Francesca Soncin

Epithelial-mesenchymal transition events during human embryonic stem cell differentiation.

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.

Directed differentiation of human embryonic stem cells toward chondrocytes

We report a chemically defined, efficient, scalable and reproducible protocol for differentiation of human embryonic stem cells (hESCs) toward chondrocytes. HESCs are directed through intermediate developmental stages using substrates of known matrix proteins and chemically defined media supplemented with exogenous growth factors. Gene expression analysis suggests that the hESCs progress through primitive streak or mesendoderm to mesoderm, before differentiating into a chondrocytic culture comprising cell aggregates. At this final stage, 74% (HUES1 cells) and up to 95–97% (HUES7 and HUES8 cells) express the chondrogenic transcription factor SOX9. The cell aggregates also express cell surface CD44 and aggrecan and deposit a sulfated glycosaminoglycan and cartilage-specific collagen II matrix, but show very low or no expression of genes and proteins associated with nontarget cell types. Our protocol should facilitate studies of chondrocyte differentiation and of cell replacement therapies for cartilage repair.

Abrogation of E-cadherin-mediated cell-cell contact in mouse embryonic stem cells results in reversible LIF-independent self-renewal.

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

The Function of E-Cadherin in Stem Cell Pluripotency and Self-Renewal

Embryonic stem (ES) and induced-pluripotent stem (iPS) cells can be grown indefinitely under appropriate conditions whilst retaining the ability to differentiate to cells representative of the three primary germ layers. Such cells have the potential to revolutionize medicine by offering treatment options for a wide range of diseases and disorders as well as providing a model system for elucidating mechanisms involved in development and disease. In recent years, evidence for the function of E-cadherin in regulating pluripotent and self-renewal signaling pathways in ES and iPS cells has emerged. In this review, we discuss the function of E-cadherin and its interacting partners in the context of development and disease. We then describe relevant literature highlighting the function of E-cadherin in establishing and maintaining pluripotent and self-renewal properties of ES and iPS cells. In addition, we present experimental data demonstrating that exposure of human ES cells to the E-cadherin neutralizing antibody SHE78.7 allows culture of these cells in the absence of FGF2-supplemented medium.

E-Cadherin Acts as a Regulator of Transcripts Associated with a Wide Range of Cellular Processes in Mouse Embryonic Stem Cells

Background We have recently shown that expression of the cell adhesion molecule E-cadherin is required for LIF-dependent pluripotency of mouse embryonic stem (ES) cells. Methodology In this study, we have assessed global transcript expression in E-cadherin null (Ecad-/-) ES cells cultured in either the presence or absence of LIF and compared these to the parental cell line wtD3. Results We show that LIF has little effect on the transcript profile of Ecad-/- ES cells, with statistically significant transcript alterations observed only for Sp8 and Stat3. Comparison of Ecad-/- and wtD3 ES cells cultured in LIF demonstrated significant alterations in the transcript profile, with effects not only confined to cell adhesion and motility but also affecting, for example, primary metabolic processes, catabolism and genes associated with apoptosis. Ecad-/- ES cells share similar, although not identical, gene expression profiles to epiblast-derived pluripotent stem cells, suggesting that E-cadherin expression may inhibit inner cell mass to epiblast transition. We further show that Ecad-/- ES cells maintain a functional β-catenin pool that is able to induce β-catenin/TCF-mediated transactivation but, contrary to previous findings, do not display endogenous β-catenin/TCF-mediated transactivation. We conclude that loss of E-cadherin in mouse ES cells leads to significant transcript alterations independently of β-catenin/TCF transactivation.

Novel cell lines isolated from mES cells exhibiting de novo methylation of the E-cadherin promoter.

Mouse embryonic stem cells (mESCs) and epiblast stem cells represent the naïve and primed pluripotent states, respectively. These cells self‐renew via distinct signaling pathways and can transition between the two states in the presence of appropriate growth factors. Manipulation of signaling pathways has therefore allowed the isolation of novel pluripotent cell types such as Fibroblast growth factor, Activin and BIO‐derived stem cells and IESCs. However, the effect of cell seeding density on pluripotency remains unexplored. In this study, we have examined whether mESCs can epigenetically regulate E‐cadherin to enter a primed‐like state in response to low cell seeding density. We show that low density seeding in the absence of leukaemia inhibitory factor (LIF) induces decreased apoptosis and maintenance of pluripotency via Activin/Nodal, concomitant with loss of E‐cadherin, Signal transducer and activator of transcription phosphorylation, and chimera‐forming ability. These cells, E‐cadherin negative proliferating stem cells (ENPSCs) can be reverted to a naïve phenotype by addition of LIF or forced E‐cadherin expression. However, prolonged culture of ENPSCs without LIF leads to methylation of the E‐cadherin promoter (ENPSCM), which cannot be reversed by LIF supplementation, and increased histone H3K27 and decreased H3K4 trimethylation. Transcript analysis of ENPSCM revealed a primed‐like phenotype and their differentiation leads to enrichment of neuroectoderm cells. The generation of ENPSCs is similar to tumorigenesis as ENPSCs exhibit transcript alterations associated with neoplasia, hyperplasia, carcinoma, and metastasis. We therefore describe a novel cell model to elucidate the role of E‐cadherin in pluripotency and to investigate epigenetic regulation of this gene during mESC differentiation and tumor metastasis. Stem Cells 2014;32:2869–2879

Novel Cell Lines Isolated From Mouse Embryonic Stem Cells Exhibiting De Novo Methylation of the E-Cadherin Promoter

Mouse embryonic stem cells (mESCs) and epiblast stem cells represent the naïve and primed pluripotent states, respectively. These cells self‐renew via distinct signaling pathways and can transition between the two states in the presence of appropriate growth factors. Manipulation of signaling pathways has therefore allowed the isolation of novel pluripotent cell types such as Fibroblast growth factor, Activin and BIO‐derived stem cells and IESCs. However, the effect of cell seeding density on pluripotency remains unexplored. In this study, we have examined whether mESCs can epigenetically regulate E‐cadherin to enter a primed‐like state in response to low cell seeding density. We show that low density seeding in the absence of leukaemia inhibitory factor (LIF) induces decreased apoptosis and maintenance of pluripotency via Activin/Nodal, concomitant with loss of E‐cadherin, Signal transducer and activator of transcription phosphorylation, and chimera‐forming ability. These cells, E‐cadherin negative proliferating stem cells (ENPSCs) can be reverted to a naïve phenotype by addition of LIF or forced E‐cadherin expression. However, prolonged culture of ENPSCs without LIF leads to methylation of the E‐cadherin promoter (ENPSCM), which cannot be reversed by LIF supplementation, and increased histone H3K27 and decreased H3K4 trimethylation. Transcript analysis of ENPSCM revealed a primed‐like phenotype and their differentiation leads to enrichment of neuroectoderm cells. The generation of ENPSCs is similar to tumorigenesis as ENPSCs exhibit transcript alterations associated with neoplasia, hyperplasia, carcinoma, and metastasis. We therefore describe a novel cell model to elucidate the role of E‐cadherin in pluripotency and to investigate epigenetic regulation of this gene during mESC differentiation and tumor metastasis. Stem Cells 2014;32:2869–2879