Characterization and co-culture of novel nontransformed cell lines derived from rat endometrial epithelium and stroma

Summary Normal and neoplastic growth of epithelial cells depends on mutual interactions between epithelial and stromal cells. As a tool for the study of the underlying molecular mechanisms, we have developed temperature-sensitive, nontransformed cell lines derived from rat uterine epithelium and stroma by transfecting primary cultures with a temperature-sensitive mutant of the SV40 large T antigen. The epithelial and stromal cell lines obtained shared relevant morphological characteristics with the primary cells from which they were derived. Immunocytochemical analysis showed that the epithelial cell lines expressed the intermediate filament cytokeratin, whereas the stromal lines expressed the intermediate filament vimentin. Alkaline phosphatase activity was present in all cell lines examined. All cell lines were anchorage dependent and did not form foci. One epithelial cell line expressed oxytocin mRNA, a gene product recently shown to be highly expressed in vivo in the uterine epithelium at term. If grown on Matrigel, this cell line formed domelike structures, a further characteristic of its differentiated phenotype. In an attempt to reconstitute an endometrium in vitro, epithelial cells were seeded on top of a layer of stromal cells. Paraffin cross sections showed that this in vitro system consisted of a bilayer structure. Four to five cuboidal epithelial cells were typically anchored atop one stromal cell, forming an endometriumlike tissue. The present in vitro system should provide a useful model for further studies on endometrial functions and epithelial/stromal cell interactions at a molecular level.     

Growth of neonatal rat uterine luminal epithelium on extracellular matrix

Summary We have developed a tissue culture system using an extract of basement membrane (extracellular matrix) which promotes the in vitro growth and development of uterine luminal epithelium from the 5-day-old rat. Uterine luminal epithelium, free of stroma, was obtained as short tubes by trypsinization of uterine segments followed by mechanical separation. Epithelial segments were grown in a serum-free medium on culture dishes coated with an extracellular matrix. After 2 days, rapid cell growth resulted in monolayer cultures, which subsequently formed organoid structures similar to differentiated uterine glands present in uterine tissue taken from older rats. Electron microscopy of cultures revealed columnar cells with basally located nuclei, apical microvilli, lateral membranes with interdigitations, desmosomes, and secretory Golgi complexes, all features found in functioning uterine epithelium in vivo. This model will allow the in vitro investigation of the development of uterine epithelium-specific functions free of the influence of stromal cell factors.     

Early expression of vimenti in human mammary cultures

Summary The intermediate filaments of most epithelial cells in vivo consist solely of cytokeratins. Using monoclonal antibodies to vimentin or keratin, we have examined the expression of vimentin in homologous specimens of frozen tissue sections and primary cultures of normal human mammary epithelium. In frozen sections, only epithelial cells reacted with the antikeratin antibody, whereas antivimentin reactivity was associated with stromal cells. All epithelial cultures were positive for cytokeratin and in addition coexpressed vimentin as strongly as cultured fibroblasts and as early as the 4th d after initiation of the culture. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis of cytoskeletal preparations of secondary cultures of normal mammary epithelium have also demonstrated the appearance of a moiety identical to the vimentin found in cultured fibroblasts. Our observations are consistent with the hypothesis that vimentin expression is induced, possibly as a result of changes in cell shape or growth rate, when cells are freed from three-dimensional restirctions imposed by the tissue of origin     

Identification and characterization of monolayer cultures of sheep trophoblast cells maintained in bicameral culture chambers

Enriched epithelial cell and fibroblast fractions were isolated from ovine placentomes by isopycnic centrifugation of collagenase/DNAse-dispersed cells through a density gradient of 45% Percoll. The epithelial cells formed confluent monolayers when plated onto filters impregnated with a 50-microns layer of Matrigel in medium containing 10% fetal bovine serum. These cells were maintained in dual environment culture chambers in serum-free medium for at least 12 days. The epithelium had a polarized appearance similar to that found in vivo only when cells were plated at high density (10(7)/cells/cm2). The epithelial monolayer consisted predominantly of a single population of uninucleate cells with intracellular features similar to those previously described for ovine trophoblast both in vivo and in vitro. These cells stained positively with an antiserum to alpha-keratin, a marker specific to epithelial cells, and no staining was observed with antisera raised against binucleate cells or leucocyte-common antigen. Binucleate cells were detected by microscopy and immunostaining in the pellet of cells obtained from the Percoll gradient but were rarely seen in the epithelium. The epithelial monolayer excluded 3H-inulin, added to the basal chamber, from the apical chamber, thus demonstrating the formation of a permeability barrier similar to that found in vivo. The maintenance of a monolayer of pure ovine trophoblast cells in vitro, which retain the characteristics of the epithelium in vivo, will enable the study of many cellular functions of the trophoblast.     

