Selective adherence of neurons and glial cells from dissociated cerebral and spinal cord microcarrier cultures.

In stationary cultures of dissociated brain and spinal cord grown on microcarriers (MCs), the neuronal and ependymal cells attached to the MCs forming floating aggregates in which they grow in a three-dimensional pattern. The glial and meningeal elements on the contrary, tend to dissociate from the aggregates and adhere to the plastic dish where they divide to form a monolayer. This different behavior of CNS components is not observed in rotating cultures in which all CNS cells remain attached to the MCs and develop into mature floating structures. This cell separation in stationary MC-cultures which is documented here by SEM and immunocytochemistry, may be useful for analysis and evaluation of the metabolic biochemical events of each of the cellular components derived from the same culture.     

Extracellular Matrix Penetration by Epithelial Cells Is Influenced by Quantitative Changes in Basement Membrane Components and Growth Factors

We have previously shown that isolated mouse fetal choroid plexus epithelial (CPE) cells penetrate a basement membrane matrix (Matrigel) substratein vitroto form single-layered epithelial vesicles embedded within the matrix. To determine which properties of the matrix are important for inducing or permitting cells to penetrate the substrate and organize into multicellular vesicles we have made quantitative changes to the basement membrane components and growth factors in cell cultures. Matrigel diluted to 33 or 10% with a collagen I gel was not permissive to cell invasion, and CPE cells formed a polarized epithelial monolayer on the substrate surface which had ultrastructural characteristics similar to those of CPE vesicles. Cells in these monolayers proliferated more rapidly than cells in epithelial vesicles. When deliberately embedded within a 33 or 10% Matrigel matrix, CPE cells were able to form vesicles, indicating that a dilute matrix is nonpermissive to cell invasion but promotes epithelial polarization and organization into vesicles. Cells embedded within a 100% collagen I matrix did not proliferate or form epithelial vesicles and the majority of cells did not remain viable. Addition of laminin to the collagen I gel promoted cell adhesion and cell survival, but did not promote the formation of extensive monolayers on the substrate nor the formation of epithelial vesicles within the matrix. Cell invasion into the 33% Matrigel matrix was induced by addition of laminin, nidogen, or a laminin–nidogen complex to the substrate or by addition of TGFβ2 to the culture medium, but not TGFβ1 or PDGF. These studies show that CPE cells are sensitive to quantitative changes in matrix composition, which influences their survival and proliferation and also their ability to penetrate the matrix and organize into multicellular epithelial vesicles.     

Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes

Dissociated normal mammary epithelial cells from prelactating mice were plated on different substrates in various medium-serum-hormone combinations to find conditions that would permit maintenance of morphological differentiation. Cells cultured on floating collagen membranes in medium containing insulin, hydrocortisone and prolactin maintain differentiation through 1 month in culture. The surface cells form a continous epithelial pavement. Some epithelial cells below the surface layer rearrange themselves to form alveolus-like structures. Cells at both sites display surface polarization; microvilli and tight junctions are present at their medium-facing of luminal surface and a basal lamina separates the epithelial components from the gel and stromal cells. Occasional myoepithelial cells, characterized by myofilaments and plasmalemmmal vesicles, are identified at the basal surface of the secretory epithelium. In contrast, cells cultured on plastic, glass or collagen gels attached to Petri dishes form a confluent epithelial sheet showing surface polarization, but lose secretory and myoepithelial specializations. If these dedifferentiated cells are subsequently maintained on floating collagen membranes, they redifferentiate. There is little DNA synthesis in cells on collagen gels, in contrast to Petri-dish controls. Protein synthesis in cells on floating collagen membranes increases over TO values and remains constant through 7 days in culture whereas it decreases on attached gels; however, if the gels are freed to float, protein synthesis increases sharply and parallels that seen on floating membranes.     

Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes

Summary Dissociated normal mammary epithelial cells from prelactating mice were plated on different substrates in various medium-serum-hormone combinations to find conditions that would permit maintenance of morphological differentiation. Cells cultured on floating collagen membranes in medium containing insulin, hydrocortisone and prolactin maintain differentiation through 1 month in culture. The surface cells form a continuous epithelial pavement. Some epithelial cells below the surface layer rearrange themselves to form alveolus-like structures. Cells at both sites display surface polarization; microvilli and tight junctions are present at their medium-facing or luminal surface and a basal lamina separates the epithelial components from the gel and stromal cells. Occasinal myoepithelial cells, characterized by myofilaments and plasmalemmal vesicles, are identified at the basal surface of the secretory epithelium. In contrast, cells cultured on plastic, glass or collagen gels attached to Petri dishes form a confluent epithelial sheet showing surface polarization, but lose secretory and myoepithelial specializations. If these dedifferentiated cells are subsequently maintained on floating collagen membranes, they redifferentiate. There is little DNA synthesis in cells on collagen gels, in contrast to Petri-dish controls. Protein synthesis in cells on floating collagen membranes increases over T₀ values and remains constant through 7 days in culture whereas it decreases on attached gels; however, if the gels are freed to float, protein synthesis increases sharply and parallels that seen on floating membranes. The work was supported by USPHS Grants CA-05388 and CA-05045 from the National Cancer Institute, DHEW.     

Emergent cell and tissue dynamics from subcellular modeling of active biomechanical processes

Cells and the tissues they form are not passive material bodies. Cells change their behavior in response to external biochemical and biomechanical cues. Behavioral changes, such as morphological deformation, proliferation and migration, are striking in many multicellular processes such as morphogenesis, wound healing and cancer progression. Cell-based modeling of these phenomena requires algorithms that can capture active cell behavior and their emergent tissue-level phenotypes. In this paper, we report on extensions of the subcellular element model to model active biomechanical subcellular processes. These processes lead to emergent cell and tissue level phenotypes at larger scales, including (i) adaptive shape deformations in cells responding to slow stretching, (ii) viscous flow of embryonic tissues, and (iii) streaming patterns of chemotactic cells in epithelial-like sheets. In each case, we connect our simulation results to recent experiments.     

Immunology of gangliosides

This review discusses the immunology of gangliosides from the perspective of tumor, neuronal and general immunology. Antiganglioside antibodies in human sera are invariably IgM and are found in healthy individuals. Their titers decline with age. Persistent high titer of IgM is associated with several diseases, particularly neuropathies. Membrane-bound gangliosides are important tumor-associated antigens and targets for immune attack. Cells enriched with gangliosides can be used as cancer vaccines. Efficacy of these vaccines depends on the viability of whole cells, integrity of the cell membranes, adjuvants and topography of the tumor-associated antigens. The role of antiganglioside IgM is to eliminate the immunosuppressive gangliosides shed from tissues during ageing, degeneration of neural and extraneural tissues, and tumor growth and necrosis. In addition, in vitro observations with human and murine monoclonal antibodies suggest that they are capable of complement dependent cytotoxicity and apoptosis.     

Fibroblast quiescence in floating collagen matrices: decrease in serum activation of MEK and Raf but not Ras

Fibroblasts synthesize, organize, and maintain connective tissues during development and in response to injury and fibrotic disease. Studies on cells in three-dimensional collagen matrices have shown that fibroblasts switch between proliferative and quiescence phenotypes, depending upon whether matrices are attached or floating during matrix remodeling. Previous work showed that cell signaling through the ERK pathway was decreased in fibroblasts in floating matrices. In the current research, we extend the previous findings to show that serum stimulation of fibroblasts in floating matrices does not result in ERK translocation to the nucleus. In addition, there was decreased serum activation of upstream members of the ERK signaling pathway, MEK and Raf, even though Ras became GTP loaded. The findings suggest that quiescence of fibroblasts in floating collagen matrices may result from a defect in Ras coupling to its downstream effectors.     

