Isolation of the choanocyte in the fresh water sponge, Ephydatia fluviatilis and its lineage marker, Ef annexin

In order to investigate the cellular system of the freshwater sponge, Ephydatia fluviatilis, we isolated a molecular marker for the most prominent cell type, the choanocyte. After feeding sponge with fluorescent beads, fluorescent-labeled choanocytes were collected by fluorescence activated cell sorting (FACS). By protein profiling choanocyte and archeocyte (stem cell)-rich fractions, proteins characteristic of choanocyte were identified. The partial amino-acid sequence of one of the proteins characteristic of choanocyte matches the deduced amino-acid sequence of sponge expression tag (EST) clones and mouse annexin VII. These EST clones overlap and encode a protein, designated Ef annexin, which includes four annexin domains. Whole mount in situ hybridization shows Ef annexin expression in chamber-forming choanocytes in 7-day-old sponge, leading us to conclude that Ef annexin can be used as a choanocyte marker. In the early development stage, Ef annexin expression can be detected in both large single cells, characteristic of archeocytes, and cells forming 2-, 4- and multiple-cell clusters. These results indicate that Ef annexin is initially expressed in the choanocyte-committed archeocyte which then undergoes several mitotic cell divisions to form a choanocyte chamber. This suggests that the single choanocyte chamber essentially originates from a single archeocyte.     

The active stem cell specific expression of sponge Musashi homolog EflMsiA suggests its involvement in maintaining the stem cell state

A hallmark of stem cells is the ability to sustainably generate stem cells themselves (self-renew) as well as differentiated cells. Although a full understanding of this ability will require clarifying underlying the primordial molecular and cellular mechanisms, how stem cells maintain their stem state and their population in the evolutionarily oldest extant multicellular organisms, sponges, is poorly understood. Here, we report the identification of the first stem cell-specific gene in demosponges, a homolog of Musashi (an evolutionarily conserved RNA binding protein that regulates the stem cell state in various organisms). EflMsiA, a Musashi paralog, is specifically expressed in stem cells (archeocytes) in the freshwater sponge Ephydatia fluviatilis. EflMsiA protein is localized predominantly in the nucleus, with a small fraction in the cytoplasm, in archeocytes. When archeocytes enter M-phase, EflMsiA protein diffuses into the cytoplasm, probably because of the breakdown of the nuclear membrane. In the present study, the existence of two types of M-phase archeocytes [(M)-archeocytes] was revealed by a precise analysis of the expression levels of EflMsiA mRNA and protein. In Type I (M)-archeocytes, presumably archeocytes undergoing self-renewal, the expression levels of EflMsiA mRNA and protein were high. In Type II (M)-archeocytes, presumably archeocytes committed to differentiate (committed archeocytes), the expression levels of EflMsiA mRNA and protein were about 60% and 30% lower than those in Type I (M)-archeocytes. From these results, archeocytes can be molecularly defined for the first time as EflMsiA-mRNA-expressing cells. Furthermore, these findings shed light on the mode of cell division of archeocytes and suggest that archeocytes divide symmetrically for both self-renewal and differentiation.     

Ultrastructural evidence of extruding exocytosis of residual bodies in the freshwater sponge Ephydatia fluviatilis

Exocytosis of residual bodies by choanocytes, archeocytes and endopinacocytes lining the aquiferous system of Ephydatia fluviatilis has been demonstrated using calibrated latex beads and Escherichia coli as tracers. In passing into the mesohyl or the lumen of the exhalant aquiferous canals, beads, and altered bacteria were enveloped by the plasma membrane of the cell containing them. The membrane constricted at a neck region to form extruding vacuoles. This process appeared first in choanocytes and later in other cell types. The occurrence of these buds increased with the length of incubation time, as did the number of particles they contained. Acid phosphatase activity was frequently associated with the particles budding from the cell membrane, confirming that this process followed digestive activity. Membranous vacuoles were recovered from the external medium and observed by TEM and those adhering to the substratum were seen by SEM. These observations proved that vacuoles were released from the sponges. This membrane-consuming mechanism of exoctyosis implies intense membrane replacement in the digestive cells of the sponge.     

