Efficient Differentiation of Embryonic Stem Cells into Mesodermal Precursors by BMP, Retinoic Acid and Notch Signalling

The ability to direct differentiation of mouse embryonic stem (ES) cells into specific lineages not only provides new insights into the pathways that regulate lineage selection but also has translational applications, for example in drug discovery. We set out to develop a method of differentiating ES cells into mesodermal cells at high efficiency without first having to induce embryoid body formation. ES cells were plated on a feeder layer of PA6 cells, which have membrane-associated stromal-derived inducing activity (SDIA), the molecular basis of which is currently unknown. Stimulation of ES/PA6 co-cultures with Bone Morphogenetic Protein 4 (BMP4) both favoured self-renewal of ES cells and induced differentiation into a Desmin and Nestin double positive cell population. Combined stimulation with BMP4 and all-trans Retinoic Acid (RA) inhibited self-renewal and resulted in 90% of cells expressing Desmin and Nestin. Quantitative reverse transcription-polymerase chain reaction (qPCR) analysis confirmed that the cells were of mesodermal origin and expressed markers of mesenchymal and smooth muscle cells. BMP4 activation of a MAD-homolog (Smad)-dependent reporter in undifferentiated ES cells was attenuated by co-stimulation with RA and co-culture with PA6 cells. The Notch ligand Jag1 was expressed in PA6 cells and inhibition of Notch signalling blocked the differentiation inducing activity of PA6 cells. Our data suggest that mesodermal differentiation is regulated by the level of Smad activity as a result of inputs from BMP4, RA and the Notch pathway

Use of hospitalisation history (lookback) to determine prevalence of chronic diseases: impact on modelling of risk factors for haemorrhage in pregnancy

<p>Abstract</p> <p>Background</p> <p>Concern about the completeness of comorbidity information in hospital records has been raised as a limitation of using hospital discharge data for research. The aim of this study is to assess the impact of additional comorbidity information from prior hospital admissions on estimation of prevalence and modelling of risk factors for obstetric haemorrhage.</p> <p>Methods</p> <p>A range of chronic disease prevalence for 53,438 women who had their first birth in New South Wales (NSW), Australia, 2005-2006, were ascertained for up to five years prior to the birth admission (for pregnancy, 2-, 3-, 4- and 5-year periods) and obstetric haemorrhage was identified from maternal hospital records for 2005 and 2006.</p> <p>Results</p> <p>The ascertainment of chronic disease prevalence increased with increasing length of lookback. However, the rate of the increase was slower after 2 to 3 years than for the more recent periods. The effect size of chronic diseases on obstetric haemorrhage risk decreased with the increased case ascertainment associated with longer lookback. Furthermore, longer lookback did not improve the predictive capacity (C-statistic: 0.624) of a model that was based only on the birth admission records.</p> <p>Conclusions</p> <p>Longer ascertainment periods resulted in improved identification of chronic disease history among pregnant women, but the additional information from prior admissions did little to improve the modelling of risk factors for obstetric haemorrhage.</p

A Conserved Mechanism for Control of Human and Mouse Embryonic Stem Cell Pluripotency and Differentiation by Shp2 Tyrosine Phosphatase

Recent studies have suggested distinctive biological properties and signaling mechanisms between human and mouse embryonic stem cells (hESCs and mESCs). Herein we report that Shp2, a protein tyrosine phosphatase with two SH2 domains, has a conserved role in orchestration of intracellular signaling cascades resulting in initiation of differentiation in both hESCs and mESCs. Homozygous deletion of Shp2 in mESCs inhibited differentiation into all three germ layers, and siRNA-mediated knockdown of Shp2 expression in hESCs led to a similar phenotype of impaired differentiation. A small molecule inhibitor of Shp2 enzyme suppressed both hESC and mESC differentiation capacity. Shp2 modulates Erk, Stat3 and Smad pathways in ES cells and, in particular, Shp2 regulates BMP4-Smad pathway bi-directionally in mESCs and hESCs. These results reveal a common signaling mechanism shared by human and mouse ESCs via Shp2 modulation of overlapping and divergent pathways

Human breast cancer-derived soluble factors facilitate CCL19-induced chemotaxis of human dendritic cells

