Signals causing change in morphological phenotype,growth mode,and gene expression of vascular endothelial cells

Comparison of three different lines of bovine aortal endothelial cells provides a clear demonstration of reversible morphologic phenotype coincidental with change in expression and growth mode. These phenotypic forms can be externally controlled so that cells may exist either in an epithelioid contact-inhibitable state or as a fibroblastoid non-contact-inhibitable state. Clonal cell line N (normal) shows a strong tendency to maintain the epithelioid phenotype. Clonal cell line Sp (sprout) can readily and reversibly adopt the epithelioid or fibroblastoid phenotype. A factor in normal serum is responsible for maintaining the cells in the epithelioid phenotype. This factor could be a growth factor since several polypeptide growth factors are shown to drive cells from the fibroblastoid phenotype to the epithelioid phenotype within 11 hours. This growth factor-induced change is not mediated through induced DNA synthesis. Clonal cell line V (variant) normally maintains the fibroblastoid phenotype but can be directed to the epithelioid phenotype provided cells are on an appropriate collagenous matrix. Associated with these changes in morphological phenotype are depression of the expression of the pro α₂ chain of collagen type I which may be characteristic of the contact-inhibited state and of an 80,000 mol wt polypeptide synthesized only by cells in the fibroblastoid phenotype. An endothelial cell collagen ECl (mol wt 177,000) was synthesized by all cell lines regardless of phenotype whereas a suspected breakdown product EC3 (mol wt 100,000) was found only in the epithelioid phenotype. Other differences and similarities between cell lines include expression of a 135,000 mol wt glycoprotein GP (V and N), the procollagen of collagen type III (N) of fibronectin (N, V, Sp), and of the pro α₁ chain of collagen type I (Sp, V). The characteristic expression of each line and its response to signals controlling morphologic phenotype impinges on the question of whether there exist several distinct types of vascular endothelial cells with different functional potentials controlled by extracellular signals.     

Subpopulations of fibroblasts from mouse skeletal muscle defined by clonal variation for 5' nucleotidase expression

A primary cloning technique has been employed for the isolation of nine spontaneously transformed cell lines from mouse skeletal muscle. Four of these lines were isolated after selection for partial resistance to the purine (adenine) analog 2'6'diaminopurine and five were isolated from non-selected control dishes. Four of the nonselected lines and three of the selected lines demonstrated a fibroblastoid morphology in vitro. The other two cell lines (one from each group) were epithelioid. Two of the three selected fibroblastoid lines were found to contain significant quantities of the enzyme 5'nucleotidase (EC3.1.3.5), whereas the four nonselected fibroblastlike lines, one selected fibroblastlike line, and the two epithelioid lines did not. In the two cell lines expressing 5'nucleotidase activity, this expression was stable in the absence of selective pressure. Histochemical staining of mouse skeletal muscle for 5'nucleotidase activity demonstrated positive staining in the cells of small blood vessels and in a subset of the connective tissue cells. The bulk of the skeletal muscle tissue, however, had no detectable 5'nucleotidase activity. We propose that the two cultivatable types of fibroblastoid cell lines represent distinct classes of fibroblastlike cells in vivo, reflecting alternative states of stable cellular differentiation involving 5'nucleotidase expression.     

Clonal preadipocyte cell lines with different phenotypes derived from murine marrow stroma: Factors influencing growth and Adipogenesis in vitro

We have isolated continuously growing cell lines derived from mouse bone marrow stroma. These cell lines were independently obtained, and though they showed morphologies ranging from the epithelioid to the fibroblastoid patterns, they all differentiated into adipocytes. Subclones obtained from two cell lines had a very high frequency (90-100%) of differentiation into adipocytes after two or three weeks of arrested growth. Though extensive accumulation of lipid often mechanically impaired mitosis, the cells committed to adipocytes did not suffer an irreversible loss of proliferative capacity. Adipogenesis was obtained in conditions similar to those required for fat cell formation in long-term bone marrow culture. The cell lines were found to be insensitive to insulin as a signal of adipocyte differentiation. The ultrastructural characteristics of the preadipocytes and fat cells are also similar to those of the fat cells developing in long-term bone marrow culture. As such, these cell lines should prove useful for analysing cell/cell interactions in haemopoiesis.     

