Substratum attachment of embryonic mesoderm cells in culture

The cell-substratum adhesive characteristics of cultured chick embryo primary mesoderm cells have been examined by interference reflection microscopy and transmission electron microscopy under various conditions. Correlations were drawn between the type of adhesion and the degree of motility shown by the cells. During the rapid spreading and motility of cells cultured on fibronectin-containing substrata, focal contacts (10 to 15-nm gap) were rare and close contacts (about 30-nm gap) were predominant. By contrast, when the cells were immobile, after 5 d in culture, extensive focal contacts were present, together with stress fibers. The results indicate that tight cell-substratum contact is incompatible with rapid cell motility and that fibronectin acts by inducing the formation of close contacts rather than focal contacts.     

Combination of small molecules enhances differentiation of mouse embryonic stem cells into intermediate mesoderm through BMP7-positive cells

Embryonic stem cells (ESCs) are potentially powerful tools for regenerative medicine and establishment of disease models. The recent progress in ESC technologies is noteworthy, but ESC differentiation into renal lineages is relatively less established. The present study aims to differentiate mouse ESCs (mESCs) into a renal progenitor pool, the intermediate mesoderm (IM), without addition of exogenous cytokines and embryoid formation. First, we treated mESCs with a combination of small molecules (Janus-associated tyrosine kinase inhibitor 1, LY294002, and CCG1423) and differentiated them into BMP7-positive cells, BMP7 being the presumed inducing factor for IM. When these cells were cultured with adding retinoic acid, expression of odd-skipped related 1 (Osr1), which is essential to IM differentiation, was enhanced. To simplify the differentiation protocol, the abovementioned four small molecules (including retinoic acid) were combined and added to the culture. Under this condition, more than one-half of the cells were positive for Osr1, and at the same time, Pax2 (another IM marker) was detected by real-time PCR. Expressions of ectodermal marker and endodermal marker were not enhanced, while mesodermal marker changed. Moreover, expression of genes indispensable to kidney development, i.e., Lim1 and WT1, was detected by RT-PCR. These results indicate the establishment of a specific, effective method for differentiation of the ESC monolayer into IM using a combination of small molecules, resulting in an attractive cell source that could be experimentally differentiated to understand nephrogenic mechanisms and cell-to-cell interactions in embryogenesis.     

Transferrin and iron requirements of embryonic mesoderm cells cultured in hydrated collagen matrices

Summary Very early embryonic mesoderm cells were taken from the primitive streak-stage chick embryo and cultured in a matrix of type I collagen in the presence of serum. Previous work has shown that under these conditions cells do not leave the explant and move in the collagen in the absence of supplemented avian transferrin. Cells explanted onto tissue culture plastic in the presence of serum do not require this transferrin supplement. These observations were investigated further by culturing cells in collagen in the presence of the lipophilic iron chelator, ferric pyridoxal isonicotinoyl hydrazone (FePIH), which can replace transferrin as an iron-delivery agent. Under conditions in which FePIH could effectively stimulate chick embryo myoblast growth, no such long-term stimulation was obtained with the early mesoderm cells in collagen. This suggested that for mesoderm cells, FePIH could not replace transferrin. Antibody to the transferrin receptor and to transferrin itself inhibited growth of myoblasts in collagen and on plastic, and of mesoderm cells in collagen. Mesoderm cells on plastic, however, were refractory to the presence of the antibody directed to the receptor and seemed to show a low dependency on transferrin-delivered iron under these conditions, inasmuch as antiserum to transferrin itself only caused a partial inhibition of outgrowth. The results suggest that mesoderm cells in collagen require transferrin for both iron uptake and for another unspecified function. It is consistent with the results to propose that transferrin binding might modulate the cells' attachment to collagen, thus influencing outgrowth. The distribution of the actin cytoskeleton in mesoderm cells actively migrating in collagen, such as in the presence of transferrin, suggests a stronger attachment to the collagen than nonmigrating cells. This work was supported by an operating grant from the Medical Research Council of Canada.     

The role of cellular adhesion molecules in virus attachment and entry

As obligate intracellular parasites, viruses must traverse the host-cell plasma membrane to initiate infection. This presents a formidable barrier, which they have evolved diverse strategies to overcome. Common to all entry pathways, however, is a mechanism of specific attachment to cell-surface macromolecules or ‘receptors’. Receptor usage frequently defines viral tropism, and consequently, the evolutionary changes in receptor specificity can lead to emergence of new strains exhibiting altered pathogenicity or host range. Several classes of molecules are exploited as receptors by diverse groups of viruses, including, for example, sialic acid moieties and integrins. In particular, many cell-adhesion molecules that belong to the immunoglobulin-like superfamily of proteins (IgSF CAMs) have been identified as viral receptors. Structural analysis of the interactions between viruses and IgSF CAM receptors has not shown binding to specific features, implying that the Ig-like fold may not be key. Both proteinaceous and enveloped viruses exploit these proteins, however, suggesting convergent evolution of this trait. Their use is surprising given the usually occluded position of CAMs on the cell surface, such as at tight junctions. Nonetheless, the reason for their widespread involvement in virus entry most probably originates in their functional rather than structural characteristics.     

