Hemidesmosome formation by embryonic chick corneal epithelium in vitro

This study was undertaken in order to determine whether 15-day embryonic chick corneal epithelial cells can form hemidesmosomes when cultured on a variety of substrata. It was found that hemidesmosomes were formed on gelatin films, hydrated collagen gels, lens capsule, scraped corneal stroma, matrix produced by corneal endothelial cells and untreated tissue culture plastic. Hemidesmosomes were found after 5 days in cultures produced from either dissociated epithelial cells or whole epithelial explants. Hemidesmosomes occurred both singly and in groups and their morphology varied between well-defined structures with attachment plaques, sub-basal dense plates and connections to intracellular filamentous networks, and more rudimentary forms. The presence of extracellular material was often associated with the hemidesmosomes, although it was also possible to find hemidesmosomes where this material was absent. This work suggests that, in the embryonic chick cornea, extracellular structures such as anchoring filaments and anchoring fibres often associated with mature hemidesmosomes are not essential for hemidesmosome formation.     

Obplacental giant cells of the domestic rabbit: development, morphology, and intermediate filament composition

Obplacental giant cells are large (less than or equal to 210 microns) polyploid cells that appear in the stroma of the pregnant uterus of the rabbit following ovoimplantation. Histological examination of a complete developmental series indicates that obplacental giant cells arise from trophoblastic knobs that have traversed the uterine epithelium during early implantation. During maturation, the cells undergo a massive (approximately 6,000%) increase in volume and penetrate deeply into the uterine stroma and myometrium, where they often become associated with blood vessels and smooth muscle cells. Giant cells at mid-gestation contain one or two large nuclei with prominent nucleoli and appear to be amitotic. They are rich in Golgi complexes, RER, SER, and cortically distributed cytoplasmic filaments, and contain intracellular canaliculi lined by microvilli. Giant cells vary with respect to the occurrence of lipid droplets, phagocytotic inclusions, lysosomal structures, and electron-dense granules. Immunocytochemistry demonstrates that the giant cells exhibit intermediate filaments related to cytokeratin and vimentin, but are negative for desmin and for an endothelial cell marker, Factor VIII-related antigen. The cells are positive for cytokeratin from their inception, but only become vimentin-positive between Days 12 and 15 of pregnancy, a change seemingly related to their detachment from epithelial tissue to take on an independent existence. Our findings indicate that the giant cells originate from obplacental trophoblast and, at maturity, exhibit cytoskeletal characteristics of isolated epithelial cells, as well as a complement of organelles suggestive of synthetic activity.     

Collagen family of proteins

Collagen molecules are structural macro-molecules of the extracellular matrix that include in their structure one or several domains that have a characteristic triple helical conformation. They have been classified by types that define distinct sets of polypeptide chains that can form homo- and heterotrimeric assemblies. All the collagen molecules participate in supramolecular aggregates that are stabilized in part by interactions between triple helical domains. Fourteen collagen types have been defined so far. They form a wide range of structures. Most notable are 1) fibrils that are found in most connective tissues and are made by alloys of fibrillar collagens (types I, II, III, V, and XI) and 2) sheets constituting basement membranes (type IV collagen), Descemet's membrane (type VIII collagen), worm cuticle, and organic exoskeleton of sponges. Other collagens, present in smaller quantities in tissues, play the role of connecting elements between these major structures and other tissue components. The fibril-associated collagens with interrupted triple helices (FACITs) (types IX, XII, and XIV) appear to connect fibrils to other matrix elements. Type VII collagen assemble into anchoring fibrils that bind epithelial basement membranes and entrap collagen fibrils from the underlying stroma to glue the two structures together. Type VI collagen forms thin-beaded filaments that may interact with fibrils and cells.     