Characterization of a reptilian epithelioid skin cell line derived from the green sea turtle,Chelonia mydas

Summary A continuous line of epithelioid cells was established from explant skin tissues of the green sea turtle,Chelonia mydas. These cells, designated GTS, have been subcultured more than 60 times in commercially available mammalian cell culture medium supplemented with 5% bovine calf serum. Of those temperatures tested, optimal growth was achieved at 30°C although replication occurred between 16 and 37°C. These cells may be held as monolayers at 8°C or stored frozen in growth medium containing 10% dimethylsulfoxide at −70 or −196°C. The modal number of 55 chromosomes per cell is in agreement with the heterogametic female diploid number of this species. The GTS line represents the first established culture of normal epithelioid skin cells to be reported for a poikilothermic species.     

Relation between ionic coupling and morphology of established cells in culture

Ionic coupling was found in all investigated fibroblastoid cells of 7 permanent cell lines in culture, whereas in 7 epithelioid cell lines no coupling could be detected. These established lines consisted of cells of normal or malignant origin as well as cells that were able to, or failed to, produce tumors, but the only relation with ionic coupling appeared to be morphology. The ionic coupling between fibroblastoid cells was unaffected by the presence of fetal calf serum instead of calf serum; culturing in media conditioned by non-coupled cells; variation of the potential difference and phase of the cell cycle. Coupled cells could be depolarized by decreasing the bicarbonate concentration in the media; non-coupled cells were unaffected.     

Long-term cell culture of two differentiated cell types from the liver of larval and adult Xenopus laevis

Summary Two distinct types of cells were derived from organ cultures of liver from adult and larval Xenopus laevis. Each type was isolated in clonal cell culture. Several media were compared with respect to support of epithelioid outgrowths from explants and support of growth of epithelioid colonies in cell culture. Ultracentrifuged embryo extract promotes the growth of all cell types, but the particulate fraction is also required for the maintenance of the epithelioid morphology of larval cells. In these media it was possible to maintain some epithelioid cell cultures for over 6 months. The identity and retention of some specialized functions of both cell types were demonstrated on larval cells. One cell type contained PAS-stainable, amylase-sensitive granules that increased in amount after treatment with glucocorticoids. This same type was shown by histochemical methods to contain phosphorylase, glucose-6-phosphatase, and dexamethasone-inducible tyrosine aminotransferase, and is considered to be a hepatocyte. The second type appears to be a sinusoidal cell, since it phagocytosed trypan blue and stained positively for acid phosphatase.     

Establishment of permanent cell lines purified from human mesothelioma: morphological aspects,new marker expression and karyotypic analysis

This study reports the establishment of three major subtypes of human mesothelioma cells in tissue culture, i.e. the epithelioid, sarcomatoid and biphasic forms, and compares their phenotypic and biological characteristics. Primary cells isolated from biopsies or pleural exudates were subcultured for over 50 passages. We evaluated immunoreactivity using various mesothelial markers related to histological patterns of these cell lines. For epithelioid cells, calretinin and cytokeratin were found to be useful and easily interpretable markers as for control mesothelial cells. The biphasic form was only partially positive and the sarcomatoid type negative. Vimentin was expressed by all cell lines. BerEP4, a specific marker for adenocarcinoma, was negative. Interestingly, while the macrophage marker CD14 was negative, immunoreactivity for a mature macrophage marker (CD68) was expressed by all cell types, suggesting that this marker might constitute an additional tool useful in the differential diagnosis of mesothelioma. At the ultrastructural level, a cell surface rich in microvilli confirmed their mesothelial origin. PCR analysis revealed that none of the cell lines contained SV40 DNA. Karyotypic analyses showed more complex abnormalities in the epithelioid subtype than in the sarcomatoid form. These cell lines may be useful in the study of cellular, molecular and genetic aspects of the disease.     

