Origin of metazoan stem cell system in sponges: first approach to establish the model (Suberites domuncula)

It is established that Porifera (sponges) represent the earliest phylum which branched off from the common ancestor of all multicellular animals, the Urmetazoa. In the present study, the hypothesis is tested if, during this transition, pluripotent stem cells were formed which are provided-similar to the totipotent cells (archaeocytes/germ cells)-with a self-renewal capacity. As a model system, primmorphs from the sponge Suberites domuncula were used. These 3D-cell aggregates were cultivated in medium (RPMI 1640/seawater) either lacking silicate and ferric iron or in medium which was supplemented with these 'morphogenetic' factors. As molecular markers for the potential existence of stem cells in primmorphs, two genes which encode proteins found in stem cells of higher metazoan species, were cloned from S. domuncula. First, the noggin gene, which is present in the Spemann organizer of amphibians and whose translation product acts during the formation of dorsal mesoderm derivatives. The second gene encodes the mesenchymal stem cell-like protein. Both cDNAs were used to study their expression in primmorphs in dependence on the incubation conditions. It was found that noggin expression is strongly upregulated in primmorphs kept in the presence of silicate and ferric iron, while the expression of the mesenchymal stem cell-like protein was downregulated. These data are discussed with respect to the existence of stem cells in sponges.     

Purification and in vitro cultivation of archaeocytes (stem cells) of the marine sponge <Emphasis Type="Italic">Hymeniacidon perleve</Emphasis> (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-Ca²⁺/Mg²⁺ 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. The authors are grateful for the financial support of the Chinese Academy of Sciences under the “100 Talent Project”, the “Innovation Fund” from the Dalian Institute of Chemical Physics, the “Hi-Tech Research and Development Program of China” (2001AA620404), and the European Commission (project: Silicon Biotechnology).     

Germinal granules in archaeocytes of the sponge <Emphasis Type="Italic">Oscarella malakhovi</Emphasis> Ereskovsky, 2006

The morphology of archaeocytes, sponge stem cells, was studied in Oscarella malakhovi during asexual reproduction (budding) using light and electron microscopy. Electron-dense germinal granules, which are ultrastructural markers and key organelles of metazoan germline cells and potentially gametogenic stem cells of asexually reproducing invertebrates, were found in the perinuclear cytoplasm of the sponge archaeocyte for the first time. Common features of the morphological and functional organization of the “primary” stem cells in asexually reproducing invertebrates and the germline cells in Metazoa are discussed.     

Cellular location of (2R, 3R, 7Z)-2-aminotetradec-7-ene-1, 3-diol, a potent antimicrobial metabolite produced by the Caribbean sponge Haliclona vansoesti

The Caribbean sponge Haliclona vansoesti has been found to contain large amounts of a new sphingosine derivative, (2R, 3R, 7Z)-2-aminotetradec-7-ene-1, 3-diol (compound 1). To determine the localization of this compound within the organism, cell distribution and quantitative determination of the aminodiol content of cell fractions obtained by differential centrifugation have been performed. Results show that choanocytes and archaeocytes are the major sponge cell types and that H. vansoesti harbour small photosynthetic symbionts (cyanobacteria) and few heterotrophic bacteria. Reverse-phase HPLC analyses of the cell fractions reveal that the aminodiol 1 is not associated with the prokaryotic endobionts but with the sponge cells, in particular the archaeocytes. This is clearly established by the positive significant correlation existing between the numbers of archaeocytes and the amounts of aminodiol 1. The mean aminodiol concentration is estimated to be 2 microg/10(5) archaeocytes. The aminodiol 1 is also found in substantial amounts in primary cell cultures, so that cell culture can be envisaged as an option for its production. Sponge cell suspensions display potent antibacterial and antiyeast activities, in correlation with their aminodiol content, indicating that this compound is at least in part responsible for these activities in the sponge. The release of the aminodiol I into the external medium suggests that this substance may be involved in the defence mechanisms of the sponge.     

