AP182C Sigma-AldrichDonkey Anti-Rabbit IgG Antibody, Cy3 conjugate, Species Adsorbed

Mesenchymal stem cells (MSC) have the potential to differentiate into multiple cell lineages and their therapeutic potential has become obvious. In the liver, MSC are represented by stellate cells which have the potential to differentiate into hepatocytes after stimulation with growth factors. Since bile acids can promote liver regeneration, their influence on liver-resident and bone marrow-derived MSC was investigated. Physiological concentrations of bile acids such as tauroursodeoxycholic acid were able to initiate hepatic differentiation of MSC via the farnesoid X receptor and transmembrane G-protein-coupled bile acid receptor 5 as investigated with knockout mice. Notch, hedgehog, transforming growth factor-β/bone morphogenic protein family and non-canonical Wnt signalling were also essential for bile acid-mediated differentiation, whereas β-catenin-dependent Wnt signalling was able to attenuate this process. Our findings reveal bile acid-mediated signalling as an alternative way to induce hepatic differentiaion of stem cells and highlight bile acids as important signalling molecules during liver regeneration.

Endurance athletes exhibit sinus bradycardia, that is a slow resting heart rate, associated with a higher incidence of sinus node (pacemaker) disease and electronic pacemaker implantation. Here we show that training-induced bradycardia is not a consequence of changes in the activity of the autonomic nervous system but is caused by intrinsic electrophysiological changes in the sinus node. We demonstrate that training-induced bradycardia persists after blockade of the autonomous nervous system in vivo in mice and in vitro in the denervated sinus node. We also show that a widespread remodelling of pacemaker ion channels, notably a downregulation of HCN4 and the corresponding ionic current, If. Block of If abolishes the difference in heart rate between trained and sedentary animals in vivo and in vitro. We further observe training-induced downregulation of Tbx3 and upregulation of NRSF and miR-1 (transcriptional regulators) that explains the downregulation of HCN4. Our findings provide a molecular explanation for the potentially pathological heart rate adaptation to exercise training.

High-throughput imaging-based hepatotoxicity studies capable of analyzing individual cells in situ hold enormous promise for drug safety testing but are frequently limited by a lack of sufficient metabolically competent human cells. This study examined cryopreserved HepaRG cells, a human liver cell line which differentiates into both hepatocytes and biliary epithelial cells, to determine if these cells may represent a suitable metabolically competent cellular model for novel High Content Analysis (HCA) applications. Characterization studies showed that these cells retain many features characteristic of primary human hepatocytes and display significant CYP3A4 and CYP1A2 induction, unlike the HepG2 cell line commonly utilized for HCA studies. Furthermore, this study demonstrates that CYP3A4 induction can be quantified via a simple image analysis-based method, using HepaRG cells as a model system. Additionally, data demonstrate that the hepatocyte and biliary epithelial subpopulations characteristic of HepaRG cultures can be separated during analysis simply on the basis of nuclear size measurements. Proof of concept studies with fluorescent cell function reagents indicated that further multiparametric image-based assessment is achievable with HepaRG. In summary, image-based screening of metabolically competent human hepatocyte models cells such as HepaRG offers novel approaches for hepatotoxicity assessment and improved drug screening tools.

Retinoid-storing hepatic stellate cells (HSCs) have recently been described as a liver-resident mesenchymal stem cell (MSC) population; however, it is not clear whether these cells contribute to liver regeneration or serve as a progenitor cell population with hepatobiliary characteristics. Here, we purified HSCs with retinoid-dependent fluorescence-activated cell sorting from eGFP-expressing rats and transplanted these GFP(+) HSCs into wild-type (WT) rats that had undergone partial hepatectomy in the presence of 2-acetylaminofluorene (2AAF) or retrorsine, both of which are injury models that favor stem cell-based liver repair. Transplanted HSCs contributed to liver regeneration in host animals by forming mesenchymal tissue, progenitor cells, hepatocytes, and cholangiocytes and elevated direct bilirubin levels in blood sera of GUNN rats, indicating recovery from the hepatic bilirubin-handling defect in these animals. Transplanted HSCs engrafted within the bone marrow (BM) of host animals, and HSC-derived cells were isolated from BM and successfully retransplanted into new hosts with injured liver. Cultured HSCs transiently adopted an expression profile similar to that of progenitor cells during differentiation into bile acid-synthesizing and -transporting hepatocytes, suggesting that stellate cells represent a source of liver progenitor cells. This concept connects seemingly contradictory studies that favor either progenitor cells or MSCs as important players in stem cell-based liver regeneration.

