An apicosome initiates self-organizing morphogenesis of human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) self-organize into apicobasally polarized cysts, reminiscent of the lumenal epiblast stage, providing a model to explore key morphogenic processes in early human embryos. Here, we show that apical polarization begins on the interior of single hPSCs through the dynamic formation of a highly organized perinuclear apicosome structure. The membrane surrounding the apicosome is enriched in apical markers and displays microvilli and a primary cilium; its lumenal space is rich in Ca2+ Time-lapse imaging of isolated hPSCs reveals that the apicosome forms de novo in interphase, retains its structure during mitosis, is asymmetrically inherited after mitosis, and relocates to the recently formed cytokinetic plane, where it establishes a fully polarized lumen. In a multicellular aggregate of hPSCs, intracellular apicosomes from multiple cells are trafficked to generate a common lumenal cavity. Thus, the apicosome is a unique preassembled apical structure that can be rapidly used in single or clustered hPSCs to initiate self-organized apical polarization and lumenogenesis.

Comparison of the Ultrastructures of Primed and Naïve Mouse Embryonic Stem Cells.

Culture conditions have been established to maintain the pluripotency of mouse naïve and primed embryonic stem cells (ESCs) using human amnion epithelial cells (hAECs) as the feeder layer. In this study, the ultrastructures of mouse primed ESCs grown on hAECs were analyzed by transmission electron microscopy. Consistent with mouse naïve ESCs, the undifferentiated mouse primed ESC line ESD-EpiSC [ESC-derived epiblast stem cells (EpiSCs)] revealed typical characteristics, including large nuclei, reticulated nucleoli, scanty cytoplasm, and low cytoplasm-to-nuclear ratios. Cells had prominent Golgi apparatus and well-developed endoplasmic reticulum. Adjacent cells were tightly in contact with dense junction desmosomes. However, in EpiSC colonies, cell contact was no longer close like naïve ESCs, and differentiated cells existed. The differentiated cells had small nucleoli with large cytoplasm, which represented primitive mesenchyme. Phagosomes or apoptotic cells were also common in the cytoplasm of differentiated cells, which suggests a differentiation potential. When exposed to leukemia inhibitory factor (LIF), ESD-EpiSCs could convert to naïve-like cells. We further analyzed the ultrastructure of converted EpiSCs (cESCs). As compared to ESD-EpiSCs, cESCs showed similar ultrastructural characteristics as naïve ESCs. These findings suggest that ultrastructure could be used to evaluate the pluripotency of ESCs.

Ultrastructural and molecular distinctions between the porcine inner cell mass and epiblast reveal unique pluripotent cell states.

Characterization of the pluripotent cell populations within the porcine embryo is essential for understanding pluripotency and self-renewal regulation in the inner cell mass (ICM) and epiblast. In this study, we perform detailed ultrastructural and molecular characterization of the developing pluripotent cell population as it develops from the ICM to the late epiblast. The ultrastructural observations revealed that the outer cells of the ICM have a high nuclear:cytoplasmic ratio but are transcriptionally inactive and contain mitochondria with few cristae. In contrast, the epiblast cells have a reduced nuclear:cytoplasmic ratio, are more transcriptionally active, and contain abundant cellular organelles. This study also revealed cavitation and potential unfolding of the epiblast. As the ICM forms the epiblast, SSEA1 is lost and VIMENTIN is lost and re-expressed. The D6 blastocyst expressed high levels of STELLA, TERF1, and GDF3, and the epiblast expressed epithelial markers, MUC1 and E-CADHERIN, and the pluripotency markers, DNMT3B and CRIPTO.

Global chromatin architecture reflects pluripotency and lineage commitment in the early mouse embryo.

An open chromatin architecture devoid of compact chromatin is thought to be associated with pluripotency in embryonic stem cells. Establishing this distinct epigenetic state may also be required for somatic cell reprogramming. However, there has been little direct examination of global structural domains of chromatin during the founding and loss of pluripotency that occurs in preimplantation mouse development. Here, we used electron spectroscopic imaging to examine large-scale chromatin structural changes during the transition from one-cell to early postimplantation stage embryos. In one-cell embryos chromatin was extensively dispersed with no noticeable accumulation at the nuclear envelope. Major changes were observed from one-cell to two-cell stage embryos, where chromatin became confined to discrete blocks of compaction and with an increased concentration at the nuclear envelope. In eight-cell embryos and pluripotent epiblast cells, chromatin was primarily distributed as an extended meshwork of uncompacted fibres and was indistinguishable from chromatin organization in embryonic stem cells. In contrast, lineage-committed trophectoderm and primitive endoderm cells, and the stem cell lines derived from these tissues, displayed higher levels of chromatin compaction, suggesting an association between developmental potential and chromatin organisation. We examined this association in vivo and found that deletion of Oct4, a factor required for pluripotency, caused the formation of large blocks of compact chromatin in putative epiblast cells. Together, these studies show that an open chromatin architecture is established in the embryonic lineages during development and is sufficient to distinguish pluripotent cells from tissue-restricted progenitor cells.