Ultrastructural study of differentiation processes during aggregation of purified sponge archaeocytes

Archaeocytes from the spongeEphydatia fluviatilis were dissociated and then isolated on Ficoll density gradients. Their aggregation and reconstitution processes were studied by transmission electron microscopy to determine their capabilities for differentiation.Archaeocyte aggregates follow a well defined sequence of differentiation to generate the characteristic structures of a sponge. Pinacoderm is the first structure to be regenerated and appears progressively at the surface of the 12 h aggregates. Pinacocytes which have differentiated in archaeocyte aggregates are identical to native ones except that the nucleolus remains in most cells. The choanocytes appear only after 24 h by a two step process. First, small cells (choanoblasts) are formed from archaeocytes by mitosis. These cells then transform into fully differentiated choanocytes possessing collars and flagella. The early choanocyte chambers are small, irregular and randomly dispersed in the aggregates. Finally, collencytes and sclerocytes begin to appear just before the aggregates spread on the substrate.The differentiation of a suspension of pure archaeocytes is a unique model system to study sponge cell differentiation and has allowed us to demonstrate that archaeocytes isolated from developed sponges maintain the capacity to differentiate even though this capacity is not usually expressed.     

繁茂膜海绵原细胞富集细胞团培养过程中的细胞迁移规律

海绵是重要的生物活性物质来源, 近10年来, 从海绵中发现的具有生物活性的新化合物占海洋生物来源的30%以上, 并且大多具有显著的抗肿瘤, 抗艾滋病病毒的活性。但是, 由于海绵生物量不能满足这些活性物质进一步研究和商业化的需求, 目前仅有一种活性物质被成功的商业化, 这不仅是商业开发的损失, 也是提高人类生活质量活动的一种损失。为了解决海绵供给不足的问题, 人们进行了包括化学合成、海绵养殖以及海绵细胞培养在内的多种尝试,目前的研究结果表明, 海绵细胞离体培养技术是最有可能彻底解决海绵供给不足的途径之一。但是由于海绵自身的特殊性, 还没有人成功的建立起海绵细胞系以满足生产需要。人们发现, 海绵细胞的相互接触对于离体海绵细胞长期培养至关重要。经过多年的探索, 大连化物所海洋生物产品工程组建立了开发出了海绵原细胞富集细胞团培养技术, 通过对海绵组织内的原细胞进行富集来获得可长期培养的海绵细胞。海绵原细胞是海绵组织内的“干细胞”, 具有很强的分化、增殖潜力, 同时也是海绵组织内负责消化的主要细胞类型。为了探索海绵原细胞的增殖、分化规律, 本研究基于海绵原细胞富集细胞团培养体系, 构建了海绵细胞培养实时观测平台, 对繁茂膜海绵原细胞、领细胞、上皮细胞3类主要海绵细胞类型在海绵细胞团形成及生长的全过程进行观察, 了解不同类型细胞迁移规律的变化。通过对视频记录进行分析,发现离散的海绵细胞与细胞团内的海绵细胞具有截然相反的运动规律, 海绵细胞的运动具有很强的协同性。伴随原细胞在细胞团内不停息的迁移, 还观察到海绵细胞团内新生骨针的迁移以及细胞间进行颗粒物质的传递。这些信息的获得, 将有助于进一步了解不同细胞的功能与作用, 也有助于在此基础上探索海绵细胞的增殖、分化控制规律。     

Cellular origin of chlorinated diketopiperazines in the dictyoceratid sponge Dysidea herbacea (Keller)

The tropical marine sponge Dysidea herbacea (Keller) contains the filamentous unicellular cyanobacterium Oscillatoria spongeliae (Schulze) Hauck as an endosymbiont, plus numerous bacteria, both intracellular and extracellular. Archaeocytes and choanocytes are the major sponge cell types present. Density gradient centrifugation of glutaraldehyde-fixed cells with Percoll as the support medium has been used to separate the cyanobacterial symbiont from the sponge cells on the basis of their differing densities. The protocol also has the advantage of separating broken from intact cells of O. spongeliae. The lighter cell preparations contain archaeocytes and choanocytes together with damaged cyanobacterial cells, whereas heavier cell preparations contain intact cyanobacterial cells, with less than 1% contamination by sponge cells. Gas chromatography/mass spectrometry analysis has revealed that the terpene spirodysin is concentrated in preparations containing archaeocytes and choanocytes, whereas nuclear magnetic resonance analysis of the symbiont cell preparations has shown that they usually contain the chlorinated diketopiperazines, dihydrodysamide C and didechlorodihydrodysamide C, which are the characteristic metabolites of the sponge/symbiont association. However, one symbiont preparation, partitioned by a second Percoll gradient, has been found to be devoid of chlorinated diketopiperazines. The capability to synthesize secondary metabolites may depend on the physiological state of the symbiont; alternatively, there may be two closely related cyanobacterial strains within the sponge tissue.     