Cell Volume Fluctuations in MDCK Monolayers

Cells moving collectively in tissues constitute a form of active matter, in which collective motion depends strongly on driven fluctuations at the single-cell scale. Fluctuations in cell area and number density are often seen in monolayers, yet their role in collective migration is not known. Here we study density fluctuations at the single- and multicell level, finding that single-cell volumes oscillate with a timescale of 4 h and an amplitude of 20%; the timescale and amplitude are found to depend on cytoskeletal activity. At the multicellular scale, density fluctuations violate the central limit theorem, highlighting the role of nonequilibrium driving forces in multicellular density fluctuations.     

Mechanism of migration of olfactory bulb interneurons

Neuroblasts in the subventricular zone of the walls of the lateral ventricle in the brain of young and adult rodents migrate into the olfactory bulb where they differentiate into local interneurons. These cells move closely associated with each other, forming chains without radial glial or axonal guidance. The migrating neuroblasts express PSA-NCAM on their surface and PSA residues are crucial for cell-cell interaction during chain migration. This migration occurs throughout the lateral wall of the lateral ventricle, where the precursors form an extensive network of chains. Cells remain organized as chains until they reach the olfactory bulb, where they disperse organized as chains until they reach the olfactory bulb, where they disperse radially as individual cells. Chain migration defines a novel form of neuronal precursor translocation which is based on homotypic interactions between cells.     

Establishment of a peritubular myoid-like cell line and interactions between established testicular cell lines in culture

Established cell lines and primary cultures derived from somatic cells of the testis have been used to study cell-cell interactions. Primary cultures of Sertoli cells or Sertoli-derived cell lines from the mouse (TM4) and rat (TR-ST) will aggregate when plated on monolayers of primary cultures of peritubular myoid cells or a rat (TR-M) cell line which has many properties of peritubular myoid cells. Time-lapse cinematography and scanning and transmission electron microscopy reveal that Sertoli cells formed aggregates after 1 day in coculture, display surface activity and move on the monolayer. When these aggregates touch one another, they rapidly combine. By the 4th day of culture, spherical aggregates are composed of 50 to 200 cells. They do not display surface activity or movement on the myoid monolayer. On the 5th and 6th day of culture most spherical aggregates have flattened to form dome-shaped aggregates in close association with the monolayer. Cells in the aggregates are characterized by long microvilli and some ruffles. In large aggregates, cells sometimes form close associations within the aggregates although junctions are seldom observed. Sertoli-derived cell lines will not aggregate on monolayers of Leydig-derived (TM3) or testicular endothelial-derived (TR-1) cell lines. Neither TM3 nor TR-1 cells will aggregate when plated on myoid monolayers. The TR-M cells produced an extensive extracellular matrix beneath the cells which contains collagen, an amorphous globular material resembling elastin and a fibrous noncollagenous component. Sertoli cells plated on this matrix will not aggregate. Thus the aggregation of Sertoli cells on myoid cell monolayers is cell type, but not species dependent and not determined solely by extracellular matrix components produced by TR-M cells.     

Neuronal nicotinic alpha-bungarotoxin sites

At the vertebrate neuromuscular junction and in the electroplax of eel and electric fish, the nicotinic alpha-bungarotoxin site and the nicotinic receptor involved in synaptic transmission are very tightly coupled and, indeed, appear to be the same molecular component. On the other hand, the nature of the relationship between the nicotinic receptor mediating synaptic events and the nicotinic alpha-bungarotoxin binding site in nervous tissue has been a matter of controversy over the last few years. Experimental studies have been accumulating which suggest that in many neuronal tissues these two components are distinct molecular entities with their own unique regulation. However, it also appears that in other nervous tissues, possibly in species lower on the evolutionary scale, the toxin binding site is part of the nicotinic receptor. An evaluation of all available evidence would point to the conclusion that, in neuronal tissues, the nicotinic acetylcholine receptor involved in synaptic events and the nicotinic alpha-bungarotoxin site can exist both in a tightly coupled form and one in which the two sites are mutually distinct. The possible physiological significance of the nicotinic alpha-bungarotoxin site is discussed in light of current experimental data. Evidence is available which may imply that the alpha-toxin site, whether it is present as a distinct entity or in association with the nicotinic acetylcholine receptor, is involved in trophic or growth related activities, as well as in other cellular functions. The possibility of an endogenous ligand for the nicotinic alpha-bungarotoxin site is also discussed.     