Breast cancer remains as a challenging disease with high mortality in women. Increasing evidence points the importance of understanding a crosstalk between breast cancers and immune cells, but little is known about the effect of breast cancer-derived factors on the migratory properties of dendritic cells (DCs) and their consequent capability in inducing T cell immune responses. Utilizing a unique 3D microfluidic device, we here showed that breast cancers (MCF-7, MDA-MB-231, MDA-MB-436 and SK-BR-3)-derived soluble factors increase the migration of DCs toward CCL19. The enhanced migration of DCs was mainly mediated via the highly activated JNK/c-Jun signaling pathway, increasing their directional persistence, while the velocity of DCs was not influenced, particularly when they were co-cultured with triple negative breast cancer cells (TNBCs or MDA-MB-231 and MDA-MB-436). The DCs up-regulated inflammatory cytokines IL-1?? and IL-6 and induced T cells more proliferative and resistant against activation-induced cell death (AICD), which secret high levels of inflammatory cytokines IL-1??, IL-6 and IFN-??. This study demonstrated new possible evasion strategy of TNBCs utilizing their soluble factors that exploit the directionality of DCs toward chemokine responses, leading to the building of inflammatory milieu which may support their own growth.ope

Generation of integration-free neural progenitor cells from cells in human urine

Human neural stem cells hold great promise for research and therapy in neural disease. We describe the generation of integration-free and expandable human neural progenitor cells (NPCs). We combined an episomal system to deliver reprogramming factors with a chemically defined culture medium to reprogram epithelial-like cells from human urine into NPCs (hUiNPCs). These transgene-free hUiNPCs can self-renew and can differentiate into multiple functional neuronal subtypes and glial cells in vitro. Although functional in vivo analysis is still needed, we report that the cells survive and differentiate upon transplant into newborn rat brain.postprin

Association between RUNX3 promoter methylation and gastric cancer: a meta-analysis

<p>Abstract</p> <p>Background</p> <p>Runt-related transcription factor 3 (RUNX3) is a member of the runt-domain family of transcription factors and has been reported to be a candidate tumor suppressor in gastric cancer. However, the association between RUNX3 promoter methylation and gastric cancer remains unclear.</p> <p>Methods</p> <p>We systematically reviewed studies of RUNX3 promoter methylation and gastric cancer published in English or Chinese from January 2000 to January 2011, and quantified the association between RUNX3 promoter methylation and gastric cancer using meta-analysis methods.</p> <p>Results</p> <p>A total of 1740 samples in 974 participants from seventeen studies were included in the meta-analysis. A significant association was observed between RUNX3 promoter methylation and gastric cancer, with an aggregated odds ratio (OR) of 5.63 (95%CI 3.15, 10.07). There was obvious heterogeneity among studies. Subgroup analyses (including by tissue origin, country and age), meta-regression were performed to determine the source of the heterogeneity. Meta-regression showed that the trend in ORs was inversely correlated with age. No publication bias was detected. The ORs for RUNX3 methylation in well-differentiated <it>vs </it>undifferentiated gastric cancers, and in intestinal-type <it>vs </it>diffuse-type carcinomas were 0.59 (95%CI: 0.30, 1.16) and 2.62 (95%CI: 1.33, 5.14), respectively. There were no significant differences in RUNX3 methylation in cancer tissues in relation to age, gender, TNM stage, invasion of tumors into blood vessel or lymphatic ducts, or tumor stage.</p> <p>Conclusions</p> <p>This meta-analysis identified a strong association between methylation of the RUNX3 promoter and gastric cancer, confirming the role of RUNX3 as a tumor suppressor gene.</p

Microtubular Stability Affects pVHL-Mediated Regulation of HIF-1alpha via the p38/MAPK Pathway in Hypoxic Cardiomyocytes

BACKGROUND: Our previous research found that structural changes of the microtubule network influence glycolysis in cardiomyocytes by regulating the hypoxia-inducible factor (HIF)-1α during the early stages of hypoxia. However, little is known about the underlying regulatory mechanism of the changes of HIF-1α caused by microtubule network alternation. The von Hippel-Lindau tumor suppressor protein (pVHL), as a ubiquitin ligase, is best understood as a negative regulator of HIF-1α. METHODOLOGY/PRINCIPAL FINDINGS: In primary rat cardiomyocytes and H9c2 cardiac cells, microtubule-stabilization was achieved by pretreating with paclitaxel or transfection of microtubule-associated protein 4 (MAP4) overexpression plasmids and microtubule-depolymerization was achieved by pretreating with colchicine or transfection of MAP4 siRNA before hypoxia treatment. Recombinant adenovirus vectors for overexpressing pVHL or silencing of pVHL expression were constructed and transfected in primary rat cardiomyocytes and H9c2 cells. With different microtubule-stabilizing and -depolymerizing treaments, we demonstrated that the protein levels of HIF-1α were down-regulated through overexpression of pVHL and were up-regulated through knockdown of pVHL in hypoxic cardiomyocytes. Importantly, microtubular structure breakdown activated p38/MAPK pathway, accompanied with the upregulation of pVHL. In coincidence, we found that SB203580, a p38/MAPK inhibitor decreased pVHL while MKK6 (Glu) overexpression increased pVHL in the microtubule network altered-hypoxic cardiomyocytes and H9c2 cells. CONCLUSIONS/SIGNIFICANCE: This study suggests that pVHL plays an important role in the regulation of HIF-1α caused by the changes of microtubular structure and the p38/MAPK pathway participates in the process of pVHL change following microtubule network alteration in hypoxic cardiomyocytes