Allograft rejection, autograft fusion and inflammatory responses to injury in Callyspongia diffusa (Porifera; Demospongia)

Sponges exhibit a variety of swift, cellular defence responses to protect self integrity. The sponge Callyspongia diffusa has been used to characterize the cytological changes that occur during allograft rejection, autograft fusion, and inflammation. Allogeneic contact results in fusion of the two exopinacoderms followed by an infiltration of mesohyl cells into the graft zone. As mesohyl cells accumulate, they form tissue bridges that span the graft interface. After a few days, the tissue bridges and the nearby cellular infiltrate become necrotic and slough off, which separates the allogeneic tissues. Autograft fusion begins similarly but cellular infiltration does not follow exopinacoderm fusion. Contacted exopinacocytes are redistributed, the endopinacoderms and choanosomes come into contact, and the grafted sponge tissues merge. Tissue damage exposes internal regions of the sponge to the external environment. In many areas of injury, exposed choanosome is sealed by infiltrating mesohyl cells. In other areas, exposed endopinacoderm appears to serve as new exopinacoderm. Cellular debris is removed by phagocytic archaeocytes and new exopinacoderm is regenerated over the damaged choanosome. No scars remain once the inflammatory infiltrate has dispersed. In general, mesohyl cells are involved in defence responses without an observed enrichment of any specific cell type. However, archaeocytes from rejecting sponges appear to line both sides of the allogeneic interface.     

Pluripotent versus totipotent plant stem cells: dependence versus autonomy?

Little is known of the mechanisms that induce the dedifferentiation of a single somatic cell into a totipotent embryogenic cell that can either be regenerated or develop into an embryo and subsequently an entire plant. In this Opinion article, we examine the cellular, physiological and molecular similarities and differences between different plant stem cell types. We propose to extend the plant stem cell concept to include single embryogenic cells as a totipotent stem cell based on their capacity to regenerate or develop into an embryo under certain conditions. Our survey suggests that differences in chromatin structure might ensure that meristem-localized stem cells have supervised freedom and are pluripotent, and that embryogenic stem cells are unsupervised, autonomous and, hence, freely totipotent.     

Differenzierungsvorgänge in der keimenden Gemmula vonEphydatia fluviatilis

Zusammenfassung Die beschalten Dauerstadien (Gemmulae) des SüßwasserschwammesEphydatia fluviatilis enthalten uniforme, totipotente Statocyten (Thésocyten), aus denen sich im Keimungsverlauf Archaeocyten (ein- und zweikernige) und Histoblasten differenzieren. Letztere treten nach einem gewissen Inkubationszeitraum in der zapfenartigen Zone unter der Mikropyle auf, während sich die übrigen Zelltypen zu einem an der Schalenöffnung orientierten, dreidimensionalen Muster gefälleartig anordnen.Nach Ausbildung eines einschichtigen Pinacocyten-Epithels (primäres Pinacoderm) aus peripher gelegenen, einkernigen Zellen schlüpft das nunmehr im Kapselinneren entstandene Primordium durch die offene Mikropyle und nimmt mit dem Substrat Verbindung auf.Das Primordium entwickelt sich zum frühen Jungschwamm, in den die restlichen Archaeocyten, Histoblasten und auch vereinzelt Skleroblasten einwandern.

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Differentiation processes in the germinating Gemmula ofEphydatia fluviatilis

Summary The dormant shelled gemmulae of the fresh water spongeEphydatia fluviatilis contain uniform, totipotent statocytes (thésocytes), which can differentiate either into archaeocytes (mono- and binucleated) or into histoblasts. The histoblasts accumulate at the villus near the micropyle. The other cell types orientate in a three-dimensional pattern at the micropyle, according to a developing gradient.After the primary pinacoderm is formed, the sponge primordium is released through the open micropyle. The primordium develops into a new sponge, into which archaeocytes, histoblasts and scleroblasts migrate.

