatic β-cells, hepatocytes, and intestinal epithelia. Importantly, EP cells are nontumorigenic in vivo. Thus, EP cells represent a powerful tool to study endoderm specification and offer a potentially safe source of endodermal-derived tissues for transplantation therapies.     

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).     

Cellular microenvironment influences the ability of mammary epithelia to undergo cell cycle

The use of cell culture models is a principal and fundamental technology used in understanding how mammalian cells work. However, for some cell types such as mammary epithelia, the lines selected for extended culture are often transformed or have chromosomal abnormalities, while primary cultures have such a curtailed lifespan that their use is restricted. For example, mammary luminal epithelial cells (MECs) are used to study mechanisms of breast cancer, but the proliferation of primary cell cultures is highly limited. Here we describe the establishment of a new culture system to allow extended analysis of cultures of primary mouse MECs. In 2D monolayer culture, primary MECs showed a burst of proliferation 2-3 days post isolation, after which cell cycle decreased substantially. Addition of mammary epithelial growth factors, such as Epidermal Growth Factor, Fibroblast Growth Factor-2, Hepatocyte Growth Factor, and Receptor Activator for Nuclear Factor κB Ligand, or extracellular matrix proteins did not maintain their proliferation potential, neither did replating the cells to increase the mitogenic response. However, culturing MECs directly after tissue extraction in a 3D microenvironment consisting of basement membrane proteins, extended the time in culture in which the cells could proliferate. Our data reveal that the cellular microenvironment has profound effects on the proliferative properties of the mammary epithelia and is dominant over growth factors. Moreover, manipulating the cellular environment using this novel method can maintain the proliferative potential of primary MECs, thus enabling cell cycle to be studied as an endpoint after gene transfer or gene deletion experiments.     

COMPARISON OF THE GROWTH AND DIFFERENTIATION OF EPITHELIAL CELLS FROM NORMAL AND HYPERPLASTIC LENSES OF THE CHICK: STUDIES OF IN VITRO CELL CULTURES

Epithelial cells from hyperplastic lenses of a strain of chicks (Hy-1) selected for high growth rate were dissociated and cultured in vitro and compared with lens epithelial cells from a normal strain (N) in similar conditions. The hyperplastic lens cells showed remarkable motility and adhesiveness after dissociation and formed cell aggregates of various sizes before attaching to the substrate, giving a rather low plating efficiency. The lens structures (lentoid bodies) developed in partially confluent cultures of Hy-1 cells at least three days earlier than those in the cultures from normal control cells, in which the lens structures developed only after the cultures reached confluence. The results of culture at low cell density showed that the Hy-1 cell population consisted of at least two cell types different from each other in growth capacity. These striking differences in in vitro behaviour of dissociated cells from normal and hyperplastic lens epithelia and the results of clonal culture are discussed in relation to the possible mechanisms of abnormal morphogenesis and growth which are likely to be involved in the development of the hyperplastic lens in situ .     

Age-related expression of PEDF/EPC-1 in human endometrial stromal fibroblasts: implications for interactive senescence

Aging is the major risk factor for many cancers, and age-related changes in the tissue microenvironment can facilitate tumor growth. This study uses human endometrial cells to begin to test the hypothesis that age-related changes in pigment epithelium-derived factor/early population doubling cDNA-1 (PEDF/EPC-1) levels create an environment that is more permissive to tumor growth. Endometrial stromal fibroblasts (ESF) are the predominant cell type in the human endometrium and exert regulatory control over the glandular epithelial cells, which are the source of most tumors. As ESF age in vitro, their ability to regulate appropriate growth and differentiation of epithelial cells declines. Endometrial epithelial cells in primary culture expressed relatively low levels of PEDF/EPC-1 mRNA. In contrast, early passage quiescent ESF from adult donors produce higher levels of the 1.5-kb PEDF/EPC-1 mRNA and 50-kDa secreted protein than epithelial cells. As ESF age in vitro the relative abundance of PEDF/EPC-1 mRNA declines, as does the level of PEDF/EPC-1 protein secreted into cell culture medium. Treatment with PEDF/EPC-1 protein had no effect on ESF proliferation but did inhibit anchorage-dependent and anchorage-independent proliferation of endometrial carcinoma cells in a dose- and time-dependent manner. These findings imply that an age-related loss of PEDF/EPC-1 expression by ESF could eliminate a negative regulator of cancer cell growth and, thereby, contribute to the age-related increase in cancer incidence.