Low expression of activin a in mouse and human embryonic teratocarcinoma cells

TGFβ family factors play an important role in regulating the balance of self-renewal and differentiation of mouse and human pluripotent stem and embryonic teratocarcinoma cells. The expression patterns of TGFβ family signaling ligands and functional roles of these signaling pathways differ significantly in mouse and human embryonic stem cells, but the activity and functional role of these factors in mouse and human embryonic teratocarcinoma cells were not sufficiently investigated. Comparative quantitative real-time PCR analysis of the expression of TGFβ family factors in mouse embryonic stem, embryonic germ, and embryonic teratocarcinoma cells showed that embryonic teratocarcinoma cells express lower ActivinA than pluripotent stem cells but similar levels of factors Nodal, Lefty1, TGFβ1, BMP4, and GDF3. In human nullipotent embryonic teratocarcinoma PA-1 cells, most factors of the TGFβ family (ACTIVINA, NODAL, LEFTY1, BMP4, and GDF3) are expressed at lower levels than in human embryonic stem cells. Thus, in mouse and human nullipotent teratocarcinoma cells, the expression of ActivinA is significantly reduced compared with embryonic stem cells. Presumably, these differences may be associated with changes in the functional activity of the respective signaling pathways and deregulation of proliferative and antiproliferative mechanisms in embryonic teratocarcinoma cells.     

Inhibition of Activin/Nodal signaling promotes specification of human embryonic stem cells into neuroectoderm

Nodal, a member of the TGF-β family of signaling molecules, has been implicated in pluripotency in human embryonic stem cells (hESCs) [Vallier, L., Reynolds, D., Pedersen, R.A., 2004a. Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway. Dev. Biol. 275, 403-421], a finding that seems paradoxical given Nodal's central role in mesoderm/endoderm specification during gastrulation. In this study, we sought to clarify the role of Nodal signaling during hESC differentiation by constitutive overexpression of the endogenous Nodal inhibitors Lefty2 (Lefty) and truncated Cerberus (Cerb-S) and by pharmacological interference using the Nodal receptor antagonist SB431542. Compared to wildtype (WT) controls, embryoid bodies (EBs) derived from either Lefty or Cerb-S overexpressing hESCs showed increased expression of neuroectoderm markers Sox1, Sox3, and Nestin. Conversely, they were negative for a definitive endoderm marker (Sox17) and did not generate beating cardiomyocyte structures in conditions that allowed mesendoderm differentiation from WT hESCs. EBs derived from either Lefty or Cerb-S expressing hESCs also contained a greater abundance of neural rosette structures as compared to controls. Differentiating EBs derived from Lefty expressing hESCs generated a dense network of β-tubulin III positive neurites, and when Lefty expressing hESCs were grown as a monolayer and allowed to differentiate, they generated significantly higher numbers of β-tubulin positive neurons as compared to wildtype hESCs. SB431542 treatments reproduced the neuralising effects of Lefty overexpression in hESCs. These results show that inhibition of Nodal signaling promotes neuronal specification, indicating a role for this pathway in controlling early neural development of pluripotent cells.     

Intestinal lineage commitment of embryonic stem cells

Generating lineage-committed intestinal stem cells from embryonic stem cells (ESCs) could provide a tractable experimental system for understanding intestinal differentiation pathways and may ultimately provide cells for regenerating damaged intestinal tissue. We tested a two-step differentiation procedure in which ESCs were first cultured with activin A to favor formation of definitive endoderm, and then treated with fibroblast-conditioned medium with or without Wnt3A. The definitive endoderm expressed a number of genes associated with gut-tube development through mouse embryonic day 8.5 (Sox17, Foxa2, and Gata4 expressed and Id2 silent). The intestinal stem cell marker Lgr5 gene was also activated in the endodermal cells, whereas the Msi1, Ephb2, and Dcamkl1 intestinal stem cell markers were not. Exposure of the endoderm to fibroblast-conditioned medium with Wnt3A resulted in the activation of Id2, the remaining intestinal stem cell markers and the later gut markers Cdx2, Fabp2, and Muc2. Interestingly, genes associated with distal gut-associated mesoderm (Foxf2, Hlx, and Hoxd8) were also simulated by Wnt3A. The two-step differentiation protocol generated gut bodies with crypt-like structures that included regions of Lgr5-expressing proliferating cells and regions of cell differentiation. These gut bodies also had a smooth muscle component and some underwent peristaltic movement. The ability of the definitive endoderm to differentiate into intestinal epithelium was supported by the vivo engraftment of these cells into mouse colonic mucosa. These findings demonstrate that definitive endoderm derived from ESCs can carry out intestinal cell differentiation pathways and may provide cells to restore damaged intestinal tissue.     

