Graphical abstract

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 , 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 b family signaling in the self-renewal of Ecad 2/2 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 selfrenewal and that multiple self-renewal signaling networks subsist in ES cells, with activity dependent upon the cellular context.

The pathological potential of human astroglia in Alzheimer's disease (AD) was analysed in vitro using induced pluripotent stem cell (iPSC) technology. Here, we report development of a human iPSC-derived astrocyte model created from healthy individuals and patients with either early-onset familial AD (FAD) or the late-onset sporadic form of AD (SAD). Our chemically defined and highly efficient model provides >95% homogeneous populations of human astrocytes within 30 days of differentiation from cortical neural progenitor cells (NPCs). All astrocytes expressed functional markers including glial fibrillary acidic protein (GFAP), excitatory amino acid transporter-1 (EAAT1), S100B and glutamine synthetase (GS) comparable to that of adult astrocytes in vivo. However, induced astrocytes derived from both SAD and FAD patients exhibit a pronounced pathological phenotype, with a significantly less complex morphological appearance, overall atrophic profiles and abnormal localisation of key functional astroglial markers. Furthermore, NPCs derived from identical patients did not show any differences, therefore, validating that remodelled astroglia are not as a result of defective neural intermediates. This work not only presents a novel model to study the mechanisms of human astrocytes in vitro, but also provides an ideal platform for further interrogation of early astroglial cell autonomous events in AD and the possibility of identification of novel therapeutic targets for the treatment of AD.

Leukemia inhibitory factor plays a major role in the uterus and in its absence embryos fail to implant. Our knowledge of the targets for LIF and the consequences of its absence is still very incomplete. In this study, we have examined the ultrastructure of the potential implantation site in LIF-null MF1 female mice compared to that of wild type animals. We also compared expression of proteins associated with implantation in luminal epithelium and stroma. Luminal epithelial cells (LE) of null animals failed to develop apical pinopods, had increased glycocalyx, and retained a columnar shape during the peri-implantation period. Stromal cells of LIF-null animals showed no evidence of decidual giant cell formation even by day 6 of pregnancy. A number of proteins normally expressed in decidualizing stroma did not increase in abundance in the LIF-null animals including desmin, tenascin, Cox-2, bone morphogenetic protein (BMP)-2 and -7, and Hoxa-10. In wild type animals, the IL-6 family member Oncostatin M (OSM) was found to be transiently expressed in the luminal epithelium on late day 4 and then in the stroma at the attachment site on days 5-6 of pregnancy, with a similar but not identical pattern to that of Cox-2. In the LIF-null animals, no OSM protein was detected in either LE or stroma adjacent to the embryo, indicating that expression requires uterine LIF in addition to a blastocyst signal. Fucosylated epitopes: the H-type-1 antigen and those recognized by lectins from Ulex europaeus-1 and Tetragonolobus purpureus were enhanced on apical LE on day 4 of pregnancy. H-type-1 antigen remained higher on day 5, and was not reduced even by day 6 in contrast to wild type uterus. These data point to a profound disturbance of normal luminal epithelial and stromal differentiation during early pregnancy in LIF-nulls. On this background, we also obtained less than a Mendelian ratio of null offspring suggesting developmental failure.

Prolactin (PRL) is an important hormone in mammary tumorigenesis in rodents but its involvement in human breast cancer has been controversial. A role for locally produced PRL in breast carcinogenesis is suggested by its mitogenic action on breast cancer cells and the expression of both PRL and its receptor (PRL-R) in breast carcinomas. Our objective was to examine whether PRL, overexpressed by breast cancer cells, forms an autocrine/paracrine loop that confers a growth advantage for tumors. MDA-MB-435 breast cancer cells overexpressing 23K human PRL were generated, and PRL production and secretion by the clones were confirmed by RT-PCR, western blotting, and the Nb2 bioassay; control clones contain vector only. In vitro the 23K PRL clones proliferated faster and expressed higher levels of the PRL-R protein than controls only when incubated in charcoal-stripped serum (CSS) devoid of lactogenic hormones. When injected into the mammary fatpad of female nude mice or subcutaneously into males, the PRL-overexpressing clones formed tumors that grew 2-4-fold faster than tumors derived from control clones or wild type MDA-MB-435 cells. Western analysis demonstrated significantly higher PRL, PRL-R, and bcl-2 levels in the tumors overexpressing PRL compared to control tumors. These data support a role for breast PRL as a growth/anti-apoptotic factor and suggest that it may serve as a novel therapeutic target for the treatment of breast cancer.

