The outgrowth of parietal endoderm from mouse teratocarcinoma stem-cell embryoid bodies

Teratocarcinoma stem cells can be used to study certain events occurring during early mouse embryogenesis. We report that the outgrowth of parietal endoderm from teratocarcinoma stem-cell embryoid bodies in vitro is analogous to the same process in vivo in terms of the spatial distribution of endoderm types: only parietal endoderm migrates away from the aggregate, whereas visceral endoderm remains associated with the embryoid body. The outgrowths generated on a substrate of type-I collagen from PSA-1 and retinoic-acid-treated F 9 embryoid bodies were found to be comparable, even though these aggregates express different endoderm types. We demonstrated that retinoic-acid-treated F 9 embryoid bodies that contain essentially only visceral endoderm in suspension culture can nonetheless generate parietal-endoderm outgrowth when plated on type-I collagen, suggesting that substrate interaction plays an important role in inducing parietal-endoderm differentiation. These data indicate the usefulness and relevance of studying endoderm differentiation and outgrowth in vitro employing the teratocarcinoma model system.     

Plasminogen activator expression in F9 teratocarcinoma embryoid bodies and their endoderm derivatives

Plasminogen activators are believed to play an important role in tissue remodeling and cell migration. During mouse embryogenesis, visceral endoderm secretes urokinase-type plasminogen activator (uPA) whereas parietal endoderm secretes tissue-type plasminogen activator (tPA). Visceral endoderm from F9 embryoid bodies can transdifferentiate into parietal endoderm under the appropriate culture conditions. We have examined at the protein and mRNA levels the type of plasminogen activator expressed in whole embryoid bodies, visceral endoderm and its parietal endoderm derivatives. Our experiments show that the visceral endoderm on F9 embryoid bodies synthesizes and secretes substantial amounts of both tPA and uPA. In contrast, the parietal endoderm derived directly from the visceral endoderm secretes dramatically increased levels of tPA and decreases production of uPA to low or below detectable levels. These data support the finding that visceral endoderm can transdifferentiate to parietal endoderm. In addition, this transition provides an excellent model for studying the molecular basis of the coincident down- and upregulation of the two plasminogen activators as well as their potential function during embryogenesis.     

Indian hedgehog signaling in extraembryonic endoderm and ectoderm differentiation in ES embryoid bodies

We previously demonstrated that a member of the Hedgehog gene family, Indian hedgehog (Ihh), is expressed in the visceral endoderm of EC and ES cell embryoid bodies and mouse embryos. Overexpression studies suggested that Ihh was involved in visceral endoderm differentiation. We now provide evidence for a Hh response in the embryoid body core and in the mesothelial layer of the visceral yolk sac. We also demonstrate that treatment of ES embryoid bodies with the Hh antagonists cAMP and forskolin results in downregulation of the Hh response and altered embryoid body differentiation. The outer endoderm layer undergoes a transition to parietal endoderm while formation of an embryonic ectoderm layer surrounding a cavity is inhibited. These treatments also result in a decrease in the expression of markers for the mesoderm derivatives, blood and endothelial cells. We present a model to explain how Ihh and BMP signaling may regulate extraembryonic endoderm and embryonic ectoderm differentiation.     

Nucleolus-like bodies in embryonal carcinoma cells of the embryoid bodies isolated from mouse teratocarcinoma

Undifferentiated embryonal carcinoma cells (EC cells) in the embryoid bodies isolated from mouse teratocarcinoma contained nucleolus-like bodies (NLBs) of smaller sizes in their cytoplasm (their sectional area averaged about 0.036 μm²). At the onset of EC cell differentiation, the average sectional area of NLBs significantly increased (about 0.107 μm²). When EC cells had differentiated into mesenchymal cells and endothelial cells of primitive blood vessels, NLBs decreased dramatically both in size and number. The possible role of NLBs in the differentiation process of EC cells is discussed.     

The development of cystic embryoid bodies in vitro from clonal teratocarcinoma stem cells

Previous findings on exogenous RNA-induced heart muscle differentiation in the non-heartforming cultured explants of the chick blastoderm, the postnodal pieces, were reexamined. Some of the changes that characterized the transition in the host tissues were: (i) the formation of highly ordered myofibrils; (ii) the appearance of characteristic cytoplasmic glycogen particles; (iii) a 2.5- and 3.5-fold increase in actin and myosin-like polypetides respectively; (iv) an increase in acetylcholinesterase activity; and (v) the acquisition of spontaneous and rhythmic pulsations. These specific changes appeared only in those explants that received a poly(A)-containing RNA fraction obtained from the 16-day-old chick embryonic heart. Neither synthetic polynucleotides nor a variety of RNA from several sources could replace the RNA from chick embryonic heart as an inducer of heart muscle differentiation.     

