Ultrastructure and surface topography of aggregates of human limb mesenchymal cells (HLM15) in vitro

Summary Tissue-like aggregates of human embryo fibroblasts can be created in vitro by limited aspiration of cells released from substrate during subcultivation. Aggregates increase in size, exhibit intercellular junctions, display a surface topography characteristic of cellular movement, elaborate an extracellular matrix and possess features of cellular death and phagocytosis. These cells, when introduced to a new culture environment, do not migrate away from one another as is common when a primary culture is started from tissue fragments. Instead, cells exhibit continued contact with each other, and develop complex junctional structures during that association. Cellular aggregates generated in this manner may provide a useful system for providing further information on cellular adhesion, intercellular communication, morphogenetic cell movements and the mechanisms of cell death. Dr. Kelley is the recipient of a Research Career Development Award (HD70407).     

A model for the kinetics of homotypic cellular aggregation under static conditions.

We present the formulation and testing of a mathematical model for the kinetics of homotypic cellular aggregation. The model considers cellular aggregation under no-flow conditions as a two-step process. Individual cells and cell aggregates 1) move on the tissue culture surface and 2) collide with other cells (or aggregates). These collisions lead to the formation of intercellular bonds. The aggregation kinetics are described by a system of coupled, nonlinear ordinary differential equations, and the collision frequency kernel is derived by extending Smoluchowski's colloidal flocculation theory to cell migration and aggregation on a two-dimensional surface. Our results indicate that aggregation rates strongly depend upon the motility of cells and cell aggregates, the frequency of cell-cell collisions, and the strength of intercellular bonds. Model predictions agree well with data from homotypic lymphocyte aggregation experiments using Jurkat cells activated by 33B6, an antibody to the beta 1 integrin. Since cell migration speeds and all the other model parameters can be independently measured, the aggregation model provides a quantitative methodology by which we can accurately evaluate the adhesivity and aggregation behavior of cells.     

Transformation-induced alterations in adhesion : Binding of pre-formed cell aggregates to cell layers

The formation of intercellular adhesions by mouse 3T3 cells and their SV40-transformed derivatives is analysed by measuring the binding of pre-formed aggregates of these cells to cell layers or to a plastic substratum. The rationale for this procedure is to reduce the effects of cell dissociation on quantitative assessments of adhesive interactions. The fibroblasts within the aggregates retain the growth characteristics these cells show in monolayer culture. The proportion of aggregates binding is independent of the number of aggregates added and changes with time in a manner consistent with a first-order process, allowing the percent aggregates binding per unit time to serve as a parameter of intercellular adhesion. The rate of binding in homologous adhesive interactions is slower than in heterologous ones, binding in 3T3SV interactions is slower than in 3T3 interactions, and binding to cellular substrata is slower than to plastic. Binding of 3T3SV aggregates is readily distinguished from binding of 3T3 aggregates by the presence of a brief lag in binding rate, the formation of irregular projections from the bound aggregate, and a differential effect on binding rates of varying the temperature or of treating a single reactant with glutaraldehyde. Thus, there are quantitative and qualitative differences in the adhesive interactions of normal and transformed cells. The distinct binding properties of 3T3SV aggregates and the greater binding rates in heterologous interactions may be relevant to the invasive behavior of transformed cells in vivo.     

Changes in Cell Walls Associated with Cell Separation in Suspension Cultures of Paul's Scarlet Rose

Studies of the formation and separation of cellular aggregatesin Paul's Scarlet rose suspension cultures have been conductedin an attempt to resolve the basis for intercellular adherence.During the typical growth cycle, cellular aggregates begin tofragment spontaneously at about day 12 in culture. This fragmentationis accompanied by a sharp decrease in the galactose contentof wall polysaccharides. At approximately this same time, thereis an increase in ß-glucosidase and ß-galactosidaseactivity associated with the cell surface. The presence of aß-glucosidase inhibitor, nojirimycin, inhibits thesurface ß-glycosidase activity, reduces the extentto which cell wall galactose is decreased, and prevents theseparation of cells. No other cell wall component examined changesin a manner consistent with cell separation. The results suggestthat a galactose-containing wall polysaccharide may be involvedin the maintenance of intercellular coherency and that partialremoval of this component may be causally related to the spontaneousseparation of cells in culture.     

