Interaction of three human malignant cell lines with chick hypoblast in culture

The hypoblast (lower layer) was dissected from young chick blastoderms and explanted in vitro, where it formed an epitheloid sheet. Cells from the following malignant lines were explanted on top of the sheet both as aggregates and as cell suspensions: Hu456 human bladder carcinoma, SAOS-2 human osteosarcoma, LICR(LOND)-HN-4 laryngeal carcinoma. The interaction of the malignant cells with the hypoblast was studied by time lapse cinephotography, light microscopy, and transmission electron microscopy. All malignant cells penetrated through the hypoblast, so that a gradually enlarging hole formed in it. Apart from this common pattern of behaviour, the three types of malignant cells differed in their interactions with the hypoblast in the following ways. 1) Both the Hu456 and to a lesser extent the SAOS-2 cells brought about an initial retraction of the hypoblast so that a temporary cell-free space was formed. No such retraction occurred in response to the LICR-(LOND)-HN-4 cells. 2) Each of the three types of malignant cells migrated for some distance beneath the hypoblast, and in this area of underlap, there were differences in the amount and disposition of extracellular material. Thus, there was more extracellular material between the hypoblast and underlying SAOS-2 cells than between the hypoblast and underlying Hu456 cells, whilst there was no extracellular material between the hypoblast and underlying LICR(LOND)-HN-4 cells. Indeed, the hypoblast and LICR(LOND)-HN-4 cells often shared desmosomes. 3) When explanted as aggregates on hypoblast Hu456 and SAOS-2 cells left the corona and migrated as solitary cells underneath the hypoblast in contrast with control aggregates explanted on plastic. These cells which had migrated beneath the hypoblast were flatter than their corresponding control cells which had spread on the plastic substrate. The flatter cells appeared to have been using the extracellular materials as a substrate, rather than the plastic. Such differences in the migratory behaviour between experimental and control cultures were not observed with LICR(LOND)-HN-4 cells.     

Phagocytic capacity of invasive malignant cells in three-dimensional culture

To study the phagocytic capacity of invasive malignant cells, fragments of the hypoblast from chick blastoderms were confronted in three-dimensional culture with spheroidal aggregates of 1) malignant virally transformed C3H mouse cells (MO4), 2) HeLa cells and 3) embryonic chick heart cells. The hypoblast was used because it contains yolk, a marker that is absent in the confronting cells and that can be identified histologically and ultrastructurally. The confronting tissues were incubated on semi-solid agar-agar medium or in fluid medium on a gyrotory shaker. Cultures were followed for 1 to 7 days by stereomicroscopy, cinemicrophotography, light and transmission electron microscopy. Confrontation with MO4 cells of HeLa cells, known to be invasive in vitro, led to complete disappearance of the hypoblast. The fragments of hypoblast were well conserved when cultured alone or confronted with aggregates of chick heart cells. Degeneration of the hypoblast is shown at the area of contact with MO4-cell or HeLa-cell aggregates, in contrast to heart cells. Filopodia-like extensions from the MO4 or HeLa cells penetrate intercellularly, transcellularly and intracellularly into the hypoblast. Phagosomes, containing yolk and unidentified debris are observed in MO4 cells and in HeLa cells, but not in heart cells. These observations demonstrate the phagocytic capacity of invasive malignant cells.     

An electron-microscopical analysis of embryonic chick tissues explanted in culture

Summary Three types of tissue (hypoblast, germ wall and epiblast) were dissected from early chick embryos and explanted on Falcon plastic dishes. After they had settled and spread, the explants were fixed, usually within 18–24 h after explantation, and sections were cut through the tissue and the Falcon dish. The closeness of the cells to the substrate varied even within the same explant, but the epiblast tended to be closer to the substrate than did the hypoblast or germ wall. Plaques were present in all three tissues in regions where the cell processes contacted the substrate. Extensive desmosomes were visible in the epiblast explants, small desmosomes were present in the germ wall explants, but desmosomes were never seen in hypoblast explants. These differences in cell/substrate and cell/cell morphology are discussed in relation to the different behavioural characteristics of the three tissues. Some mixed cultures were also examined by electron microscopy. When the epiblast was confronted with either hypoblast or germ wall, it underlapped them at the region of contact.     

