Genetic targeting of the endoderm with claudin-6CreER

A full description of the ontogeny of the β cell would guide efforts to generate β cells from embryonic stem cells (ESCs). The first step requires an understanding of definitive endoderm: the genes and signals responsible for its specification, proliferation, and patterning. This report describes a global marker of definitive endoderm, Claudin-6 (Cldn6). We report its expression in early development with particular attention to definitive endoderm derivatives. To create a genetic system to drive gene expression throughout the definitive endoderm with both spatial and temporal control, we target the endogenous locus with an inducible Cre recombinase (Cre-ERT2) cassette. Cldn6 null mice are viable and fertile with no obvious phenotypic abnormalities. We also report a lineage analysis of the fate of Cldn6-expressing embryonic cells, which is relevant to the development of the pancreas, lung, and liver

In vitro chondrogenic commitment of human Wharton's jelly stem cells by co-culture with human articular chondrocytes.

Wharton's jelly stem cells (WJSCs) are a potential source of transplantable stem cells in cartilage-regenerative strategies, due to their highly proliferative and multilineage differentiation capacity. We hypothesized that a non-direct co-culture system with human articular chondrocytes (hACs) could enhance the potential chondrogenic phenotype of hWJSCs during the expansion phase compared to those expanded in monoculture conditions. Primary hWJSCs were cultured in the bottom of a multiwell plate separated by a porous transwell membrane insert seeded with hACs. No statistically significant differences in hWJSCs duplication number were observed under either of the culture conditions during the expansion phase. hWJSCs under co-culture conditions show upregulations of collagen type I and II, COMP, TGFβ1 and aggrecan, as well as of the main cartilage transcription factor, SOX9, when compared to those cultured in the absence of chondrocytes. Chondrogenic differentiation of hWJSCs, previously expanded in co-culture and monoculture conditions, was evaluated for each cellular passage using the micromass culture model. Cells expanded in co-culture showed higher accumulation of glycosaminoglycans (GAGs) compared to cells in monoculture, and immunohistochemistry for localization of collagen type I revealed a strong detection signal when hWJSCs were expanded under monoculture conditions. In contrast, type II collagen was detected when cells were expanded under co-culture conditions, where numerous round-shaped cell clusters were observed. Using a micromass differentiation model, hWJSCs, previously exposed to soluble factors secreted by hACs, were able to express higher levels of chondrogenic genes with deposition of cartilage extracellular matrix components, suggesting their use as an alternative cell source for treating degenerated cartilage.European Union-funded Network of Excellence’EXPERTISSUES’(Grant No. NMP3-CT-2004-500283)

Transcriptional regulation of chondrogenesis by coactivator Tip60 via chromatin association with Sox9 and Sox5

Sox9 is a transcription factor of the SRY family required for several steps of chondrogenesis. It activates the expression of various chondrocyte-specific genes, but the mechanisms and role of cofactors involved in Sox9-regulated gene transcription are not fully understood. Here, we report on the characterization of a Tat interactive protein-60 (Tip60) as Sox9-associated protein identified in a yeast two-hybrid screen. Both in vitro and in vivo assays confirmed the specificity of interactions between Sox9 and Tip60 including the existence of an endogenous complex containing both polypeptides in chondrocytes. Gel shift assays showed the presence of a complex containing Sox9, Tip60 and the DNA of an enhancer region of the Col2a1 promoter. Reporter assays using a Col2a1 promoter with multimerized Col2a1 Sox9-binding sites indicated that Tip60 enhanced the transcriptional activity of Sox9. A larger Col2a1 promoter showed that Tip60 increased the activity of this promoter in the presence of both Sox9 and Sox5. Ectopic expression of Sox9 and transient-cotransfection with Tip60 in COS7 cells showed a more diffuse subnuclear colocalization, suggesting changes in the chromatin structure. Chromatin immunoprecipitation assays showed that Tip60, Sox9 and Sox5 associated with the same Col2a1 enhancer region. Consistent with a role of Tip60 in chondrogenesis, addition of Tip60 siRNA to limb-bud micromass cultures delayed chondrocyte differention. Tip60 enhances acetylation of Sox9 mainly through K61, 253, 398 residues; however, the K61/253/398A mutant of Sox9 still exhibited enhanced transcriptional activity by Tip60. Our results support the hypothesis that Tip60 is a coactivator of Sox9 in chondrocytes

