Epithelial-mesenchymal transition (EMT) occurs during embryonic development and may also be associated with the metastatic spread of epithelial tumors. During EMT, E-cadherin is down-regulated and this correlates with increased motility and invasion of cells. We show that differentiation of human embryonic stem (ES) cells in monolayer culture is associated with an E- to N-cadherin switch, increased vimentin expression, up-regulation of E-cadherin repressor molecules (Snail and Slug proteins), and increased gelatinase (matrix metalloproteinases; MMP-2 and MMP-9) activity and cellular motility, all characteristic EMT events. The 5T4 oncofetal antigen, previously shown to be associated with early human ES cell differentiation, is also part of this process. Abrogation of E-cadherin–mediated cell-cell contact in undifferentiated ES cells using neutralizing antibody (nAb) SHE78.7 resulted in increased cellular motility, altered actin cytoskeleton arrangement and a mesenchymal phenotype together with presentation of the 5T4 antigen at the cell surface. nAb-treated ES cells remained in an undifferentiated state, as assessed by OCT-4 protein expression, and did not express EMT-associated transcripts. Removal of nAb from ES cells resulted in the restoration of cell-cell contact, absence of cell surface 5T4, decreased mesenchymal cellular morphology and motility, and enabled the differentiation of the cells to the three germ layers upon their removal from the fibroblast feeder layer. We conclude that E-cadherin functions in human ES cells to stabilize the cortical actin cyoskeletal arrangement and this prevents cell surface localization of the 5T4 antigen. Furthermore, human ES cells represent a useful model system with which to study EMT events relevant to embryonic development and tumor cell metastasis. [Cancer Res 2007;67(23):11254–62]

In preimplantation mammalian development the transcription factor Sox2 (SRY-related HMG-box gene 2) forms a complex with Oct4 and functions in maintenance of self-renewal of the pluripotent inner cell mass (ICM). Previously it was shown that Sox2−/− embryos die soon after implantation. However, maternal Sox2 transcripts may mask an earlier phenotype. We investigated whether Sox2 is involved in controlling cell fate decisions at an earlier stage. We addressed the question of an earlier role for Sox2 using RNAi, which removes both maternal and embryonic Sox2 mRNA present during the preimplantation period. By depleting both maternal and embryonic Sox2 mRNA at the 2-cell stage and monitoring embryo development in vitro we show that, in the absence of Sox2, embryos arrest at the morula stage and fail to form trophectoderm (TE) or cavitate. Following knock-down of Sox2 via three different short interfering RNA (siRNA) constructs in 2-cell stage mouse embryos, we have shown that the majority of embryos (76%) arrest at the morula stage or slightly earlier and only 18.7–21% form blastocysts compared to 76.2–83% in control groups. In Sox2 siRNA-treated embryos expression of pluripotency associated markers Oct4 and Nanog remained unaffected, whereas TE associated markers Tead4, Yap, Cdx2, Eomes, Fgfr2, as well as Fgf4, were downregulated in the absence of Sox2. Apoptosis was also increased in Sox2 knock-down embryos. Rescue experiments using cell-permeant Sox2 protein resulted in increased blastocyst formation from 18.7% to 62.6% and restoration of Sox2, Oct4, Cdx2 and Yap protein levels in the rescued Sox2-siRNA blastocysts. We conclude that the first essential function of Sox2 in the preimplantation mouse embryo is to facilitate establishment of the trophectoderm lineage. Our findings provide a novel insight into the first differentiation event within the preimplantation embryo, namely the segregation of the ICM and TE lineages.

Effective gene therapy for diseases of the circulation requires vectors capable of systemic delivery. The molecular weight of poly(l-lysine) (pLL) has a significant effect on the circulation of pLL/DNA complexes in mice, with pLL211/DNA complexes displaying up to 20 times greater levels in the blood after 30 minutes compared with pLL20/DNA. It is shown that pLL20/DNA complexes fix mouse complement C3 in vitro, independent of immunoglobulin binding; are less soluble in the blood in vivo; bind erythrocytes; are rapidly removed by the liver, where they associate predominantly with Kupffer cells; and result in a rapid increase in hepatic leukocytes expressing high levels of complement receptor 3 (CR3). The circulation properties of these complexes are also dependent on the type of DNA used, with circular plasmid DNA complexes exhibiting increased circulation compared with linear DNA. PLL211/DNA complexes bind erythrocytes and associate with Kupffer cells but, in contrast, do not fix mouse complement in vitro and are unaffected by the type of DNA used. In rats, both types of complexes produce hematuria and are rapidly removed from the circulation. Correlation of in vivo and in vitro results suggests that the solubility of complexes in physiological saline and species-matched complement fixation and erythrocyte lysis may correlate with systemic circulation. Analysis using human blood in vitro shows no hemolysis, but both types of complexes fix complement and bind IgG, suggesting that pLL/DNA complexes may be rapidly cleared from the human circulation.