Invasion of malignant C3H mouse fibroblasts from aggregates transplanted into the auricles of syngenic mice

Spheroid aggregates of malignant fibroblasts (MO4), shown to be invasive in vitro, were implanted subcutaneously into the auricle of the external ear of syngenic C3H mice. The course of early invasion into the surrounding tissues and the formation of tumours was studied in serial sections of auricles fixed 6 h to 30 days after implantation. MO4 cells are first observed to make contact with the surrounding tissues after 6 h. They exhibit, cytoplasmic extensions and spread from the original aggregate. During the first day invading MO4 cells preferentially follow tissue crevices created by the inoculation procedure. Later they also invade the surrounding tissues. Polymorphonuclear leucocytes, and later monocytes infiltrate the aggregate, which is completely destroyed after 4 days. Palpable tumours arise from MO4 cells that have left the original implant and invaded the tissues of the auricles. These observations indicate that invasion of malignant cells is important for the take of a transplant.     

Cell junctions in explanted tissues from early chick embryos

Summary Hypoblast and definitive endoblast derived from young chick embryos were explanted and grown for 24 h in culture. The junctional complexes which characterise these tissues were studied on freeze-fracture replicas and thin sections. Cell membranes of the hypoblast displayed tight junctions only, disposed in randomly arranged strands or narrow belts which included many discontinuous strands. The definitive endoblast showed tight and gap junctions as well as desmosomes in close association with the tight junctions. It is suggested that the differences between the two types of tissue may be related to cell cohesiveness, which appears to be relatively low in the hypoblast and high in the definitive endoblast.     

Contact inhibition of locomotion and the structure of homotypic and heterotypic intercellular contacts in embryonic epithelial cultures

Epithelia from the early chick embryo have been grown in culture and then fixed for electron microscopy so that the ultrastructure of intercellular contacts could be examined. Epithelia were used which showed various forms of contact inhibition of locomotion upon confrontation with one another. Confrontations of hypoblast with hypoblast after 6 days, and endoblast with endoblast after 24 h showed type 1 contact inhibition and formed desmosomes and zonulae adhaerentes with extensive microfilament collinearity between apposed cells. Hypoblast-hypoblast confrontations after 24 h resulted in type 2 contact inhibition with considerable ruffling and position shifting. In this case desmosomes were absent and microfilament collinearity was restricted. Endoblast cells after 24 h in culture show type 2 inhibition with respect to hypoblast monolayers which they infiltrated upon confrontation. Examination of these heterotypic contacts also showed an absence of desmosomes and reduced adhaerens junctions. Intermediate filaments accumulated at all contact sites examined. It is concluded that whereas type 1 contact inhibition of locomotion in these epithelial cells is accompanied by desmosome formation and extensive zonula adhaerens junctions, type 2 inhibition is not. These junctional deficiences may be responsible in part for the cell motility characteristically observed in monolayers of type 2 inhibited cells.     

Hypoblast cells of chick pre-streak stage embryos invaded basement membrane analogues in vitro: Implications for hypoblast layer formation

In early chick blastodermal morphogenesis, the hypoblast layer is organized beneath the epiblast and induces an axial structure. However, the origin of hypoblast cells and the mechanism of hypoblast layer formation are poorly understood. We hypothesized that the hypoblast layer is formed by an invasive process across the basement membrane of the juxtaposing epiblast, and tested the idea in vitro . Primary and secondary hypoblast cells from embryos at various pre-streak stages were dissociated into single cells and cultured on reconstituted basement membrane gel, laminin gel or fibronectin gel in the culture medium with or without serum for 24–48 h. As a result, we found that after 24 h of serum-supplemented culture, up to 35% of the hypoblast cells dissolved the gel and made holes on it. Similarly, up to 36% of the hypoblast cells showed invasiveness after 48 h in the serum-free culture. Furthermore, it was observed that Koller's sickle cells, which are regarded to be the progenitors of secondary hypoblast cells, penetrated those gels on which they were seeded. The posterior epiblast cells covering Koller's sickle were also invasive. These results suggest that the presumptive primary hypoblast cells that are known to mingle with epiblast cells invade through the basement membrane to form the hypoblast layer. Furthermore, the present results imply that invasion through the basement membrane may be involved in the formation of Koller's sickle, the anlage of secondary hypoblast.     

