Quiescent epithelial cell rests of Malassez can differentiate into ameloblast-like cells

Epithelial cell rests of Malassez (ERM) are quiescent epithelial remnants of Hertwig's epithelial root sheath (HERS) that are involved in the formation of tooth roots. After completion of crown formation, HERS are converted from cervical loop cells, which have the potential to generate enamel for tooth crown formation. Cervical loop cells have the potential to differentiate into ameloblasts. Generally, no new ameloblasts can be generated from HERS, however this study demonstrated that subcultured ERM can differentiate into ameloblast-like cells and generate enamel-like tissues in combination with dental pulp cells at the crown formation stage. Porcine ERM were obtained from periodontal ligament tissue by explant culture and were subcultured with non-serum medium. Thereafter, subcultured ERM were expanded on 3T3-J2 feeder cell layers until the tenth passage. The in vitro mRNA expression pattern of the subcultured ERM after four passages was found to be different from that of enamel organ epithelial cells and oral gingival epithelial cells after the fourth passage using the same expansion technique. When subcultured ERM were combined with subcultured dental pulp cells, ERM expressed cytokeratin14 and amelogenin proteins in vitro. In addition, subcultured ERM combined with primary dental pulp cells seeded onto scaffolds showed enamel-like tissues at 8 weeks post-transplantation. Moreover, positive staining for amelogenin was observed in the enamel-like tissues, indicating the presence of well-developed ameloblasts in the implants. These results suggest that ERM can differentiate into ameloblast-like cells.     

Effect of the extracellular matrix molecules fibronectin and laminin on the adhesion and growth of primary renal cortical epithelial cells

Primary tubular epithelial cells were isolated from renal cortex following enzymatic dissociation with collagenase. These cells were then grown in chemically defined media containing insulin, transferrin, selenium, tri-iodothyronine and either fibronectin or laminin. The tubular epithelial cells were studied ultrastructurally and compared to another epithelial cell type present in the renal cortex, the glomerular epithelial cell. In contrast to the constant morphology of glomerular epithelial cells grown in chemically defined media, tubular epithelial cell morphology depended on whether the cells were placed in fibronectin or laminin and on the age of the donor animal used for culture. Primary tubular cells grown in laminin formed colonies; cells grown from young animals were rounded, whereas cells grown from adult animals were flattened. Primary tubular cells grown in fibronectin were flattened regardless of age, but cells from young animals formed colonies while those from adult animals formed a monolayer. Despite these differences in gross morphology, scanning and transmission electron microscopy revealed similar ultrastructural features in primary tubular cells from young and adult animals grown in fibronectin or laminin. Quantitative adhesion studies demonstrated that secondary subcultured tubular cells adhered equally well to dimeric and multimeric forms of fibronectin, but not to laminin. Quantitative colony growth studies of subcultured secondary tubular cells showed that laminin supports colony formation of trypsinized tubular cells, while previous work has demonstrated that fibronectin supports colony formation of glomerular cells. These results are consistent with the hypothesis that different extracellular matrix molecules are involved in colony formation of different cell types, with fibronectin stimulating growth of glomerular cells and laminin supporting growth of tubular cells.     

Isolation, cell culture and immunocytochemical characterization of oviduct epithelial cells of the cow

Incubation of cow oviducts flushed with 0.1 mg collagenase/ml, for 90 min helped to dislodge large numbers of ciliated and secretory cells. About 90-95% of the isolated epithelial cells were viable. The epithelial cells suspended in DMEM:F-12 + 10% serum attached to the plastic culture dish in 18-20 h after seeding. The ciliated cells which attached to the plastic dish lost their cilia after 4-5 days in culture. The attached cells, which proliferated to form a confluent monolayer 8-10 days after seeding in a 35-mm dish, could be subcultured at least 3 successive times. Some cell aggregates which did not attach to the culture dish proliferated into floating balls of cells. The ciliated cells in the unattached floating colonies maintained the ciliary movement for 9-10 days in the same culture medium. The primary cultures of the ciliated and the secretory cells maintained most of the histoarchitecture observed in intact epithelium. The secretory cells maintained their secretory activity of specific proteins in culture as indicated by immunocytology. The cultured cells contained keratin, a specific cytoskeletal component of epithelial cells.     

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 .     