An autocrine/paracrine loop linking keratin 14 aggregates to tumor necrosis factor alpha-mediated cytotoxicity in a keratinocyte model of epidermolysis bullosa simplex

Epidermolysis bullosa simplex (EBS) is a blistering cutaneous disease featuring protein aggregates. Here we investigate the molecular mechanisms linking protein aggregates to cell death in a cellular model of EBS in which HaCaT keratinocytes are transfected with plasmids expressing various mutant forms of keratin 14 (K14). In HaCaT cells, mutant K14 was found to form ubiquitinated protein aggregates that suppressed 20 S proteasome function instead of being degraded by 20 S proteasome. Keratinocytes with mutant K14-induced phosphorylation of the stress-activated kinase c-Jun, as well as up-regulation of unfolding protein Bip, indicates induction of endoplasmic reticulum stress. HaCaT cells were susceptible to apoptosis by activation of caspases-3, and -8, but not caspase-9 or -12. Tumor necrosis factor-alpha (TNFalpha) in the culture medium was increased in keratinocytes with mutant K14 compared with wild K14, and the addition of neutralizing anti-TNFalpha antibody to the culture medium rescued keratinocytes from cell death. Thus, TNFalpha release and the subsequent activation of the TNFalpha receptor by an autocrine/paracrine pathway links protein aggregates to cell death in this keratinocyte EBS cellular model. Furthermore, mutation in K14 reduced its affinity to TNFalpha receptor-associated death domain (TRADD), suggesting that the susceptibility of keratinocytes to caspase-8-mediated apoptosis is increased in mutated K14 because of impairment of the cytoprotective mechanism mediated by K14-TRADD interaction.     

CELL SORTING IN THE PRESENCE OF CYTOCHALASIN B

The ability of cytochalasin B to inhibit ruffled membrane activity and cellular locomotion of vertebrate cells in monolayer culture prompted its use to study the necessity for this kind of active cellular locomotion in cell sorting in heterotypic cell aggregates. Cell sorting was inhibited in chick embryo heart-pigmented retina aggregates but a remarkable degree of sorting did occur in neural retina-pigmented retina aggregates. In these experiments, the levels of cytochalasin B employed (5 or 10 µg/ml) are sufficient to inhibit completely locomotion of these cell types in monolayer culture. It is proposed that the degree of cell movement achieved during sorting in neural retina-pigmented retina aggregates in the presence of cytochalasin B is the result of changes in cell contact resulting from adhesive interaction of cells. The effect of cytochalasin B on the initial aggregation of dissociated cells was also tested. With the cell types used in this study (chick embryo neural retina and limb bud), aggregation was not affected for a period of several hours.     

Effect of cell-to-cell contact on in vitro deoxyribonucleic acid synthesis and apoptosis responses of bovine granulosa cells to insulin-like growth factor-I and epidermal growth factor

Follicle development is the result of a balanced ratio between cell proliferation and cell death. Previous studies demonstrated differential mitotic responses to insulin-like growth factor (IGF)-I and epidermal growth factor (EGF) of cumulus cells (CC) and mural granulosa cells (MGC). Because cell-to-cell contact seems to modulate the occurrence of programmed cell death, the present experiments investigated the role of cell association in mediating apoptosis and the mitogenic responses to these growth factors of CC and MGC. Cumulus cells were cultured either as intact cumulus-oocyte complexes (COC) or after dissociation with EGTA + sucrose, in the presence of 50 ng/ml IGF-I, 5 ng/ml EGF, or both. Mural granulosa cells from the same follicles were similarly cultured either as cell aggregates or as dissociated cells. Synthesis of DNA was assessed by measurement of [(3)H]thymidine incorporation during the last 6 h of a 24-h culture in TCM199. Percentages of cells undergoing apoptosis were determined immunohistochemically in intact COC and GC aggregates, before and after dissociation as well as after the culture period. Epidermal growth factor and IGF-I stimulated DNA synthesis in both cell types; however, EGF inhibited the action of IGF-I in intact COC but not in MGC. Compared to nondissociated cells, dissociation resulted in a reduction of the mitogenic response of CC to both growth factors and of MGC to EGF. Unlike the response of intact COC to combined treatment with the two growth factors, dissociated CC displayed additive responses to the two growth factors in combination. Addition of denuded oocytes to cultures of dissociated CC enhanced both basal and growth factor-stimulated DNA synthesis but did not restore the inhibitory effect of EGF on the IGF-I response characteristic of intact COC. A significant proportion of intact MGC aggregates underwent apoptosis after 24 h of culture, while no increase of apoptotic cells was observed in intact COC. A dramatic increase in the percentage of apoptotic cells was observed in both CC and MGC when cell-cell contact was interrupted, and EGF and IGF-I were able to partially prevent its occurrence. Taken together these data showed that CC and MGC exhibit qualitatively and quantitatively different responses to IGF-I when cultured in the presence of EGF both in terms of DNA synthesis and onset of apoptosis. Moreover, the disruption of cell-cell contact was a major factor reducing cell proliferation and inducing apoptosis among both subsets of GC.     