The sorting-out of thymidine-labelled chick hypoblast cells in mixed epiblast--hypoblast aggregates.

Tritium-labelled disaggregated chick hypoblast cells were mixed with non-labelled epiblast cells and vice-versa. The mixtures were allowed to aggregate in a gyratory shaker and were transferred on to a solid culture medium for further incubation. The aggregates were fixed after various incubation times, sectioned and examined for sorting-out. There was already a tendency to sort out after 10 h of incubation, a process which was completed after 25 h. The hypoblast cells formed a continuous layer adjacent to the vitelline membrane, while the epiblast cells moved out to form the upper external layer. The position of the two layers was normal as far as the substrate and external environment are concerned, and reversed in relation to their relative position to the vitelline membrane. The hypoblast cells tended to migrate to the margins of the aggregate. The latter phenomenon seems to parallel the migration of hypoblast cells towards the extra-embryonal area during the formation of the primitive streak.     

The Sorting-out of Thymidine-labelled Chick Hypoblast Cells in Mixed Epiblast–Hypoblast Aggregates

Tritium-labelled disaggregated chick hypoblast cells were mixed with non-labelled epiblast cells and vice-versa . The mixtures were allowed to aggregate in a gyratory shaker and were transferred on to a solid culture medium for further incubation. The aggregates were fixed after various incubation times, sectioned and examined for sorting-out. There was already a tendency to sort out after 10 h of incubation, a process which was completed after 25 h. The hypoblast cells formed a continuous layer adjacent to the vitelline membrane, while the epiblast cells moved out to form the upper external layer. The position of the two layers was normal as far as the substrate and external environment are concerned, and reversed in relation to their relative position to the vitelline membrane. The hypoblast cells tended to migrate to the margins of the aggregate. The latter phenomenon seems to parallel the migration of hypoblast cells towards the extra-embryonal area during the formation of the primitive streak.     

Primary hypoblast development in the chick

Summary Scanning electron microscopy (SEM) indicates that the primary hypoblast forms beneath the area pellucida during the first 8 h of incubation mainly by establishment of contact among cells which move downward out of the epiblast. This movement, polyingression, begins posteriorly and continues antero-laterally during the period of primary hypoblast formation. Polyingression produces many pits and possibly a crescentic fold in the embryo upper surface with corresponding cell clusters and a ridge on the lower surface. Fixationin situ helps prevent formation of artifactual folds and wrinkles facilitating interpretation of the SEM images.Formation of intercellular adhesions which lead to development of an epithelial primary hypoblast proceeds in a posterior to anterior direction along with polyingression. This epithelialization begins with elaboration of numerous filamentous processes by cells as they arrive from the epiblast, and continues with ongoing input of cells, merging of cells and cell clusters, and cell flattening. We have also shown (Weinberger and Brick 1982) that proliferation of ingressing cells provides additional cells for hypoblast development.     

Transfer of extracellular matrix components between germ layers in chimaeric chicken-quail blastoderms

Summary A chemical basis for the transmission of signals during gastrulation has been investigated by using chimaeric embryos resulting from the combination of ³H-glucosamine-labelled and unlabelled hypoblast with epiblast taken from chicken and quail embryos at stage 3 of Vakaet (1970). The ability to distinguish chicken from quail cells on the basis of their different nuclear distribution of heterochromatin after Feulgen staining made it possible to determine the origin of the cells in the chimaerae. Tritiated quail hypoblast (after incubation of the embryo in the presence of ³H-glucosamine) was transplanted onto unlabelled chicken blastoderm deprived of its hypoblast. After culture of the chimaera for 5 h, the autoradiographic pattern shows silver grains not only over the graft, but also at the ventral surface of the epiblast of the host. Transfer of label may occur to mesoblast cells, but not between chicken and quail hypoblast cells. Chase experiments exclude the possibility that unprocessed, tritiated glucosamine is transferred. Chemical fixation of the host before transplantation of a labelled quail hypoblast also allows visualization of a transfer of macromolecules from hypoblast to the basement membrane of the epiblast, suggesting that an intervention of the epiblast cells in this process is not necessary. The morphology of the chimaeric embryos, as studied by scanning electron microscopy, suggests a direct deposition of these macromolecules by filopodia of the dorsal surface of the hypoblast. The possibility of diffusion of free macromolecules has been considered and can reasonably be discarded on the basis of several observations. The reverse experiment, in which unlabelled quail hypoblast and possibly some mesoblast have been combined with a tritiated host deprived of its hypoblast, also shows the transfer of label from the host to the cellular surface of the graft. A two-way exchange of glucosamine-containing molecules thus occurs in the blastoderm. It is hypothesized that: (1) low molecular weight compounds, macromolecular material, and/or catabolic products, are exchanged between the different germ layers during gastrulation; (2) the components of the extracellular matrix turn over and are continuously changing; (3) this transfer is a possible mechanism of transmission for developmental or inductive signals during embryonic development. The present results also demonstrate the participation of underlying tissue in the biosynthesis of basement membrane components of an epithelium.     