Effects of substrata on the polarization of bovine endometrial epithelial cells in vitro

Summary Epithelial-cell function requires cellular polarity in which apical membrane surfaces have unique characteristics and cellular organelles are stratified. Physiological investigations of endometrial, epithelial cells would be enhanced greatly by the ability of a method to polarize cells in culture. This study investigates the effects of different substrata on polarization of cultured bovine endometrial epithelial cells. Fetal bovine endometrial epithelial-cell lines were developed from explant outgrowth. Epithelial monolayers were subcultured onto amniotic membranes, Millicell-HA membranes, or Millicell-CM membranes coated with rat-tail collagen, Matrigel, laminin, Vitrogen,or fibronectin. Cultures on these substrata were maintained at the air/liquid interface. Cells grown on either collagen-coated or uncoated Milli-cell membranes also were maintained submerged in medium. Excellent polarized morphology was attained in cultures grown at the air/liquid interface on amniotic membranes and rat-tail collagen-coated membranes. Lectin-binding patterns, to apical membranes of polarized epithelial cell cultures paralleled patterns of binding to bovine endometrial surfaces in vivo. Cultures on rat-tail collagen were maintained for several weeks. These methods provide a valuable system for studying the endometrium in vitro.     

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.     

Isolation,culture, and characterization of equine oviduct epithelial cells in vitro

Oviduct epithelial cells (OEC) increasingly are used to support embryonic development and to study gamete interactions with the female reproductive tract in vitro. This series of experiments was designed to characterize monolayers derived from oviduct epithelium. Epithelial cells harvested from the isthmus and ampulla of the oviducts of five estrous mares were cultured with or without the basal lamina extract, Matrigel. Within each group OEC were cultured in the presence of either estradiol-17β or a carrier control. All groups were subcultured three times. Epithelial cell morphology and function were examined by microscopy, analysis of secreted proteins, and immunocytochemistry. Epithelial cells attached more rapidly and reached confluence sooner when cultured on Matrigel than in uncoated wells. Cells showed variable evidence of ciliary activity up to 12 days in primary culture. Cells grown on Matrigel had a more polarized appearance in primary culture than those in uncoated wells, although no morphologic difference between anatomic site of origin or between steroid treated groups was noted. Anatomic site of origin had no effect, and steroid treatment had minimal effects, on patterns of secreted proteins. However, some differences were noted in protein secretion between cells grown with or without Matrigel. These data suggest that culture substrate may affect structure and function of OEC monolayers. © 1995 Wiley-Liss, Inc.     

Characterization of Long-Term Cultured Murine Submandibular Gland Epithelial Cells

Purpose

Human and rat salivary gland cell lines derived from tumors or genetic modification are currently available for research. Here, we attempted to culture and characterize long-term cultured cells spontaneously derived from wild type murine submandibular glands (SGs).

Methods

SGs were removed from 3-week-old C57B/6J female mice and dissociated by collagenase type 1 and hyaluronidase digestion. Isolated SG epithelial cells were cultured in low calcium, serum-free growth media in the presence of cholera toxin (CT) during early passages. Single-cell colonies were isolated by limiting dilution culture after 25 passages. Early- and late-stage cell cultures were characterized for keratin 14, keratin 18, α-smooth muscle actin, and p63 by immunostaining and quantitative real-time PCR analysis.

Results

SG epithelial cells cultured in optimized media maintained their proliferative ability and morphology for over 80 passages. Long-term cultured cells expressed keratin 14, keratin 18, and p63, indicative of an epithelial phenotype.

Conclusions

Epithelial cells originating from wild type murine SGs could be cultured for longer periods of time and remain phenotypically similar to ductal basal epithelium.     

Growth of seminal vesicle epithelial cells in serum-free collagen gel culture

Summary Epithelial cells from mouse seminal vesicles were enzymatically dissociated enriched by gradient centrifugation, and maintained in collagen gel cultures with defined (serum-free) media. The epithelial origin of the cells was determined morhologically, immunocytochemically, and biochemically. Cells formed three-dimensional colonies with a lumen in collagen gels. Cell number was increased eight-fold within a 8 to 12-d culture period in a medium supplemented with epidermal growth factor (EGF) (10 ng/ml), insulin (10 μg/ml), transferrin (10 μg/ml), cholera toxin (10 ng/ml), and hydrocortisone (0.1 μg/ml). The cells required eGF and insulin; the growth-promoting effects of these two peptide hormones were optimized by transferrin, cholera toxin, and hydrocortisone. Fetal bovine serum did not support growth; rather, it suppressed the stimulated growth observed in serum-free media. A time-course study revealed that a lag period preceded rapi growth. The collagen gel, serum-free culture provides a powerful tool to study the effects of hormones on proliferation and differentiation of androgen sensitive cells.     