Initiation and characterization of five embryonic cell lines from the cotton boll weevil anthonomus grandis in a commercial serum- free medium

Summary Five continuous cell lines were initiated from embryonic tissue of the cotton boll weevilAnthonomus grandis Boheman in a commercially available, serum-free medium (Excell 401) and have undergone in excess of 60 passages. Isoenzyme analysis confirmed that the lines originated from boll weevil tissue. Four of the lines grew as single attached cells of either epithelioid or fibroblastoid morphology. The fifth line, BRL-AG-2, grew primarily as cell aggregates and was found to release ecdysteroids (primarily ecdysone) into the culture medium. Evidence was also obtained suggesting that line BRL-AG-2 synthesizes chitin. Three lines, BRL-AG-1, BRL-AG-3A, and BRL-AG-3C, could be induced to produce an antibacterial factor(s) which was released into the culture medium.     

Comparison of tissue-cultured bovine endothelial cells from aorta and saphenous vein

Endothelial cells were harvested from bovine aorta and saphenous vein with collagenase and cultured in McCoy's 5a medium (modified GIBCO) supplemented with 10% fetal bovine serum. The cells were subcultured through 17 passages over 4 to 5 months. The growth properties in culture of the two cell types were compared. Morphological comparisons included phase microscopy and scanning and transmission electron microscopy. Comparisons with cultured aortic smooth-muscle cells were made using phase and scanning electron microscopy. No differences were found between cultured endothelial cells from aorta and saphenous vein. Differences in growth patterns in culture clearly distinguished both endothelial cell types from smooth-muscle cells. The presence of Weibel-Palade bodies identified the cells from both sources as endothelial.     

Hepatopancreas cell cultures from mud crab, <Emphasis Type="Italic">Scylla paramamosain</Emphasis>

Hepatopancreas is an important digestive and endocrine organ in crustacean. However, there are few reports on cell cultures from crabs. Here, the cell cultures of hepatopancreas from Scylla paramamosain was studied in vitro. Both the primary cell culture and subculture were grown in Leibovitz’ L-15 medium, M199 medium, or a specially designed medium for S. paramamosain (MSP). The results showed that hepatopancreas cells in vitro grew in compact clusters in 2–3 d. Four types of cells could be identified. They were embryo cells, fibrillar cells, resorptive cells, and blister-like cells, respectively. Some of these cells could be subcultured for three generations. The MSP supported the best survival of these hepatopancreas cells, while M199 medium was the least effective of these three media. Fetal bovine serum and crab muscle extracts as supplements stimulated growth, but the crab hemolymph inhibited cell growth. Taken together, MSP is an appropriate medium for hepatopancreas cell cultures from S. paramamosain and can support cultures through several passages.     

Comparison of tissue-cultured bovine endothelial cells from aorta and sphenous vein

Summary Endothelial cells were harvested from bovine aorta and saphenous vein with collagenase and cultured in McCoy's 5a medium (modified GIBCO) supplemented with 10% fetal bovine serum. The cells were subcultured through 17 passages over 4 to 5 months. The growth properties in culture of the two cell types were compared. Morphological comparisons included phase microscopy and scanning and transmission electron microscopy. Comparisons with cultured aortic smooth-muscle cells were made using phase and scanning electron microscopy. No differences were found between cultured endothelial cells from aorta and saphenous vein. Differences in growth patterns in culture clearly distinguished both endothelial cell types from smooth-muscle cells. The presence of Weibel-Palade bodies identified the cells from both sources as endothelial. This work was supproted by Grants HL-1330 and HL-17269 from NIH.     

The effects of incubation of marrow in tissue fragments in vitro on the growth of adherent cells from this marrow

Femoral marrow was either cultured as a single cell suspension immediately following collection from the donor mouse or following 4 day incubation in vitro of the whole marrow shaft. Several parameters of growth of adherent, i.e. composed of fibroblastoid cells and macrophages, colonies were determined following 14 day culture in Dulbecco medium at 37 degrees C. These included: number and diameter of macroscopic colonies, number of macrophages per fibroblastoid cell inside the colonies and per eyefield in intercolony spaces, number of cells in supernatant from the culture. The 4 day incubation of marrow fragments in vitro (Dulbecco medium, 37 degrees C) doubled the number of adherent colonies grown from this marrow and, moreover, the colonies formed were increased in size. Other parameters of cell growth in these cultures were unchanged. These data suggest that under conditions of in vitro incubation of marrow shaft (close cell-to-cell contact) marrow fibroblastoid colony forming units (MF-CFU) are stimulated to self-renewal.     