Development of Foreign Mammary Epithelial Morphology in the Stroma of Immunodeficient Mice

Systemic growth and branching stimuli, and appropriate interactions with the host stroma are essential for the development of foreign epithelia in the mammary gland of immunodeficient mice. These factors were manipulated to promote and investigate the generation of representative bovine epithelial morphology in the transplanted mouse mammary stroma. The bovine mammary epithelium is unique in its commitment to rapid proliferation and high rate of differentiation. Its morphological organization within a fibrotic stroma resembles that of the human breast, and differs significantly from the rudimentary ductal network that penetrates a fatty stroma in mice. Transplantation of bovine mammary epithelial cells into the cleared mammary fat pad of NOD-SCID mice led to continuous growth of epithelial structures. Multilayered hollow spheres developed within fibrotic areas, but in contrast to mice, no epithelial organization was formed between adipocytes. The multilayered spheres shared characteristics with the heifer gland’s epithelium, including lumen size, cell proliferation, cytokeratin orientation, estrogen/progesterone receptor expression and localization, and milk protein synthesis. However, they did not extend into the mouse fat pad via ductal morphology. Pre-transplantation of fibroblasts increased the number of spheres, but did not promote extension of bovine morphology. The bovine cells preserved their fate and rarely participated in chimeric mouse–bovine outgrowths. Nevertheless, a single case of terminal ductal lobuloalveolar unit (TDLU) development was recorded in mice treated with estrogen and progesterone, implying the feasibility of this representative bovine morphology’s development. In vitro extension of these studies revealed paracrine inhibition of bovine epithelial mammosphere development by adipocytes, which was also generalized to breast epithelial mammosphere formation. The rescue of mammosphere development by fibroblast growth factor administration evidences an active equilibrium between inhibitory and supportive effects exerted by the adipose and fibrotic regions of the stroma, respectively, which determines the development of foreign epithelium.     

Ultrastructures of rabbit corneal stroma: Mapping of optical and morphological anisotropies

Pole figures of optic and morphological structures of rabbit corneal stroma have been determined. The birefringence of stroma is non-uniform, but tends to increase in the directions of nose to ear and of periphery to the vertex. There is no obvious symmetry in the local optic axes. Its direction changes from limbus to limbus. Small angle laser scattering showed stroma to have sheaf-like morphology. This texture is consistent with bundles of collagen fibrils which divide and anastomose. The average size of the scattering entities in rabbit corneal stroma is 19–23 μm with sector angles β ranging from 1 to 15°.     

Macrophage and dendritic cell infiltration in head and neck squamous-cell carcinoma; an immunohistochemical study

A study was undertaken to help us reach a better understanding of the tumor-infiltrating pattern of lymphoid cells and in particular of monocyte-derived cells, namely the CD68⁺, acid-phosphatase-expressing scavenger macrophages and the MHC-class-II- and S100-antigen-presenting dendritic cells in head and neck squamous-cell carcinoma. In the stroma of the tumors distinctive small fields of lymphocytes were found, the T cell areas of these fields being intermingled with dendritic cells. Intra-epithelial dendritic cell infiltration was low. The infiltrative pattern of macrophages was similar to patterns described in earlier studies with substantial stromal invasion and inconsistent intra-epithelial invasion, but small granuloma-like structures of CD68⁺ macrophage-like cells, found in the stroma of tumors, have not been reported before. The histochemical localization of the tumor-infiltrated dendritic cells and macrophages supports the view that the former cells are involved in the sensitization to tumor antigens, whereas the latter cells are involved in tumor cytotoxicity/scavenging of tumor cell debris. Although it has been shown in the past that transmembranal (TM) factors (p15E-like factors) present in the serum and tumor of patients with cancer of the head and neck have suppressive effects on monocyte/macrophage/dendritic cell function, a relationship between the intensity of epithelial staining for TM factors and the infiltrative pattern of monocytes/macrophages/dendritic cells could not be demonstrated.     