We have previously demonstrated that differentiation of embryonic stem (ES) cells is associated with downregulation of cell surface E-cadherin. In this study, we assessed the function of E-cadherin in mouse ES cell pluripotency and differentiation. We show that inhibition of E-cadherin-mediated cell–cell contact in ES cells using gene knockout (Ecad^−/−), RNA interference (EcadRNAi), or a transhomodimerization-inhibiting peptide (CHAVC) results in cellular proliferation and maintenance of an undifferentiated phenotype in fetal bovine serum-supplemented medium in the absence of leukemia inhibitory factor (LIF). Re-expression of E-cadherin in Ecad^−/−, EcadRNAi, and CHAVC-treated ES cells restores cellular dependence to LIF supplementation. Although reversal of the LIF-independent phenotype in Ecad^−/− ES cells is dependent on the β-catenin binding domain of E-cadherin, we show that β-catenin null (βcat^−/−) ES cells also remain undifferentiated in the absence of LIF. This suggests that LIF-independent self-renewal of Ecad^−/− ES cells is unlikely to be via β-catenin signaling. Exposure of Ecad^−/−, EcadRNAi, and CHAVC-treated ES cells to the activin receptor-like kinase inhibitor SB431542 led to differentiation of the cells, which could be prevented by re-expression of E-cadherin. To confirm the role of transforming growth factor β family signaling in the self-renewal of Ecad^−/− ES cells, we show that these cells maintain an undifferentiated phenotype when cultured in serum-free medium supplemented with Activin A and Nodal, with fibroblast growth factor 2 required for cellular proliferation. We conclude that transhomodimerization of E-cadherin protein is required for LIF-dependent ES cell self-renewal and that multiple self-renewal signaling networks subsist in ES cells, with activity dependent upon the cellular context. STEM CELLS 2009;27:2069–2080

Embryonic stem (ES) cells can be cultured in conditions that either maintain pluripotency or allow differentiation to the three embryonic germ layers. Heparan sulfate (HS), a highly polymorphic glycosaminoglycan, is a critical cell surface coreceptor in embryogenesis, and in this paper we describe its structural transition from an unusually low-sulfated variant in ES cells to a more highly sulfated form in fluorescence-activated cell sorting-purified neural progenitor cells. The characteristic domain structure of HS was retained during this transformation. However, qualitative variations in surface sulfation patterns between ES and differentiated cells were revealed using HS epitope-specific antibodies and the HS-binding growth factor fibroblast growth factor 2 (FGF-2). Expression profiles of the HS modification enzymes indicated that both “early” (N-sulfotransferases) and “late” (6O- and 3O-sulfotransferases) sulfotransferases contributed to the alterations in sulfation patterning. An HS-null ES line was used to demonstrate the necessity for HS in neural differentiation. HS is a coreceptor for many of the protein effectors implicated in pluripotency and differentiation (e.g., members of the FGF family, bone morphogenic proteins, and fibronectin). We suggest that the stage-specific activities of these proteins are finely regulated by dynamic changes in sulfation motifs in HS chains. Disclosure of potential conflicts of interest is found at the end of this article.

The regulation of gene expression is central to developmental programs and largely depends on the binding of sequence-specific transcription factors with cis-regulatory elements in the genome. Hox transcription factors specify the spatial coordinates of the body axis in all animals with bilateral symmetry, but a detailed knowledge of their molecular function in instructing cell fates is lacking. Here, we used chromatin immunoprecipitation with massively parallel sequencing (ChIP-seq) to identify Hoxa2 genomic locations in a time and space when it is actively instructing embryonic development in mouse. Our data reveals that Hoxa2 has large genome coverage and potentially regulates thousands of genes. Sequence analysis of Hoxa2-bound regions identifies high occurrence of two main classes of motifs, corresponding to Hox and Pbx–Hox recognition sequences. Examination of the binding targets of Hoxa2 faithfully captures the processes regulated by Hoxa2 during embryonic development; in addition, it uncovers a large cluster of potential targets involved in the Wnt-signaling pathway. In vivo examination of canonical Wnt–β-catenin signaling reveals activity specifically in Hoxa2 domain of expression, and this is undetectable in Hoxa2 mutant embryos. The comprehensive mapping of Hoxa2-binding sites provides a framework to study Hox regulatory networks in vertebrate developmental processes.