In vitro culture of epicardial cells from adult zebrafish heart on a fibrin matrix

We describe here a protocol for culturing epicardial cells from adult zebrafish hearts, which have a unique regenerative capacity after injury. Briefly, zebrafish hearts first undergo ventricular amputation or sham operation. Next, the hearts are excised and explanted onto fibrin gels prepared in advance in a multiwell tissue culture plate. The procedure allows the epicardial cells to outgrow from the ventricle onto a fibrin matrix in vitro. This protocol differs from those used in other organisms by using a fibrin gel to mimic blood clots that normally form after injury and that are essential for proper cell migration. The culture procedure can be accomplished within 5 h; epicardial cells can be obtained within 24-48 h and can be maintained in culture for 5-6 d. This protocol can be used to investigate the mechanisms underlying epicardial cell migration, proliferation and epithelial-to-mesenchymal transition during heart regeneration, homeostatic cardiac growth or other physiological processes.     

Isolation and monolayer culture of guinea pig pancreatic duct epithelial cells

Summary Monolayers of cultured epithelial cells have been prepared from fragments of guinea pig pancreatic excretory ducts isolated by a simple procedure employing collagenase digestion and manual selection, through which virtually all of the ductal system can be recovered. The isolated fragments were cultured in enriched Waymouth's medium on extracellular matrices of various composition and thickness, including: thin (<5 μm) and thick (0.5 mm) layers of rat tail collagen; thin layers of human placental collagen; thin layers of Matrigel (a reconstituted basement membrane material); uncoated tissue culture plastic; and the cellulose ester membranes of Millipore Millicells. Cells spread rapidly from duct fragments cultured on uncoated plastic or on plastic coated with thin layers of rat tail collagen or human placental collagen and formed epithelial monolayers. However, these cells were squamous and lacked the abundant basolateral membrane amplification and apical microvilli characteristic of freshly isolated duct epithelial cells. Cells did not spread from duct fragments cultured on Matrigel. In contrast, when fragments of pancreatic ducts were explanted onto either a thick layer of rat tail collagen or onto Millicell membranes, cells readily spread and formed confluent monolayers of cuboidal epithelial cells characterized by abundant mitochondria, apical microvilli, and basolateral plasma membrane elaboration. These results demonstrate that different forms of extracellular matrix modulate the growth and differentiation of pancreatic duct epithelial cells, and that culture on a permeable substrate markedly enhances the maintenance of differentiated characteristics in this cell type. The monolayers formed on Millicell membranes should provide a useful model system for physiologic analysis of the regulation of electrolyte secretion by this epithelium. This research was supported by grants DK32994 and DK35912 from the National Institutes of Health, Bethesda, MD.     

Aggregation of Cells from Early Chick Blastoderms

The aggregation of dissociated cells from chick blastoderms at Hamburger and Hamilton stages 1–5 was studied. Aggregation was measured during the first 4 h of culture by determination of the proportion of single cells in the medium. No difference in aggregation was found when cells dissociated by either trypsin or EDTA were studied. Similarly the presence or absence of serum in the medium had no appreciable effect on early phases of aggregation, although at 24 h and thereafter, aggregate size was reduced in serum-free cultures. It was found that at all of the stages studied, cells aggregate and sort out into two groups. One group forms a continuous phase of loosely associated cells while the other segregates into several localised areas of closely associated cells within the aggregate. Examination of aggregates up to 7 days in culture showed progressive differentiation within each phase and several identifiable cell types were observed. Basal laminae were present at the boundary between the compact phase and the loose phase.     