We have recently shown that loss of E-cadherin in mouse embryonic stem cells (mESCs) results in significant alterations to both the transcriptome and hierarchy of pluripotency-associated signaling pathways. Here, we show that E-cadherin promotes kruppel-like factor 4 (Klf4) and Nanog transcript and protein expression in mESCs via STAT3 phosphorylation and that b-catenin, and its binding region in E-cadherin, is required for this function. To further investigate the role of E-cadherin in leukemia inhibitory factor (LIF)-dependent pluripotency, E-cadherin null (Ecad 2/2 ) mESCs were cultured in LIF/bone morphogenetic protein supplemented medium. Under these conditions, Ecad 2/2 mESCs exhibited partial restoration of cell-cell contact and STAT3 phosphorylation and upregulated Klf4, Nanog, and N-cadherin transcripts and protein.Abrogation of N-cadherin using an inhibitory peptide caused loss of phospho STAT3, Klf4, and Nanog in these cells, demonstrating that N-cadherin supports LIF-dependent pluripotency in this context. We therefore identify a novel molecular mechanism linking E-and N-cadherin to the core circuitry of pluripotency in mESCs. This mechanism may explain the recently documented role of E-cadherin in efficient induced pluripotent stem cell reprogramming.

E-cadherin is the primary cell adhesion molecule within the epithelium, and loss of this protein is associated with a more aggressive tumour phenotype and poorer patient prognosis in many cancers. Loss of E-cadherin is a defining characteristic of epithelial-mesenchymal transition (EMT), a process associated with tumour cell metastasis. We have previously demonstrated an EMT event during embryonic stem (ES) cell differentiation, and that loss of E-cadherin in these cells results in altered growth factor response and changes in cell surface localisation of promigratory molecules. We discuss the implication of loss of E-cadherin in ES cells within the context of cancer stem cells and current models of tumorigenesis. We propose that aberrant E-cadherin expression is a critical contributing factor to neoplasia and the early stages of tumorigenesis in the absence of EMT by altering growth factor response of the cells, resulting in increased proliferation, decreased apoptosis, and acquisition of a stem cell-like phenotype.

BackgroundA fundamental requirement for the exploitation of embryonic stem (ES) cells in regenerative medicine is the ability to reproducibly derive sufficient numbers of cells of a consistent quality in a cost-effective manner. However, undifferentiated ES cells are not ideally suited to suspension culture due to the formation of cellular aggregates, ultimately limiting scalability. Significant advances have been made in recent years in the culture of ES cells, including automated adherent culture and suspension microcarrier or embryoid body bioreactor culture. However, each of these methods exhibits specific disadvantages, such as high cost, additional downstream processes or reduced cell doubling times.Methodology/Principal FindingsHere we show that abrogation of the cell surface protein E-cadherin, using either gene knockout (Ecad-/-) or the neutralising antibody DECMA-1 (EcadAb), allows culture of mouse ES cells as a near-single cell suspension in scalable shake flask culture over prolonged periods without additional media supplements. Both Ecad-/- and EcadAb ES cells exhibited adaptation phases in suspension culture, with optimal doubling times of 7.3 h±0.9 and 15.6 h±4.7 respectively and mean-fold increase in viable cell number of 95.1±2.0 and 16±0.9-fold over 48 h. EcadAb ES cells propagated as a dispersed cell suspension for 15 d maintained expression of pluripotent markers, exhibited a normal karyotype and high viability. Subsequent differentiation of EcadAb ES cells resulted in expression of transcripts and proteins associated with the three primary germ layers.Conclusions/SignificanceThis is the first demonstration of the culture of pluripotent ES cells as a near-single cell suspension in a manual fed-batch shake flask bioreactor and represents a significant improvement on current ES cell culture techniques. Whilst this proof-of-principle method would be useful for the culture of human ES and iPS cells, further steps are necessary to increase cell viability of hES cells in suspension.