Globular and pre-fibrillar prion aggregates are toxic to neuronal cells and perturb their electrophysiology

Prion diseases are characterised at autopsy by neuronal loss and accumulation of amorphous protein aggregates and/or amyloid fibrils in the brains of humans and animals. These protein deposits result from the conversion of the cellular, mainly alpha-helical prion protein (PrP(C)) to the beta-sheet-rich isoform (PrP(Sc)). Although the pathogenic mechanism of prion diseases is not fully understood, it appears that protein aggregation is itself neurotoxic and not the product of cell death. The precise nature of the neurotoxic species and mechanism of cell death are yet to be determined, although recent studies with other amyloidogenic proteins suggest that ordered pre-fibrillar or oligomeric forms may be responsible for cellular dysfunction. In this study we have refolded recombinant prion protein (rPrP) to two distinct forms rich in beta-sheet structure with an intact disulphide bond. Here we report on the structural properties of globular aggregates and pre-fibrils of rPrP and show that both states are toxic to neuronal cells in culture. We show that exogenous rPrP aggregates are internalised by neuronal cells and found in the cytoplasm. We also measured the changes in electrophysiological properties of cultured neuronal cells on exposure to exogenous prion aggregates and discuss the implications of these findings.     

Intercellular adhesive selectivity. I. An improved assay for the measurement of embryonic chick intercellular adhesion (liver and other tissues)

An improved assay for measuring intercellular adhesive selectivity of embryonic chick liver cells is described. Three major improvements over earlier procedures are noted: (a) enhanced reproducibility of liver cell-liver cell aggregate adhesion (homotypic adhesion) was achieved; (b) 25-70% of the input cells adhered to the collecting aggregates during the course of routine experiments as compared to the 0.25% in earlier assays. This increase in cellular adhesion suggests that the observed cell pick-up is a characteristic of the majority of the dissociated liver cell population; (c) the rate of intercellular adhesion was increased 1,000-fold. The main feature of the assay is that it measures the tissue adhesive selectivities of the dissociated cell population. Studies were undertaken on three embryonic chick tissues (liver, neural retina, and mesencephalon) to determine the tissue selectivity of intercellular adhesion of these dissociated cell types. Some general properties of liver cell homotypic adhesion have been studied and are reported.     

Protein Folding, Misfolding, and Aggregation. Formation of Inclusion Bodies and Aggresomes

In this review the mechanisms of protein folding, misfolding, and aggregation as well as the mechanisms of cell defense against toxic protein aggregates are considered. Misfolded and aggregated proteins in cells are exposed to chaperone-mediated refolding and are degraded by proteasomes if refolding is impossible. Proteolysis-stable protein aggregates accumulate, forming inclusion bodies. In eucaryotic cells, protein aggregates form structures in the pericentrosomal area that have been termed "aggresomes". Formation of aggresomes in cells is a general cellular response to the presence of misfolded proteins when the degrading capacity of the cells is exceeded. The role of aggresomes in disturbance of the proteasomal system operation and in cellular death, particularly in the so-called "protein conformational diseases", is discussed.     

The topographical regulation of embryonic stem cell differentiation

The potential use of pluripotent stem cells for tissue repair or replacement is now well recognized. While the ability of embryonic stem (ES) cells to differentiate into all cells of the body is undisputed, their use is currently restricted by our limited knowledge of the mechanisms controlling their differentiation. This review discusses recent work by ourselves and others investigating the intercellular signalling events that occur within aggregates of mouse ES cells. The work illustrates that the processes of ES cell differentiation, epithelialization and programmed cell death are dependent upon their location within the aggregates and coordinated by the extracellular matrix. Establishment of the mechanisms involved in these events is not only of use for the manipulation of ES cells themselves, but it also throws light on the ways in which differentiation is coordinated during embryogenesis.     