Importance of cell aggregation for expression of liver functions and regeneration demonstrated with primary cultured hepatocytes

Adult rat hepatocytes aggregated to form floating multicellular spheroids when cultured in Primaria dishes, which have a positively charged surface, in serum-free Williams' medium E (WE) supplemented with insulin and epidermal growth factor (EGF). These hormones were essential for maintenance of the spheroids, whereas the size of the spheroids depended on the inoculum cell density. The spheroids retained in vivo levels of expressions of albumin and glucokinase and synthesized scarcely any DNA even in the presence of insulin and EGF. On transfer to type I collagen-coated dishes, the spheroids gradually disaggregated and the cells formed monolayers, in which the expressions of albumin and glucokinase were suppressed and DNA synthesis and hexokinase activity were increased. DNA synthesis of hepatocytes in monolayer culture was maximal 24 hr after transfer of the spheroids, ～80% of the hepatocyte nuclei were labelled with bromodeoxyuridine during culture for 48 hr, and the mitotic index was ～70% after 60 hr. These results suggest that, in spheroids, hepatocytes remained in the G₀ phase, but that when they formed monolayers, they progressed to the G₁ phase and proceeded through the cell cycle in the presence of insulin and EGF. This work shows that the cell cycle of hepatocytes in culture can be manipulated by providing conditions for quiescence as spheroids or growth as monolayers and that the shape of hepatocytes is important for regulating their growth and liver-specific functions. © 1993 Wiley-Liss, Inc.     

BAR domains, amphipathic helices and membrane-anchored proteins use the same mechanism to sense membrane curvature

The internal membranes of eukaryotic cells are all twists and bends characterized by high curvature. During recent years it has become clear that specific proteins sustain these curvatures while others simply recognize membrane shape and use it as “molecular information” to organize cellular processes in space and time. Here we discuss this new important recognition process termed membrane curvature sensing (MCS). First, we review a new fluorescence-based experimental method that allows characterization of MCS using measurements on single vesicles and compare it to sensing assays that use bulk/ensemble liposome samples of different mean diameter. Next, we describe two different MCS protein motifs (amphipathic helices and BAR domains) and suggest that in both cases curvature sensitive membrane binding results from asymmetric insertion of hydrophobic amino acids in the lipid membrane. This mechanism can be extended to include the insertion of alkyl chain in the lipid membrane and consequently palmitoylated and myristoylated proteins are predicted to display similar curvature sensitive binding. Surprisingly, in all the aforementioned cases, MCS is predominantly mediated by a higher density of binding sites on curved membranes instead of higher affinity as assumed so far. Finally, we integrate these new insights into the debate about which motifs are involved in sensing versus induction of membrane curvature and what role MCS proteins may play in biology.     