Basic fibroblast growth factor and its receptors in human embryonic stem cells

Human embryonic stem cells (hESCs) are pluripotent stem cells with long-lasting capacity to self-renew and differentiate into various cell types of endodermal, ectodermal or mesodermal origin. Unlike mouse ESCs (mESCs), which can be maintained in an undifferentiated state simply by adding leukemia inhibitory factor (LIF) into the culture medium, hESCs are notorious for the sustained willingness to differentiate and not yet clearly defined signaling pathways that are crucial for their "stemness". Presently, our knowledge involves only limited number of growth factor signaling pathways that appear to be biologically relevant for stem cell functions in vitro. These include BMP, TGFbeta, Wnt, and FGF signaling pathway. The purpose of this review is to summarize recent data on the expression of FGFs and their receptors in hESCs, and critically evaluate the potential effects of FGF signals for their undifferentiated growth and/or differentiation in context with our current understanding of FGF/FGFR biology.     

Dissecting signaling pathways that govern self-renewal of rabbit embryonic stem cells

The pluripotency and self-renewal of embryonic stem cells (ESC) are regulated by a variety of cytokines/growth factors with some species differences. We reported previously that rabbit ESC (rESC) are more similar to primate ESC than to mouse ESC. However, the signaling pathways that regulate rESC self-renewal had not been identified. Here we show that inhibition of the transforming growth factor beta (TGFbeta), fibroblast growth factor (FGF), and canonical Wnt/beta-catenin (Wnt) pathways results in enhanced differentiation of rESC accompanied by down-regulation of Smad2/3 phosphorylation and beta-catenin expression and up-regulation of phosphorylation of Smad1 and beta-catenin. These results imply that the TGFbeta, FGF, and Wnt pathways are required for rESC self-renewal. Inhibition of the MAPK/ERK and PI3K/AKT pathways, which lie downstream of the FGF pathway, led to differentiation of rESC accompanied by down-regulation of phosphorylation of ERK1/2 or AKT, respectively. Long-term self-renewal of rESC could be achieved by adding a mixture of TGFbeta ligands (activin A, Nodal, or TGFbeta1) plus basic FGF (bFGF) and Noggin in the absence of serum and feeder cells. Our findings also suggest that there is a regulatory network consisting of the FGF, Wnt, and TGFbeta pathways that controls rESC pluripotency and self-renewal. We conclude that bFGF controls the stem cell properties of rESC both directly and indirectly through TGFbeta or other pathways, whereas the effect of Wnt on rESC might be mediated by the TGFbeta pathway.     

Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells

TET family enzymes convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. Here, we show that Tet1 and Tet2 are Oct4-regulated enzymes that together sustain 5hmC in mouse embryonic stem cells (ESCs) and are induced concomitantly with 5hmC during reprogramming of fibroblasts to induced pluripotent stem cells. ESCs depleted of Tet1 by RNAi show diminished expression of the Nodal antagonist Lefty1 and display hyperactive Nodal signaling and skewed differentiation into the endoderm-mesoderm lineage in embryoid bodies in?vitro. In Fgf4- and heparin-supplemented culture conditions, Tet1-depleted ESCs activate the trophoblast stem cell lineage determinant Elf5 and can colonize the placenta in midgestation embryo chimeras. Consistent with these findings, Tet1-depleted ESCs?form aggressive hemorrhagic teratomas with increased endoderm, reduced neuroectoderm, and ectopic appearance of trophoblastic giant cells. Thus, 5hmC is an epigenetic modification associated with the pluripotent state, and Tet1 functions to regulate the lineage differentiation potential of ESCs.     

The location and expression of fibroblast growth factor (FGF) in F9 visceral and parietal embryonic cells after retinoic acid-induced differentiation

It is well-established that fibroblast growth factors (FGFs) participate in mesoderm formation and patterning in the developing embryo. To identify cells in mammalian embryos that produce and/or respond to FGFs, we utilized the F9 teratocarcinoma cell system. Undifferentiated F9 cells resemble inner cell mass (ICM) cells of the mouse blastocyst by several criteria including having a characteristic high nuclear to cytoplasmic ratio and by their expression of stage-specific embryonic antigens. F9 stem cells differ from ICM cells by their low spontaneous rate of differentiation and their differentiation potential. ICM cells are heterogeneous with a proportion of the cells maintaining totipotency. In contrast, F9 stem cells appear capable of forming only endodermal derivatives. Retinoic acid (RA) treatment of F9 stem cells is required for them to differentiate, and under different culturing conditions the F9 cells will form either extraembryonic parietal or visceral endoderm. We have previously shown that FGF is synthesized by F9 parietal endoderm, but not by F9 stem cells. Our present study demonstrates that F9 aggregate cultures that contain visceral endoderm cells produce cell-associated-heparin-binding mitogens for 3T3 and endothelial cells, factors with characteristics of FGFs. Furthermore, our studies detect endothelial cell-mitogens within the extracellular matrix (ECM) of F9 parietal endoderm cells, not detected within F9 stem cell 'matrices'. Parietal endoderm cell matrix mitogens could be removed by prior treatment of the ECM with buffers containing heparin or 2 M NaCl, and could be neutralized by basic FGF antibodies.     