Purification and direct transformation of epithelial progenitor cells from primary human prostate

Epithelial cell transformation has been demonstrated in numerous animal models for the study of solid tumor biology. However, little evidence exists for human epithelial cell transformation without previous immortalization via genetic influences such as SV40 T-antigen, thus limiting our knowledge of the events that can transform naive human epithelium. Here we describe a system developed in our laboratory to directly transform freshly isolated primary human prostate epithelial cells without previous culture or immortalization. Prostate tissue is obtained from patients and benign tissue is separated from malignant tissue. Benign and malignant tissues are mechanically and enzymatically dissociated to single cells overnight, and immune cells and epithelial subsets are isolated on the basis of differential expression of surface antigens. Epithelial progenitor cells are transduced with lentiviruses expressing oncogenes and combined with inductive stroma for in vivo studies. At 8-16 weeks after transplantation into immune-deficient mice, the development of lesions, histologically classified as benign prostate, prostatic intraepithelial neoplasia and adenocarcinoma, can be evaluated.     

In vitro isolation and cultivation of rabbit tracheal epithelial cells using tissue explant technique

Epithelial cells from tracheal mucosa offer significant potential as a cell source in development of tissue-engineered trachea. The purpose of this study was to investigate and optimize a suitable culture system for tracheal epithelial cells, including the methods of primary culture, passage, identification, and cryopreservation. Epithelial cells were isolated from rabbit tracheal mucosa using tissue explant technique and were subjected to immunohistochemistry, immunofluorescence, and cryopreservation after purification. Epithelial cells reached confluency at 14–15 d. Immunohistochemical staining for cytokeratin showed brown yellow-positive cytoplasm and blue-counterstained nuclei, while immunofluorescence staining for cytokeratin showed green-positive cytoplasm and clear cell outline, indicating that the cultured cells had properties of epithelial cells. After recovery, epithelial cells exhibited high survival and viability. The results demonstrated that in vitro isolation and cultivation model was successfully established to provide high proliferative capacity, typical morphology and characteristics of tracheal epithelial cells from trachea mucosa by the use of the tissue explant technique.     

Influence of Donor Age on Establishing Well-Differentiated Clonal Cultures from Embryonic Chicken Retinal Pigmented Epithelium

Retinal pigmented epithelia (RPE) isolated from chicken embryos of various developmental stages were dissociated into single cells, and their ability to re-express defferentiated characteristics in clonal culture was investigated. The lighty pigmented, columnar cells isolated from stage 25 to 29 embryos dissociated more easily than the heavily pigmeted, cuboidal cells from embryos of stages 30 to 34. The yield of RPE cells per embryo increased with donor age, paralleling the growth of the epithelium in vivo . However, the potential these cells to attach, to proliferate, and to form typical, welldifferentiated RPE colonies declined with donor age. Cells from stage 25 embryos developed exclusively into large, typical epithelial colonies which expressed all stages of differentiation from flat, unpigmented cells at the margin to cuboidal, pigmented cells in the centre. At the other end of the spectrum, cells from stage 34 embryos frequently formed small, atypical colonies of unpigmented cells, in addition to typical but relatively small colonies. The plating efficiency (calculated on the basis of pigmented colonies formed within 3 weeks) dropped from more than 2% at stage 25 to 0.01% at stage 34.     

Enrichment and characterization of clonogenic epithelial cells from adult rat liver and initiation of epithelial cell strains

Summary A highly efficient method is described for obtaining prolifertive epithelial cells from adult rat livers for the reproducible establishment of liver epithelial cell strains. When cells were isolated from livers of 10-to 15-wk-old male Fischer 344 rats by a collagenase-perfusion method, collected by centrifugation at 50×g for 5 min, and cultured in Williams' medium E containing fetal bovine serum and dexamethasone, colonies of epithelial cells different in size and morphology from hepatocytes were obtained. Sequential perfusion with collagenase and dispase yielded numerous epithelial cell colonies. When isolated cells were fractionated by differential centrifugation, the great majority of hepatocytes were sedimented at 50 ×g for 1 min, whereas many non-hepatocytic cells remiined in the supernatant and could be sedimented by a second centrifugation at 50×g for 5 min. Culture of the two fractions revealed that almost all the epithelial cell colonies were derived from cells in the non-hepatocytic cell fraction. The epithelial cells were cytochemically negative for γ-glutamyl transpeptidase activity, whereas an increase in the activity was detected in hepatocytes with duration in culture. Ultrastructural characteristics of hepatocytes were not found in the cells of newly established cell strains. These results suggest that adult rat liver epithelial cells propagable in culture were derived from a cell type other than the hepatocyte.     