An epithelial precursor is regulated by the ureteric bud and by the renal stroma

Kidney epithelia develop from the metanephric mesenchyme after receiving inductive signals from the ureteric bud and from the renal stroma. However, it is not clear how these signals induce the different types of epithelia that make up the nephron. To investigate inductive signaling, we have isolated clusters of epithelial progenitors from the metanephric mesenchyme, thereby separating them from the renal stroma. When the isolated progenitors were treated with the ureteric bud factor LIF, they expressed epithelial proteins (ZO-1, E-cadherin, laminin alpha(5)) and produced nephrons (36 glomeruli with 58 tubules), indicating that they are the target of inductive signaling from the ureteric bud, and that renal stroma is not absolutely required for epithelial development in vitro. In fact, stroma-depleted epithelial progenitors produced sevenfold more glomeruli than did intact metanephric mesenchyme (5 glomeruli, 127 tubules). Conversely, when epithelial progenitors were treated with both LIF and proteins secreted from a renal stromal cell line, glomerulogenesis was abolished but tubular epithelia were expanded (0 glomeruli, 47 tubules). Hence, by isolating epithelial progenitors from the metanephric mesenchyme, we show that they are targeted by factors from the ureteric bud and from the renal stroma, and that epithelial diversification is stimulated by the ureteric bud and limited by renal stroma.     

Airway branching morphogenesis in three dimensional culture

Background

Lungs develop from the fetal digestive tract where epithelium invades the vascular rich stroma in a process called branching morphogenesis. In organogenesis, endothelial cells have been shown to be important for morphogenesis and the maintenance of organ structure. The aim of this study was to recapitulate human lung morphogenesis in vitro by establishing a three dimensional (3D) co-culture model where lung epithelial cells were cultured in endothelial-rich stroma.

Methods

We used a human bronchial epithelial cell line (VA10) recently developed in our laboratory. This cell line cell line maintains a predominant basal cell phenotype, expressing p63 and other basal markers such as cytokeratin-5 and -14. Here, we cultured VA10 with human umbilical vein endothelial cells (HUVECs), to mimic the close interaction between these cell types during lung development. Morphogenesis and differentiation was monitored by phase contrast microscopy, immunostainings and confocal imaging.

Results

We found that in co-culture with endothelial cells, the VA10 cells generated bronchioalveolar like structures, suggesting that lung epithelial branching is facilitated by the presence of endothelial cells. The VA10 derived epithelial structures display various complex patterns of branching and show partial alveolar type-II differentiation with pro-Surfactant-C expression. The epithelial origin of the branching VA10 colonies was confirmed by immunostaining. These bronchioalveolar-like structures were polarized with respect to integrin expression at the cell-matrix interface. The endothelial-induced branching was mediated by soluble factors. Furthermore, fibroblast growth factor receptor-2 (FGFR-2) and sprouty-2 were expressed at the growing tips of the branching structures and the branching was inhibited by the FGFR-small molecule inhibitor SU5402.

Discussion

In this study we show that a human lung epithelial cell line can be induced by endothelial cells to form branching bronchioalveolar-like structures in 3-D culture. This novel model of human airway morphogenesis can be used to study critical events in human lung development and suggests a supportive role for the endothelium in promoting branching of airway epithelium.     

Occurrence of a microcanalicular system within the ossicles and dermal tissue of Asterias rubens and Marthasterias glacialis (Echinodermata: Asteroidea)

Summary A microcanalicular network is demonstrated within the ossicle stroma and the dermal tissue of two asteroid species. Microcanaliculi are presumed to be mesodermal structures. They consist of convoluted tubular ducts lined by epithelial cells associated with scattered basiepithelial nervous processes. Such a microcanalicular system has not been reported previously from any echinoderm species. Its discovery in asteroids entails some conceptual changes, especially considering the physiology of the body wall.Research assistants of the National Fund for Scientific Research (NFSR, Belgium)     

