Cultured cell lines from human teratomas provide models to study molecules which might regulate tumour growth or normal embryonic development. Here, we describe efforts to delineate the role of transcription factors, extracellular matrix, and growth factors in controlling growth and differentiation of human embryonal carcinoma and yolk sac tumour.

The biology of human germ cell neoplasia has been the subject of increasing investigation during the past decade. The objectives of such investigations include better understanding of the pathogenesis of the diseases, improvement in patient management, and insights into cancer cell differentiation and human development. Many of the approaches to human germ cell tumour biology have been strongly influenced by work in the mouse. Here we review some comparative aspects of germ cell tumours in mouse and man. There are striking differences between the two species in the mechanisms of tumour development, the role of genetic background in tumour susceptibility, the cytogenetics of the tumours, and the phenotype of the embryonal carcinoma stem cells. Despite these differences, certain common themes emerge from a survey of work on growth factors and their receptors, extracellular matrix molecules and transcriptional regulators. Both mouse and human embryonal carcinoma cells produce growth factors, but strong evidence for autocrine regulation of stem cell growth is lacking in both species. Differentiated derivatives (primarily extraembryonic endoderm) of mouse embryonal carcinoma cells may respond to factors produced by the stem cells. These differentiated cells also secrete high levels of growth factors and extracellular matrix molecules in both species, supporting a role for paracrine growth interactions in peri-implantation development. The requirements of embryonal carcinoma stem cells for cell substrate adhesion molecules are specific and differ in mouse and man. There are some similarities in the induction of putative transcriptional regulators by retinoids in human and mouse embryonal carcinoma cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The molecular mechanisms controlling DNA-damage-induced apoptosis of human embryonic stem cells (hESC) are poorly understood. Here we investigate the role of p53 in etoposide-induced apoptosis. We show that p53 is constitutively expressed at high levels in the cytoplasm of hESC. Etoposide treatment results in a rapid and extensive induction of apoptosis and leads to a further increase in p53 and PUMA expression as well as Bax processing. p53 both translocates to the nucleus and associates with the mitochondria, accompanied by colocalization of Bax with Mcl1. hESC stably transduced with p53 shRNA display 80% reduction of endogenous p53 and exhibit an 80% reduction in etoposide-induced apoptosis accompanied by constitutive downregulation of Bax and an attenuated upregulation of PUMA. Our data further show that undifferentiated hESC that express Oct4 are much more sensitive to etoposide-induced apoptosis than their more differentiated progeny. Our study demonstrates that p53 is required for etoposide-induced apoptosis of hESC and reveals, at least in part, the molecular mechanism of DNA-damage-induced apoptosis in hESC.

The fifth harmonic (162 nm, 11 fs), generated in a short argon cell from 12 fs Ti-sapphire laser pulses, was used to excite C 2H 4 and C 2D 4 in the maximum of the first pipi* transition. Around 10% of the molecules were excited to the pi3s Rydberg state instead. The subsequent motion of the wave packet, moving over the potentials from the Franck-Condon region down to the ground state, was monitored by nonresonant ionization at 810 nm with mass-selective detection of the ion yield. Five time constants (from approximately 20 fs in excited states to 0.6-11 ps in the hot ground state) and four coherent oscillations (CC stretch and torsion vibrations or hindered free rotation) were determined for each isotopomer. The initial relaxation follows a superposition of CC twist and stretch coordinates; this explains a surprisingly small deuterium isotope effect of the initial time constant (21 versus 24 fs). Also the vibrations in the Franck-Condon region have such a mixed character and a correspondingly small isotope shift. From the perpendicular minimum the wave packet reaches (within 17 or 21 fs for the two isotopomers) a conical intersection via a direction that also involves partial hydrogen migration. This is concluded from the detection of ethylidene (CH 3CH), formed simultaneously with ground-state ethylene. This carbene isomerizes in the ground state within 0.6 ps (1.6 ps for CD 3CD) to ethylene. Two time constants for dissociation (4.5 and 11 ps) in the hot ground state were also identified. The small yields of bimolecular reactions (photodimerization, addition reactions involving a "suddenly polarized" excited state, carbene reactions) are interpreted in terms of the short lifetimes. It is pointed out that the relaxation path starting from the Rydberg state merges into that from the pipi* state; nevertheless, there is a wavelength dependence in the photochemistry of olefins, because due to a momentum effect the wave packet remembers from which state it came.