Modeling human extraembryonic mesoderm cells using naive pluripotent stem cells

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Electric discharge plasmas influence attachment of cultured CHO K1 cells

Non-thermal plasmas can be generated by electric discharges in gases. These plasmas are reactive media, capable of superficial treatment of various materials. A novel non-thermal atmospheric plasma source (plasma needle) has been developed and tested. Plasma appears at the end of a metal pin as a submillimetre glow. We investigate the possibility of applying the plasma needle directly to living tissues; the final goal is controlled cell treatment in microsurgery. To resolve plasma effects on cells, we study cultured Chinese hamster ovarian cells (CHO-K1) as a model system. When these are exposed to the plasma, instantaneous detachment of cells from the surface and loss of cell-cell interaction is observed. This occurs in the power range 0.1-0.2 W. Cell viability is assessed using propidium iodide (PI) and cell tracker green (CTG) fluorescent staining utilizing confocal laser scanning microscopy (CLSM). Detached cells remain alive. Use of higher doses (plasma power >0.2 W) results in cell necrosis. In all cases, plasma-influenced cells are strictly localized in submillimetre areas, while no reaction in surrounding cells is observed. Due to its extreme precision, plasma treatment may be applicable in refined tissue modification.     

Unidirectional and phase-gated signaling synchronizes murine presomitic mesoderm cells

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Dual functions of the heartless fibroblast growth factor receptor in development of the Drosophila embryonic mesoderm

The Drosophila embryonic mesoderm forms by invagination of the ventral-most blastoderm cells. Subsequent development of this germ layer involves the dorsolateral migration of the internalized cells and expansion by cell division, followed by the specification of particular cell fates through the coordinate actions of both intrinsic and extrinsic regulatory mechanisms. The latter include several intercellular signals that function across germ layers. These processes combine to generate a diversity of mesodermal subtypes, including the cardial and pericardial cells of the heart or dorsal vessel, a complete set of somatic muscle founders each with its unique identity, a population of cells that form the visceral musculature, and other cells that develop into hemocytes and the fat body. Here, we review recent evidence for the involvement of a fibroblast growth factor receptor (FGFR) encoded by the heartless (htl) gene in early directional migration of the Drosophila mesoderm. In addition, we provide new data that 1) demonstrate a second role for Htl in promoting the specification of the precursors to certain cardiac and somatic muscle cells in the Drosophila embryo, independent of its cell migration function, 2) suggest that Ras and at least one other signal transduction pathway act downstream of Htl, and 3) establish a functional relationship between the Ras pathway and Tinman (Tin), a homeodomain factor that is essential for specifying some of the same dorsal mesodermal cells that are dependent on Htl. Finally, parallels between requirements for FGFR signaling in Drosophila and vertebrate mesoderm development are considered. Dev. Genet. 22:212–229, 1998. © 1998 Wiley-Liss, Inc.     

Engineering tissue from human embryonic stem cells

Recent advances in human embryonic stem cell (hESC) biology now offer an alternative cell source for tissue engineers, as these cells are capable of proliferating indefinitely and differentiating to many clinically relevant cell types. Novel culture methods capable of exerting spatial and temporal control over the stem cell microenvironment allow for more efficient expansion of hESCs, and significant advances have been made toward improving our understanding of the biophysical and biochemical cues that direct stem cell fate choices. Effective production of lineage specific progenitors or terminally differentiated cells enables researchers to incorporate hESC derivatives into engineered tissue constructs. Here, we describe current efforts using hESCs as a cell source for tissue engineering applications, highlighting potential advantages of hESCs over current practices as well as challenges which must be overcome.     

Development of embryonic stem cells in recombinant kidneys

Embryonic stem cells (ESC) are self-renewing and can generate all cell types during normal development. Previous studies have begun to explore fates of ESCs and their mesodermal derivatives after injection into explanted intact metanephric kidneys and neonatal kidneys maturing in vivo. Here, we exploited a recently described recombinant organ culture model, mixing fluorescent quantum dot labeled mouse exogenous cells with host metanephric cells. We compared abilities of undifferentiated ESCs with ESC-derived mesodermal or non-mesodermal cells to contribute to tissue compartments within recombinant, chimeric metanephroi. ESC-derived mesodermal cells downregulated Oct4, a marker of undifferentiated cells, and, as assessed by locations of quantum dots, contributed to Wilms’ tumor 1-expressing forming nephrons, synaptopodin-expressing glomeruli, and organic ion-transporting tubular epithelia. Similar results were observed when labeled native metanephric cells were recombined with host cells. In striking contrast, non-mesodermal ESC-derived cells strongly inhibited growth of embryonic kidneys, while undifferentiated ESCs predominantly formed Oct4 expressing colonies between forming nephrons and glomeruli. These findings clarify the conclusion that ESC-derived mesodermal cells have functional nephrogenic potential, supporting the idea that they could potentially replace damaged epithelia in diseased kidneys. On the other hand, undifferentiated ESCs and non-mesodermal precursors derived from ESCs would appear to be less suitable materials for use in kidney cell therapies.     