Influence of substrate composition on human embryonic stem cell differentiation and extracellular matrix production in embryoid bodies

Stem cells reside in specialized niches in vivo. Specific factors, including the extracellular matrix (ECM), in these niches are directly responsible for maintaining the stem cell population. During development, components of the stem cell microenvironment also control differentiation with precise spatial and temporal organization. The stem cell microenvironment is dynamically regulated by the cellular component, including stem cells themselves. Thus, a mechanism exists whereby stem cells modify the ECM, which in turn affects the fate of the stem cell. In this study, we investigated whether the type of ECM initially adsorbed to the culture substrate can influence the composition of the ECM deposited by human embryonic stem cells (hESCs) differentiating in embryoid bodies, and whether different ECM composition and deposition profiles elicit distinct differentiation fates. We have shown that the initial ECM environment hESCs are exposed to affects the fate decisions of those cells and that this initial ECM environment is constantly modified during the differentiation process. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:212–219, 2015     

The role of cell interactions in the differentiation of teratocarcinoma-derived parietal and visceral endoderm

Cell interactions have been implicated in the differentiation of visceral and parietal endoderm in the developing mouse embryo. Embryoid bodies formed from F9 embryonal carcinoma cells have been useful in characterizing the events which lead to endoderm formation. As part of our effort to specify the interactions which may be involved in this process we have isolated visceral endoderm-like cells (VE) from F9 embryoid bodies and cultured them under various conditions. Using a combination of immunoprecipitation and enzyme-linked immunosorbent assay, we demonstrate that monolayer culture of these cells on a number of different substrates leads to a dramatic decrease in the level of alphafetoprotein (AFP), a VE-specific marker. Northern blot analysis of AFP mRNA indicates very low levels of this message are present after 48 hr in monolayer culture. Coincident with the drop in AFP levels is an increase in the levels of the cytokeratin Endo C and tissue plasminogen activator, both markers for parietal endoderm (PE). Morphological evidence at the ultrastructural level supports a transition from VE to PE. In contrast, the VE phenotype can be maintained in vitro by interaction with aggregates, but not monolayers, of stem cells. In addition, culturing the cells on the curved surface of gelatin-coated dextran beads, but not on a flat gelatin surface facilitates AFP expression and the cells are morphologically intermediate between VE and PE cells. The potential role of junctional complexes and cell shape are discussed.     

A role for p21 (WAF1) in the cAMP-dependent differentiation of F9 teratocarcinoma cells into parietal endoderm

Combined treatment of teratocarcinoma F9 cells with retinoic acid and dibutyryl-cAMP induces the differentiation into cells with a phenotype resembling parietal endoderm. We show that the levels of cyclin-dependent kinase inhibitor p21/WAF1/Cip1 (p21) protein and mRNA are dramatically elevated at the end of this differentiation, concomitantly with the appearance of p21 in the immunoprecipitated CDK2-cyclin E complex. The induction of differentiation markers could not be achieved by expression of ectopic p21 alone and still required treatment with differentiation agents. Clones of F9 cells transfected with sense or antisense p21 cDNA constructs revealed, upon differentiation, upregulated levels of mRNA for thrombomodulin, a parietal endoderm-specific marker, or increased fraction of cells in sub-G1 phase of the cell cycle, respectively. Consistent with this observation, whereas p21 was strictly nuclear in undifferentiated cells, a large proportion of differentiated cells had p21 localized also in the cytoplasm, a site associated with the antiapoptotic function of p21. Furthermore, p21 activated the thrombomodulin promoter in transient reporter assays and the p21 mutant defective in binding to cyclin E was equally efficient in activation. The promoter activity in differentiated cells was reduced by cotransfection of p21-specific siRNA or antisense cDNA. Coexpression of p21 increased the activity of the GAL-p300(1-1303) fusion protein on the GAL sites-containing TM promoter. This implies that p21 might act through a derepression of the p300 N-terminal-residing repression domain, thereby enhancing the p300 coactivator function. As differentiation of F9 cells into parietal endoderm-like cells requires the cAMP signaling, the results together suggest that the cyclin-dependent kinase inhibitor p21 may promote specifically this pathway in F9 cells.     