Contact-inhibited chemotaxis in de novo and sprouting blood-vessel growth

Blood vessels form either when dispersed endothelial cells (the cells lining the inner walls of fully formed blood vessels) organize into a vessel network (vasculogenesis), or by sprouting or splitting of existing blood vessels (angiogenesis). Although they are closely related biologically, no current model explains both phenomena with a single biophysical mechanism. Most computational models describe sprouting at the level of the blood vessel, ignoring how cell behavior drives branch splitting during sprouting. We present a cell-based, Glazier-Graner-Hogeweg model (also called Cellular Potts Model) simulation of the initial patterning before the vascular cords form lumens, based on plausible behaviors of endothelial cells. The endothelial cells secrete a chemoattractant, which attracts other endothelial cells. As in the classic Keller-Segel model, chemotaxis by itself causes cells to aggregate into isolated clusters. However, including experimentally observed VE-cadherin-mediated contact inhibition of chemotaxis in the simulation causes randomly distributed cells to organize into networks and cell aggregates to sprout, reproducing aspects of both de novo and sprouting blood-vessel growth. We discuss two branching instabilities responsible for our results. Cells at the surfaces of cell clusters attempting to migrate to the centers of the clusters produce a buckling instability. In a model variant that eliminates the surface-normal force, a dissipative mechanism drives sprouting, with the secreted chemical acting both as a chemoattractant and as an inhibitor of pseudopod extension. Both mechanisms would also apply if force transmission through the extracellular matrix rather than chemical signaling mediated cell-cell interactions. The branching instabilities responsible for our results, which result from contact inhibition of chemotaxis, are both generic developmental mechanisms and interesting examples of unusual patterning instabilities.     

Insulin-like growth factor-I (IGF-I) stimulates protein synthesis and collagen gene expression in monolayer and lattice cultures of fibroblasts.

Fibroblasts cultivated in three-dimensional tissue-like matrices are characterized by a slowed metabolism and a decrease of protein synthesis, unless they are submitted to physical tensions. We checked the effects of insulin like growth factor-I (IGF-I), known as a potent stimulator of mitogenesis and protein synthesis for many cell types, in various models of cultures: confluent monolayers, collagen lattices, non-retracting or retracting fibrin lattices. IGF-I (1-100 ng.ml-1) had no effect on cell divisions in lattice cultures. It was able to stimulate collagen lattice retraction when the medium was supplemented with low concentrations of serum. IGF-I at 10 or 100 ng.ml-1 stimulated collagen and non-collagen syntheses in all culture systems, but stimulation of collagen synthesis only began at the highest concentration (100 ng.ml-1) in retracted lattices. Northern blot and dot-blot analyses of mRNAs extracted from monolayer cultures of fibroblasts showed that IGF-I stimulated pro alpha 1(I) collagen synthesis at the pretranslational level. Cycloheximide (7.5 micrograms.ml-1) completely inhibited pro alpha 1(I) collagen gene expression induced by IGF-I. These results show that IGF-I is a potent stimulus for protein synthesis and collagen gene expression in monolayers and tridimensional cultures of fibroblasts, but that it exerts no mitogenic activity in tridimensional lattices. Synergistic associations of IGF-I with other growth factors will have to be found in order to reverse the quiescent status of fibroblasts in lattices.     

Characteristics of human embryonic neuronal cells procured by non-enzyme method

Isolation and culturing of human neuronal progenitor cells is of significant value for both fundamental research and therapeutic purposes. In this work, human embryonic neuronal cells were characterized as a heterogeneous population of progenitor cells with various differentiation potentials. During in vitro culturing the cells are capable of re-inoculating, proliferating, differentiating and migrating. While differentiating, these cells form neurons and glial cells. The present research demonstrates that depending upon the culturing conditions the embryonic neuronal cells may either form floating aggregates (incubation with embryonic serum), attach (incubation without serum), proliferate, or form neurospheres. Besides, peculiarities of aggregate differentiation during their incubation under various media are described.     

Further portrayal of epithelial monolayers emergent de novo from extirpated ascidians palleal buds