Origin of primordial germ cells in the prestreak chick embryo

The temporal and spatial pattern of segregation of the avian germline from the formation of the area pellucida to the beginning of primitive streak formation (stages VII–XIV, EG&K) was investigated using the culture of whole embryos and central and peripheral embryo fragments on vilelline membranes at stages VII–IX, immunohistological analysis of whole mount embryos and sections with monoclonal antibodies MC-480 against stage-specific embryonic antigen-1 (SSEA-1) and EMA-1, and with the culture of dispersed blastoderms at stages IX–XIV with and without an STO feeder layer. Whole embryos at intrauterine stages developed up to the formation of the primitive streak despite the absence of area pellucida expansion. Primordial germ cells (PGCs) appeared in the cultures of whole embryos and only in central fragments containing a partially formed area pellucida at stages VII–IX. When individual stage IX–XIV embryos were dispersed and cultured without a feeder layer, 25–45 PGCs/embryo were detected only with stage X–XIV, but not with stage IX blastoderms. However, the culture of dispersed cells from the area pellucida of stages IX–XIII on STO feeder layers yielded about 150 PGCs/embryo. The carbohydrate epitopes recognized by anti-SSEA-1 and EMA-1 first appeared at stage X on cells in association with polyingressing cells on the ventral surface of the epiblast and later on the dorsal surface of the hypoblast. The SSEA-1-positive hypoblast cells gave rise to chicken PGCs when cultured on a feeder layer of quail blastodermal cells. From these observations, we propose that the segregation and development of avian germline is a gradual, epigenetic process associated with the translocation of SSEA-1/EMA-1-positive cells from the ventral surface of the area pellucida at stage X to the dorsal side of the hypoblast at stages XI–XIV. © 1996 Wiley-Liss, Inc.     

The determination of myogenic and cartilage cells in the early chick embryo and the modifying effect of retinoic acid

Summary The time of determination of cartilage and skeletal muscle was studied by making chimeric grafts or explants of small tissue pieces from several stages of early chick or quail embryos. Chondrogenesis was assessed by histology or with antibodies directed against type II collagen or cartilage proteoglycan, while myogenesis was detected immunohistochemically with antibodies directed against 3 different muscle markers, including muscle myosin. Grafts from Hensen's node, primitive streak and segmental plate of donor embryos of Stage 3–5 (Hamburger and Hamilton) were transplanted under the ectoderm in the extraembryonic area of Stage 12 host embryos. In addition, explants and mesodermal cells were cultured on glass in DMEM+F12 medium supplemented with 10% FCS. The results showed that determined myogenic cells could first be detected in Hensen's node and the primitive streak at Stage 3⁺–4 and that they developed from mesodermal cells located between the epiblast and hypoblast. Myogenic cells also appeared in grafted and explanted segmental plate with or without notochord from Stage 5 embryos. On the other hand, cartilage cells only formed in grafted and explanted segmental plate that also contained notochord. RA (1 ng/ml) could induce the formation of cartilage cells in the explanted primitive streak without Hensen's node or notochord taken from Stage 3–5 embryos and could also promote the differentiation of myogenic cells in primitive streak from Stage 3 embryo. Thus RA can substitute for Hensen's node or the notochord in the induction of cartilage cells and has some stimulatory effects on the differentiation of myogenic cells. Additional evidence indicates that the hypoblast might play an inductive role in the formation of the notochord which may subsequently promote the differentiation of cartilage cells. Offprint requests to: M. Solursh     

Gastrulation: Is It Analogous to Malignant Invasion

The process of gastrulation has often been compared with thatof malignant invasion. In this paper, the terms "malignant"and "invasion" are denned and the characteristics of malignantcells are discussed. One of the best examples of invasion duringgastrulation takes place during the formation of the endodermin the chick, when the definitive endoblast invades the hypoblast.Experiments are described in which the hypoblast is invadedby a) definitive endoblast, b) other normal embryonic cells,and c) three types of human malignant cells. It was found thatnot only does the hypoblast react differently to normal andmalignant cells, but that the cell interactions differ alsoaccording to the type of malignant cells. In particular, thereare differences in the behaviour of the cells and in the amountof extracellular material laid down between the hypoblast andmalignant cells. It is concluded that even within the limitsof this experiment, chick gastrulation is not wholly analogousto malignant invasion.     