Therapeutic potential of transplanted placental mesenchymal stem cells in treating Chinese miniature pigs with acute liver failure

<p>Abstract</p> <p>Background</p> <p>Stem cell-based therapy to treat liver diseases is a focus of current research worldwide. So far, most such studies depend on rodent hepatic failure models. The purpose of this study was to isolate mesenchymal stem cells from human placenta (hPMSCs) and determine their therapeutic potential for treating Chinese experimental miniature pigs with acute liver failure (ALF).</p> <p>Methods</p> <p>hPMSCs were isolated and analyzed for their purity and differentiation potential before being employed as the donor cells for transplantation. ALF models of Chinese experimental miniature pigs were established and divided into four groups: no cell transplantation; hPMSCs transplantation via the jugular vein; X-ray-treated hPMSCs transplantation via the portal vein; and hPMSCs transplantation via the portal vein. The restoration of biological functions of the livers receiving transplantation was assessed via a variety of approaches such as mortality rate determination, serum biochemical analysis, and histological, immunohistochemical, and genetic analysis.</p> <p>Results</p> <p>hPMSCs expressed high levels of CD29, CD73, CD13, and CD90, had adipogenic, osteogenic, and hepatic differentiation potential. They improved liver functions <it>in vivo </it>after transplantation into the D-galactosamine-injured pig livers as evidenced by the fact that ALT, AST, ALP, CHE, TBIL, and TBA concentrations returned to normal levels in recipient ALF pigs. Meanwhile, histological data revealed that transplantation of hPMSCs via the portal vein reduced liver inflammation, decreased hepatic denaturation and necrosis, and promoted liver regeneration. These ameliorations were not found in the other three groups. The result of 7-day survival rates suggested that hPMSCs transplantation via the portal vein was able to significantly prolong the survival of ALF pigs compared with the other three groups. Histochemistry and RT-PCR results confirmed the presence of transplanted human cells in recipient pig livers (Groups III, IV).</p> <p>Conclusions</p> <p>Our data revealed that hPMSCs could not only differentiate into hepatocyte-like cells <it>in vitro </it>and <it>in vivo</it>, but could also prolong the survival time of ALF pigs. Regarding the transplantation pathways, the left branch of the portal vein inside the liver was superior to the jugular vein pathway. Thus, hPMSCs transplantation through the portal vein by B-ultrasonography may represent a superior approach for treating liver diseases.</p

CDK5 Is Essential for Soluble Amyloid β-Induced Degradation of GKAP and Remodeling of the Synaptic Actin Cytoskeleton

The early stages of Alzheimer's disease are marked by synaptic dysfunction and loss. This process results from the disassembly and degradation of synaptic components, in particular of scaffolding proteins that compose the post-synaptic density (PSD), namely PSD95, Homer and Shank. Here we investigated in rat frontal cortex dissociated culture the mechanisms involved in the downregulation of GKAP (SAPAP1), which links the PSD95 complex to the Shank complex and cytoskeletal structures within the PSD. We show that Aβ causes the rapid loss of GKAP from synapses through a pathway that critically requires cdk5 activity, and is set in motion by NMDAR activity and Ca2+ influx. We show that GKAP is a direct substrate of cdk5 and that its phosphorylation results in polyubiquitination and proteasomal degradation of GKAP and remodeling (collapse) of the synaptic actin cytoskeleton; the latter effect is abolished in neurons expressing GKAP mutants that are resistant to phosphorylation by cdk5. Given that cdk5 also regulates degradation of PSD95, these results underscore the central position of cdk5 in mediating Aβ-induced PSD disassembly and synapse loss