A fucosyltransferase in teratocarcinoma stem cells. Decreased activity accompanying differentiation to parietal endoderm cells

Teratocarcinoma stem cell F9 expressed a potent fucosyltransferase activity acting on asialofetuin. A majority of the product was susceptible to alpha-L-fucosidase I from almond emulsin, indicating that the linkage formed was mainly Fuc alpha 1 leads to 3GlcNAc. The specific activity of the transferase decreased when the stem cells were induced to differentiate into parietal endoderm cells by retinoic acid and dibutyryl cyclic AMP. Furthermore, PYS-2 cell, a parietal endoderm cell line virtually lacked the transferase. The change in the fucosyltransferase activity could be correlated with cell surface changes occurring during differentiation.     

Localization of phosphatase activities on mouse teratocarcinoma embryoid bodies

Non-specific alkaline phosphatase and Mg²⁺-dependent adenosine triphosphatase activities were ultracytochemically investigated on embryoid bodies of murine teratocarcinomas, in order to find markers of endodermal cell differentiation of early embryonic cells. The former was localized mainly on the cell surface of inner embryonal carcinoma cells, as already shown by other workers, and weakly on the bound surface of outer endodermal cells of embryoid bodies. The latter, however, was found only on the outer free surface of endodermal cells and never on the surface of embryonal carcinoma cells. It suggests that Mg²⁺-dependent ATP activity might become the marker for early differentiation of embryonal carcinoma cells.     

Hormonal induction of differentiation in teratocarcinoma stem cells: generation of parietal endoderm by retinoic acid and dibutyryl cAMP

It has previously been shown that retinoic acid induces multiple phenotypic changes in cultures of F9 teratocarcinoma stem cells. In this paper we demonstrate that these retinoid-generated cells can be converted to yet another cell type by compounds that elevate cAMP concentrations. The phenotype of the new cell type is characterized by the synthesis of plasminogen activator, laminin and type IV collagen, and by very low levels of alkaline phosphatase and lactate dehydrogenase. The secretion of plasminogen activator and type IV collagen, and low levels of alkaline phosphatase and lactate dehydrogenase, have been previously shown to be properties of parietal endoderm, an extraembryonic cell which is generated early in mouse embryonesis. We show here that parietal endoderm also synthesizes laminin. The cell type generated by retinoic acid and dibutyryl cAMP treatment is therefore indistinguishable from definitive parietal endoderm. Analysis of the final phenotype indicates that it is not dependent upon the continued presence of either compound, and that cAMP agents are active only on cells that have been treated with retinoic acid.     

The planar cell polarity pathway directs parietal endoderm migration

Parietal endoderm (PE) contributes to the yolk sac and is the first migratory cell type in the mammalian embryo. We can visualize PE migration in vitro using the F9 teratocarcinoma derived embryoid body outgrowth system and, show here that PE migration is directed by the non-canonical Wnt planar cell polarity (PCP) pathway via Rho/ROCK. Based on golgi apparatus localization and microtubule orientation, 68.6% of cells in control outgrowths are oriented in the direction of migration. Perturbation of Wnt signaling via sFRP treatment results in a loss of orientation coupled with an increase in cell migration. Inhibition of the PCP pathway at the level of Daam1 also results in a loss of cell orientation along with an increase in cell migration, as seen with sFRP treatment. Constitutively active Daam can inhibit the loss of orientation that occurs with sFRP treatment. We previously demonstrated that ROCK inhibition leads to an increase in cell migration, and we now show that these cells also lack oriented migration. Canonical Wnt signaling or the Rac arm of the PCP pathway does not appear to play a role in PE oriented migration. These data suggest the PCP pathway via Rho/ROCK modulates migration of PE.     

BMPs regulate differentiation of a putative visceral endoderm layer within human embryonic stem-cell-derived embryoid bodies.