Characterisation of bovine epiblast-derived outgrowth colonies.

The aim of the present study was to characterise bovine epiblast-derived outgrowth colonies (OCs) with respect to the embryonic origin of their cellular components. Epiblasts were isolated mechanically from bovine Day 12 embryos. Epiblasts were cultured on feeder layers of SNL cells (neomycin-resistant leukaemia inhibitory factor (LIF)-producing STO cells) in Dulbecco's modified Eagle's medium (DMEM)/F12 medium supplemented with 15% fetal calf serum, 5% KnockOut Serum Replacement, LIF, basic fibroblast growth factor, non-essential amino acids (NEAA) and nucleosides. Samples were fixed on Days 4, 6 and 8 of culture and processed for immunocytochemistry and transmission electron microscopy. Epiblasts formed OCs consisting of a central core of epiblast-like cells with a basal plate of flattened cells extending outwards from the core. The cells of the core showed nuclear octamer-binding transcription factor 4 (OCT4) staining, indicating an epiblast origin, and some also stained positive for cytoplasmic vimentin. Adjacent cells were linked by tight junctions towards the surface of the colony and rested on a basal lamina. The cells of the basal plate predominantly stained for alpha1-fetoprotein (AFP), indicative of a possible hypoblast origin. Only a few cells scattered within the basal plate exhibited cytokeratin 8 staining, indicating a trophectoderm nature. The intensity of OCT4 and vimentin staining within the core had decreased by Day 8 of culture. In conclusion, OCs derived from bovine Day 12 epiblasts display a central core of OCT4-stained cells of a potential epiblast origin surrounded by a basal plate of mainly AFP-stained cells of a potential hypoblast nature.

Substantial variation in the cardiac differentiation of human embryonic stem cell lines derived and propagated under the same conditions--a comparison of multiple cell lines.

AIM: The differentiation efficiencies of human embryonic stem cell (hESC) lines differ from each other. To assess this in more detail we studied the cardiac differentiation of eight hESC lines derived in the same laboratory. RESULTS: Substantial variation in growth and in the ability to form beating areas was seen between the different hESC lines; line HS346 gave the best efficiency (9.4%), while HS293 did not differentiate into beating colonies at all. Nine germ layer and differentiation markers were quantified during early differentiation in four hESC lines. The expression levels of Brachyury T, MESP1 and NKX2.5 were highest in the most efficient cardiac line (HS346). A systematic characterization of the beating cells revealed proper cardiac marker expression, electrophysiological activity, and pharmacological response. CONCLUSIONS: The hESC lines derived in the same laboratory varied considerably in their potential to differentiate into beating cardiomyocytes. None of the expression markers could clearly predict cardiac differentiation potential, although the expression of early cardiomyogenic genes was upregulated in the best cardiac line. The proper cardiomyocyte characteristics and pharmacological response indicate that these cells could be used as a model for human cardiomyocytes in pharmacological and toxicological analyses when investigating new heart medications or cardiac side-effects.

Tensile forces govern germ-layer organization in zebrafish.

Understanding the factors that direct tissue organization during development is one of the most fundamental goals in developmental biology. Various hypotheses explain cell sorting and tissue organization on the basis of the adhesive and mechanical properties of the constituent cells. However, validating these hypotheses has been difficult due to the lack of appropriate tools to measure these parameters. Here we use atomic force microscopy (AFM) to quantify the adhesive and mechanical properties of individual ectoderm, mesoderm and endoderm progenitor cells from gastrulating zebrafish embryos. Combining these data with tissue self-assembly in vitro and the sorting behaviour of progenitors in vivo, we have shown that differential actomyosin-dependent cell-cortex tension, regulated by Nodal/TGFbeta-signalling (transforming growth factor beta), constitutes a key factor that directs progenitor-cell sorting. These results demonstrate a previously unrecognized role for Nodal-controlled cell-cortex tension in germ-layer organization during gastrulation.

