Sponge Cells Research Articles

Cellular localisation of secondary metabolites isolated from the Caribbean sponge Plakortis simplex

The Caribbean sponge, Plakortis simplex, is known to contain a large array of secondary metabolites, including the antimalarial polyketide plakortin, several unusual glycolipids, and some hopanoids, which closely resemble typical bacterial metabolites. The hypothesis that they could be products of bacterial metabolism was tested by localizing specific metabolites in cells using physical separation of sponge cells, bacterial symbionts and supernatant by differential centrifugation. The obtained fractions were analysed separately for the typical P. simplex metabolites by NMR and mass spectrometry, and most of them were shown to be present in the bacterial cells but not in the sponge cells. In addition, PCR screening showed that the biosynthetic pathway for glycosphingolipids was present in the bacterial cells. Isolation of a Sphingomonas strain PS193 from P. simplex and subsequent glycosphingolipid analysis resulted in the detection of a known glycosphingolipid, GSL-1, that did, however, not match the glycosphingolipid profile of P. simplex. Therefore, it is unlikely that Sphingomonas strain PS193 is an abundant member of the microbial community associated with P. simplex. Other glycosphingolipid producing bacteria in P. simplex remain to be identified. In conclusion, this study provides experimental evidence that the glycolipids and hopanoids and possibly also the polyketide plakortin are produced by microbial symbionts rather than the sponge from which the metabolites were originally isolated.

Enhanced proliferation and osteogenic differentiation of rat mesenchymal stem cells in collagen sponge reinforced with different poly(ethylene terephthalate) fibers

The objective of this study was to investigate the feasibility of collagen sponges mechanically reinforced by the incorporation of poly(ethylene terephthalate) (PET) fibers in stem cell culture. A collagen solution with homogeneously dispersed PET fibers was freeze-dried, followed by dehydrothermal cross-linking to obtain the collagen sponge incorporating PET fibers. By scanning electron microscopy observation, the collagen sponges exhibited isotropic and interconnected pore structures with an average size of 200 μm, irrespective of PET fiber incorporation. As expected, PET fibers incorporation significantly enhanced the compression strength of collagen sponge. When used for rat mesenchymal stem cells (MSC), the collagen sponge incorporating PET fibers was superior to the original collagen sponge without PET fibers incorporation in terms of the initial attachment, proliferation and osteogenic differentiation of cells, irrespective of the amount and diameter of fibers incorporated. The shrinkage of sponges during cell culture was significantly suppressed by the fiber incorporation. It is possible that the shrinkage suppression maintains the three-dimensional inner pore structure of collagen sponges without impairing the cell compatibility, resulting in the superior MSC attachment and the subsequent osteogenic differentiation in the sponge incorporating PET fiber.

Purification and in vitro cultivation of archaeocytes (stem cells) of the marine sponge Hymeniacidon perleve (Demospongiae)

Marine sponges (Porifera) are the best source of marine bioactive metabolites for drug discovery and development, although the sustainable production of most sponge-derived metabolites remains a difficult task. In vitro cultivation of sponge cells in bioreactors has been proposed as a promising technology. However, no continuous cell line has as yet been developed. Archaeocytes are considered to be toti/multipotent stem cells in sponges and, when purified, may allow the development of continuous sponge cell lines. As a prerequisite, we have developed a novel four-step protocol for the purification of archaeocytes from a marine sponge, Hymeniacidon perleve: (1) differential centrifugation to separate large sponge cells including archaeocytes; (2) selective agglomeration in low-Ca(2+)/Mg(2+) artificial seawater in which living archaeocytes form small loose aggregates with some pinacocytes and collencytes; (3) differential adherence to remove anchorage-dependent pinacocytes, collencytes and other mesohyl cells; (4) Ficoll-Vrografin density gradient centrifugation to purify archaeocytes. The final purity of archaeocytes is greater than 80%. The proliferation potential of the archaeocytes has been demonstrated by high levels of BrdU incorporation, PCNA expression and telomerase activity. In 4-day primary cultures, the purified archaeocytes show a 2.5-fold increase in total cell number. This study opens an important avenue towards developing sponge cell cultures for the commercial exploitation of sponge-derived drugs.