Analysis of pancreatic development using a cell lineage label

We have devised a new culture system for in vitro culture of pancreatic buds from mouse embryos which enables the organ to grow as a flat branched structure suitable for wholemount immunostaining. This system has been used to analyze pancreatic development. We have also used the ROSA-26 gene trap mouse strain as a source of tissue which expresses lacZ in a stable manner, in all cell types, during in vitro culture. Combinations of lacZ epithelium and unlabeled mesenchyme show that both exocrine and endocrine cells arise from the epithelium, and smooth muscle cells from the mesenchyme. Although previously suspected, this is the first formal proof that both exocrine and endocrine cells are of endodermal origin. Combinations of lacZ epithelium with unlabeled stomach mesenchyme give similar results and show that stomach mesenchyme has the same trophic effect as pancreatic mesenchyme. When a lacZ and an unlabeled epithelium are combined with an unlabeled mesenchyme, both acini and islets in the resulting culture can be of mixed cell composition. This shows that neither of the chief structural units of the pancreas is formed by clonal growth from a single cell.     

Keratinocyte differentiation of human buccal mucosa in vitro

The process of differentiation in keratinocytes of human buccal mucosa is accompanied by a number of specific membrane and cytoplasmic changes. Using simple tissue culture techniques, it has been possible to establish an in vitro model for keratinocyte differentiation which closely resembles the in vivo situation. A multi-layered structure is formed with maturation towards the surface. The superficial cells are characterized by a thickening of the plasma membrane, an increased concentration of tonofilaments, loss of intercellular adhesions and the presence of membrane-coated granules. It is concluded that this represents a suitable in vitro model for the biological and pathological study of both normal and abnormal oral epithelium.     

Human vaginal epithelial multilayer tissue culture

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

维胺酸对体外转化人支气管上皮细胞及体内构建人支气管上皮组织的抑制作用

癌前改变是肿瘤演变过程中的关键阶段。许多研究显示维甲类化合物对动物肿瘤及体外恶性细胞系具有抑制作用,但尚未见其对肺癌前病变作用的实验室研究报道。人类肺癌的绝大部分起源于支气管上皮,为研究维胺酸对体外转化人支气管上皮M细胞系以及在大鼠气管构建后移植到裸鼠体内生长的具有癌前病变特点的人支气管上皮组织的抑制作用,采用上皮细胞无血清培养技术,人支气管上皮组织大鼠气管内构建／裸鼠皮下移植生长技术,流式细胞学分析,免疫组化、凋亡细胞原位末端标记以及病理学检查等研究方法发现,维胺酸可抑制体外培养的转化人支气管上皮细胞的增殖,使S期细胞比例下降,以及细胞增殖标志Ki-67、mpm-2阳性反应细胞比例下降;明显诱导细胞凋亡。裸鼠腹腔注射给予维胺酸也可使大鼠气管内构建后移植到裸鼠体内生长的癌前期人支气管上皮组织的生长率明显降低,病变程度明显减轻;同样可以诱导细胞凋亡。研究结果提示,维胺酸对体外培养的转化人支气管上皮细胞系及大鼠气管构建／裸鼠体内移植生长的人支气管上皮组织均有明显的抑制作用,是有希望的肺癌化学预防药物。     

Outgrowth in vitro of cervical epithelium separated from the uterovaginal anlage of newborn mice