Long-term primary culture of epithelial cells from rainbow trout (Oncorhynchus mykiss) liver

Summary Long-term primary cultures of epithelial cells from rainbow trout (Oncorhynchus mykiss) liver have been established. Nearly homogenous (>97%) populations of hepatocytes were placed into primary culture and remained viable and proliferative for at least 70 d. In addition to hepatocytes, proliferative biliary cells persisted in the cultures for at least 30 d. Finally, a third type of epithelial cell, which we have termed a “spindle cell,” consistently appeared and proliferated to confluence in these cultures. The confluent cultures of spindle cells were successfully subcultured and passaged. The initial behavior, growth, and optimization of serum and media requirements for these cells is described. All three cell types proliferated as measured by thymidine incorporation, autoradiography, proliferating cellular nuclear antigen analysis, and propidium iodine staining. Further efforts to characterize the cells included western blotting and immunohistochemical staining with antibodies to cytokeratins previously reported in fish liver. From these data, it appears that all three cell populations are epithelial in nature. Furthermore, significant changes in actin organization, often indicative of transformation or pluripotent cells, were observed with increased time in primary culture.     

Regulation of the cytoskeleton in mesothelial cells: Reversible loss of keratin and increase in vimentin during rapid growth in culture

Human mesothelial cells grew rapidly in culture when provided with serum, EGF, and hydrocortisone, adopting a fibroblastoid shape and forming parallel, multilayered arrays at saturation density. In the absence of EGF, the cells grew slowly to a flat, epithelioid monolayer similar to their normal pattern in vivo. Mesothelial cells normally have a high keratin and a low vimentin content in vivo. In culture, rapidly growing cells greatly reduced synthesis and content of their four major keratins to levels undetectable by immunofluorescence in most cells, but keratin synthesis and content returned to high levels whenever growth slowed. Vimentin synthesis and content was high during serial culture, but decreased several-fold in nondividing cells. The unique ability of the mesothelial cell to reversibly alter its morphology and intermediate filament composition is of unknown function and mechanism, but accounts for the morphological heterogeneity and the presence of keratin-negative cells in mesotheliomas.     

Epithelioid cell cultures from rat small intestine. Characterization by morphologic and immunologic criteria

Rat small intestinal epithelial cell lines have been established in vitro and subcultured serially for periods up to 6 mo. These cells have an epithelioid morphology, grow as monolayers of closely opposed polygonal cells, and during the logarithmic phase of growth have a population doubling time of 19--22 h. Ultrastructural studies revealed the presence of microvilli, tight junctions, an extensive Golgi complex, and the presence of extracellular amorphous material similar in appearance to isolated basement membrane. These cells exhibit a number of features characteristic of normal cells in culture; namely, a normal rat diploid karyotype, strong density inhibition of growth, lack of growth in soft agar, and a low plating efficiency when seeded at low density. They did not produce tumors when injected in syngeneic animals. Immunochemical studies were performed to determine their origin using antisera prepared against rat small intestinal crypt cell plasma membrane, brush border membrane of villus cells and isolated sucrase-isomaltase complex. Antigenic determinants specific for small intestinal epithelial (crypt and villus) cells were demonstrated on the surface of the epithelioid cells, but they lacked immunological determinants specific for differentiated villus cells. An antiserum specifically staining extracellular material surrounding the cells cultured in vitro demonstrated cross-reactivity to basement membrane in rat intestinal frozen sections. It is concluded that the cultured epithelioid cells have features of undifferentiated small intestinal crypt cells.     