Morphological study of anomalous "laminated" brood capsules in cystic echinococcosis in humans and sheep.

"Anomalous", as yet unreported forms of brood capsules (BC) in cystic echinococcosis larvae from humans and sheep are described, thought to be the likely effect of degenerative involution of the parasite. Morphological examination showed an inner laminated layer (LL) in the brood capsules in intimate contact with the internal wall surface, suggesting the term "laminated brood capsules" (LBC). Analysis conducted on LBC from cysts of different typology invariably revealed them to contain just dead and highly degenerated protoscoleces (PSC). Structural and ultrastructural aspects of these peculiar forms are described and possible processes of formation discussed. Comparison of germinal layer (GL), BC wall and PSC organization recognize a "tegumental" and a "non-tegumental" pole, coinciding in GL and PSC and inverted in BC. A quite reversible PSC-or-LL switch governed by complex mechanisms is proposed, of which PSC viability/death is assumed to be an essential part, that under certain conditions makes in vivo LL production inwards possible in BC.

Adherens junction breakdown in the periderm following cadmium administration in the chick embryo: distribution of cadherins and associated molecules.

BACKGROUND: The teratogenic metal cadmium (Cd) has been found to cause ventral body wall defects similar to human omphalocele when administered to post-gastrulation chick embryos prior to body wall folding. From 4h after Cd, affected embryos demonstrate varying degrees of cell junction breakdown and desquamation in the periderm. We examined the effect of Cd on tissue and cell distribution of cadherins and their intracellular associates. METHODS: Chicks were explanted and given 50microl of 50microM Cd solution at 60h incubation (Hamburger-Hamilton stage 16-17). To examine peridermal junctions, embryos were processed into resin and ultra-thin sections examined by transmission electron microscopy (TEM). Tissue was processed into paraffin and 6microm sections treated according to standard protocols for immunohistochemical detection of L-CAM, pan-cadherin, beta-catenin, alpha-1 and alpha-2 catenin. To examine actin distribution, frozen sections were cut at 10-20microm, stained with oragon green phalloidin and nuclei counter-stained with propidium iodide. RESULTS: The overall tissue distribution of L-CAM, pan-cadherin and the alpha-catenins did not appear to be altered following Cd. However, beta-catenin changed from its normal sub-membranous location to a more general cytoplasmic distribution, with translocation to the nucleus in both peridermal and ectodermal cells. Similarly, actin distribution in the periderm in embryos demonstrating cell junction breakdown was markedly altered, with clumping and disorganization after 4h. CONCLUSIONS: Although L-CAM is distributed normally after Cd, post-translational modification may occur causing breakdown of its normal association with the catenins and actin, and allowing beta-catenin to translocate to the nucleus in peri-ectodermal tissue, mimicking the canonical Wnt pathway.

Cryopreservation of mouse embryoid bodies.

Cryopreservation of embryonic stem (ES) cells is essential to establish them as a resource for regenerative therapy. We evaluated survival adhesion rate, cell structure, gene expression, and multipotency of frozen and thawed embryoid bodies (EBs) derived from mouse ES cells. EBs were cryopreserved using the BICELL and the Program Freezer. After one week the EBs were thawed and cultured. EBs prepared in the Program Freezer had the highest survival adhesion (Program Freezer; 55-69%, BICELL; 30-38%). Though many cells in the thawed EBs were damaged, some were not, especially those prepared in the Program Freezer. RT-PCR analysis showed that genes characteristic of the three embryonic germ layers were expressed in thawed EBs cultured for one week. EBs transplanted into mice formed teratomas consisting of cells derived from the three germ layers. In conclusion, EBs frozen in the Program Freezer had higher survival adhesion rates compared to the BICELL and formed differentiated cells characteristic of the three embryonic germ layers.

Human embryonic stem cells and their spontaneous differentiation.