Cold stress defense in the freshwater sponge Lubomirskia baicalensis

The endemic freshwater sponge Lubomirskia baicalensis lives in Lake Baikal in winter (samples from March have been studied) under complete ice cover at near 0 degrees C, and in summer in open water at 17 degrees C (September). In March, specimens show high metabolic activity as reflected by the production of gametes. L. baicalensis lives in symbiosis with green dinoflagellates, which are related to Gymnodinium sanguineum. Here we show that these dinoflagellates produce the toxin okadaic acid (OA), which is present as a free molecule as well as in a protein-bound state. In metazoans OA inhibits both protein phosphatase-2A and protein phosphatase-1 (PP1). Only cDNA corresponding to PP1 could be identified in L. baicalensis and subsequently isolated from a L. baicalensis cDNA library. The deduced polypeptide has a molecular mass of 36 802 Da and shares the characteristic domains known from other protein phosphatases. As determined by western blot analysis, the relative amount of PP1 is almost the same in March (under ice) and September (summer). PP1 is not inhibited by low OA concentrations (100 nm); concentrations above 300 nm are required for inhibition. A sponge cell culture system (primmorphs) was used to show that at low temperatures (4 degrees C) expression of hsp70 is strongly induced and hsp70 synthesis is augmented after incubation with 100 nm OA to levels measured at 17 degrees C. In the enriched extract, PP1 activity at 4 degrees C is close to that measured at 17 degrees C. Immunoabsorption experiments revealed that hsp70 contributes to the high protein phosphatase activity at 4 degrees C. From these data we conclude that the toxin OA is required for the expression of hsp70 at low temperature, and therefore contributes to the cold resistance of the sponge.

Stress response in Baikalian sponges exposed to pollutants

In a first biomarker-based study in Lake Baikal, we demonstrated that the heat shock protein Hsp70, a biomarker of exposure, is present in various endemic sponge species of the lake. The expression of Hsp70 was induced following heat stress in tissue or dissociated cells of the Baikalian sponges, Baikalospongia intermedia, Lubomirskia fusifera, and Lubomirskia abietina. Increased Hsp70 levels were still visible after a recovery period of 24 h. Exposure of B. intermedia to lead (Pb) and zinc (Zn) caused a strong induction of Hsp70 in this sponge, while copper (Cu) was not effective. However, all these heavy metals were able to induce DNA damage, a biomarker of effect, in B. intermedia, as determined by Fast Micromethod. Induction of DNA damage in B. intermedia was also observed after exposure to the organochlorines tetrachloroguaiacol and pentachlorophenol, which are released by pulp bleaching. The Baikalsk Pulp and Paper Plant (BPPP) is considered to be one main potential source of pollution of the lake. Investigations of the influence of the waste water from the final refinement and aeration pond of BPPP revealed a concentration-dependent increase in Hsp70 expression in B. intermedia. In addition, the effluents of the pond were able to induce DNA damage. The basal level of DNA single-strand breaks/alkali-labile sites in B. intermedia collected close to the site where the BPPP effluents are discharged into the lake was higher than that in sponges collected at an unpolluted site. Our results suggest that Baikalian sponges are suitable bioindicators to monitor water quality and ecosystem health of the lake.

Glycohistochemistry of a marine sponge, Chondrilla nucula (Porifera, Desmospongiae), with remarks on a possibly related antimicrobial defense strategy and a note on exopinacoderm function