Cortical efferent and afferent fibers are arranged in a stereotyped pattern in the intermediate zone (IZ). Here, we studied the mechanism of axonal pathway formation by identifying a molecule that is expressed in a subset of cortical axons in the rat. We found that T-cadherin (T-cad), a member of the cadherin family, is expressed in deep-layer cell axons projecting to subcortical structures, but not in upper layer callosal axons projecting to the contralateral cortex. Ectopic expression of T-cad in upper layer cells induced axons to project toward subcortical structures via the upper part of the IZ. Moreover, the axons of deep-layer cells in which T-cad expression was suppressed by RNAi projected towards the contralateral cortex via an aberrant route. These results suggest that T-cad is involved in axonal pathway formation in the developing cortex.

Chondroitin sulfate proteoglycan (CSPG) inhibits neurite outgrowth of various neuronal cell types, and CSPG-associated inhibition of neurite outgrowth is mediated by the Rho/ROCK pathway. Mesenchymal stromal/stem cells (MSCs) have the potential to differentiate into neuron-like cells under specific conditions and have been shown to differentiate into neuron-like cells by co-treatment with the ROCK inhibitor Y27632 and the hypoxia condition mimicking agent CoCl2. In this study, we addressed the hypothesis that a ROCK inhibitor might be beneficial to regenerate neurons during stem cell therapy by preventing transplanted MSCs from inhibition by CSPG in damaged tissues. Indeed, dose-dependent inhibition by CSPG pretreatment was observed during morphological changes of Wharton's jelly-derived MSCs (WJ-MSCs) induced by Y27632 alone. The formation of neurite-like structures was significantly inhibited when WJ-MSCs were pre-treated with CSPG before induction under Y27632 plus CoCl2 conditions, and pretreatment with a protein kinase C inhibitor reversed such inhibition. However, CSPG treatment resulted in no significant inhibition of the WJ-MSC morphological changes into neuron-like cells after initiating induction by Y27632 plus CoCl2. No marked changes were detected in expression levels of neuronal markers induced by Y27632 plus CoCl2 upon CSPG treatment. CSPG also blocked the morphological changes of human bone marrow-derived MSCs into neuron-like cells under other neuronal induction condition without the ROCK inhibitor, and Y27632 pre-treatment blocked the inhibitory effect of CSPG. These results suggest that a ROCK inhibitor can be efficiently used in stem cell therapy for neuronal induction by avoiding hindrance from CSPG.

The mammalian neocortex is composed of various types of neurons that reflect its laminar and area structures. It has been suggested that not only intrinsic but also afferent-derived extrinsic factors are involved in neuronal differentiation during development. However, the role and molecular mechanism of such extrinsic factors are almost unknown. Here, we attempted to identify molecules that are expressed in the thalamus and affect cortical cell development. First, thalamus-specific molecules were sought by comparing gene expression profiles of the developing rat thalamus and cortex using microarrays, and by constructing a thalamus-enriched subtraction cDNA library. A systematic screening by in situ hybridization showed that several genes encoding extracellular molecules were strongly expressed in sensory thalamic nuclei. Exogenous and endogenous protein localization further demonstrated that two extracellular molecules, Neuritin-1 (NRN1) and VGF, were transported to thalamic axon terminals. Application of NRN1 and VGF to dissociated cell culture promoted the dendritic growth. An organotypic slice culture experiment further showed that the number of primary dendrites in multipolar stellate neurons increased in response to NRN1 and VGF, whereas dendritic growth of pyramidal neurons was not promoted. These molecules also increased neuronal survival of multipolar neurons. Taken together, these results suggest that the thalamus-specific molecules NRN1 and VGF play an important role in the dendritic growth and survival of cortical neurons in a cell type-specific manner.