Fibroblasts share mesenchymal phenotypes with stem cells, but lack their differentiation and colony-forming potential

Background information. Although MSCs (mesenchymal stem cells) and fibroblasts have been well studied, differences between these two cell types are not fully understood. We therefore comparatively analysed antigen and gene profiles, colony‐forming ability and differentiation potential of four human cell types in vitro: commercially available skin‐derived fibroblasts [hSDFs (human skin‐derived fibroblasts)], adipose tissue‐derived stem cells [hASCs (human adipose tissue‐derived stem cells)], embryonic lung fibroblasts (WI38) and dermal microvascular endothelial cells [hECs (human dermal microvascular endothelial cells)]. Results. hSDFs, hASCs and WI38 exhibited a similar spindle‐like morphology and expressed same antigen profiles: positive for MSC markers (CD44, CD73 and CD105) and fibroblastic markers [collagen I, HSP47 (heat shock protein 47), vimentin, FSP (fibroblast surface protein) and αSMA (α smooth muscle actin)], and negative for endothelial cell marker CD31 and haemopoietic lineage markers (CD14 and CD45). We further analysed 90 stem cell‐associated gene expressions by performing real‐time PCR and found a more similar gene expression pattern between hASCs and hSDFs than between hSDFs and WI38. The expression of embryonic stem cell markers [OCT4, KLF4, NANOG, LIN28, FGF4 (fibroblast growth factor 4) and REST] in hASCs and hSDFs was observed to differ more than 2.5‐fold as compared with WI38. In addition, hSDFs and hASCs were able to form colonies and differentiate into adipocytes, osteoblasts and chondrocytes in vitro, but not WI38. Moreover, single cell‐derived hSDFs and hASCs obtained by clonal expansion were able to differentiate into adipocytes and osteoblasts. However, CD31 positive hECs did not show differentiation potential. Conclusions. These findings suggest that (i) so‐called commercially available fibroblast preparations from skin (hSDFs) consist of a significant number of cells with differentiation potential apart from terminally differentiated fibroblasts; (ii) colony‐forming capacity and differentiation potential are specific important properties that discriminate MSCs from fibroblasts (WI38), while conventional stem cell properties such as plastic adherence and the expression of CD44, CD90 and CD105 are unspecific for stem cells.     

Mesenchymal differentiation propensity of a human embryonic stem cell line

Objectives: To characterize basal differentiation tendencies of a human embryonic stem (hES) cell line, KCL‐002. Materials and methods: In vitro specification and differentiation of hES cells were carried out using embryoid body (EB) cultures and tests of pluripotency and in vivo differentiation were performed by teratoma assays in SCID mice. Real‐time PCR, immunohistochemistry, flow cytometry and histological analyses were used to identify expression of genes and proteins associated with the ectodermal, endodermal and mesodermal germ layers. Results: Undifferentiated KCL‐002 cells expressed characteristic markers of pluripotent stem cells such as Nanog, Sox‐2, Oct‐4 and TRA 1‐60. When differentiated in vitro as EB cultures, expression of pluripotency, endodermal and ectodermal markers decreased rapidly. In contrast, mesodermal and mesenchymal markers such as VEGFR‐2, α‐actin and vimentin increased during EB differentiation as shown by qPCR, immunostaining and flow cytometric analyses. Teratoma formation in SCID mice demonstrated the potential to form all germ layers in vivo with a greater proportion of the tumours containing mesenchymal derivatives. Conclusions: The data presented suggest that the KCL‐002 hES cell line is pluripotent and harbours a bias in basal differentiation tendencies towards mesodermal and mesenchymal lineage cells. Characterizing innate differentiation propensities of hES cell lines is important for understanding heterogeneity between different cell lines and for further studies aimed at deriving specific lineages from hES cells.     

Feulgen microspectrophotometric analysis of deoxyribonucleoprotein organization in larval and adult freshwater sponge nuclei.

Microspectrophotometric measurements of nuclei of adult choanocytes and archeocytes and larval archeocytes and flagellated ectodermal epithelia from stages III and IV were undertaken on tissues from the freshwater sponge, Eunapius fragilis (Leidy). The condensed nuclei of differentiated choanocytes and state IV flagellated epithelial cells presented integrated extinction values about 64% of those obtained for either adult or larval archeocytes. This suggests that such measurements represent sensitive indicators of deoxyribonucleoprotein-complex organization; thus, nuclear differentiation. Further, no large population of G2 (4C) nuclei was found among these populations as has been reported in two classes of coelenterates.     

CD45+/CD133+ positive cells expanded from umbilical cord blood expressing PDX‐1 and markers of pluripotency

UCB (human umbilical cord blood) contains cells able to differentiate into non‐haematopoietic cell lineages. It also contains cells similar to primitive ESCs (embryonic stem cells) that can differentiate into pancreatic‐like cells. However, few data have been reported regarding the possibility of expanding these cells or the differential gene expression occurring in vitro. In this study, we expanded formerly frozen UCB cells by treatment with SCF (stem cell factor) and GM‐CSF (granulocyte–macrophage colony stimulating factor) in the presence of VPA (valproic acid). Gene expression profiles for beta cell differentiation and pluripotency (embryo stem cell phenotype) were analysed by RT‐PCR and immunocytochemistry. The results show a dramatic expansion (>150‐fold) of haematopoietic progenitors (CD45⁺/CD133⁺) which also expressed embryo markers of pluripotency (nanog, kfl‐4, sox‐2, oct‐3/4 andc‐myc), nestin, and pancreatic markers such as pax‐4, ngn‐3, pdx‐1 and syt‐1 (that is regulated by pdx‐1 and provides the cells with a Ca⁺⁺ regulation mechanism essential for insulin exocytosis). Our results show that UCB cells can be expanded to produce large numbers of cells of haematopoietic lineage that naturally (without the need of retroviral vectors or transposons) express a gene pattern compatible with endocrine pancreatic precursors and markers of pluripotency. Further investigations are necessary to clarify, first, whether in this context, the embryogenes expressed are functional or not, and secondly, since these cells are safer than cells transfected with retroviral vectors or transposons, whether they would represent a potential tool for clinical application.     

