The development of the central nervous system is known to result from two sequential events. First, an inductive event of the mesoderm on the overlying ectoderm that generates a neural plate that, after rolling into a neural tube, acts as the main source of neural progenitors. Second, the axial regionalization of the neural plate that will result in the specification of neurons with different anteroposterior identities. Although this description of the process applies with ease to amphibians and fish, it is more difficult to confirm in amniote embryos. Here, a specialized population of cells emerges at the end of gastrulation that, under the influence of Wnt and FGF signalling, expands and generates the spinal cord and the paraxial mesoderm. This population is known as the long-term neuromesodermal precursor (NMp). Here, we show that controlled increases of Wnt/β-catenin and FGF signalling during adherent culture differentiation of mouse embryonic stem cells (mESCs) generates a population with many of the properties of the NMp. A single-cell analysis of gene expression within this population reveals signatures that are characteristic of stem cell populations. Furthermore, when this activation is triggered in three-dimensional aggregates of mESCs, the population self-organizes macroscopically and undergoes growth and axial elongation that mimics some of the features of the embryonic spinal cord and paraxial mesoderm. We use both adherent and three-dimensional cultures of mESCs to probe the establishment and maintenance of NMps and their differentiation.

The maintenance of pluripotency in mouse embryonic stem cells (mESCs) relies on the activity of a transcriptional network that is fuelled by the activity of three transcription factors (Nanog, Oct4 and Sox2) and balanced by the repressive activity of Tcf3. Extracellular signals modulate the activity of the network and regulate the differentiation capacity of the cells. Wnt/β-catenin signaling has emerged as a significant potentiator of pluripotency: increases in the levels of β-catenin regulate the activity of Oct4 and Nanog, and enhance pluripotency. A recent report shows that β-catenin achieves some of these effects by modulating the activity of Tcf3, and that this effect does not require its transcriptional activation domain. Here, we show that during self-renewal there is negligible transcriptional activity of β-catenin and that this is due to its tight association with membranes, where we find it in a complex with Oct4 and E-cadherin. Differentiation triggers a burst of Wnt/β-catenin transcriptional activity that coincides with the disassembly of the complex. Our results establish that β-catenin, but not its transcriptional activity, is central to pluripotency acting through a β-catenin/Oct4 complex.

We determined the antitumor properties of a newly available hemocyanin obtained from the Chilean gastropod Concholepas concholepas (Biosonda Corp., Santiago, Chile) in a syngeneic heterotopic mouse bladder carcinoma model. Since keyhole limpet hemocyanin (Pierce, Rockford, Illinois) is used increasingly in biomedicine as a carrier for vaccines and an immunotherapeutic agent for bladder transitional cell carcinoma, there is a growing interest in finding new substances that share its potent immunomodulatory properties. Considering that keyhole limpet hemocyanin and Concholepas concholepas hemocyanin differ significantly, it was not possible to predict a priori the antitumor properties of Concholepas concholepas hemocyanin. C3H/He mice were primed with Concholepas concholepas hemocyanin before subcutaneous implantation of mouse bladder tumor-2 cells. Treatment consisted of a subcutaneous dose of Concholepas concholepas hemocyanin (1 mg or 100 mug) at different intervals after implantation. Keyhole limpet hemocyanin and phosphate buffered saline served as positive and negative controls, respectively. In addition, experiments were designed to determine which elements of the immune response were involved in its adjuvant immunostimulatory effect. Mice treated with Concholepas concholepas hemocyanin showed a significant antitumor effect, as demonstrated by decreased tumor growth and incidence, prolonged survival and lack of toxic effects. These effects were similar to those achieved with keyhole limpet hemocyanin. We found that each hemocyanin increased natural killer cell activity but the effect of Concholepas concholepas hemocyanin was stronger. Analysis of serum from treated mice showed an increased interferon-gamma and low interleukin-4, which correlated with antibody isotypes, confirming that hemocyanins induce a T helper type 1 cytokine profile. To our knowledge our results are the first demonstration of the antitumor effect of a hemocyanin other than keyhole limpet hemocyanin. They suggest that this is an ancient conserved immunogenic mechanism shared by those hemocyanins that is able to enhance T helper type 1 immunity and lead to antitumor activity. Therefore, Concholepas concholepas hemocyanin may be an alternative candidate for providing safe and effective immunotherapy for human superficial bladder cancer.

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We studied the reactivity of mouse monoclonal antibodies (MAbs) against the hemocyanin from the Chilean marine gastropod Concholepas concholepas (CCH). This protein has been successfully used as a carrier to produce antibodies to haptens and peptides. All MAbs (13) belonging to IgG subclass exhibit dissociation constants (K(d)) from 1 x 10(-7) M to 1 x 10(-9) M. MAbs were characterized by enzyme-linked immunosorbant assay (ELISA) using CCH treated with different procedures, including dissociation into CCH-A and CCH-B subunits, Western blot, enzymatic digestion, chemical deglycosylation, and thermal denaturation. MAbs were classified into three categories, according to subunit specificity by ELISA. The epitope distribution shows that CCH subunits display common epitopes (group I, 5 MAbs, 1H5, 2A8, 3A5, 3B3, and 3E3), as well as specific epitopes for CCH-A subunits (group II, 3 MAbs, 1B8, 4D8, and 8E5) and for CCH-B subunits (group III, 5 MAbs, 1A4, 1E4, 2H10, 3B7, and 7B4). The results can be summarized as follows: (1). six antibodies react with thermal denatured CCH, suggesting that they recognize linear epitopes, whereas seven recognize conformational epitopes; (2). oxidation of carbohydrate moieties does not affect the binding of the MAbs; (3). enzymatic digestion of CCH decreases the reactivity of all antibodies irrespective of the protease used (elastase or trypsin); (4). bringing together the above data, in addition to epitopic complementarity analysis, we identified 12 different epitopes on the CCH molecule recognized by these MAbs. The anti-CCH MAbs presented here can be useful tools to understand the subunit organization of the CCH and its complex structure, which can explain its immunogenic and immunostimulating properties in mammals.