Combination of microwell structures and direct oxygenation enables efficient and size‐regulated aggregate formation of an insulin‐secreting pancreatic β‐cell line

Spherical three‐dimensional (3D) cellular aggregates are valuable for various applications such as regenerative medicine or cell‐based assays due to their stable and high functionality. However, previous methods to form aggregates have shown drawbacks, being labor‐intensive, showing low productivity per unit area or volume and difficulty to form homogeneous aggregates. We proposed a novel strategy based on oxygen‐permeable polydimethylsiloxane (PDMS) honeycomb microwell sheets, which can theoretically supply about 80 times as much oxygen as conventional polystyrene culture dishes, to produce recoverable aggregates in controllable sizes using mouse insulinoma cells (MIN6‐m9). In 48 hours of culture, the PDMS sheets produced aggregates whose diameters were strictly controlled (?32, 60, 90, 150 and 280 mm) even at an inoculum density eight times higher (8.0×105 cells/cm²) than that of normal confluent monolayers (1.0×105 cells/cm²). Measurement of the oxygen tension near the cell layer and glucose/lactate analysis clearly showed that cells exhibit aerobic respiration on the PDMS‐based culture system. Glucose‐responsive insulin secretion of the recovered aggregates showed that the aggregates around 90 mm in diameter secreted the largest amounts of insulin. This confirmed the advantages of 3D cellular organization and the existence of a suitable aggregate size, above which excess organization leads to a decreased metabolic response. These results demonstrated that this microwell‐based PDMS culture system provides a promising method to form size‐regulated and better functioning 3D cellular aggregates of various kinds of cells with a high yield per surface area. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:178–187, 2014     

Electron microscopic observations on the formation of elastic fibres in cultures of aortic medial cells and adventitial cells

The formation of elastic fibres was observed in the cultured cells derived from the tunica media and the tunica adventitia of mouse aorta. Bundles of myofilaments with dense bodies were abundantly observed in the cytoplasm of the cultured medial cells, and numerous bundles of microfibrillar components were present in the intercellular spaces. Fine granules of approximately 50 nm in diameter were observed in the bundles of microfibrillar components. It was supposed that these fine granules of elastin fused with each other and formed elastic aggregates and then formed large elastic clumps. Numerous bundles of microfibrillar components were also present in the intercellular spaces of the cultured adventitial cells. Elastic aggregates were scarcely observed in the bundles of microfibrillar components. However, large elastic clumps as observed in the medial cell culture could not be found in the adventitial cell culture. It is suggested that the formation of large elastic clumps might be related to the sheet structures or lamellae of elastic fibres in the tunica media.     

Protein aggregates: an aging factor involved in cell death

In a previous study, we demonstrated the presence of protein aggregates in an exponentially grown Escherichia coli culture. In light of these observations, protein aggregates could be considered damage to cells that is able to pass from one generation to the next. Based on the assumption that the amount of aggregate protein could represent an aging factor, we monitored this amount in a bacterial culture during senescence. In doing so, we observed (i) a significant increase in the amount of aggregate protein over time, (ii) a proportional relationship between the amount of aggregate protein and the level of dead cells, (iii) a larger amount in dead cells than in culturable cells, (iv) a heterogeneous distribution of different amounts within a homogenous population of culturable cells entering stasis, and (v) that the initial amount of aggregate protein within a culturable population conditioned the death rate of the culture. Together, the results presented in this study suggest that protein aggregates indeed represent one aging factor leading to bacterial cell death.     

Intercellular bridges in vertebrate gastrulation

The developing zebrafish embryo has been the subject of many studies of regional patterning, stereotypical cell movements and changes in cell shape. To better study the morphological features of cells during gastrulation, we generated mosaic embryos expressing membrane attached Dendra2 to highlight cellular boundaries. We find that intercellular bridges join a significant fraction of epiblast cells in the zebrafish embryo, reaching several cell diameters in length and spanning across different regions of the developing embryos. These intercellular bridges are distinct from the cellular protrusions previously reported as extending from hypoblast cells (1-2 cellular diameters in length) or epiblast cells (which were shorter). Most of the intercellular bridges were formed at pre-gastrula stages by the daughters of a dividing cell maintaining a membrane tether as they move apart after mitosis. These intercellular bridges persist during gastrulation and can mediate the transfer of proteins between distant cells. These findings reveal a surprising feature of the cellular landscape in zebrafish embryos and open new possibilities for cell-cell communication during gastrulation, with implications for modeling, cellular mechanics, and morphogenetic signaling.     