Human embryonic stem cells (HESCs), pluripotent cells derived from the inner cell mass (ICM) of human blastocysts, represent a novel tool for the study of early human developmental events. When cultured in suspension with serum, HESCs form spherical structures resembling embryoid bodies (EBs). We show that differentiation of HESCs within EBs occurs radially, with central cells then undergoing apoptosis in association with EB cavitation. Cells within the outer layer of cavitating EBs display stage-specific immunoreactivity to pan-keratin, cytokeratin-8, GATA6, alpha-fetoprotein, and transthyretin specific antibodies, and hybridization to disabled-2, GATA4, and GATA6 specific riboprobes. Transmission electron microscopy of these cells reveals clathrin-coated micropinocytotic vesicles, microvilli, and many vacuoles, a phenotype consistent with mouse visceral endoderm (VE) rather than mouse definitive or parietal endoderm. When cultured in media supplemented with the BMP inhibitor noggin, or in the absence of serum, HESC derivatives do not develop the mouse VE-like phenotype. The addition of BMP-4 to noggin-treated HESCs cultured in serum or in serum-free conditions reconstituted development of the VE-like phenotype. These data demonstrate that human EBs undergo developmental events similar to those of mouse EBs and that in vitro BMP signalling induces derivatives of the human ICM to express a phenotype similar to mouse VE.     

Paracrine promotion of cardiomyogenesis in embryoid bodies by LIF modulated endoderm

In the vertebrate embryo the heart is the first organ to form. Embryonic and extra-embryonic tissues are supposed to contribute to cardiac lineage commitment before and during gastrulation in a paracrine fashion. Evidence has accumulated that factors secreted by the anterior lateral endoderm and extra-embryonic endoderm contribute to cardiomyogenesis. Here we exploit in vitro differentiation of embryonic stem cells in embryoid bodies to study differentiation of the extraembryonic endodermal lineage, gastrulation-like processes, and the influence of endoderm on cardiomyogenesis. We demonstrate that in embryoid bodies primitive endoderm differentiates to visceral and parietal endoderm and that parietal endoderm influences onset of cardiomyogenesis in a concentration-dependent manner. Both increased concentrations of leukemia inhibitory factor and its absence in lif-/- embryoid bodies hampered parietal endoderm formation. Reduced differentiation of parietal endoderm correlated with an attenuation of cardiomyogenesis even in the presence of LIE These and previous results suggest that leukemia inhibitory factor is directly and indirectly, via endoderm formation, involved in the regulation of cardiomyogenesis. Increased proliferation of parietal endoderm in lifr -/- embryoid bodies and addition of conditioned lif -/- cell culture supernatant promoted cardiomyogenesis, demonstrating for the first time that parietal endoderm also contributes to cardiomyogenesis in embryoid bodies in a paracrine and leukemia inhibitory factor and its receptor independent pathway. New factors signaling independently of the leukemia inhibitory-factor receptor pathway may sustain cardiomyocyte cell proliferation and thus be a future target for gene therapy of cardiomyopathies and cell therapy of the myocardium.     

BMP signaling induces visceral endoderm differentiation of XEN cells and parietal endoderm

The extraembryonic endoderm of mammals is essential for nutritive support of the fetus and patterning of the early embryo. Visceral and parietal endoderm are major subtypes of this lineage with the former exhibiting most, if not all, of the embryonic patterning properties. Extraembryonic endoderm (XEN) cell lines derived from the primitive endoderm of mouse blastocysts represent a cell culture model of this lineage, but are biased towards parietal endoderm in culture and in chimeras. In an effort to promote XEN cells to adopt visceral endoderm character we have mimicked different aspects of the in vivo environment. We found that BMP signaling promoted a mesenchymal-to-epithelial transition of XEN cells with up-regulation of E-cadherin and down-regulation of vimentin. Gene expression analysis showed the differentiated XEN cells most resembled extraembryonic visceral endoderm (exVE), a subtype of VE covering the extraembryonic ectoderm in the early embryo, and during gastrulation it combines with extraembryonic mesoderm to form the definitive yolk sac. We found that laminin, a major component of the extracellular matrix in the early embryo, synergised with BMP to promote highly efficient conversion of XEN cells to exVE. Inhibition of BMP signaling with the chemical inhibitor, Dorsomorphin, prevented this conversion suggesting that Smad1/5/8 activity is critical for exVE induction of XEN cells. Finally, we show that applying our new culture conditions to freshly isolated parietal endoderm (PE) from Reichert's membrane promoted VE differentiation showing that the PE is developmentally plastic and can be reprogrammed to a VE state in response to BMP. Generation of visceral endoderm from XEN cells uncovers the true potential of these blastocyst-derived cells and is a significant step towards modelling early developmental events ex vivo.     

Effect of ascitic liquid on growth in vitro of embryoid bodies derived from teratocarcinoma.