Summary After gentle trypsinization, the pseudostratified columnar Müllerian epithelium that lines the uterine cervix of newborn mice could be separated from the enclosing stromal tissue. Pure epithelial tubes explanted in vitro and were allowed to grow in a standard medium for 3–4 days forming a confluent colony of rather closely-fitting cells. The cell sheet was studied by a preparatory technique that allows examination of a large number of cells with preserved intercellular spatial orientation. Attempts were made to identify cultured cells according to the morphology of cell types in the cervicovaginal epithelium in vivo.Electron micrographs revealed that, close to the explant, the cultured cell sheet exhibited several features similar to the Müllerian epithelium in vivo. Outside these central areas of the colony was a broad transitional zone consisting of thin platelike cells distinguished by an abundance of microfilaments. At the periphery of the colonies, bulky cells possessing microvilli and a vacuolated cytoplasm tended to overlap adjoining platelike cells. These bulky cells had a morphology resembling that of the superficial cells seen in the upper vagina and common cervical canal of immature and diestrous animals. The epithelial development in the cultures apparently simulated the transformation in vivo from a pseudostratified Müllerian epithelium in the newborn to a stratified epithelium resembling that of the uppermost vagina and common cervical canal of immature animals. Judged by morphological and cytochemical criteria, the Müllerian cells in the outgrowth obviously had many changed features. It thus seems questionable whether the cells grown in vitro are comparable with the corresponding cells in vivo when used for experiments requiring the controlled conditions of the culture environment.Supported by grants from the Norwegian Research Council for Science and the Humanities and from the Norwegian Cancer Society     

Pancreatic tissue formation from murine embryonic stem cells in vitro

The in vitro formation of organs and/or tissues is a major goal for regenerative medicine that would also provide a powerful tool for analyzing both the mechanisms of development and disease processes for each target organ. Here, we present a method whereby pancreatic tissues can be formed in vitro from mouse embryonic stem (ES) cells. Embryoid body-like spheres (EBSs) induced from ES cell colonies were treated with retinoic acid (RA) and activin, which are candidate regulators of pancreatic development in vivo. These induced tissues had decreased expression of the sonic hedgehog (shh) gene and expressed several pancreatic marker genes. ES cell-derived pancreatic tissue was composed of exocrine cells, endocrine cells, and pancreatic duct-like structures. In addition, the ratio of exocrine to endocrine cells in the induced tissue was found to be sensitive to the concentrations of RA and activin in the present experiment.     

Successful transplantation of three tissue-engineered cell types using capsule induction technique and fibrin glue as a delivery vehicle

Recent advances in cell biology and tissue engineering have used various delivery vehicles for transplanting varying cell cultures with limited success. These techniques are frequently complicated by tissue necrosis, infection, and resorption. The purpose of this study was to investigate whether urothelium cells, tracheal epithelial cells, and preadipocytes cultured in vitro could be successfully transplanted onto a prefabricated capsule surface by using fibrin glue as a delivery vehicle, with the ultimate goal for use in reconstruction. In the first step of the animal study, tissue specimens (bladder urothelium, tracheal epithelial cells, epididymal fat pad) were harvested for in vitro cell culturing, and a silicone block was implanted subcutaneously or within the anterior rectus sheath to induce capsule formation. After 6 to 10 days, when primary cultures were confluent, the animals were re-anesthetized, the newly formed capsule pouches were incised, and the suspensions of cultured urothelia cells (n = 40), tracheal epithelial cells (n = 32), and preadipocytes (n = 40) were implanted onto the capsule surface in two groups, one using standard culture medium as a delivery vehicle and the second using fibrin glue. Histologic sections were taken, and different histomorphologic studies were performed according to tissue type. Consistently in all animals, a highly vascularized capsule was induced by the silicon material. In all animals in which the authors used fibrin glue as a delivery vehicle, they could demonstrate a successful reimplantation of cultured urothelium cells, tracheal epithelial cells, or preadipocytes. Their animal studies showed that capsule induction in combination with fibrin glue as a delivery vehicle is a successful model for transplantation of different in vivo cultured tissue types.     

Distinction between the G0 and the late G1 phase of rat cells in vivo and in vitro with the nuclear QDH-fluorescence pattern

The quinacrine dihydrochloride (QDH) staining and the [3H]thymidine incorporation patterns were simultaneously analyzed in nuclei of rat cells from a proliferating (granulation tissue) and a nonproliferating tissue (liver). Nuclei from freshly isolated and cultured cells of the rapidly proliferating subcutaneous granulation tissue showed a cell cycle-related pattern similar to that previously described with growing fibroblast-like cells in vitro. Nuclei of liver cells in smears from biopsies and in histological sections showed a fluorescence pattern similar to that of serum-deprived arrested G0 cells from established cell lines. Treatment of primary cultured rat hepatocytes with phenobarbital altered their degree of chromatin condensation similar to that seen after treatment of rats in vivo. The data indicate that the QDH staining pattern is an early marker, suitable for detecting the cell cycle-promoting activity of chemicals (e.g., of tumor promoters) in nonproliferating cells from various tissues in vivo and in vitro.     