Interdependent fibroblast growth factor and activin A signaling promotes the expression of endodermal genes in differentiating mouse embryonic stem cells expressing Src Homology 2-domain inactive Shb

Abstract The mechanisms controlling endodermal development during stem cell differentiation have been only partly elucidated, although previous studies have suggested the participation of fibroblast growth factor (FGF) and activin A in these processes. Shb is a Src homology 2 (SH2) domain-containing adapter protein that has been implicated in FGF receptor 1 (FGFR1) signaling. To study the putative crosstalk between activin A and Shb-dependent FGF signaling in the differentiation of endoderm from embryonic stem (ES) cells, embryoid bodies (EBs) derived from mouse ES cells overexpressing wild-type Shb or Shb with a mutated SH2 domain (R522K-Shb) were cultured in the presence of activin A. We show that expression of R522K-Shb results in up-regulation of FGFR1 and FGF2 in EBs. Addition of activin A to the cultures enhances the expression of endodermal genes primarily in EBs expressing mutant Shb. Inhibition of FGF signaling by the addition of the FGFR1 inhibitor SU5402 completely counteracts the synergistic effects of R522K-Shb and activin A. In conclusion, the present results suggest that expression of R522K-Shb enhances certain signaling pathways downstream of FGF and that an interplay between FGF and activin A participates in ES cell differentiation to endoderm.     

Expression of TGFβ family factors and FGF2 in mouse and human embryonic stem cells maintained in different culture systems

Mouse and human embryonic stem cells are in different states of pluripotency (naive/ground and primed states). Mechanisms of signaling regulation in cells with ground and primed states of pluripotency are considerably different. In order to understand the contribution of endogenous and exogenous factors in the maintenance of a metastable state of the cells in different phases of pluripotency, we examined the expression of TGFβ family factors (ActivinA, Nodal, Lefty1, TGFβ1, GDF3, BMP4) and FGF2 initiating the appropriate signaling pathways in mouse and human embryonic stem cells (mESCs, hESCs) and supporting feeder cells. Quantitative real-time PCR analysis of gene expression showed that the expression patterns of endogenous factors studied were considerably different in mESCs and hESCs. The most significant differences were found in the levels of endogenous expression of TGFβ1, BMP4 and ActivinA. The sources of exogenous factors ActivnA, TGFβ1, and FGF2 for hESCs are feeder cells (mouse and human embryonic fibroblasts) expressing high levels of these factors, as well as low levels of BMP4. Thus, our data demonstrated that the in vitro maintenance of metastable state of undifferentiated pluripotent cells is achieved in mESCs and hESCs using different schemes of the regulations of ActivinA/Nodal/Lefty/Smad2/3 and BMP/Smad1/5/8 endogenous branches of TGFβ signaling. The requirement for exogenous stimulation or inhibition of these signaling pathways is due to different patterns of endogenous expression of TGFβ family factors and FGF2 in the mESCs and hESCs. For the hESCs, enhanced activity of ActivinA/Nodal/Lefty/Smad2/3 signaling by exogenous factor stimulation is necessary to mitigate the effects of BMP/Smad1/5/8 signaling pathways that promote cell differentiation into the extraembryonic structures. Significant differences in endogenous FGF2 expression in the cells in the ground and primed states of pluripotency demonstrate diverse involvement of this factor in the regulation of the pluripotent cell self-renewal.     

Disabled-2 (Dab2) is required for transforming growth factor beta-induced epithelial to mesenchymal transition (EMT)

Transforming growth factor beta (TGFbeta) induces an epithelial to mesenchymal transition (EMT) during both physiological and pathological processes; however, the mechanism underlying this transition is not fully elucidated. Here, we have demonstrated that TGFbeta induces the expression of the adaptor molecule disabled-2 (Dab2) concomitant with the promotion of EMT. We show that TGFbeta induces a transient accumulation of Dab2 to the membrane and increases Dab2 binding to beta1 integrin. Furthermore, small interfering RNA (siRNA)-mediated silencing of Dab2 expression in mouse mammary gland epithelial cells results in inhibition of integrin activation, shown by a decrease of both TGFbeta-induced focal adhesion kinase phosphorylation and cellular adherence, leading to apoptosis and inhibition of EMT. Forced re-expression of human Dab2, not targeted by the mouse siRNA sequence, rescues cells from apoptosis and restores TGFbeta-mediated integrin activation and EMT. These results are confirmed in the F9 teratocarcinoma cell line, a model for retinoic acid-induced visceral endoderm differentiation in which we demonstrate that ablation of retinoic acid-induced Dab2 expression levels, by stable siRNA silencing of Dab2, blocks visceral endoderm differentiation. Our findings indicate that Dab2 plays an important regulatory role during cellular differentiation and that induction of differentiation in the absence of Dab2 expression commits the cell to apoptosis.     