Background Intravenous immunoglobulins (IVIg) and cytomegalovirus immunoglobulins (CMVIg) are currently finding increased acceptance in clinical states of high immune activity and in transplant recipients. A rare side-effect of their application is intravascular thrombosis, which is thought to be related to pre-existing hyperviscosity. In a previous study we have shown that rabbit antithymocyte globulin causes platelet aggregation in vitro via the Fc IgG receptor (CD32). Objectives To investigate if IVIg and CMVIg have the potential to cause CD32-dependent platelet aggregation. Methods The influence of CMVIg or IVIg on platelets pre-incubated with or without monoclonal antibody AT10 was studied in an aggregometer. Expression of platelet surface activation marker CD62P was determined by fluorescence-activated cell sorting analysis and presence of soluble CD40L (sCD40L) was evaluated by enzyme-linked immunosorbent assay. All in vitro experiments were performed using platelet concentrates from the blood bank, at therapeutic concentrations of immunoglobulins. Results Incubation of platelets with CMVIg and IVIg markedly induced platelet aggregation, and increased expression of CD62P and secretion of sCD40L. The capacity of CMVIg and IVIg to induce platelet aggregation was completely abrogated by adding the blocking antibody AT10 directed against the low-affinity Fc IgG receptor (CD32). Conclusions Our results suggest that CMVIg and IVIg solutions with activating Fc domains are able to bind CD32 on platelets and cause platelet aggregation in vitro. These results indicate a mechanism by which in vivo intravascular thrombosis may be explained and suggest caution with concomitant use of packed platelets and IVIg in autoimmune diseases in the clinical setting.

Genes have to be expressed in specific cell types at appropriate times of development dependent on external signals. cAMP signaling occurs in all cells, thus raising the question of how this signal transduction pattern is integrated into mechanisms determining cell-specific gene expression. We have analyzed expression of the tyrosine aminotransferase gene as a model to study the basis of this cell type specificity of hormone induction. We found that cell-type-specific expression is generated by combined action of cAMP signal-dependent and liver cell-specific transcription factors. The interdependence of the cAMP response element and an element determining liver cell specificity enables a gene to respond to an ubiquitous signal in a cell-specific manner.

Complete and isolated herniation of the urinary bladder is extremely rare, and the consecutive appearance of bilateral urethral obstruction and renal failure is even rarer. We report about a 73 year old male presenting with massive nausea and muscular weakness. On physical examination he showed a giant inguinal hernia with involvement of the entire bladder along with evidence of bilateral hydronephrosis. His serum creatinine and potassium levels were markedly elevated most likely leading to his presenting symptoms of azotemia (nausea) and hyperkalemia (weakness). After transscrotal drainage and decompression of the bladder, a transurethral catheter was inserted. After gaining full renal recovery, the hernia was repaired successfully performing the Lichtenstein procedure.

Current methods to analyze gene expression measure steady-state levels of mRNA. To specifically analyze mRNA transcription, we have developed a technique that can be applied in vivo in intact cells and animals. Our method makes use of the cellular pyrimidine salvage pathway and is based on affinity-chromatographic isolation of thiolated mRNA. When combined with data on mRNA steady-state levels, this method is able to assess the relative contributions of mRNA synthesis and degradation/stabilization. It overcomes limitations associated with currently available methods such as mechanistic intervention that disrupts cellular physiology, or the inability to apply the techniques in vivo. Our method was first tested in serum response of cultured fibroblast cells and then applied to the study of renal ischemia reperfusion injury, demonstrating its applicability for whole organs in vivo. Combined with data on mRNA steady-state levels, this method provided a detailed analysis of regulatory mechanisms of mRNA expression and the relative contributions of RNA synthesis and turnover within distinct pathways, and identification of genes expressed at low abundance at the transcriptional level.