Embryoid bodies (EB) derived from teratocarcinoma (TC) OTT6050 were cultured with ascitic liquids (AL) from animals carrying 16-, 22- and 35-day evolved EB. At the same time the presence of fibronectin (FN) in AL were analyzed by immunoblotting. Results indicate the probable existence of growth-stimulatory factors for EB, as well as the presence of FN in the 22-day AL.     

Expression of c-fos in parietal endoderm, amnion and differentiating F9 teratocarcinoma cells

The expression of the cellular proto-oncogene, c-fos, in extra-embryonic tissues of the mouse was investigated using a v-fos DNA probe and an affinity-purified antiserum raised against a C-terminal synthetic peptide. At 13.5 days of development, parietal endoderm--a tissue not previously studied using these methods--was found to express c-fos RNA at a higher level than the amnion or placenta. The previously reported dramatic increase in c-fos RNA levels in extra-embryonic membranes during gestation was found to be confined to the amnion. The antipeptide serum specifically recovered proteins with Mr values of 46,000 and 39,000 from extracts of parietal endoderm and amnion cells labelled for 15 min with 35S-methionine. On sodium-dodecyl-sulphate/polyacrylamide gel electrophoresis these proteins co-migrated with proteins immunoprecipitated using serum from rats inoculated with FBJ-MuSV-transformed cells (tumour-bearing rat serum). Pulse-chasing and 32P-labelling experiments showed that the protein with an Mr of 46,000 was rapidly converted into higher-molecular-weight phosphorylated derivatives. F9 teratocarcinoma stem cells differentiated into parietal-endoderm-like cells in response to treatment with retinoic acid and dibutyryl cyclic AMP. However, this differentiation was not accompanied by any large transient increase in c-fos RNA expression.     

Mouse mutants provide new insights into the role of extracellular matrix in cell migration and differentiation

Migratory cell populations in the developing embryo disperse, localize and eventually differentiate in environments rich in extracellular matrix material. The extracellular matrix provides both a substratum for migration and a source of differentiative cues for the developing cells. Mutations in mice and other animals that alter embryonic interstitial environments are now providing information about the role of the extracellular matrix in these early developmental processes.     

Cell autonomous sorting and surface positioning in the formation of primitive endoderm in embryoid bodies

The differentiation and formation of the primitive endoderm in early embryos can be mimicked in vitro by the aggregation of embryonic stem cells to form embryoid bodies. We present morphological evidence that primitive endoderm cells often first locate in the interior of embryoid bodies and subsequently migrate to the surface. Cell mixing experiments indicate that surface positioning is an intrinsic property of endoderm epithelial cells. Moreover, Disabled-2 (Dab2) is required for surface sorting and positioning of the endoderm cells: when Dab2 expression was eliminated, the differentiated endoderm epithelial cells distributed throughout the interior of the embryoid bodies. Surprisingly, E-cadherin is dispensable for primitive endoderm differentiation and surface sorting in embryoid bodies. These results support the model that primitive endoderm cells first emerge in the interior of the inner cell mass and are subsequently sorted to the surface to form the primitive endoderm.     

Localization and expression of integrin subunits in the embryoid bodies of F9 teratocarcinoma cells

F9 embryonal carcinoma cells can differentiate in vitro into either parietal (PE) or visceral (VE) endoderm, depending upon specific retinoic acid (RA) treatment and growth conditions. In differentiated aggregates of F9 cells (EB), the VE is a polarized monolayer surrounding a core of undifferentiated cells. Within 7 days of treatment the cells organize their cytoskeleton and synthesize large amounts of extracellular matrix proteins to form a basal lamina under the newly formed epithelium. All these changes are likely to involve integrin expression and organization. In this study we have analyzed the spatio-temporal changes in the pattern and level of expression of beta1, beta4, alpha5, alpha6A, and alpha6B integrin subunits. We found that the organization of the VE monolayer in F9 aggregates involves both qualitative and quantitative changes in integrin expression. beta1 is downregulated and accumulates in the forming epithelium. The same occurs for alpha5, although its location on the surface of the aggregate appears to be transient as in fully differentiated EB its distribution is uniform. beta4 and alpha6A are also mainly localized in the VE but they are undetectable in undifferentiated aggregates and their expression is induced by RA treatment. An important exception is represented by alpha6B whose distribution and expression remain almost unchanged throughout treatment.