Based on carbohydrate histochemistry, including the use of lectins, and TEM, the study describes the distribution of terminal sugars in different structures of the demosponge Chondrilla nucula. The results of the general and specific carbohydrate histochemical approaches confirmed the presence of acidic and neutral glycoconjugates in the cells, and, with declining amounts from the ectosome to the mesohyl, in the extracellular matrix (ECM). AB-PAS staining indicated acidic complex carbohydrates particularly in the exopinacoderm, and more neutral ones in the cells and the ECM of the mesohyl. The PO-lectins applied demonstrated a general spectrum of free saccharide residues (α-d-mannose, α-/β-d-N-acetylglucosamine, α-d-N-acetylgalactosamine, α-d-galactose, β-d-galactose) in both sponge parts; α-l-fucose was only distinct in the ectosome. Sialic acids [siaα(2,3)-galactose, siaα(2,6)-N-acetylgalactosamine] were dominant in the very thin exopinacoderm, indicating O-linked high molecular weight glycoproteins. In this way a glycophysiologically ‘rigid’ outer mucus cover is developed as protection against mechanical hazards. Some of the free sugars (α-d-mannose, N-acetylglucosamine, N-acetylgalactosamine β-d-galactose, α-l-fucose) are known to prevent the adherence of different bacteria and fungi to cellular surfaces. Thus a high concentration of such sugars, may impede massive attacks of micro-inhabitants on mobile sponge cells, pinacocytes, and the exopinacoderm layer.

Proteoglycan Mechanics Studied by Single-molecule Force Spectroscopy of Allotypic Cell Adhesion Glycans

Early Metazoans had to evolve the first cell adhesion system addressed to maintaining stable interactions between cells constituting different individuals. As the oldest extant multicellular animals, sponges are good candidates to have remnants of the molecules responsible for that crucial innovation. Sponge cells associate in a species-specific process through multivalent calcium-dependent interactions of carbohydrate structures on an extracellular membrane-bound proteoglycan termed aggregation factor. Single-molecule force spectroscopy studies of the mechanics of aggregation factor self-binding indicate the existence of intermolecular carbohydrate adhesion domains. A 200-kDa aggregation factor glycan (g200) involved in cell adhesion exhibits interindividual differences in size and epitope content which suggest the existence of allelic variants. We have purified two of these g200 distinct forms from two individuals of the same sponge species. Comparison of allotypic versus isotypic g200 binding forces reveals significant differences. Surface plasmon resonance measurements show that g200 self-adhesion is much stronger than its binding to other unrelated glycans such as chondroitin sulfate. This adhesive specificity through multiple carbohydrate binding domains is a type of cooperative interaction that can contribute to explain some functions of modular proteoglycans in general. From our results it can be deduced that the binding strength/surface area between two aggregation factor molecules is comparable with that of focal contacts in vertebrate cells, indicating that strong carbohydrate-based cell adhesions evolved at the very start of Metazoan history.

Biology of the Porifera: cell culture

The discovery that dissociated sponge cells will reaggregate to form a functional organism was the basis for the establishment of sponge cell cultures that have been used as a model for the study of fundamental processes in developmental biology and immunology. More recent is the discovery of unique bioactive compounds in marine sponges, and the feasibility of in vitro production of these chemicals is being evaluated. Techniques are well established for cell dissociation; development of several nutrient media formulations has resulted in improvements in viability and cell division; and molecular approaches to identification of genes responsible for regulation of cell cycling may provide unique perspectives in culture optimization. The use of novel substrates for immobilization of cells offers alternatives for proliferation and scale-up. All of these results support the potential for development of a model system for the study of basic metabolic processes involved in cell differentiation, as well as an in vitro production system for sponge-derived bioactive compounds. Perhaps more important, however, is the development of cell lines of these "simple" metazoans to facilitate basic cell physiology and molecular biology research that may be applied to understanding more complex metazoan systems, including humans.

Novel mechanism for the radiation-induced bystander effect: Nitric oxide and ethylene determine the response in sponge cells

Until now the bystander effect had only been described in vertebrates. In the present study the existence of this effect has been demonstrated for the phylogenetically oldest metazoan phylum, the Porifera. We used the demosponge Suberites domuncula for the experiments in the two-chamber-system. The lower dish contained irradiated “donor” cells (single cells) and the upper dish the primmorphs (“recipient” primmorphs). The “donor” cells were treated with UV-B light (40 mJ/cm 2) and 100 μM hydrogen peroxide (H 2O 2), factors that exist also in the natural marine aquatic environment of sponges; these factors caused a high level of DNA strand breaks followed by a reduced viability of the cells. If these cells were added to the “recipient” primmorphs these 3D-cell cultures started to undergo apoptosis. This effect could be abolished by the NO-specific scavenger PTIO and ethylene. The conclusion that NO is synthesized by the UV-B/H 2O 2-treated cells was supported analytically. The cDNA encoding the enzyme dimethylarginine dimethylaminohydrolase (DDAH) was isolated from the “donor” cells. High levels of DDAH transcripts were measured in UV-B/H 2O 2-treated “donor” cells while after ethylene treatment the steady-state level of expression drops drastically. We conclude that in the absence of ethylene the concentration of the physiological inhibitor for the NO synthase ADMA is low, due to the high level of DDAH. In consequence, high amounts of NO are released from “donor” cells which cause apoptosis in “recipient” primmorphs. In contrast, ethylene reduces the DDAH expression with the consequence of higher levels of ADMA which prevent the formation of larger amounts of NO. This study describes the radiation-induced bystander effect also for the most basal metazoans and demonstrates that this effect is controlled by the two gasses NO and ethylene.