Derivation of Chondrogenically-Committed Cells from Human Embryonic Cells for Cartilage Tissue Regeneration

Background: Heterogeneous and uncontrolled differentiation of human embryonic stem cells (hESCs) in embryoid bodies (EBs) limits the potential use of hESCs for cell-based therapies. More efficient strategies are needed for the commitment and differentiation of hESCs to produce a homogeneous population of specific cell types for tissue regeneration applications. Methodology/Principal Findings: We report here that significant chondrocytic commitment of feeder-free cultured human embryonic stem cells (FF-hESCs), as determined by gene expression and immunostaining analysis, was induced by coculture with primary chondrocytes. Furthermore, a dynamic expression profile of chondrocyte-specific genes was observed during monolayer expansion of the chondrogenically-committed cells. Chondrogenically-committed cells synergistically responded to transforming growth factor-b1 (TGF-b1) and b1-integrin activating antibody by increasing tissue mass in pellet culture. In addition, when encapsulated in hydrogels, these cells formed cartilage tissue both in vitro and in vivo. In contrast, the absence of chondrocyte co-culture did not result in an expandable cell population from FF-hESCs. Conclusions/Significance: The direct chondrocytic commitment of FF-hESCs can be induced by morphogenetic factor

Proline-rich antimicrobial peptide, PR-39 gene transduction altered invasive activity and actin structure in human hepatocellular carcinoma cells

PR-39 is an endogenous proline-rich antimicrobial peptide which induces the synthesis of syndecan-1, a transmembrane heparan sulphate proteoglycan involved in cell-to-matrix interactions and wound healing. Previously, we revealed that the expression of syndecan-1 was reduced in human hepatocellular carcinomas with high metastatic potential and speculated that syndecan-1 played an important role in inhibition of invasion and metastasis. It is assumed that a modification of this process with PR-39 and syndecan-1 may result in a new strategy by which it can inhibit the invasion and metastasis. Therefore, we transduced a gene of PR-39 into human hepatocellular carcinoma cell line HLF, which shows a low expression of syndecan-1 and a high in vitro invasive activity, and examined whether this procedure could reduce the invasive activity of tumour cells. In two transfectants with PR-39 gene, the syndecan-1 expression was induced and the invasive activity in type I collagen-coated chamber was inhibited. Moreover, these transfectants showed the suppression of motile activity assayed by phagokinetic tracks in addition to the disorganization of actin filaments observed by a confocal imaging system. In contrast, five transfectants with syndecan-1 gene in the HLF cells revealed suppression of invasive activity but did not alter the motile activity and actin structures of the cell. These results suggest that PR-39 has functions involved in the suppression of motile activity and alteration of actin structure on human hepatocellular carcinoma cells in addition to the suppression of invasive activity which might result from the induction of syndecan-1 expression. © 1999 Cancer Research Campaig

Syndecan-1 Enhances Proliferation, Migration and Metastasis of HT-1080 Cells in Cooperation with Syndecan-2

Syndecans are transmembrane heparan sulphate proteoglycans. Their role in the development of the malignant phenotype is ambiguous and depends upon the particular type of cancer. Nevertheless, syndecans are promising targets in cancer therapy, and it is important to elucidate the mechanisms controlling their various cellular effects. According to earlier studies, both syndecan-1 and syndecan-2 promote malignancy of HT-1080 human fibrosarcoma cells, by increasing the proliferation rate and the metastatic potential and migratory ability, respectively. To better understand their tumour promoter role in this cell line, syndecan expression levels were modulated in HT-1080 cells and the growth rate, chemotaxis and invasion capacity were studied. For in vivo testing, syndecan-1 overexpressing cells were also inoculated into mice. Overexpression of full length or truncated syndecan-1 lacking the entire ectodomain but containing the four juxtamembrane amino acids promoted proliferation and chemotaxis. These effects were accompanied by a marked increase in syndecan-2 protein expression. The pro-migratory and pro-proliferative effects of truncated syndecan-1 were not observable when syndecan-2 was silenced. Antisense silencing of syndecan-2, but not that of syndecan-1, inhibited cell migration. In vivo, both full length and truncated syndecan-1 increased tumour growth and metastatic rate. Based on our in vitro results, we conclude that the tumour promoter role of syndecan-1 observed in HT-1080 cells is independent of its ectodomain; however, in vivo the presence of the ectodomain further increases tumour proliferation. The enhanced migratory ability induced by syndecan-1 overexpression is mediated by syndecan-2. Overexpression of syndecan-1 also leads to activation of IGF1R and increased expression of Ets-1. These changes were not evident when syndecan-2 was overexpressed. These findings suggest the involvement of IGF1R and Ets-1 in the induction of syndecan-2 synthesis and stimulation of proliferation by syndecan-1. This is the first report demonstrating that syndecan-1 enhances malignancy of a mesenchymal tumour cell line, via induction of syndecan-2 expression