Growth of normal human mammary cells in culture

Summary Reduction mammoplasty tissue was used to obtain short-term cultures of human epithelial cell populations. Digestion of tissue with collagenase and hyaluronidase resulted in cell clusters (organoids) resembling ductal and alveolar structures; these could be separated by filtration from the stromal components. Epithelial outgrowth from these organoids was greatly enhanced by the addition of conditioned medium from other human epithelial and myoepithelial cell lines. Additionally, the mammary epithelial growth was stimulated by insulin, hydrocortisone, epidermal growth factor, and steroid hormones. With this enriched nutritional environment, active cell division could be maintained for 1 to 3 months and cells could be serially subcultured 1 to 4 times. This research was supported by Grant PDT-72 from the American Cancer Society and Grant CP-70510 from the National Institutes of Health.     

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

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

Eicosanoid release from human bronchial epithelial cells upon exposure to toluene diisocyanate in vitro

Epithelial injury and inflammation are involved in airway hyperresponsiveness and asthma induced by toluene diisocyanate. In that isocyanates are insoluble and highly reactive compounds, bronchial epithelial cells may represent the most important target cells of their toxic effect. We hypothesized that damage to airway epithelium by toluene diisocyanate may result in the release of metabolites of arachidonic acid, which are known to promote inflammation and to alter epithelial cell function and airway smooth muscle responsiveness. To test this hypothesis we examined eicosanoid products in the culture media of bronchial epithelial cells exposed in vitro to 8 and 18 ppb toluene diisocyanate. Epithelial cells derived from human bronchi obtained at surgery were cultured to confluency on collagen-coated microporous membranes. Those cells, which expressed differentiated characteristics of epithelial cells (they showed keratin-containing filaments and had a cobblestone appearance), were alternatively exposed to toluene diisocyanate or air for 30 min in a specially designed in vitro chamber. The production of metabolites of arachidonic acid was assessed by measuring the release of immunoreactive products into the cell medium at the end of the exposure and during a 2 hr period after exposure. This method revealed a predominant isocyanate-induced release of immunoreactive 15-hydroxyeicosatetraenoic acid. Release rate of this compound tended to be dose-related and was associated with cell damage as assessed by the release of lactate dehydrogenase in the medium.     

Growth and differentiation of the cultured secretory cells of the cow oviduct on reconstituted basement membrane

Isolated bovine oviduct epithelial cells were cultured on plastic precoated with matrigel. The epithelial cells seeded on 10 mg/ml matrigel often organized themselves into hollow tubes or spheres with microvilli directed towards the lumen. This is the first report of describing the spontaneous tube formation of oviduct epithelial cells in vitro. The epithelial cells growing on this substratum became fully differentiated with the formation of junctional complexes and the production of secretory vesicles which migrated to apical plasmalemma. Epithelial cells seeded on 2 mg/ml matrigel (dry film) formed a subconfluent monolayer in 15-20 days after seeding. The histoarchitecture of the secretory cells growing on the matrigel dry film closely resembled that of intact epithelial cells. Occasional ciliated cells containing large numbers of mitochondria were observed in cell colonies growing on 2 mg/ml matrigel but they possessed very few intact cilia. The monolayer linearly incorporated 35S-methionine into proteins up to 8 hr in presence of estradiol or progesterone. The fluorography of the newly synthesized proteins indicated that the cell extracts of estradiol-stimulated cells contained an additional protein of approximate molecular weight of 60 kd as compared to the extracts of cells incubated without steroids or incubated with progesterone.     