Synthesis of basement membrane proteins by rat mammary epithelial cells

A mammary epithelial cell line, Rama 25, growing on plastic, deposits fibronectin, type IV collagen, and laminin in punctate structures located beneath the basal surface of the cells. When grown on the surface of collagen gels, Rama 25 cells deposit these basement membrane proteins in a continuous layer between the basal surface of the cells and the surface of the collagen matrix. Rama 25 cells also penetrate the collagen matrix forming rudimentary duct-like structures. These structures are surrounded by a discontinuous layer of basement membrane proteins. The ducts of fetal and neonatal rat mammary glands contain few mature myoepithelial cells and our results suggest that some mammary epithelial cells, in contact with a collagenous stroma, are capable of synthesizing a basal lamina-like structure.     

Prostate epithelial differentiation is dictated by its surrounding stroma

Prostate epithelial differentiation is dictated by its surrounding stroma which determines androgen induced growth responsiveness and expression of specific secretory proteins in normal prostate gland. During neoplastic progression, organ specific stroma has been shown to determine the rate of neoplastic progression from androgen-dependent to androgen-independent and metastatic states. Although growth factors and extracellular matrix are recognized as important contributors to prostate epithelial growth, hormonal responsiveness, and neoplastic progression, the exact mechanism of intercellular communication between stromal and epithelial cells remains undefined. In addition to the importance of defining the reciprocal interaction between stromal and epithelial interaction in the prostate, clonal interaction between two dissimilar prostate epithelial cells is also recognized to contribute to disease progression. In this review, we summarized recent advances made in delineating molecular mechanisms underlying stromal epithelial interaction and clonal interaction between androgen-dependent and androgen-independent prostate cancer cells in vivo and in culture. Understanding cellular interaction between prostate epithelium and its surrounding stroma could help us in developing metastatic models of prostate carcinogenesis. This concept will allow us to define epithelial-specific markers, markers induced as the result of stromal-epithelial interaction, and stroma-associated markers. These markers together will assist us in diagnosing, preventing, prognosing and treating prostate cancer more efficaciously in the future.     

The selection of thin epithelial layers: a method for single cell preparations of very small biopsies from the vocal cords

Precancerous lesions in biopsy material very rarely contain more than a thin epithelial layer of atypical cells overlaying stromal parts. This makes it difficult to produce cell monolayers for cytometry from archival material and necessitates a technique which excludes the stromal areas. We have developed a method by which it is possible to separate epithelial layers from the stroma with a thickness of 100-200 microns, using fluorescence microscopy and dissection under a stereo microscope. The stroma can be used as an internal reference in IOD measurements. Our method insures maximum control when dealing with very small lesions of interest (down to 0.02 mm2) that have to be separated from the rest of the biopsy. The method has been applied to normal, dysplastic and cancerous epithelial parts of biopsies of the vocal chords.     

In vivo gene manipulations of epithelial cell sheets: a novel model to study epithelial-to-mesenchymal transition

Embryonic cells are classified into two types of cells by their morphology, epithelial and mesenchymal cells. During dynamic morphogenesis in development, epithelial cells often switch to mesenchymal by the process known as epithelial-to-mesenchymal transition (EMT). EMT is a central issue in cancer metastasis where epithelial-derived tumor cells are converted to mesenchymal with high mobility. Although many molecules have been identified to be involved in the EMT mostly by in vitro studies, in vivo model systems have been limited. We here established a novel model with which EMT can be analyzed directly in the living body. By an electroporation technique, we targeted a portion of the lateral plate mesoderm that forms epithelial cell sheets delineating the kidney region, called nephric coelomic epithelium (Neph-CE). Enhanced green fluorescent protein-electroporated Neph-CE retained the epithelial integrity without invading into the underling stroma (mesonephros). The Neph-CE transgenesis further allowed us to explore EMT inducers in vivo, and to find that Ras-Raf and RhoA signals were potent inducers. Live-imaging confocal microscopy revealed that during EMT processes cells started extending cellular protrusions toward the stroma, followed by translocation of their cell bodies. Furthermore, we established a long-term tracing of EMT-induced cells, which were dynamically relocated within the kidney stroma. The Neph-CE-transgenesis will open a way to study cellular and molecular mechanisms underlying EMT directly in actual body.     