With the advent of induced pluripotent stem cell (iPSC) technology, it is now feasible to generate iPSCs with a defined genotype or disease state. When coupled with direct differentiation to a defined lineage, such as hepatic endoderm (HE), iPSCs would revolutionize the way we study human liver biology and generate efficient "off the shelf" models of human liver disease. Here, we show the "proof of concept" that iPSC lines representing both male and female sexes and two ethnic origins can be differentiated to HE at efficiencies of between 70%-90%, using a method mimicking physiological relevant condition. The iPSC-derived HE exhibited hepatic morphology and expressed the hepatic markers albumin and E-cadherin, as assessed by immunohistochemistry. They also expressed alpha-fetoprotein, hepatocyte nuclear factor-4a, and a metabolic marker, cytochrome P450 7A1 (Cyp7A1), demonstrating a definitive endodermal lineage differentiation. Furthermore, iPSC-derived hepatocytes produced and secreted the plasma proteins, fibrinogen, fibronectin, transthyretin, and alpha-fetoprotein, an essential feature for functional HE. Additionally iPSC-derived HE supported both CYP1A2 and CYP3A4 metabolism, which is essential for drug and toxicology testing. Conclusion: This work is first to demonstrate the efficient generation of hepatic endodermal lineage from human iPSCs that exhibits key attributes of hepatocytes, and the potential application of iPSC-derived HE in studying human liver biology. In particular, iPSCs from individuals representing highly polymorphic variants in metabolic genes and different ethnic groups will provide pharmaceutical development and toxicology studies a unique opportunity to revolutionize predictive drug toxicology assays and allow the creation of in vitro hepatic disease models.(HEPATOLOGY 2009.).

BACKGROUND: The extraembryonic endoderm (ExEn) defines the yolk sac, a set of membranes that provide essential support for mammalian embryos. Recent findings suggest that the committed ExEn precursor is present already in the embryonic Inner Cell Mass (ICM) as a group of cells that intermingles with the closely related epiblast precursor. All ICM cells contain Oct4, a key transcription factor that is first expressed at the morula stage. In vitro, the epiblast precursor is most closely represented by the well-characterized embryonic stem (ES) cell lines that maintain the expression of Oct4, but analogous ExEn precursor cell lines are not known and it is unclear if they would express Oct4. METHODOLOGY/PRINCIPAL FINDINGS: Here we report the isolation and characterization of permanently proliferating Oct4-expressing rat cell lines ("XEN-P cell lines"), which closely resemble the ExEn precursor. We isolated the XEN-P cell lines from blastocysts and characterized them by plating and gene expression assays as well as by injection into embryos. Like ES cells, the XEN-P cells express Oct4 and SSEA1 at high levels and their growth is stimulated by leukemia inhibitory factor, but instead of the epiblast determinant Nanog, they express the ExEn determinants Gata6 and Gata4. Further, they lack markers characteristic of the more differentiated primitive/visceral and parietal ExEn stages, but exclusively differentiate into these stages in vitro and contribute to them in vivo. CONCLUSIONS/SIGNIFICANCE: Our findings (i) suggest strongly that the ExEn precursor is a self-renewable entity,(ii) indicate that active Oct4 gene expression (transcription plus translation) is part of its molecular identity, and (iii) provide an in vitro model of early ExEn differentiation.

The ultrastructure of endoderm cells of the area pellucida has been analysed in the chick embryo by stereological methods. These cells show a specific subcellular evolution which can be correlated with several aspects of morphogenetic behaviour. The cell form coefficient (CFc) changes notably from stage 5 (0.683) to stage 8 (0.446) accompanying the transformation of this layer into a squamous epithelium. An increase of the nuclear surface density is observed and is discussed in relation to the control of nucleocytoplasmic interchange. The mitochondrial volume and surface densities remain constant (3.12% of cellular volume and 0.727 mitochondria/mu(3) respectively). The endodermal cells possess higher levels of vitelline reserves (lipid bodies, 6.97% and yolk droplets, 8.90%) than other cellular types of the chick embryo. This fact is discussed with respect to the role of the endoderm in the phagocytosis of yolk. The RER length density shows an increase that could be related to some specific changes of the extracellular matrix during this period, but this fact remains to be demonstrated in relation to changes of Golgi membranes.