Canonical Wnt/beta-catenin signaling regulates stem/progenitor cells and, when perturbed, induces many human cancers. A significant proportion of human breast cancer is associated with loss of secreted Wnt antagonists and mice expressing MMTV-Wnt1 and MMTV-DeltaN89beta-catenin develop mammary adenocarcinomas. Many studies have assumed these mouse models of breast cancer to be equivalent. Here we show that MMTV-Wnt1 and MMTV-DeltaN89beta-catenin transgenes induce tumors with different phenotypes. Using axin2/conductin reporter genes we show that MMTV-Wnt1 and MMTV-DeltaN89beta-catenin activate canonical Wnt signaling within distinct cell-types. DeltaN89beta-catenin activated signaling within a luminal subpopulation scattered along ducts that exhibited a K18(+)ER(-)PR(-)CD24(high)CD49f(low) profile and progenitor properties. In contrast, MMTV-Wnt1 induced canonical signaling in K14(+) basal cells with CD24/CD49f profiles characteristic of two distinct stem/progenitor cell-types. MMTV-Wnt1 produced additional profound effects on multiple cell-types that correlated with focal activation of the Hedgehog pathway. We document that large melanocytic nevi are a hitherto unreported hallmark of early hyperplastic Wnt1 glands. These nevi formed along the primary mammary ducts and were associated with Hedgehog pathway activity within a subset of melanocytes and surrounding stroma. Hh pathway activity also occurred within tumor-associated stromal and K14(+)/p63(+) subpopulations in a manner correlated with Wnt1 tumor onset. These data show MMTV-Wnt1 and MMTV-DeltaN89beta-catenin induce canonical signaling in distinct progenitors and that Hedgehog pathway activation is linked to melanocytic nevi and mammary tumor onset arising from excess Wnt1 ligand. They further suggest that Hedgehog pathway activation maybe a critical component and useful indicator of breast tumors arising from unopposed Wnt1 ligand.

Enhanced green fluorescent protein (GFP) irreversibly loses not only fluorescence but also antigenicity recognized with conventional anti-GFP antibodies by heat denaturation. This hinders combinatory applications of the GFP immunodetection technique with heat-requiring procedures, such as in situ hybridization histochemistry, antigen retrieval, and Western blot. Here we produced new rabbit and guinea pig antibodies against heat-denatured GFP. The polyclonal antibodies affinity-purified with the antigen column detected a single band corresponding to the molecular size of GFP in Western blot analysis, with mouse brain expressing GFP from the GAD67 locus. By immunofluorescence labeling, the new antibodies detected GFP molecules in heat (greater than or = 70 degrees C)-treated sections but not in untreated sections of the mouse brain. When the sections were incubated at greater than or = 37 degrees C with in situ hybridization buffer containing 50% formamide, a denaturing reagent, the sections lost immunoreactivity with the conventional anti-GFP antibodies but acquired immunoreactivity with the new antibodies to heat-denatured GFP. Finally, GFP immunofluorescence was successfully visualized with the new antibodies in sections of the GFP-expressing mice labeled by fluorescence in situ hybridization histochemistry against GAD67 mRNA. Thus, the antibodies produced in this study may provide an opportunity to combine GFP immunodetection with procedures requiring heat treatment. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

In the bovine synepitheliochorial placenta, restricted trophoblast invasion requires complex interactions of integrin receptors with proteins of the extracellular matrix (ECM) and integrin receptors of neighboring cells. Activated integrins assemble to focal adhesions and are linked to the actin cytoskeleton via signaling molecules including alpha-actinin (ACTN), focal adhesion kinase (PTK2 or FAK), phosphotyrosine, and talin (TLN1). Aims of this study were to assess integrin activation and focal adhesion assembly within epithelial cells of bovine placentomes and low-passage (not transformed) placentomal caruncular epithelial cells cultured on dishes coated with ECM proteins. Immunofluorescence analysis was performed to colocalize the signaling molecules ACTN, PTK2, phosphotyrosine, and TLN1 with each other and with beta(1)-integrin (ITGB1) in placentomal cryosections throughout pregnancy and in caruncular epithelial cells in vitro. Antibody specificity was confirmed by Western blot. Cells were cultured on uncoated dishes, and the dishes were coated with fibronectin (FN), laminin (LAMA), and collagen type IV (COL4), thereby statistically assessing cell number and qualitatively assessing the expression pattern of ITGB1, phosphotyrosine, and TLN1. Results demonstrated integrin activation and focal adhesion assembly in the placentome and that low-passage caruncular epithelial cells maintain integrin-associated properties observed in vivo. Expression and/or colocalization of signaling molecules with ITGB1 confirmed, for the first time, integrin activation and participation in "outside-in" and "inside-out" signaling pathways. The prominent role of ECM, and FN in particular, in integrin signaling is supported by the in vitro enhancement of proliferation and focal adhesion expression. Thus, this in vitro model provides excellent potential for further mechanistic studies designed to elucidate feto-maternal interactions in the bovine placentome.