Isolation of Ef silicatein and Ef lectin as molecular markers for sclerocytes and cells involved in innate immunity in the freshwater sponge Ephydatia fluviatilis

Sponges (phylum Porifera) have remarkable regenerative and reconstitutive abilities and represent evolutionarily the oldest metazoans. To investigate sponge stem cell differentiation, we have focused on the asexual reproductive system in the freshwater sponge Ephydatia fluviatilis. During germination, thousands of stem cells proliferate and differentiate to form a fully functional sponge. As an initial step of our investigation of stem cell (archeocyte) differentiation, we isolated molecular markers for two differentiated cell types: spicule-making sclerocyte cells, and cells involved in innate immunity. Sclerocyte lineage-specific Ef silicatein shares 45% to 62% identity with other sponge silicateins. As in situ hybridization of Ef silicatein specifically detects archeocytes possibly committed to sclerocytes, as well as sclerocytes with an immature or mature spicule, therefore covering all the developmental stages, we conclude that Ef silicatein is a suitable sclerocyte lineage marker. Ef lectin, a marker for the cell type involved in innate immunity, shares 59% to 65% identity with the marine sponge Suberites domuncula galactose-binding protein (Sd GBP) and horseshoe crab Tachypleus tridentatus tachylectin1/lectinL6. Since Sd GBP and tachylectin1 are known to bind to bacterial lipopolysaccharides and inhibit the growth of bacteria, Ef lectin may have a similar function and be expressed in a specialized type of cell involved in defense against invading bacteria. Ef lectin mRNA and protein are not expressed in early stages of development, but are detected in late stages. Therefore, Ef lectin may be specifically expressed in differentiating and/or differentiated cells. We suggest Ef lectin as a marker for cells that assume innate immunity in freshwater sponges.     

Immunolocalization of the Toxin Latrunculin B within the Red Sea Sponge Negombata magnifica (Demospongiae, Latrunculiidae)

The location of latrunculin B, the major toxin of the Red Sea sponge Negombata magnifica, was revealed using specific antibodies. Antibodies from rabbits immunized with a conjugate of latrunculin B with keyhole limpet hemocyanin (KLH) were purified over a latrunculin B–Sepharose affinity column. Analysis of immunohistochemical and immunogold-stained sponge sections, using light and transmission electron microscopy, revealed latrunculin B labeling mostly beneath the sponge cortex at the border between the external (ectosome) and internal (endosome) layers (ectosome-endosome border). The endosome was less labeled than the border. Immunogold localization revealed latrunculin B in the sponge cells but not in its prokaryotic symbionts. Archeocytes and choanocytes were significantly more labeled than other cells. The antibodies primarily labeled membrane-limited vacuoles within archeocytes and choanocytes that are perhaps latrunculin B secretory or storage vesicles. Peripheral latrunculin B may have a role in defense against external epibionts, predators, and competitors. Received December 21, 1999; accepted March 5, 2000.     

Stem cells in asexual reproduction of Enchytraeus japonensis (Oligochaeta, Annelid): proliferation and migration of neoblasts

Enchytraeus japonensis is a small oligochaete that reproduces mainly asexually by fragmentation (autotomy) and regeneration. As sexual reproduction can also be induced, it is a good animal model for the study of both somatic and germline stem cells. To clarify the features of stem cells in regeneration, we investigated the proliferation and lineage of stem cells in E. japonensis. Neoblasts, which have the morphological characteristics of undifferentiated cells, were found to firmly adhere to the posterior surface of septa in each trunk segment. Also, smaller neoblast‐like cells, which are designated as N‐cells in this study, were located dorsal to the neoblasts on the septa. By conducting 5‐bromo‐2′‐deoxyuridine (BrdU)‐labeling‐experiments, we have shown that neoblasts are slow‐cycling (or quiescent) in intact growing worms, but proliferate rapidly in response to fragmentation. N‐cells proliferate more actively than do neoblasts in intact worms. The results of pulse‐chase experiments indicated that neoblast and N‐cell lineage mesodermal cells that incorporated BrdU early in regeneration migrated toward the autotomized site to form the mesodermal region of the blastema, while the epidermal and intestinal cells also contributed to the blastema locally near the autotomized site. We have also shown that neoblasts have stem cell characteristics by expressing Ej‐vlg2 and by the activity of telomerase during regeneration. Telomerase activity was high in the early stage of regeneration and correlated with the proliferation activity in the neoblast lineage of mesodermal stem cells. Taken together, our results indicate that neoblasts are mesodermal stem cells involved in the regeneration of E. japonensis.     