Restoration of nonclonal hematopoiesis in chronic myelogenous leukemia with interferon alpha

Studies have shown that recombinant human alpha interferon (rIFN alpha) inhibits the growth of colonies of multipotential stem cells from human bone marrow. This report demonstrates that rIFN alpha inhibits the growth of such colonies from the bone marrow of patients with chronic myelogenous leukemia (CML) to a greater extent than from bone marrow of healthy individuals. It also shows that T lymphocyte colonies subcloned with interleukin 2 (IL-2) from CML mixed colonies were inhibited more by rIFN alpha than were similar colonies subcultured from normal mixed colonies. The report demonstrates that the Ph' chromosome is present in such T cell colonies subcultured from CML mixed colonies. When mixed colonies were grown from CML bone marrow in the presence of rIFN alpha, Ph' negative colonies were observed, whereas no such Ph' negative mixed colonies grew from a similar number of bone marrow cells incubated without rIFN alpha. These observations confirm that T lymphocytes derived from bone marrow stem cells are from the CML clone, and that the inhibition of growth of Ph' positive colonies, by rIFN alpha permits the growth of residual normal stem cells. The disappearance of the Ph-chromosome in subclones of T lymphocytes supports the notion of nonclonal hematopoiesis in patients with CML.     

The isolation and characterization in vitro of normal epithelial cells,endothelial cells and fibroblasts from rat urinary bladder

Epithelial cells, microvascular endothelial cells, and fibroblasts have been isolated in culture from normal urinary bladders of Fischer rats. Normal epithelial cells were cultured most efficiently when transitional epithelial sheets were plated on to collagen-coated roller flasks. The epithelial sheets were obtained by two micro-dissection techniques. In the first method, the epithelium was peeled as a large coherent sheet from the submucosal connective tissue following subepithelial injection of a collagenase solution, and after incubation of the bladders in the same enzyme solution. Epithelial sheets with intact basal cell layers were essential for culture success. On collagenous matrices, epithelial differentiation was similar to that in vivo. The in vitro transitional epithelium was composed of three cell layers, namely superficial, intermediate, and basal cells. Basal cells were attached to newly synthesized basal lamina by means of hemidesmosomes. Superficial cells were sealed at their apical lateral membranes by a junctional complex, i.e. a terminal bar. Asymmetric luminal membrane plaques were not apparent. In the second method, the epithelium was separated from the underlying connective tissue after collagenase-trypsin digestion of everted urinary bladders. Although the digest consisted mainly of epithelial cells, these rarely survived the first passage when plated on conventional plastic growth surfaces. After the third culture week, epithelial cells usually died and slowly growing colonies of fibroblasts or large flattened epitheloid cells became apparent. Epitheloid cells were identified by their typical ultrastructure as endothelial cells, showing Weibel-Palade bodies and pinocytotic caveolae. These cells were reactive with antiserum against factor VIII. The free surface of monolayer cultures was non-thrombogenic when incubated in the presence of platelets. Fibroblasts were isolated from heavily contaminated epithelial cell cultures after differential trypsinization. These three cell types represent the normal control cells of an in vitro tumor model for the study of invasiveness. All three cell types are involved in the formation and functional maintenance of the epithelial-stromal junction. The study of cell-cell and cell-matrix interactions may provide important clues for the understanding of tumor invasiveness, a process that starts at the epithelial-stromal junction and proceeds with its destruction.     

Establishment and characterization of a buffalo (Bubalus bubalis) mammary epithelial cell line

Background

The objective of this study was to establish the buffalo mammary epithelial cell line (BuMEC) and characterize its mammary specific functions.

Methodology

Buffalo mammary tissue collected from the slaughter house was processed enzymatically to obtain a heterogenous population of cells containing both epithelial and fibroblasts cells. Epithelial cells were purified by selective trypsinization and were grown in a plastic substratum. The purified mammary epithelial cells (MECs) after several passages were characterized for mammary specific functions by immunocytochemistry, RT-PCR and western blot.

Principal Findings

The established buffalo mammary epithelial cell line (BuMEC) exhibited epithelial cell characteristics by immunostaining positively with cytokeratin 18 and negatively with vimentin. The BuMEC maintained the characteristics of its functional differentiation by expression of β-casein, κ-casein, butyrophilin and lactoferrin. BuMEC had normal growth properties and maintained diploid chromosome number (2n = 50) before and after cryopreservation. A spontaneously immortalized buffalo mammary epithelial cell line was established after 20 passages and was continuously subcultured for more than 60 passages without senescence.

Conclusions

We have established a buffalo mammary epithelial cell line that can be used as a model system for studying mammary gland functions.