Human embryonic stem cells (hESCs) usually grow in saucer-shaped colonies with thickened rims and can form spherical human embryoid bodies (hEBs) under non-adherent conditions. A problem associated with ES cell culture is the spontaneous differentiation of cells into a variety of cell types representing all three germ layers, which is evident in both hESC colonies and hEBs. This presentation deals with the precise origins of hESCs and their spontaneous differentiation in vitro. We have used advanced digital microscopy, including transmission electron microscopy (TEM) to define the fine structure of these cells. We present images of undifferentiated hESCs and their spontaneous differentiation into basic embryonic cell types such as nerve, muscle, connective tissue, epithelium, and digestive tract progenitors, representing all three primary germ layers: embryonic ectoderm, mesoderm and endoderm. It appears that hESCs work in concert and interact with one another, as in tissue formation of the embryo. Our fine structural observations agree mostly with those of the Thomson group. Digital microscopy of plastic sections and TEM are invaluable tools in the precise characterization of cells forming these tissues and a combined study with immunofluorescent markers is most desirable.

Generation of embryoid bodies from mouse embryonic stem cells cultured on STO feeder cells.

Embryoid bodies, which are similar to post-implantation egg-cylinder stage embryos, provide a model for the study of embryo development and stem cell differentiation. We describe here a novel method for generating embryoid bodies from murine embryonic stem (ES) cells cultured on the STO feeder layer. The ES cells grew into compact aggregates in the first 3 days of coculture, then became simple embryoid bodies (EBs) possessing primitive endoderm on the outer layer. They finally turned into cystic embryoid bodies after being transferred to Petri dishes for 1-3 days. Evaluation of the EBs in terms of morphology and differentiating potential indicates that they were typical in structure and could generate cells derived from the three germ layers. The results show that embryoid bodies can form not only in suspension culture but also directly from ES cells cultured on the STO feeder layer.

Tetraploid embryonic stem cells contribute to the inner cell mass of mouse blastocysts.

The demonstration that mouse somatic cells can be reprogrammed following fusion with embryonic stem (ES) cells may provide an alternative to somatic cell nuclear transfer (therapeutic cloning) to generate autologous stem cells. In an attempt to produce cells with an increased pool of reprogramming factors, tetraploid ES cells were produced by polyethylene glycol mediated fusion of two ES cell lines transfected with plasmids carrying puromycin or neomycin resistance cassettes, respectively, followed by double antibiotic selection. Tetraploid ES cells retain properties characteristic of diploid ES cells, including the expression of pluripotent gene markers Oct4 and Rex1. On injection into the testis capsule of severe combined immunodeficient (SCID) mice, tetraploid ES cells are able to form teratomas containing cells representative of all three germ layers. Further, these cells demonstrated the ability to integrate into the inner cell mass of blastocysts. This study indicates that tetraploid ES cells are promising candidates as cytoplasm donors for reprogramming studies.

Cell types, lineage, and architecture of the germinal zone in the adult dentate gyrus.

New neurons continue to be born in the subgranular zone (SGZ) of the dentate gyrus in the hippocampus of adult mammals, including humans. Previous work has shown that astrocytes function as the progenitors of these new neurons through immature intermediate D cells. In the first part of the present study, we determined the structure of each of these progenitors and how they are organized in three dimensions. Serial-section reconstructions of the SGZ, using confocal and electron microscopy demonstrate that SGZ astrocytes form baskets that hold clusters of D cells, largely insulating them from the hilus. Two types of glial fibrillary acidic protein-expressing astrocytes (radial and horizontal) and three classes of doublecortin and PSA-NCAM-positive D cells (D1, D2, D3) were observed. Radial astrocytes appear to interact closely with clusters of D cells forming radial proliferative units. In the second part of this study, we show that retrovirally labeled radial astrocytes give rise to granule neurons. We also used bromodeoxyuridine and [3H]thymidine labeling to study the sequence of appearance of the different D cells after a 7-day treatment with anti-mitotics. This analysis, together with retroviral labeling data, suggest that radial astrocytes divide to generate D1 cells, which in turn divide once to form postmitotic D2 cells. D2 cells mature through a D3 stage to form new granule neurons. These observations provide a model of how the germinal zone of the adult hippocampus is organized and suggest a sequence of cellular stages in the generation of new granule neurons.

Gastrulation events in the prestreak pig embryo: ultrastructure and cell markers.