N‐glycoprotein surfaceome of human induced pluripotent stem cell derived hepatic endoderm

Using cell surface capture technology, the cell surface N‐glycoproteome of human‐induced pluripotent stem cell derived hepatic endoderm cells was assessed. Altogether, 395 cell surface N‐glycoproteins were identified, represented by 1273 N‐glycopeptides. This study identified N‐glycoproteins that are not predicted to be localized to the cell surface and provides experimental data that assist in resolving ambiguous or incorrectly annotated transmembrane topology annotations. In a proof‐of‐concept analysis, combining these data with other cell surface proteome datasets is useful for identifying potentially cell type and lineage restricted markers and drug targets to advance the use of stem cell technologies for mechanistic developmental studies, disease modeling, drug discovery, and regenerative medicine.     

Induced pluripotent stem cell‐derived melanocyte precursor cells undergoing differentiation into melanocytes

Induced pluripotent stem cell (iPSC) technology offers a novel approach for conversion of human primary fibroblasts into melanocytes. During attempts to explore various protocols for differentiation of iPSCs into melanocytes, we found a distinct and self‐renewing cell lineage that could differentiate into melanocytes, named as melanocyte precursor cells (MPCs). The MPCs exhibited a morphology distinctive from that of melanocytes, in lacking either the melanosomal structure or the melanocyte‐specific marker genes MITF, TYR, and SOX10. In addition, gene expression studies in the MPCs showed high‐level expression of WNT5A, ROR2, which are non‐canonical WNT pathway markers, and its related receptor TGFβR2. In contrast, MPC differentiation into melanocytes was achieved by activating the canonical WNT pathway using the GSK3β inhibitor. Our data demonstrated the distinct characteristic of MPCs' ability to differentiate into melanocytes, and the underlying mechanism of interfacing between canonical WNT signaling pathway and non‐canonical WNT signaling pathway.     

Stem cell dynamics in Cnidaria: are there unifying principles?

The study of stem cells in cnidarians has a history spanning hundreds of years, but it has primarily focused on the hydrozoan genus Hydra. While Hydra has a number of self-renewing cell types that act much like stem cells—in particular the interstitial cell line—finding cellular homologues outside of the Hydrozoa has been complicated by the morphological simplicity of stem cells and inconclusive gene expression data. In non-hydrozoan cnidarians, an enigmatic cell type known as the amoebocyte might play a similar role to interstitial cells, but there is little evidence that I-cells and amoebocytes are homologous. Instead, self-renewal and transdifferentiation of epithelial cells was probably more important to ancestral cnidarian development than any undifferentiated cell lineage, and only later in evolution did one or more cell types come under the regulation of a “stem” cell line. Ultimately, this hypothesis and competing ones will need to be tested by expanding genetic and developmental studies on a variety of cnidarian model systems.     

oct4‐EGFP reporter gene expression marks the stem cells in embryonic development and in adult gonads of transgenic medaka

Maintenance of pluripotency in stem cells is tightly regulated among vertebrates. One of the key genes in this process is oct4, also referred to as pou5f1 in mammals and pou2 in teleosts. Pou5f1 evolved by duplication of pou2 early in the tetrapod lineage, but only monotremes and marsupials retained both genes. Either pou2 or pou5f1 was lost from the genomes of the other tetrapods that have been analyzed to date. Consequently, these two homologous genes are often designated oct4 in functional studies. In most vertebrates oct4 is expressed in pluripotent cells of the early embryo until the blastula stage, and later persist in germline stem cells until adulthood. The isolation and analysis of stem cells from embryo or adult individuals is hampered by the need for reliable markers that can identify and define the cell populations. Here, we report the faithful expression of EGFP under the control of endogenous pou2/oct4 promoters in transgenic medaka (Oryzias latipes). In vivo imaging in oct4‐EGFP transgenic medaka reveals the temporal and spatial expression of pou2 in embryos and adults alike. We describe the temporal and spatial patterns of endogenous pou2 and oct4‐EGFP expression in medaka with respect to germline and adult stem cells, and discuss applications of oct4‐EGFP transgenic medaka in reproductive and stem cell biology. Mol. Reprod. Dev. 80: 48–58, 2013. © 2012 Wiley Periodicals, Inc.