Three‐dimensional culture of human osteoblastic cells in chitosan sponges: The effect of the degree of acetylation

In this investigation, the effect of the degree of acetylation (DA) of chitosan on the behavior of human osteoblastic MG-63 cells cultured in three-dimensional chitosan matrices was assessed. Chitosan sponges with DAs in the range of 4 to 49% were prepared and characterized in terms of microstructure, porosity, and pore size. Collagen sponges were used as 3D control. Cell proliferation was determined using the MTT assay while the retention of the osteoblastic phenotype was monitored by assaying alkaline phosphatase activity. Cell morphology, cytoskeletal organization, and viability were assessed using different microscopy techniques. Chitosan sponges showed a similar microstructure regardless the DA, except for the highest DA used, where a more heterogeneous pore distribution was observed. In terms of cell proliferation, alkaline phosphatase activity and cell viability, cells cultured in chitosan scaffolds performed as well as in the 3D control regardless the DA, except for the highest DA used, where an inhibitory effect on cell proliferation was found. However, while in sponges with DAs < or = 13% cells attached and spread displaying long cell filopodia and numerous cell-to-cell contacts, in sponges with higher DAs cells tended to remain spherical and grow into spheroid-like cellular aggregates. In the present study, the DA played a key role in determining the affinity of osteoblastic cells towards the substrates, possibly by influencing the nature of the initial adsorbed protein layer.

Perfusion Culture Enhances Osteogenic Differentiation of Rat Mesenchymal Stem Cells in Collagen Sponge Reinforced with Poly(Glycolic Acid) Fiber

The objective of this study was to obtain fundamental knowledge about in vitro culture systems to enhance the proliferation and differentiation of mesenchymal stem cells (MSCs) in collagen sponge reinforced by the incorporation of poly(glycolic acid) (PGA) fiber. A collagen solution with PGA fiber homogeneously localized at PGA:collagen weight ratios of 0.67, 1.25, 2.5, and 5 was freezedried, followed by cross-linking of combined dehydrothermal, glutaraldehyde, and ultraviolet treatment. Scanning electron microscopy revealed that collagen sponges exhibited homogeneous and interconnected pore structures with an average size of 180 microm, irrespective of PGA fiber incorporation. When rat MSCs were seeded into collagen sponge with or without PGA fiber incorporation, more attached cells were observed in collagen sponge incorporating PGA fiber than in collagen sponge without PGA fiber incorporation, irrespective of the PGA:collagen ratio. The proliferation and osteogenic differentiation of MSCs in PGA-reinforced sponge at a weight ratio of 5 were greatly influenced by the culture method and growth conditions. Alkaline phosphatase (ALP) activity and osteocalcin content of MSCs cultured in PGA-reinforced sponge by the perfusion method became maximum at a flow rate of 0.2 mL/min, although they increased with culture time period. It may be concluded that appropriate perfusion conditions enable MSCs to positively improve the extent of proliferation and differentiation.