Epithelial-to-mesenchymal transitions (EMTs) play key roles in the normal development of an organism as well as its demise following the metastasis of a malignant tumour. An EMT during early mouse development results in the differentiation of primitive endoderm into the parietal endoderm that forms part of the parietal yolk sac. In the embryo, primitive endoderm develops from cells in the inner cell mass, but the signals that instruct these cells to become specified and adopt an epithelial fate are poorly understood. The mouse F9 teratocarcinoma cell line, a model that can recapitulate the in vivo primitive to parietal endoderm EMT, has been used extensively to elucidate the signalling cascades involved in extraembryonic endoderm differentiation. Here, we identified Wnt6 as a gene up-regulated in F9 cells in response to RA and show that Wnt6 expressing cells or cells exposed to Wnt6 conditioned media form primitive endoderm. Wnt6 induction of primitive endoderm is accompanied by beta-catenin and Snail1 translocation to the nucleus and the appearance of cytokeratin intermediate filaments. Attenuating glycogen synthase kinase 3 activity using LiCl gave similar results, but the fact that cells de-differentiate when LiCl is removed reveals that other signalling pathways are required to maintain cells as primitive endoderm. Finally, Wnt6-induced primitive endodermal cells were tested to determine their competency to complete the EMT and differentiate into parietal endoderm. Towards that end, results show that up-regulating protein kinase A activity is sufficient to induce markers of parietal endoderm. Together, these findings indicate that undifferentiated F9 cells are responsive to canonical Wnt signalling, which negatively regulates glycogen synthase kinase 3 activity leading to the epithelialization and specification of primitive endoderm competent to receive additional signals required for EMT. Considering the ability of F9 cells to mimic an in vivo EMT, the identification of this Wnt6-beta-catenin-Snail signalling cascade has broad implications for understanding EMT mechanisms in embryogenesis and metastasis.

The extraembryonic endoderm of the elongating ovine conceptus was analyzed by scanning and transmission electron microscopy and by whole mount actin staining and immunofluorescence. Morphological and functional differences between the visceral endoderm (VE), the founding cell layer, and the parietal endoderm (PE) are presented. During the elongation process, the PE differentiated to fusiform multinucleated cells aligned parallel to the elongation axis of the conceptus, whereas the VE cells retained the aspect of typical epithelial cells. The multinucleated PE cells however, expressed cellular and nuclear markers typical of endodermal and polarized epithelial cells. The proteins of the extracellular matrix, laminin, and fibronectin, were specifically expressed in the PE. The presence of pairs of nuclei linked by mid-bodies positively stained with tubulin antibodies, indicated that the syncytial differentiation of the PE was due to karyokinesis which was not followed by cytokinesis rather than by cell fusion. genesis 45:709-715, 2007.(c) 2007 Wiley-Liss, Inc.

ABSTRACT: BACKGROUND: The number of genes characterized in liver development is steadily increasing, but the origin of liver precursor cells and the molecular control of liver formation remain poorly understood. Existing theories about formation of zebrafish visceral organs emphasize either their budding from the endodermal rod or formation of independent anlage followed by their later fusion, but none of these is completely satisfactory in explaining liver organogenesis in zebrafish. RESULTS: Expression of a gene encoding the retinol binding protein 4 (Rbp4) was analyzed in zebrafish. rbp4, which is expressed mainly in the liver in adults, was shown to be expressed in the yolk syncytial layer (YSL) during early embryogenesis. At 12-16 hpf rbp4 expression was restricted to the ventro-lateral YSL and later expanded to cover the posterior YSL. We demonstrated that rbp4 expression was negatively regulated by Nodal and Hedgehog (Hh) signalling and positively controlled by retinoic acid (RA). Knockdown of Rbp4 in the YSL resulted in shortened yolk extension as well as the formation of two liver buds, which could be due to impaired migration of liver progenitor cells. rbp4 appears also to regulate the extracellular matrix protein Fibronectin1 (Fn1) specifically in the ventro-lateral yolk, indicating a role of Fn1 in liver progenitor migration. Since exocrine pancreas, endocrine pancreas, intestine and heart developed normally in Rbp4 morphants, we suggest that rbp4 expression in the YSL is required only for liver development. CONCLUSION: The characteristic expression pattern of rbp4 suggests that the YSL is patterned despite its syncytial nature. YSL-expressed Rbp4 plays a role in formation of both yolk extension and liver bud, the latter may also require migration of liver progenitor cells.