Lactoferrin at basal side of mouse mammary epithelium derives in part from stroma cells

Lactoferrin is synthesized by glandular epithelial cells and neutrophils and is also present on both sides of the mammary epithelium. We have studied the origin of lactoferrin detected in the various compartments of mouse mammary tissue. As revealed by immunogold electron microscopy, lactoferrin is present in mammary epithelial cells and in the basal region of the epithelium, associated with connective tissue and stroma cells at all physiological stages studied. A perturbation of protein synthesis or transport after in vitro treatment with cycloheximide or brefeldin A does not abrogate lactoferrin labelling in the basal region of the epithelium. The expression of lactoferrin has also been observed in the fat pads of mammary glands from mice surgically depleted of epithelial cells. The sealing of one teat for 24 h is accompanied by an increase in both the number of stroma cells and the labelling of myoepithelial cells. Thus, the lactoferrin present in the interstitial space of the mouse mammary epithelium originates in part from stroma cells. Possible roles of lactoferrin at the basal side of the mammary epithelium are discussed.     

MHC class II expression and antigen presentation by human endometrial cells

It has been demonstrated previously that mixed cell suspensions from the female reproductive tract consisting of human epithelial and stromal cells were capable of presenting foreign antigen to autologous T cells. There have been, however, no reported studies examining antigen presentation by isolated epithelial cells from the human female reproductive tract. It is now shown that freshly isolated epithelial cells from the uterine endometrium constitutively express MHC class II antigen and that class II was upregulated on cultured epithelium by interferon gamma (IFNγ). Using a highly purified preparation, it was demonstrated that these epithelial cells were able to process and present tetanus toxoid recall antigen driving autologous T cell proliferation. Cells isolated from the basolateral sub-epithelium stroma were also potent antigen presenting cells in this model system. Thus, isolated endometrial epithelial cells were able to directly process and present antigen to T cells and may be responsible for the transcytosis and delivery of antigen to professional antigen presenting cells found in the sub-epithelial stroma.     

Fine needle aspiration cytology and core biopsy histology in infiltrating syringomatous adenoma of the breast. A case report

BACKGROUND: Infiltrating syringomatous adenoma is a rare tumor of the breast that can radiologically mimic invasive duct carcinoma. Detailed fine needle aspiration cytology and needle core biopsy findings on this lesion have not been previously described. CASE: The clinical, radiologic and pathologic findings of an infiltrating syringomatous adenoma of the breast in a 71-year-old female who presented with a subareolar lump are described. The cytology of the tumor was characterized by a combination of a background of plump, fibroblastoid cells and cohesive sheets of bland epithelial cells. Histologically the tumor showed infiltrating, duct-like structures with squamous metaplasia and a desmoplastic stroma. CONCLUSION: Fine needle aspiration cytology and needle core biopsy can distinguish infiltrating syringomatous adenoma from malignant disease of the breast.     

The gap junction-like form of a vacuolar proton channel component appears not to be an artifact of isolation: An immunocytochemical localization study

Gap junctional structures containing a 16-kDa intrinsic membrane protein have been isolated from the hepatopancreas of the crustacean Nephrops norvegicus. These structures are double membranes 14-15 nm thick and composed of hexagonal arrays of particles which have a central pore that is penetrated by a cationic negative stain. Membrane preparations have also been isolated from the hepatopancreas and these contain similar gap junctional regions of uniform width. Affinity purified antibodies to the 16-kDa protein bind principally to these gap junctional regions. Antiserum raised against the isolated gap junctional structures binds strongly to the lateral surfaces of the columnar epithelial cells and in particular to gap junction-like regions.     