Splenic cultures from rainbow trout,Oncorhynchus mykiss: establishment and characterisation

This study describes the culture conditions and the phenotypic features of different types of splenic cultures established from explants. Using the same culture technique it was possible to grow splenic explants from which monolayers of reticular origin, long-term haematopoietic cultures, and subcultures were obtained. The cultures were characterised by light and electron microscopy, cytochemical and immuno-cytochemical analyses, phagocytic activity and susceptibility to virus. The cultures comprised multilayers of epithelioid and fibroblastoid cells with haemopoietic foci, melanomacrophages and eosinophilic granular cells. The cytochemical and immuno-cytochemical analyses revealed that the stromal cells were always positive for ANAE activity. The stromal cells in primary cultures were negatively or weakly stained by antibodies directed against cytokeratins and S-100, but in the subcultures they were strongly stained by these antibodies. The stromal cells had very poor phagocytic activity and were susceptible to VHS virus.     

Cultured mouse marrow cell lines: Interactions between fibroblastoid cells and monocytes

Continuous cell lines were derived from primary cultures of adherent bone marrow cells from SJL/J, BALB/c, C3H/eb, RF, and nude-ICR mice. All these lines readily assumed a pure fibroblastoid appearance with the exception of the BALB/c line (MBA-14), which retained both fibroblastoid and monocytoid cells. This particular line could promote the proliferation of myeloid progenitors (CFU-C) in short-term bone-marrow cultures. The two cell types that composed the MBA-14 cell line were successfully isolated and grown separately; the monocytes as the 14M and 14M1 cell lines and the fibroblastoid cells as the 14F clones. The latter were found to be preadipocytes and accumulated fat in the absence of added hydrocortisone, in medium supplemented with fetal calf serum. Growth of the monocyte lines (14M and 14M1) was dependent upon the mononuclear phagocyte stimulator CSF-1. In the parent MBA-14 cell line the growth of monocytes seemed to depend upon stimulating factor(s) produced by the fibroblastoid cells. The 14M1 monocytes were able to process and degrade antigen as efficiently as primary macrophages. Furthermore, processed antigen produced by 14M1 cells evoked proliferative response by antigen-primed lymph-node cells. In addition to these immunological functions the 14M1 cells were capable of modulating the colony-stimulating activity and degree of adipogenesis exhibited by the fibroblastoid cells. These interactions between monocytes and fibroblastoid cells may constitute part of the mechanism controlling the activity of the hematopoietic microenvironment.     

Replacement of vertebrate serum with lipids and other factors in the culture of invertebrate cells,tissues, parasites,and pathogens

Summary Culture medium supplementation with vertebrate serum results in the selection of fibroblastoid insect cell lines and a general decline during continuous subculturing of both morphologic and functional differentiation of the surviving cells. Essential lipid mixtures can substitute for vertebrate serum in the culture of insect and some vertebrate cells, tissues, parasites, and pathogens. The provision of sterols and essential (with nonessential) polyunsaturated fatty acids as phospholipids in oxidation-protected peptoliposomes or proteoliposomes allows cells in culture to duplicate in vivo specific membranes more accurately. Such lipid-corrected membranes allow cultured cells to communicate with neighboring cells through the extracellular matrix, effectively transmit hormonal signals directly and via receptor control, and respond with various tissue-specific functions and differentiation states as directed.     

Growth and morphological characteristics of vervet monkey bone marrow-derived adherent cells

Monkey bone marrow-derived adherent cells were maintained in culture for six months. Phase contrast and scanning electron microscopy showed two cell shapes: fusiform and polygonal. No difference was observed in the cyto-chemical staining of the two shapes. Both stained positively for alpha-napthyl acetate esterase, acid phosphatase, collagens I and III, and lipids and negatively for peroxidase and factor VIII antigen. A small proportion (1.5%) were alkaline phosphatase-positive. An average of 14% of the cells were phagocytic in the primary culture, but this proportion decreased progressively with passage. Fc receptors were not detected, while C3 receptors were detected in 1% of primary cultured cells in primary culture, but were not detected in subcultured cells. Adherent cells were not evident in cultures supplemented with 10 mM ammonium acetate. These findings indicate that monkey marrow-derived adherent cells are fibroblastoid in nature.