     

Allogeneic cell interactions during graft rejection in Callyspongia diffusa (Porifera, Demospongia); a study with monoclonal antibodies

Many aspects of the cellular immune system in the marine sponge Callyspongia diffusa, have been defined by using artificially transplanted allogeneic tissues. Rejections show specificity of 'non-self' recognition, cytotoxic effector responses and short-term immunological memory. Histological investigations reveal a generalized mesohyl migration to the graft zone where archaeocytes line up at the allogeneic interface. Monoclonal antibodies (mAbs) raised to sponge cells have shown that little or no allogeneic cell mixing occurs at the graft interface and that certain mesohyl cell types do not appear to be directly involved in graft rejections. However, all mesohyl cell types are present in autograft fusion zones and in inflammatory responses to injury. The involvement of only some of the mesohyl cell types in graft rejections suggests specific interactions of an effector 'immunocyte'.     

A receptor tyrosine kinase specific to hematopoietic stem and progenitor cell-enriched populations.

To elucidate the molecular biology of the hematopoietic stem cell, we have begun to isolate genes from murine cell populations enriched in stem cell activity. One such cDNA encodes a novel receptor tyrosine kinase, designated fetal liver kinase-2 or flk-2, which is related to the W locus gene product c-kit. Expression analyses suggest an extremely restricted distribution of flk-2. It is expressed in populations enriched for stem cells and primitive uncommitted progenitors, and is absent in populations containing more mature cells. Therefore, this receptor may be a key signal transducing component in the totipotent hematopoietic stem cell and its immediate self-renewing progeny.     

Hydroxyurea: An Inhibitor of the Differentiation of Choanocytes in Fresh-Water Sponges and a Possible Agent for the Isolation of Embryonic Cells

During the development of a fresh-water sponge from its gemmules, most cell types originate from the undifferentiated archaeocytes through a few divisions, whereas each choanocyte chamber, composed of several tens of choanocytes, arises from a single archaeocyte through repeated mitoses.
This process was studied on gemmules incubated in various concentrations of hydroxyurea.
A concentration of 100 μg/ml postponed the hatching by about two days, and blocked the differentiation of the choanocytes and the morphogenesis of the aquiferous system. The resulting organism was a hollow dome of pinacoderm, stretched on spicules, the bottom of which was strewn with embryonic archaeocytes. After washing and incubation in mineral medium, the sponge differentiated its choanocytes and achieved normal development.
The incorporation of ³H-thymidine into DNA was compared throughout the development of normal and hydroxyurea-treated gemmules. Hydroxyurea delayed the first peaks of incorporation and abolished the large peak that normally occurs around 90 h, just before the formation of choanocyte chambers.
When added after 96 h incubation, hydroxyurea did not affect the differentiation of the choanocytes.
These results suggest that the differentiation of the choanocytes and the further morphogenesis of the aquiferous system depend on the repetitive divisions of the archaeocytes that normally occur around 90 h.
Furthermore, hydroxyurea-blocked sponges provide a suitable source for the isolation of pure populations of embryonic archaeocytes.     

The stem cell system in demosponges: suggested involvement of two types of cells: archeocytes (active stem cells) and choanocytes (food-entrapping flagellated cells)

Major questions about stem cell systems include what type(s) of stem cells are involved (unipotent/totipotent/pluripotent/multipotent stem cells) and how the self-renewal and differentiation of stem cells are regulated. Sponges, the sister group of all other animals and probably the earliest branching multicellular lineage of extant animals, are thought to possess totipotent stem cells. This review introduces what is known about the stem cells in sponges based on histological studies and also on recent molecular biological studies that have started to reveal the molecular and cellular mechanisms of the stem cell system in sponges (mainly in demosponges). The currently proposed model of the stem cell system in demosponges is described, and the possible applicability of this model to other classes of sponges is discussed. Finally, a possible scenario of the evolution of stem cells, including how migrating stem cells arose in the urmetazoan (the last common ancestor of metazoans) and the evolutionary origin of germ line cells in the urbilaterian (the last common ancestor of bilaterians), are discussed.     

Wnt and Frizzled RNA expression in human mesenchymal and embryonic (H7) stem cells

Background

Wnt signals are important for embryonic stem cells renewal, growth and differentiation. Although 19 Wnt, 10 Frizzled genes have been identified in mammals, their expression patterns in stem cells were largely unknown.