Astogeny in botryllid ascidians is executed by highly synchronized, repeated development and death cycles operating simultaneously on three coexisting asexually derived generations: zooids, primary buds, and secondary buds. In this study, we validated the fact that surgically removed blastogenic stage “D” primary buds cultured under in vitro conditions, away from any discrete colonial regulatory cues, exhibit intrinsic phenomena that are probably masked by astogenic controls. They produce de novo epithelial monolayers (EM), extending their lifespan from a few days to 1 mo and up to 5 mo when floating in the medium. Enhanced EM formation was documented when fibroblast growth factor (FGF) was added after at least 24 h incubation in FGF-free medium. Surprisingly, with no FGF administration, while intact isolated buds did not develop any EM, injured buds developed EM in half of the cases. Working on actin, PL10, FGF-R, P-MEK, MAP-kinase, and cadherin expressions, we documented that extirpated buds and monolayers are very active on the molecular/biochemical levels, revealing various cells and cellular organelle stains and rapid changes in the protein levels along a daily basis. Cells situated in the center of the monolayers stained differently for some proteins than peripheral cells. Cumulatively, results showed that flattened attached monolayers, as well as free-floating stage “D” buds, are highly active, not only exhibiting differential expressions of various proteins along incubation, but are also highly responsive to physical damages. These results establish a novel in vitro model system for epithelial cell development and senescence, revealing surprising rejuvenation and extended lifespan phenomena.     

Network-based functional modeling of genomics, transcriptomics and metabolism in bacteria

Molecular entities present in a cell (mRNA, proteins, metabolites,…) do not act in isolation, but rather in cooperation with each other to define an organisms form and function. Their concerted action can be viewed as networks of interacting entities that are active under certain conditions within the cell or upon certain environmental signals. A main challenge in systems biology is to model these networks, or in other words studying which entities interact to form cellular systems or accomplish similar functions. On the contrary, viewing a single entity or an experimental dataset in the light of an interaction network can reveal previous unknown insights in biological processes. In this review we give an overview of how integrated networks can be reconstructed from multiple omics data and how they can subsequently be used for network-based modeling of cellular function in bacteria.     

Synthesis of novel calcium-dependent proteins associated with mammary epithelial cell migration and differentiation

A class of proteins from mouse mammary epithelial cells has been isolated which, like the calcium-binding protein calmodulin (CaM), binds to phenothiazine in a calcium-dependent manner. These proteins do not bind to phenothiazine through binding to CaM; we infer that they are calcium-binding proteins, and that they may be related to the similarly isolated 'calcimedins' of Moore, P D & Dedman, J, J biol chem 257 (1982) 9663 [8]. In primary cultures of mouse mammary cells on collagen gels, synthesis of certain of these proteins is associated with the spreading of cells to form monolayers; failure of cells to spread and differentiate, through omission of serum from culture medium, results in the inhibition of calcium-binding protein synthesis, with the exception of CaM and a 15 kD species. The CaM/15 kD pair are prominent during all phases of culture, and are secreted during the secretory differentiation phase of culture (floating gels). We propose that these calcium-binding proteins play a specific role in the motility of mammary epithelial cells and that they may also be involved in mammary secretory differentiation.     

Mesenchymal stem cells neuronal differentiation ability: a real perspective for nervous system repair?

Mesenchymal Stem Cells (MSCs) are a bone marrow-derived population present in adult tissues that possess the important property of dividing when called upon and of differentiating into specialized cells. The evidence that MSCs were able to transdifferentiate into specialized cells of tissues different from bone marrow, in particular into nervous cells, opened up the possibility of using MSCs to substitute damaged neurons, that are normally not replaced but lost, in order to repair the Nervous System. The first neuronal differentiation protocols were based on the use of a mixture of toxic drugs which induced MSCs to rapidly acquire a neuronal-like morphology with the expression of specific neuronal markers. However, many subsequent studies demonstrated that the morphological and molecular modifications of MSCs were probably due to a stress response, rather than to a real differentiation into neuronal cells, thus throwing into question the possible use of MSCs to repair the nervous system. Currently, some papers are suggesting again that it may be possible to induce neuronal differentiation of MSCs by using several differentiation protocols, and by accompanying the morphological evidence of differentiation with functional evidence, thus demonstrating that MSC-derived cells not only seem to be neurons, but that they also function like neurons. In this review, we have attempted to shed light on the capacity of MSCs to genuinely differentiate into nervous cells, and to identify the most reliable protocols for obtaining neurons from MSCs for nervous system repair.