Cell Kinetic Studies Fail to Identify Sequentially Proliferating Progenitors as the Major Source of Epithelial Renewal in the Adult Murine Prostate

There is evidence that stem cells and their progeny play a role in the development of the prostate. Although stem cells are also considered to give rise to differentiated progeny in the adult prostate epithelium ex vivo, the cohort of adult prostate stem cells in vivo as well as the mechanisms by which the adult prostate epithelium is maintained and regenerated remain highly controversial. We have attempted to resolve this conundrum by performing in vivo tracing of serially replicating cells after the sequential administration of two thymidine analogues to mice. Our results show that, during normal prostate homeostasis, sequentially proliferating cells are detected at a rate that is consistent with a stochastic process. These findings indicate that in vivo, under steady-state conditions, most adult prostate epithelial cells do not represent the progeny of a small number of specialized progenitors that generate sequentially replicating transit-amplifying (TA) cells but are formed by stochastic cell division. Similarly, no rapidly cycling TA cells were detected during regeneration following one cycle of androgen-mediated involution/regeneration of the prostate epithelium. These findings greatly enhance our understanding of the mechanisms regulating prostate epithelial cell renewal and may have significant implications in defining the cell of origin of proliferative prostatic diseases.     

Cells from Early Chick Embryos in Culture

Just prior to streak formation (Stage XIII) the two layered chick blastoderm is formed by the one layer epiblast which needs the influence of the hypoblastic layer to develop an embryonic axis. A study has been made of this latest possible stage in the development of the chick in which one cell population, the epiblast, is still totipotential. The intention being to examine in particular the differentiation capacities of these cells in culture and at the same time to compare them with hypoblastic cells. In studying differentiation we have attempted to minimize heterogeneity of the starting cell population by culturing either hypoblastic cells or epiblastic cells. The epiblastic cells were derived from epiblasts deprived of the marginal zone and of the area opaca. Hypoblastic cells formed a one cell thick characteristic epithelium. Epiblastic cells in culture were found to evolve from a homogenous sheet to clearly demarcated areas to dome structures which resemble embryoid bodies from teratocarcinomas. Histologically three main tissue types were found in the epiblastic cultures. Sometimes the borderline between two of the tissue types was found to be clearly demarcated by a basement membrane. Both hypoblastic and epiblastic cells produced a basement membrane-like structure when cultured in vitro. The appearance of mesoderm in the epiblastic cultures was particularly interesting and it was evident by the appearance of blood islands and clearly defined endothelial-lined cavities. No complex organized embryonic structures of any kind were found in the cultures.     

Endocrine differentiation by a human rectal adenocarcinoma cell line (HRA-19)

Colorectal epithelium is composed of a variety of cell types, including absorptive, mucous and endocrine cells. All of these cell types are thought to arise from stem cells located at the base of the crypt. However, the factors which control these differentiation pathways are poorly understood. In attempts to establish differentiated in vitro systems, one approach has been to grow primary human colorectal adenocarcinomas as cell lines. Some of these cell lines retain a sufficient number of the differentiated features of their tissue of origin to make them useful experimental systems for studying differentiation. This study describes the characterisation of such a cell line, the HRA-19 line. HRA-19 cells were derived from a primary human rectal adenocarcinoma. The cells grew as monolayers in vitro on tissue-culture plastic and remained pleomorphic even after 150 passages in vitro. Some colonies of cells expressed alkaline phosphatase activity, an enzyme normally expressed in vivo by absorptive cells of the upper crypt and surface epithelium. No evidence of differentiation into goblet or endocrine cells was obtained in monolayer cultures of HRA-19 cells. Xenografts of this cell line contained cells with the ultrastructural characteristics of absorptive and endocrine cells. These endocrine cells exhibited Grimelius silver staining, displayed formaldehyde-induced fluorescence and contained many basally located, electron-dense granules. When grown as monolayers, clones of this cell line retained the heterogeneity with respect to morphology and alkaline phosphatase expression of the parent cell line. It is proposed that this cell line is derived from malignant progenitor cells which retain the ability to differentiate.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Hypoxia inducible factor 1-alpha (HIF-1 alpha) is induced during reperfusion after renal ischemia and is critical for proximal tubule cell survival