Results

We conducted RNA expression profiling for the Wnt ligands, their cellular receptors "Frizzleds" and co-receptors LRP5/6 in human embryonic stem cells (H7), human bone marrow mesenchymal cells, as well as mouse totipotent F9 teratocarcinoma embryonal cells. Except failing to express Wnt2 gene, totipotent F9 cells expressed RNA for all other 18 Wnt genes as well as all 10 members of Frizzled gene family. H7 cells expressed RNA for each of the 19 Wnt genes. In contrast, human mesenchymal cells did not display detectable RNA expression of Wnt1, Wnt8a, Wnt8b, Wnt9b, Wnt10a, and Wnt11. Analysis of Frizzled RNAs in H7 and human mesechymal cells revealed expression of 9 members of the receptor gene family, except Frizzled8. Expression of the Frizzled co-receptor LRP5 and LRP6 genes were detected in all three cell lines. Human H7 and mouse F9 cells express nearly a full complement of both Wnts and Frizzleds genes. The human mesenchymal cells, in contrast, have lost the expression of six Wnt ligands, i.e. Wnt1, 8a, 8b, 9b, 10a and 11.

Conclusion

Puripotent human H7 and mouse F9 embryonal cells express the genes for most of the Wnts and Frizzleds. In contrast, multipotent human mesenchymal cells are deficient in expression of Frizzled-8 and of 6 Wnt genes.     

Observations on the moving colonies of the genus Tethya (Demospongia,Porifera)

Summary Observations on two species of sponges, Tethya seychellensis from the Red Sea, and T. aurantium from the Mediterranean Sea revealed that young colonies are able to detach from their sites of settlement and by means of filamentous podia, to move to other sites in the vicinity. These podia are 10–16 mm long extensions of the sponge body wall that bear an adhesive knob on their distal ends. After being attached, the contracting podia pull the spherical colonies of 2.0–3.0 cm in diameter, transporting them to a new site. EM observations showed that in the podia the matrix is rich in contractile myocytes, primary archaeocytes, nucleated archaeocytes and scleroblastic cells, each of which takes part in the moving ability of the podium. It was also shown that some of the archaeocytes go over a process of ripening within the podium and produce collagenic filaments deposited in the internal matrix.     

Medaka cleavage embryos are capable of generating ES-like cell cultures

Mammalian embryos at the blastocyst stage have three major lineages, which in culture can give rise to embryonic stem (ES) cells from the inner cell mass or epiblast, trophoblast stem cells from the trophectoderm, and primitive endoderm stem cells. None of these stem cells is totipotent, because they show gene expression profiles characteristic of their sources and usually contribute only to the lineages of their origins in chimeric embryos. It is unknown whether embryos prior to the blastocyst stage can be cultivated towards totipotent stem cell cultures. Medaka is an excellent model for stem cell research. This laboratory fish has generated diploid and even haploid ES cells from the midblastula embryo with ~2000 cells. Here we report in medaka that dispersed cells from earlier embryos can survive, proliferate and attach in culture. We show that even 32-cells embryos can be dissociated into individual cells capable of producing continuously growing ES-like cultures. Our data point to the possibility to derive stable cell culture from cleavage embryos in this organism.     

胚胎干细胞生物学特性及其应用前景

胚胎干细胞是来源于着床前的囊胚内细胞团或早期胎儿的原始生殖细胞的一类未分化的全能性（多能性）干细胞,具有无限增殖和全能化的潜力,胚胎干细胞在发育生物学基础研究,动物胚胎工程研究生产和临床医学上具有诱人的应用前景。     

Cellular and developmental properties of fetal hematopoietic stem cells

We have characterized the fetal totipotent hematopoietic stem cell using a novel strategy that integrates physical analysis of cell properties and genetic analysis of in vivo developmental behavior. This approach allows the simultaneous isolation and in vivo characterization of any stem cell population. Using this procedure we demonstrate that a cell surface marker, recognized by monoclonal antibody AA4.1, defines 0.5%-1.0% of fetal liver tissue that contains the entire hierarchy of primitive hematopoietic cells. The AA4.1+ subpopulation includes multipotential in vitro progenitors, CFU-S cells, and lymphoid-myeloid stem cells that function to yield permanent and oligoclonal blood systems. Further fractionation of these cells by analysis of density, fibronectin binding, and surface antigen distribution has defined 0.1%-0.2% of fetal liver that contains the totipotent stem cell.