Three-dimensional culture of human disc cells within agarose or a collagen sponge: assessment of proteoglycan production

The objective of the present study was to assess proteoglycan production by human intervertebral disc cells cultured in vitro in selected cell carriers. Based on previous studies which evaluated disc cells seeded into collagen sponge, collagen gel, agarose, alginate or fibrin gel three-dimensional (3D) cell carriers, collagen sponge and agarose were found to provide superior microenvironments for formation of extracellular matrix (ECM). A standardized test design was used to evaluate ECM formed after 14 days of culture using the 1,9-dimethylmethylene blue (DMB) assay to assess sulfated glycosaminoglycan (S-GAG) production. Although agarose culture showed higher S-GAG levels compared to collagen sponge (2.94±2.20 (19) μg/ml S-GAG (mean±S.D. ( n)) vs. 0.94±0.77 (22), respectively, p = 0.0003 ), this is off-set by the significantly lower proliferation rate associated with culture of disc cells in agarose.

Selenium affects biosilica formation in the demosponge Suberites domuncula

Selenium is a trace element found in freshwater and the marine environment. We show that it plays a major role in spicule formation in the demosponge Suberites domuncula. If added to primmorphs, an in vitro sponge cell culture system, it stimulates the formation of siliceous spicules. Using differential display of transcripts, we demonstrate that, after a 72-h exposure of primmorphs to selenium, two genes are up-regulated; one codes for selenoprotein M and the other for a novel spicule-associated protein. The deduced protein sequence of selenoprotein M (14 kDa) shows characteristic features of metazoan selenoproteins. The spicule-associated protein (26 kDa) comprises six characteristic repeats of 20 amino acids, composed of 10 distinct hydrophobic regions ( approximately 9 amino acids in length). Recombinant proteins were prepared, and antibodies were raised against these two proteins. Both were found to stain the central axial filament, which comprises the silicatein, as well as the surface of the spicules. In the presence of selenium, only the genes for selenoprotein M and spicule-associated protein are up-regulated, whereas the expression of the silicatein gene remains unchanged. Finally we show that, in the presence of selenium, larger silica aggregates are formed. We conclude that selenium has a stimulatory effect on the formation of siliceous spicules in sponges, and it may be involved in the enzymatic synthesis of biosilica components.

Innate Immune Defense of the Sponge Suberites domuncula against Bacteria Involves a MyD88-dependent Signaling Pathway: INDUCTION OF A PERFORIN-LIKE MOLECULE

Sponges (phylum Porifera) are the phylogenetically oldest metazoa; as filter feeders, they are abundantly exposed to marine microorganisms. Here we present data indicating that the demosponge Suberites domuncula is provided with a recognition system for gram-negative bacteria. The lipopolysaccharide (LPS)-interacting protein was identified as a receptor on the sponge cell surface, which recognizes the bacterial endotoxin LPS. The cDNA was isolated, and the protein (Mr 49,937) was expressed. During binding to LPS, the protein dimerizes and interacts with MyD88, which was also identified and cloned. The sponge MyD88 (Mr 28,441) is composed of two protein interaction domains, a Toll/interleukin-1 receptor domain (found in MyD88 and in Toll-like receptors) and a death domain (present in MyD88 and interleukin-1 receptor-associated kinase). Northern blot experiments and in situ hybridization studies showed that after LPS treatment, the level of the LPS-interacting protein remains unchanged, whereas MyD88 is strongly up-regulated. A perforin-like molecule (Mr 74,171), the macrophage-expressed protein, was identified as an executing molecule of this pathway. This gene is highly expressed after LPS treatment, especially at the surfaces of the animals. The recombinant protein possesses biological activity and eliminates gram-negative bacteria; it is inactive against gram-positive bacteria. These data indicate that S. domuncula is provided with an innate immune system against gram-negative bacteria; the ligand LPS (a pathogen-associated molecular pattern) is recognized by the pattern recognition receptor (LPS-interacting protein), which interacts with MyD88. A signal transduction is established, which results in an elevated expression of MyD88 as well as of the macrophage-expressed protein as an executing protein.