Similar growth pattern of mouse mammary epithelium cultivated in collagen matrix in vivo and in vitro

Mouse mammary ductal cells cultured in type I collagen gels give rise to three-dimensional multicellular outgrowths consisting of thin spikes which are often branched, and which may have pointed or blunt ends. The significance of these spikes to normal ductal morphogenesis has been unclear, since identical structures are not known to occur in vivo; conversely, it has not been possible to maintain in gel culture the highly structured end buds which are characteristic of ductal elongation in the animal. In order to evaluate whether the pattern of radiating spikes observed in collagen gel cultures results from chemical or physical peculiarities of the culture environment, a small volume of unpolymerized type I collagen solution was injected into mammary gland-free fat pads of young adult mice. After the bubble of collagen had polymerized, an implant of mammary ductal epithelium was introduced into the center of the gel. Histological examination of the implants after 3 to 6 days of growth revealed numerous small epithelial spikes, similar to those observed in gel culture, extending into the fibrous matrix. The early stages of regeneration of mammary implants placed in gland-free fat pads were then examined without the addition of exogenous collagen. In cases where the epithelium happened to contact a fibrous region of the fatty stroma, spikes were also seen to form in these natural collagenous substrates. Whether or not exogenous collagen was used, normal end buds were formed only when epithelial spikes contacted adipocytes. It was concluded that the three-dimensional pattern of radiating tubules in collagen gels in vitro is not merely an artifact of culture, but has a counterpart in vivo whereever regenerating mammary epithelium is surrounded by fibrous stroma. A model is presented in which the pattern of epithelial outgrowth is determined by the physical characteristics of the surrounding stroma; in collagen matrix a comparatively primitive and unspecialized type of morphogenesis occurs which may not require the participation of stromal cells. In contrast, epithelial-adipocyte interactions appear to be necessary for the formation of end buds and subsequent morphogenesis of fully structured mammary ducts.     

Role of stroma in carcinogenesis of the prostate

Prostatic development is induced by androgens acting via mesenchymal-epithelial interactions. Androgens elicit their morphogenetic effects by acting through androgen receptors (ARs) in urogenital sinus mesenchyme (UGM), which induces prostatic epithelial development. In adulthood reciprocal homeostatic stromal-epithelial interactions maintain functional differentiation and growth-quiescence. Testosterone plus estradiol (T+E2) have been shown to induce prostatic carcinogenesis in animal models. Thus, tissue recombinant studies were undertaken to explore the mechanisms of prostatic carcinogenesis in BPH-1 cells in which ARs and estrogen receptors (ERs) are undetectable. For this purpose, BPH-1 cells were combined with UGM, and the UGM+BPH-1 recombinants were grafted to adult male hosts. Solid branched epithelial cords and ductal structures formed in untreated UGM+BPH-1 recombinants. Growth was modest, and tumors did not develop. UGM+BPH-1 recombinants treated with T+E2 formed invasive carcinomas. BPH-1 cells lack ARs and ERs, whereas rat UGM expresses both of these receptors. These data show that immortalized nontumorigenic human prostatic epithelial cells can undergo hormonal carcinogenesis in response to T+E2 stimulation via paracrine mechanisms and demonstrate that the stromal environment plays an important role in mediating hormonal carcinogenesis. During prostatic carcinogenesis the stroma undergoes progressive loss of smooth muscle with the appearance of carcinoma-associated fibroblasts (CAF). This altered stroma was tested for its ability to promote carcinogenesis of nontumorigenic but immortalized human prostatic epithelial cells (BPH-1). CAF+BPH-1 tissue recombinants formed large carcinomas. In contrast, recombinants composed of normal prostatic stroma+BPH-1 cells exhibited minimal growth. This stroma-induced malignant transformation was associated with additional genetic alterations and changes in gene expression. Thus, alteration in the stromal microenvironment was sufficient to promote malignant transformation of human prostatic epithelial cells.