The epithelial versus mesenchymal phenotypes of embryonic ectoderm and mesoderm cells of the prestreak stage pig embryos were examined by electron microscopy and molecular marker analysis. During this period the embryonic disc remained flat or slightly convex while becoming oval or pyriform in shape. Mesenchyme cells expressing vimentin were present between the embryonic disc and the underlying visceral endoderm before a primitive streak (or groove) was apparent. The migration of mesenchyme appeared to occur in lateral and posterior directions from a mass of quiescent cells located in the pointed end of the pyriform embryonic disc that expressed Brachyury; these cells are proposed to be the precursors of the primitive streak and/or form the equivalent of the mouse early gastrula organizer (EGO). Cells with the TEC-1 (or SSEA-1) epitope, the marker most frequently used to characterize pluripotent cells, were initially distributed randomly in the embryonic ectoderm and then were found to localize in an anterior crescent which may contain the precursor cells of ectoderm and neurectoderm. As mitotic figures were found only in the anterior crescent, it is proposed that at least some of these proliferating cells migrate toward the EGO. While cytokeratins were barely detectable in the embryonic ectoderm cells, vimentin expression was supposed to be associated with the migratory capacity of these cells. These findings indicate that the early step of gastrulation, migration of extraembryonic mesoderm, occurs at a prestreak stage during which the embryonic disc becomes polarized. genesis 38:13-25, 2004.

[Diffuse and compact chromatin in the cell nuclei from tissues derived from different germ layers]. / Diffuznyi i kompaktnyi khromatin kletochnykh iader iz tkanei--proizvodnykh razlichnykh zarodyshevykh listkov.

Chromatin structure of nuclei of histogenetically distinct cells of humans and animals under normal and pathological courses of gestation was studied by the method of optic-structural computer analysis. A constant ratio of compact and diffuse chromatin is detected in nuclei of cells originating from all the germ layers. This ratio can change to a variable degree in cases of pathological gestation.

Gastrulation in the mouse embryo: ultrastructural and molecular aspects of germ layer morphogenesis.

Ultrastructural studies and lineage analyses of gastrulating mouse embryos have revealed that different morphogenetic tissue movements are involved in the formation of the three definitive germ layers. Definitive ectoderm is formed by epibolic expansion of the pre-existing progenitor population in the embryonic ectoderm. Formation of the mesoderm and the endoderm is initiated by cellular ingression at the primitive streak. The mesodermal layer is established by cell migration and cell sheet spreading, but the endoderm is formed by replacing the original primitive endodermal population. To this date, genes that are expressed during mouse gastrulation mostly encode cell surface adhesion or signalling molecules, growth factors and their receptors, and putative transcriptional factors. Their precise role during gastrulation remains to be investigated.

Midline structures and central nervous system coordinates in zebrafish.

The embryonic zebrafish provides a relatively simple and accessible experimental system for understanding the underlying Bauplan of a vertebrate central nervous system (CNS) and for uncovering interactions critical for patterning. We show that features of CNS organization in normal, mutant, and developmentally ventralized zebrafish embryos can be explained by a three-axis hypothesis: Specifications occur according to position relative to coordinates along the anterior-posterior, dorsal-ventral, and luminal-pial axes of the neural tube. Midline tissues may be responsible for directly establishing the dorsal-ventral coordinates, but may only be indirectly involved in patterning along the anterior-posterior axis.

Vasculature to the germinal matrix in rabbit pups.

The authors report a study of the cerebral vasculature of premature rabbits pertaining to the germinal matrix (GM). A pigmented silicone material (Microfil) was injected into the carotid artery of anesthetized rabbits. Methyl methacrylate vascular casts of a similar group of premature rabbits were examined by scanning electron microscopy. The GM is supplied by arteries from both the basal and convexity surfaces of the brain. Vessels could be identified as arteries or veins by their typical patterns of branching and by the characteristic impressions made on the methyl methacrylate casts by endothelial nuclei. Specific evidence of structural weaknesses in the vasculature, which could be a site of predilection for GM bleeding, was not observed. The similarities in basal ganglia vasculature between premature rabbits and humans justifies using the rabbit model to study vascular aspects of the GM and intraventricular hemorrhage.

Stage 9 macaque embryos studied by scanning electron microscopy.

Two macaque embryos representing the beginning and the end of stage 9 were studied by scanning electron microscopy. Morphometric evaluation shows that differential growth of head plate elevation, height of the caudal region, width of the head plate and width caudal to the neural groove exceeds embryo elongation, while width of the mid-region, embryonic disc area, width of the embryonic disc, embryo height at the mid-region, length of embryonic disc, and length of the primitive streak undergo relative shrinkage. The head plate becomes topographically subdivided into the three primary segments, while the neural groove narrows, the number of somite pairs increases from one to three, and Hensen's node is obscured. Comparison with relevant rodent studies demonstrates some topographical differences and some cytological similarities.