Shear effects on suspended marine sponge cells

Fractions of viable cells, apoptotic and irreversibly damaged cells, dead whole cells and cell fragments were measured by flow cytometry during the production of freely suspended primary cells from explants of the marine sponge Axinella damicornis. The explants were disintegrated using the well-known Müller protocol [W.E.G. Müller, M. Wiens, R. Batel, R. Steffen, R. Borojevic, M.R. Custodio, Establishment of a primary cell culture from a sponge: primmorphs from Suberites domuncula, Mar. Ecol. Progr. Ser. 178 (1999) 205–219]. Supplementation of the standard Ca 2+- and Mg 2+-free artificial seawater of the Müller protocol, with the shear protectant Pluronic F68 (0.1%, w/v) greatly reduced the cell damage and enhanced the recovery of viable cells at each of the four stages of the protocol. Agitation of cells on an orbital shaker at 75 rpm essentially killed all the viable cells within 2.5 h, but no loss of viability occurred at a higher agitation speed of 100 rpm for up to 6 h when the cells were supplemented with Pluronic F68. This time-dependent loss in viability could be significantly reduced by processing at 3 °C instead of the normal 17 °C. A four-step mechanistic model was shown to describe the kinetics of cell death and fragmentation within ±10% of the measured values. The damage to cells was modeled as a web of first-order processes that did not depend on cell–cell interactions. The forces in the agitated fluid killed the viable cells by impact, which was not accompanied by cell rupture (i.e. the cell was left dead, but intact).

Inosculation of Tissue-Engineered Capillaries with the Host's Vasculature in a Reconstructed Skin Transplanted on Mice

The major limitation for the application of an autologous in vitro tissue-engineered reconstructed skin (RS) for the treatment of burnt patients is the delayed vascularization of its relatively thick dermal avascular component, which may lead to graft necrosis. We have developed a human endothelialized reconstructed skin (ERS), combining keratinocytes, fibroblasts and endothelial cells (EC) in a collagen sponge. This skin substitute then spontaneously forms a network of capillary-like structures (CLS) in vitro. After transplantation to nude mice, we demonstrated that CLS containing mouse blood were observed underneath the epidermis in the ERS in less than 4 days, a delay comparable to our human skin control. In comparison, a 14-day period was necessary to achieve a similar result with the non-endothelialized RS. Furthermore, no mouse blood vessels were ever observed close to the epidermis before 14 days in the ERS and the RS. We thus concluded that the early vascularization observed in the ERS was most probably the result of inosculation of the CLS network with the host's capillaries, rather than neovascularization, which is a slower process. These results open exciting possibilities for the clinical application of many other tissue-engineered organs requiring a rapid vascularization.

Gold Glyconanoparticles as Probes to Explore the Carbohydrate‐Mediated Self‐Recognition of Marine Sponge Cells

Cell aggregation in the red-beard marine sponge Microciona prolifera is mediated by a 2 10 kDa proteoglycan-like macromolecular aggregation factor (MAF), and is based on two highly polyvalent functional properties; a Ca-dependent proteoglycan self-interaction and a Ca-independent cell-binding activity. MAF, the first circular proteoglycan described, is composed of two N-glycosylated proteins, MAFp3 and MAFp4, with twenty units of each glycoprotein forming the central ring and the radiating arms, respectively. Each MAFp3 carries one or two copies of a 200 kDa acidic glycan, g-200, whereas each MAFp4 carries about 50 copies of a 6 kDa glycan, g-6. The MAFp4 arms of the sunburst-like proteoglycan are linked to cell-surface binding receptors, while the MAFp3 ring exposes the g-200 glycans so that they can engage in the Ca-dependent self-association (for a detailed review, see ref. [4]). By making use of MAF-specific monoclonal antibodies, it could be demonstrated that the self-association of MAF occurs through highly repetitive epitopes on the g-200 glycan. One of these epitopes was shown to be the sulfated disaccharide GlcpNAc3S(b1–3)Fucp. To gain insight into the role of carbohydrate interactions in MAF self-aggregation, we designed a challenging system for mimicking the g-200 self-association. By using the synthetic sulfated disaccharide, multivalently presented as a bovine serum albumin conjugate, and surface plasmon resonance spectroscopy, it was shown that Ca-dependent carbohydrate self-recognition is a major force in the g-200 association phenomenon. Gold glyconanoparticles have been successfully used as inert multivalent systems to explore either carbohydrate self-interactions or carbohydrate binding to proteins. In the present study, water-soluble gold glyconanoparticles coated with synthetic carbohydrates related to the sulfated disaccharide fragment (Scheme 1) were used as multivalent systems to investigate the g-200 glycan–glycan interaction by transmission electron microscopy (TEM). Very recently, an NMR study of intact MAF glycans suggested the presence of a-Fuc residues. However, earlier structural analysis of oligosaccharide fragments obtained from a partial acid hydrolysate of the g-200 glycan could not identify the anomeric configuration of the fucose residue in these fragments. 16] Therefore, gold glyconanoparticles coated with the aor the b-anomer (Au-1a and Au-1b) of the native sulfated disaccharide epitope were used in the aggregation experiments. The importance of each of the two monosaccharide units for the self-recognition process of the disaccharide epitope was determined by studying the gold glyconanoparticles Au-2 and Au-3. The three gold glyconanoparticle systems Au-4 (a-l-Fucp replaced by a-l-Galp), Au-5 (bd-GlcpNAc3S replaced by b-d-GlcpNAc), and Au-6 (b-dGlcpNAc3S replaced by b-d-Glcp3S) were used to evaluate the relevance of the modified sites in the self-recognition process. Scheme 1. Gold glyconanoparticles Au-1a/b to Au-6, related to the MAF sulfated disaccharide epitope.