Protection of human induced pluripotent stem cells against shear stress in suspension culture by Bingham plastic fluid

Suspension culture is an important method used in the industrial preparation of pluripotent stem cells (PSCs), for regenerative therapy and drug screening. Generally, a suspension culture requires agitation to keep PSC aggregates suspended and to promote mass transfer, but agitation also causes cell damage. In this study, we investigated the use of a Bingham plastic fluid, supplemented with a polysaccharide-based polymer, to preserve PSCs from cell damage in suspension culture. Rheometric analysis showed that the culture medium gained yield stress and became a Bingham plastic fluid, after supplementation with the polymer FP003. A growth/death analysis revealed that 2 days of aggregate formation and 2 days of suspension in the Bingham plastic medium improved cell growth and prevented cell death. After the initial aggregation step, whereas strong agitation (120 rpm) of a conventional culture medium resulted in massive cell death, in the Bingham plastic fluid we obtained the same growth as the normal culture with optimal agitation (90 rpm). This indicates that Bingham plastic fluid protected cells from shear stress in suspension culture and could be used to enhance their robustness when developing a large-scale.     

The effect of conditioned medium obtained from <Emphasis Type="Italic">Scenedesmus subspicatus</Emphasis> on suspension culture of <Emphasis Type="Italic">Silene vulgaris</Emphasis> (<Emphasis Type="Italic">Caryophyllaceae</Emphasis>)

The effect of culture filtrate (conditioned medium, CM) containing cell exudates obtained from green alga, Scenedesmus subspicatus, on cell suspension of dicotyledonous plant Silene vulgaris was examined. The addition of diluted CM to the modified MS medium, supplemented with dicamba and BAP, stimulates cell biomass production. The biomass was composed of association of single non-dividing cells, cells during mitosis stage and cellular aggregates. Silene cells began mitotic divisions earlier in the presence of CM in medium when compared to control treatments. Results of performed bioassay showed that some factor or factors released by green alga to the culture medium could be responsible for sustained proliferation of phylogenetically distant species cells. Although it is still unclear which culture constituent influenced most the mitotic response of Silene suspension, results point at versatile stimulatory character of green alga exudates in higher plant cell culture.     

Connexins and their channels in cell growth and cell death

Direct communication between cells, mediated by gap junctions, is nowadays considered as an indispensable mechanism in the maintenance of cellular homeostasis. In fact, gap junctional intercellular communication is actively involved in virtually all aspects of the cellular life cycle, ranging from cell growth to cell death. For a long time, it was believed that this was merely a result of the capacity of gap junctions to control the direct intercellular exchange of essential cellular messengers. However, recent data show that the picture is more complicated than initially thought, as structural precursors of gap junctions, connexins and gap junction hemichannels, can affect the cellular homeostatic balance independently of gap junctional intercellular communication. In this paper, we summarize the current knowledge concerning the roles of connexins and their channels in the control of cellular homeostasis, with the emphasis on cell growth and cell death. We also briefly discuss the role of gap junctional intercellular communication in carcinogenesis and the potential use of connexins as tools for cancer therapy.     

Embryonic tissues as elasticoviscous liquids: II. Direct evidence for cell slippage in centrifuged aggregates

Certain embryonic cell aggregates display both solid-like and liquid-like properties in organ culture. When centrifuged against solid substrata, these aggregates undergo sudden initial deformations followed by more gradual shape changes. Thus, they are either compoundviscoelastic solids (in which cells first stretch rapidly, then slowly) or elasticoviscous liquids (in which cells first quickly stretch, but then gradually slide by one another). To distinguish between these alternatives, we have examined cell shapes in centrifugally deformed chick liver aggregates. Light and electron micrographs show that initially stretched cells within flattening aggregates gradually reassume their original undisturbed shapes during prolonged centrifugation. Therefore, although the cells themselves may react to stretching forces like elastic solids, they slowly slip past one another within aggregates to relax stretching forces, endowing the aggregates with liquid properties. Slow cell slippage can account not only for temporary elastic solidity and viscous liquid flow in cell aggregates, but also for stress-free changes in the positions and configurations of tissues migrating within developing embryos. Regulation of cell slippage properties may shift the morphogenetic dependence of embryonic tissues in successive developmental stages from intracellular to intercellular force-generating mechanisms, or vice versa.