Sox17 is a Sry-related HMG-box transcription factor developmentally expressed in both the definitive endoderm and extraembryonic endoderm (ExE). Although Sox17(-/-) mouse embryos have a defective definitive gut endoderm, their developing ExE is morphologically intact. Here, we aimed to investigate the role of Sox17 in ExE development by using an in vitro differentiation system of embryonic stem cells (ESCs). Although forced Sox17 expression in ESCs did not affect ExE commitment, it facilitated the differentiation of ESC-derived primitive endoderm cells into visceral and parietal endoderm cells. This event was inhibited by the forced expression of Nanog, a negative regulator of differentiation of ESCs into the ExE. Although Sox17(-/-) ESCs could differentiate into primitive endoderm cells, further differentiation was severely impaired. These results indicate a substantial involvement of Sox17 in the late stage of ExE differentiation in vitro. Furthermore, the expression of Sox7 - another Sox factor, concomitantly expressed with Sox17 in the developing ExE - was suppressed during the in vitro differentiation of Sox17(-/-) ESCs, but it was maintained at a high level in the extraembryonic tissues of Sox17(-/-) embryos. These findings possibly explain the discrepancy between the ExE phenotype derived from Sox17(-/-) ESCs and that of Sox17(-/-) embryos.

Human embryonal carcinoma (EC) cell lines exhibit considerable heterogeneity in their levels of pluripotency. Thus, NT2/D1 cells differentiate into neural lineages upon exposure to all-trans retinoic acid (ATRA) and non-neural epithelial lineages upon exposure to Bone Morphogenetic Protein-2 (BMP-2). In contrast, 27X-1 cells differentiate into extra-embryonic endodermal (ExE) cells upon treatment with either morphogen. To understand the molecular basis for the differential responses of the two cell lines, we performed gene expression profiling at the undifferentiated EC cell line state to identify constitutive differences in gene expression. NT2/D1 cells preferentially expressed transcripts associated with neurectodermal development, while 27X-1 cells expressed high levels of transcripts associated with mesendodermal characteristics. We then determined temporal expression profiles of 27X-1 cells during ExE differentiation upon treatment with ATRA and BMP-2 and compared the data with changes in gene expression observed during BMP-2- and ATRA-induced differentiation of NT2/D1 cells. ATRA and BMP-2 induced distinct sets of transcription factors and phenotypic markers in the two EC cell lines underlying distinct lineage choices. While 27X-1 differentiation yielded comprehensive gene expression profiles of parietal endodermal lineages, we were able to use the combined analysis of 27X-1 data with data derived from yolk sac tumors for the identification of transcripts associated with visceral endoderm formation. Our results demonstrate constitutive differences in the levels of pluripotency between NT2/D1 and 27X-1 cells that correlate with linage potential. This study also demonstrates that EC cells can serve as robust models to investigate early lineage choices during both embryonic and extra-embryonic human development.

We report the differentiation potential of an immortalized non-tumorigenic human liver epithelial cell line, THLE-5b. Under basic culture conditions THLE-5b showed undifferentiated phenotypes. When grown as cell aggregates, THLE-5b exhibited a hepatocyte-like ultrastructure, ammonia metabolic activity and several other indicators that suggest hepatocytic maturation, including up-regulation or induction of liver-specific genes such as albumin and tryptophane 2,3-dioxygenase, and down-regulation of biliary cell markers such as gamma-glutamyl transpeptidase (GGT). Under these conditions, transcriptional factors such as HNF-1 and HNF-4alpha were also up-regulated or induced. In Matrigel culture, expression of GGT was up-regulated. THLE-5b expressed both albumin and alpha 1-antitrypsin, but lost expression of CK19 in severe combined immunodeficient mice. Thus, THLE-5b can be aligned with progenitor cells, which are committed to the hepatocytic or biliary epithelial cell lineage. These results imply that bipotent progenitor cell populations similar to THLE-5b cells may exist in adult human liver.

The central concept of this chapter is that derangement of microenvironment, which takes place in tumor progression, leads to the partial or full dedifferentiation of epithelial tumors. The review considers the role of intercellular communications and interaction of cells with extracellular matrix (ECM) in differentiation and tumor progression. To illustrate this point, we consider the main characteristics of normal hepatocyte differentiation and its alterations in the course of hepatocellular carcinoma progression and epithelial-to-mesenchymal transition. The control of liver differentiation is mainly implemented by hepatocyte nuclear factors (HNFs). Derangement of HNF regulatory network is clearly associated with hepatocarcinogenesis and progression. We suppose that tissue-specific factors, playing the most important role in the differentiation of particular epithelial cell types, are the preferential targets for inactivation in the progression of corresponding tumors. Moreover, these transcriptional regulators may mediate the interaction of epithelial cells with the microenvironment.