Gastrulation in Calcareous Sponges: In Search of Haeckel's Gastraea

Haeckel's studies of development in calcareous sponges (1872) led him to develop the "Gastraea Theory," which proposes that the ancestral mode of germ layer formation, or gastrulation, was by invagination to produce a functional gut. His observations that gastrulation in the Calcarea occurs by invagination of a ciliated larva upon settlement and metamorphosis were supported by remarkable photomicrographs of the stage by Hammer in 1908. Although no later work found the same stage, these concepts are repeated in texts today. We have re-examined embryogenesis and metamorphosis in Sycon sp. cf. S. raphanus in order to understand when gastrulation occurs. Almost all larvae settle on their ciliated anterior pole and metamorphose into a bilayered juvenile whose interior cells rapidly differentiate into choanocytes and other cells of the young sponge. After a four-year search we have found the transitory stage shown by Hammer in which the anterior cells invaginate into the posterior half of the larva. The hole closes and it is not until some days later that the sponge forms an osculum at its apical pole. To understand whether invagination comprises gastrulation and if the hole can be considered to be a blastopore we have carried out a review of the literature dealing with this brief moment in calcaronean sponge development. Despite the intrigue of this type of metamorphosis, we conclude that gastrulation occurs earlier, during formation of the two cellular regions of the larva, and that metamorphosis involves the reorganization of these already differentiated regions. Considering the pivotal position occupied by the Calcarea as the possible sister-group to all other Metazoa, these results call for a reassessment of germ layer formation and of the relationships of the primary germ layers among basal metazoan phyla.

Three-dimensional Culture of L929 and UMR106 Cells in Self-prepared Alginate Sponges

Three-dimensional Culture of L929 and UMR106 Cells in Self-prepared Alginate Sponges

Hepatic Organoid Formation in Collagen Sponge of Cells Isolated from Human Liver Tissues

We examined whether small hepatocytes (SHs), which are hepatic progenitor cells, could be isolated from a normal human liver and whether human hepatic cells could form hepatic organoids in a collagen sponge. Normal liver tissues were obtained from resected specimens from nine patients who underwent hepatic resection. Isolated hepatic cells were plated on dishes and a collagen sponge. More than 1 month later, SH-like cells appeared and proliferated on the dishes, whereas cell aggregates were formed in the sponge and showed characteristic tissue architecture: columnar and/or cuboidal epithelial cells lined the surface of the sponge. Clusters of epithelial cells with a large cytoplasm and ductular structures were observed under the lining cells. The lining and ductular cells were positive for cytokeratins 7 and 19, which indicated they were biliary epithelial cells (BECs), and the epithelial cells forming clusters were positive for the anti-human hepatocyte antibody, identifying them as hepatocytes. Some lining cells were positive for both the hepatic marker and the BEC markers. The cells in the collagen sponge actively proliferated and the hepatocytes excreted albumin into the medium. Thus, hepatic organoids could be reconstructed in a collagen sponge by normal human liver cells.