Inability of mesoderm cells to locomote on the modified free surface of epithelial cell sheets in vitro

Summary Previous work has shown that when chick embryo mesoderm tissue is seeded onto the free, dorsal surface of established sheets of embryonic epithelial endoblast, the former penetrates the latter and spreads on the underlying artificial substratum. In this work, the surface charge on the epithelial sheet has been altered, prior to seeding the mesoderm, to ascertain whether such a change could alter the behavior of the mesoderm with respect to the free surface of the epithelium. Charge alteration was accomplished using the polycations, poly-l-lysine and dilysine. Surface charge characteristics were examined ultrastructurally using cationized and anionized ferritin. Results showed that although surface charge changes were detectable, there was no difference in the behavior of the mesoderm with respect to the endoblast. Neuraminidase did not detectably affect the epithelial surface charge. These results are consistent with the view that changes in substratum surface charge are not necessarily correlated with changes in adhesiveness.     

The effect of cytochalasin B on chondrogenesis in chick limb-bud mesoderm cells grown in vitro

Summary Cytochalasin B (CB) has been shown to have many biological effects on cultured cells. We report that an initial 48-hr treatment of freshly plated chick embryo limb mesoderm cells with CB irreversibly inhibits chondrogenesis. A slight inhibition in the amount of matrix is seen when limb cells are allowed to grow in culture for 24 hr prior to treatment for the second 24 hr of culture. If the cells are allowed to plate-out and grow for 48 hr or longer prior to being treated with CB for 24 hr, the amount of matrix produced is essentially the same as that seen in the controls. However, if the initial 48-hr culture period is followed by a 48- or 72-hr treatment, chondrogenesis is reduced, but not to the same extent as that seen in cultures treated for the first 48 or 72 hr. The irreversible inhibition of chrondrogenesis does not appear to be due to irreversible inhibition of protein synthesis or hexose uptake because, although these are reduced during treatment, they return to control levels within 48 hr following the removal of the drug. We cannot mimic the effect of CB treatment using glucose-deficient medium, thereby eliminating the possiblity that a critical glucose level is necessary to permit chondrogenesis. Multinucleation of limb cells treated with CB is reversed within 4 to 7 days following the removal of the drug. Therefore multinucleation alone is probably not responsible for the CB effect on chondrogenesis. However, other subtle permanent changes may occur during the period of multinucleation which result in the irreversible inhibition of chondrogenesis. This work was supported in part by a grant to R. A. F. from the North Carolina United Way and a grant to C. L. P. from the General Research Support Grant RR-5404 from the National Institutes of Health. A portion of these results were presented at the 28th Annual Meeting of the Tissue Culture Association, New Orleans, Louisiana, 1977.     

Graded mesoderm assembly governs cell fate and morphogenesis of the early mammalian heart

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Adhesion of sea-urchin embryonic cells to substrata coated with cell adhesion molecules

Summary— A cell-to-substratum adhesion assay is developed to study the adhesion of sea-urchin embryonic cells to coated substrata. The involvement in this process of both carbohydrate and protein molecules is reported. Concanavalin A (Con A) increases the attachment of cells to the substratum in a dose-dependent manner and this effect is completely abolished when the incubation is carried out in the presence of the specific monocarbohydrate Con A-inhibitor, α-methyl-d -mannoside. A Con A-mediated enhancement of cell-to-substratum adhesion was also detected on cells deprived of toposome, a glycoprotein complex responsible for cell-to-cell adhesion. The involvement of other molecules as well as toposome in the process of cell-to-substratum adhesion is also investigated. Results of these in vitro experiments indicate that all the molecules tested contribute to the process of cell-to-substratum adhesion.     

Detection of teratogenic compounds using differentiating embryonic cells in culture

Summary Methods are described for screening for teratogenic compounds using differentiating neural crest and prechondrogenic limb bud mesenchyme cells in culture. Substances to be tested are either added directly to the culture medium or are combined in a dialysis bag with the postmitochondrial fraction from rat liver and certain cofactors. In the latter case, the compound and its metabolites are gradually released into the medium from the dialysis bag. The results obtained with 14 compounds demonstrate a positive relationship between teratogenicity in vivo and alterations in the growth or the differentiation of the cultured cells.     

Surface coating materials regulates the attachment and differentiation of mouse embryonic stem cell derived embryoid bodies into mesoderm at culture conditions

Cytotechnology - Pluripotent stem cells as a promising cell source with unlimited proliferation and differentiation capacity hold great promise for cell-based therapies in regenerative medicine....