Acute tubular necrosis (ATN) caused by ischemia/reperfusion (I/R) during renal transplantation delays allograft function. Identification of factors that mediate protection and/or epithelium recovery could help to improve graft outcome. We studied the expression, regulation and role of hypoxia inducible factor 1-alpha (HIF-1 α), using in vitro and in vivo experimental models of I/R as well as human post-transplant renal biopsies. We found that HIF-1 α is stabilized in proximal tubule cells during ischemia and unexpectedly in late reperfusion, when oxygen tension is normal. Both inductions lead to gene expression in vitro and in vivo. In vitro interference of HIF-1 α promoted cell death and in vivo interference exacerbated tissue damage and renal dysfunction. In pos-transplant human biopsies, HIF-1 α was expressed only in proximal tubules which exhibited normal renal structure with a significant negative correlation with ATN grade. In summary, using experimental models and human biopsies, we identified a novel HIF-1 α induction during reperfusion with a potential critical role in renal transplant.     

Establishment of esophageal-like non-keratinized stratified epithelium using normal human bronchial epithelial cells

Current experimental models of esophageal epithelium in vitro suffer from either poor differentiation or complicated culture systems. We have established a model to study stratified squamous epithelium in vitro, which is very similar to esophageal epithelium in vivo. A stratified squamous multilayer epithelium was formed by seeding primary normal human bronchial epithelial (NHBE) cells onto collagen- and fibronectin-coated trans-well inserts and then cultivating the cells under air-liquid interface (ALI) conditions in the presence of growth factors and low levels of all-trans-retinoic acid. Trans-epithelial electrical resistance (TEER) measurements revealed the presence of a tight barrier, previously only achievable with esophageal biopsies mounted in Ussing chambers. Molecular markers for desmosomes, cornified envelope, tight junctions, and mature esophageal epithelium were upregulated in the differentiating culture in parallel with functional properties, such as decreased permeability and acid resistance and restoration. Acid exposure resulted in a decrease in TEER, but following 1-h recovery the TEER values were fully restored. Treatment with all-trans-retinoic acid decreased TEER and inhibited the recovery after acid challenge. PPAR-delta agonist treatment increased TEER, and this temporary increase in TEER was consistent with an increase in involucrin mRNA. Global gene expression analysis showed that ALI-differentiated NHBE cells had expression profiles more similar to epithelial biopsies from the esophageal tissue of healthy volunteers than to any other cell line. With respect to morphology, molecular markers, barrier properties, and acid resistance, this model presents a new way to investigate barrier properties and the possible effects of different agents on human esophagus-like epithelium.     

Intestinal subepithelial myofibroblasts support in vitro and in vivo growth of human small intestinal epithelium

The intestinal crypt-niche interaction is thought to be essential to the function, maintenance, and proliferation of progenitor stem cells found at the bases of intestinal crypts. These stem cells are constantly renewing the intestinal epithelium by sending differentiated cells from the base of the crypts of Lieberkühn to the villus tips where they slough off into the intestinal lumen. The intestinal niche consists of various cell types, extracellular matrix, and growth factors and surrounds the intestinal progenitor cells. There have recently been advances in the understanding of the interactions that regulate the behavior of the intestinal epithelium and there is great interest in methods for isolating and expanding viable intestinal epithelium. However, there is no method to maintain primary human small intestinal epithelium in culture over a prolonged period of time. Similarly no method has been published that describes isolation and support of human intestinal epithelium in an in vivo model. We describe a technique to isolate and maintain human small intestinal epithelium in vitro from surgical specimens. We also describe a novel method to maintain human intestinal epithelium subcutaneously in a mouse model for a prolonged period of time. Our methods require various growth factors and the intimate interaction between intestinal sub-epithelial myofibroblasts (ISEMFs) and the intestinal epithelial cells to support the epithelial in vitro and in vivo growth. Absence of these myofibroblasts precluded successful maintenance of epithelial cell formation and proliferation beyond just a few days, even in the presence of supportive growth factors. We believe that the methods described here can be used to explore the molecular basis of human intestinal stem cell support, maintenance, and growth.