Scientific Evidence and Rationale for the Development of Curcumin and Resveratrol as Nutraceutricals for Joint Health

Interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) are key cytokines that drive the production of inflammatory mediators and matrix-degrading enzymes in osteoarthritis (OA). These proinflammatory cytokines bind to their respective cell surface receptors and activate inflammatory signaling pathways culminating with the activation of nuclear factor κB (NF-κB), a transcription factor that can be triggered by a host of stress-related stimuli including, excessive mechanical stress and ECM degradation products. Once activated, NF-κB regulates the expression of many cytokines, chemokines, adhesion molecules, inflammatory mediators, and several matrix-degrading enzymes. Therefore, proinflammatory cytokines, their cell surface receptors, NF-κB and downstream signaling pathways are therapeutic targets in OA. This paper critically reviews the recent literature and outlines the potential prophylactic properties of plant-derived phytochemicals such as curcumin and resveratrol for targeting NF-κB signaling and inflammation in OA to determine whether these phytochemicals can be used as functional foods

Psychedelics and hypnosis: Commonalities and therapeutic implications

Background Recent research on psychedelics and hypnosis demonstrates the value of both methods in the treatment of a range of psychopathologies with overlapping applications and neurophenomenological features. The potential of harnessing the power of suggestion to influence the phenomenological response to psychedelics toward more therapeutic action has remained unexplored in recent research and thereby warrants empirical attention. Aims Here we aim to elucidate the phenomenological and neurophysiological similarities and dissimilarities between psychedelic states and hypnosis in order to revisit how contemporary knowledge may inform their conjunct usage in psychotherapy. Methods We review recent advances in phenomenological and neurophysiological research on psychedelics and hypnosis and we summarize early investigations on the coupling of psychedelics and hypnosis in scientific and therapeutic contexts. Results/Outcomes We highlight commonalities and differences between psychedelics and hypnosis that point to the potential efficacy of combining the two in psychotherapy. We propose multiple research paths for coupling these two phenomena at different stages in the preparation, acute phase, and follow-up of psychedelic-assisted psychotherapy in order to prepare, guide, and integrate the psychedelic experience with the aim of enhancing therapeutic outcomes. Conclusions/Interpretation Harnessing the power of suggestion to modulate response to psychedelics could enhance their therapeutic efficacy by helping to increase the likelihood of positive responses, including mystical type experiences

Developmental malformation of the corpus callosum: a review of typical callosal development and examples of developmental disorders with callosal involvement

This review provides an overview of the involvement of the corpus callosum (CC) in a variety of developmental disorders that are currently defined exclusively by genetics, developmental insult, and/or behavior. I begin with a general review of CC development, connectivity, and function, followed by discussion of the research methods typically utilized to study the callosum. The bulk of the review concentrates on specific developmental disorders, beginning with agenesis of the corpus callosum (AgCC)—the only condition diagnosed exclusively by callosal anatomy. This is followed by a review of several genetic disorders that commonly result in social impairments and/or psychopathology similar to AgCC (neurofibromatosis-1, Turner syndrome, 22q11.2 deletion syndrome, Williams yndrome, and fragile X) and two forms of prenatal injury (premature birth, fetal alcohol syndrome) known to impact callosal development. Finally, I examine callosal involvement in several common developmental disorders defined exclusively by behavioral patterns (developmental language delay, dyslexia, attention-deficit hyperactive disorder, autism spectrum disorders, and Tourette syndrome)