Establishment of mouse teratocarcinomas stem cells line and screening genes responsible for malignancy

The sequential transplantation of embryonal carcinoma cells in vivo can accelerate the growth and malignancy of teratocarcinomas. However, the possible molecular mechanisms in this process that reflect cancer formation in the early stage are largely unknown and. To identify which genes are associated with the changes of malignancy of teratocarcinomas, we established a tumorigenesis model in which teratocarcinoma were induced via injecting embryonic stem cells into immuno-deficiency mice, isolating teratocarcinoma stem cell from a teratocarcinoma in serum-free culture medium and injecting teratocarcinoma stem cells into immune-deficient mice continuously. By using high-throughput deep sequence technology, we identified 26 differentially expressed genes related to the changes of characteristics of teratocarcinoma stem cell in which 18 out of 26 genes were down-regulated and 8 genes were up-regulated. Among these genes, several tumor-related genes such as Gata3, Arnt and Tdgf1, epigenetic associated genes such as PHC1 and Uty were identified. Pathway enrichment analysis result revealed that Wnt signaling pathway, primary immunodeficiency pathway, antigen processing and presentation pathway and allograft rejection pathway were involved in the teratocarcinoma tumorigenesis (corrected p value<0.05). In summary, our study established a tumorigenesis model and proposed some candidate genes and signaling pathways that may play a key role in the early stage of cancer occurrence.     

BMP4 induces primitive endoderm but not trophectoderm in monkey embryonic stem cells

Monkey embryonic stem (ES) cells share similar characteristics to human ES cells and provide a primate model of allotransplantation, which allows to validate efficacy and safety of cell transplantation therapy in regenerative medicine. Bone morphogenetic protein 4 (BMP4) is known to promote trophoblast differentiation in human ES cells in contrast to mouse ES cells where BMP4 synergistically maintains self-renewal with leukemia inhibitory factor (LIF), which represents a significant difference in signal transduction of self-renewal and differentiation between murine and human ES cells. As the similarity of the differentiation mechanism between monkey and human ES cells is of critical importance for their use as a primate model system, we investigated whether BMP4 induces trophoblast differentiation in monkey ES cells. Interestingly, BMP4 did not induce trophoblast differentiation, but instead induced primitive endoderm differentiation. Prominent downregulation of Sox2, which plays a pivotal role not only in pluripotency but also placenta development, was observed in cells treated with BMP4. In addition, upregulation of Hand1, Cdx2, and chorionic gonadotropin beta (CG-beta), which are markers of trophoblast, was not observed. In contrast, BMP4 induced significant upregulation of Gata6, Gata4, and LamininB1, suggesting differentiation into the primitive endoderm, visceral endoderm, and parietal endoderm, respectively. The threshold of BMP4 activity was estimated as about 10 ng/mL. These findings suggest that BMP4 induced differentiation into the primitive endoderm lineage but not into trophoblast in monkey ES cells.     

Lineage choice and differentiation in mouse embryos and embryonic stem cells

The use of embryonic stem (ES) cells for generating healthy tissues has the potential to revolutionize therapies for human disease or injury, for which there are currently no effective treatments. Strategies for manipulating stem cell differentiation should be based on knowledge of the mechanisms by which lineage decisions are made during early embryogenesis. Here, we review current research into the factors influencing lineage differentiation in the mouse embryo and the application of this knowledge to in vitro differentiation of ES cells. In the mouse embryo, specification of tissue lineages requires cell-cell interactions that are influenced by coordinated cell migration and cellular neighborhood mediated by the key WNT, FGF, and TGFbeta signaling pathways. Mimicking the cellular interactions of the embryo by providing appropriate signaling molecules in culture has enabled the differentiation of ES cells to be directed predominately toward particular lineages. Multistep strategies incorporating the provision of soluble factors known to influence lineage choices in the embryo, coculture with other cells or tissues, genetic modification, and selection for desirable cell types have allowed the production of ES cell derivatives that produce beneficial effects in animal models. Increasing the efficiency of this process can only result from a better understanding of the molecular control of cell lineage determination in the embryo.