Non-small-cell lung carcinoma cells invade human bronchial mucosa In vitro

Summary To study invasion of lung cancer in vitro a novel three-dimensional coculture assay consisting of living human tissues has been developed. Multicellular spheroids initiated from a new large-cell lung carcinoma cell line (GaL23), found to be invasive in immunodeficient mice, were confronted with precultured bronchial fragments derived from mucosal biopsies obtained during routine fiberoptic bronchoscopy. The bronchial fragments consist of a stromal core with scattered fibroblasts covered by a continuous surface epithelium resting on a basal lamina. During the first 2 wk of confrontation, a gradual retraction of the bronchial epithelium with subsequent adhesion of the tumor cells to the underlying basal lamina occurred. The following week, a limited invasion of tumor cells into the bronchial stroma was seen. To facilitate the entrance of tumor cells through the mucosal surface, the surface epithelium was removed prior to coculture by ethylenediaminetetraacetic acid (EDTA) buffer treatment. Upon confrontation, GaL23 cells then rapidly attached to and migrated on the exposed basal lamina and an increasing number of tumor cells was seen in the stroma during the first week of culture. This model offers opportunities for studying mechanisms of lung cancer adhesion, migration, and invasion using human bronchial mucosa as the natural target tissue.     

Effects of restricted nursing on milk production and collection, kid growth and plasma prolactin and growth hormone concentrations in dairy goats

The milk production of dairy goats under various regimes of mother-young contact from day 4 post partum were studied during the first 2 months of lactation, together with the prolactin (PRL) and growth hormone (GH) responses to udder stimulation. In the control group, 13 goats and their kids were left in permanent contact and did not undergo milking. In two additional groups, goats were machine milked once a day in the morning (at 0800 h) and kids were allowed 10 hours (from 1000 to 2000 h; 10H group, n = 11) or 5 h (from 1000 to 2000 h; 5H group, n = 11) of mother-young interaction per day. In the last group (MO, n = 10), mothers were permanently separated from their kids on day 4 post partum and milked once a day. Milk production during a 24-h period at 37 days post partum performed by controlled nursing and weighing of the kids (groups with kids) or by two machine milking 12 h apart (milking only group) revealed a higher production in the three groups with some mother-young contact than in the MO group. Total milk collected by milking over the 2 months of the study did not differ between the three groups that underwent milking. Kid weights at 2 months were 3.4 to 4.8 kg. lighter in the groups that underwent milking than in the control group. Hormonal profiles were significantly affected by restricted mother-young contact, with highest pre-stimulation concentrations of PRL and GH in the 5H group. Restricting mother-young contact from the first week postpartum can permit an early collection of milk without major effects on kid growth, when compared with one daily milking in goats totally separated from their young.     

Scyphozoan jellyfish's mesoglea supports attachment, spreading and migration of anthozoans' cells in vitro

Mechanically and enzymatically dissociated cells from five anthozoan species were laid on seven substrates in vitro. Cells were taken from two sea anemones (Aiptasia sp. and Anemonia sulcata), a scleractinian coral (Stylophora pistillata) and two alcyonacean corals (Heteroxenia fuscescence and Nephthea sp). Substrates tested: glass (coverslips), plastic (uncoated tissue culture plates), type IV collagen, gelatin, fibronectin, mesoglea pieces from the scyphozoan jellyfish Rhopilema nomadica and acetic acid extract of jellyfish mesoglea. Except for the mesoglea pieces, cells did not respond to any one of the other substrates, retaining their rounded shape. Following contact with mesoglea pieces, cells attached and spread. Subsequently they migrated into the mesogleal matrix at a rate of 5-10 microm/h during the first 2-5 h. No difference was found between the behavior of cells from the five different cnidarian species.