Scientific Resources

BACKGROUND In colorectal carcinoma (CRC),activation of the Raf/MEK/ERK signaling pathway is commonly observed. In addition,the commonly used 5FU-based chemotherapy in patients with metastatic CRC was found to enrich a subpopulation of CD26(+) cancer stem cells (CSCs). As activation of the Raf/MEK/ERK signaling pathway was also found in the CD26(+) CSCs and therefore,we hypothesized that an ATP-competitive pan-Raf inhibitor,Raf265,is effective in eliminating the cancer cells and the CD26(+) CSCs in CRC patients. METHODS HT29 and HCT116 cells were treated with various concentrations of Raf265 to study the anti-proliferative and apoptotic effects of Raf265. Anti-tumor effect was also demonstrated using a xenograft model. Cells were also treated with Raf265 in combination with 5FU to demonstrate the anti-migratory and invasive effects by targeting on the CD26(+) CSCs and the anti-metastatic effect of the combined treatment was shown in an orthotopic CRC model. RESULTS Raf265 was found to be highly effective in inhibiting cell proliferation and tumor growth through the inhibition of the RAF/MEK/ERK signaling pathway. In addition,anti-migratory and invasive effect was found with Raf265 treatment in combination with 5FU by targeting on the CD26(+) cells. Finally,the anti-tumor and anti-metastatic effect of Raf265 in combination with 5FU was also demonstrated. CONCLUSIONS This preclinical study demonstrates the anti-tumor and anti-metastatic activity of Raf265 in CRC,providing the basis for exploiting its potential use and combination therapy with 5FU in the clinical treatment of CRC. View Publication

RTP801 expression is induced by cellular stress and has a pro-apoptotic function in non-proliferating differentiated cells such as neurons. In several neurodegenerative disorders,including Parkinson's disease and Alzheimer's disease,elevated levels of RTP801 have been observed,which suggests a role for RTP801 in neuronal death. Neuronal death is also a pathological hallmark in Huntington's disease (HD),an inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. Currently,the exact mechanisms underlying mutant huntingtin (mhtt)-induced toxicity are still unclear. Here,we investigated whether RTP801 is involved in (mhtt)-induced cell death. Ectopic exon-1 mhtt elevated RTP801 mRNA and protein levels in nerve growth factor (NGF)-differentiated PC12 cells and in rat primary cortical neurons. In neuronal PC12 cells,mhtt also contributed to RTP801 protein elevation by reducing its proteasomal degradation rate,in addition to promoting RTP801 gene expression. Interestingly,silencing RTP801 expression with short hairpin RNAs (shRNAs) blocked mhtt-induced cell death in NGF-differentiated PC12 cells. However,RTP801 protein levels were not altered in the striatum of Hdh(Q7/Q111) and R6/1 mice,two HD models that display motor deficits but not neuronal death. Importantly,RTP801 protein levels were elevated in both neural telencephalic progenitors differentiated from HD patient-derived induced pluripotent stem cells and in the putamen and cerebellum of human HD postmortem brains. Taken together,our results suggest that RTP801 is a novel downstream effector of mhtt-induced toxicity and that it may be relevant to the human disease. View Publication

Epigenetic modifications refer to a number of biological processes which alter the structure of chromatin and its transcriptional activity such as DNA methylation and histone post-translational processing. Studies have tried to elucidate how the viral genome and its products are affected by epigenetic modifications imposed by cell machinery and how it affects the ability of the virus to either,replicate and produce a viable progeny or be driven to latency. The purpose of this study was to evaluate epigenetic modifications in PBMCs and CD4+ cells after HIV-1 infection analyzing three approaches: (i) global DNA- methylation; (ii) qPCR array and (iii) western blot. HIV-1 infection led to methylation increases in the cellular DNA regardless the activation status of PBMCs. The analysis of H3K9me3 and H3K27me3 suggested a trend towards transcriptional repression in activated cells after HIV-1 infection. Using a qPCR array,we detected genes related to epigenetic processes highly modulated in activated HIV-1 infected cells. SETDB2 and RSK2 transcripts showed highest up-regulation levels. SETDB2 signaling is related to transcriptional silencing while RSK2 is related to either silencing or activation of gene expression depending on the signaling pathway triggered down-stream. In addition,activated cells infected by HIV-1 showed lower CD69 expression and a decrease of IL-2,IFN-γ and metabolism-related factors transcripts indicating a possible functional consequence towards global transcriptional repression found in HIV-1 infected cells. Conversely,based on epigenetic markers studied here,non-stimulated cells infected by HIV-1,showed signs of global transcriptional activation. Our results suggest that HIV-1 infection exerts epigenetic modulations in activated cells that may lead these cells to transcriptional repression with important functional consequences. Moreover,non-stimulated cells seem to increase gene transcription after HIV-1 infection. Based on these observations,it is possible to speculate that the outcome of viral infections may be influenced by the cellular activation status at the moment of infection. View Publication

An investigation was conducted to demonstrate that neurodazine (Nz) and neurodazole (Nzl),two imidazole-based small molecules,promote neuronal differentiation in both neuroblastoma and fibroblast cells. The results show that differentiated cells generated by treatment with Nz and Nzl express neuron-specific markers. The ability of Nz and Nzl to induce neurogenesis of neuroblastoma and fibroblast cells was found to be comparable to those of the known neurogenic factors,retinoic acid and trichostatin A. In addition,the cells differentiated by Nz and Nzl are observed to express different isoforms of glutamate receptors. The results of signaling pathway studies reveal that two substances enhance neurogenesis in neuroblastoma cells by activating Wnt and Shh signaling pathways and neurogenesis in fibroblast cells by mainly activating the Wnt signaling pathway. Observations made in the present study suggest that Nz and Nzl will serve as chemical tools to generate specific populations of neuronal cells from readily available and simply manageable cells. View Publication

Human pluripotent stem cells (PSCs) show epiblast-type pluripotency that is maintained with ACTIVIN/FGF2 signaling. Here,we report the acquisition of a unique stem cell phenotype by both human ES cells (hESCs) and induced pluripotent stem cells (iPSCs) in response to transient (24-36 h) exposure to bone morphogenetic protein 4 (BMP4) plus inhibitors of ACTIVIN signaling (A83-01) and FGF2 (PD173074),followed by trypsin dissociation and recovery of colonies capable of growing on a gelatin substratum in standard medium for human PSCs at low but not high FGF2 concentrations. The self-renewing cell lines stain weakly for CDX2 and strongly for NANOG,can be propagated clonally on either Matrigel or gelatin,and are morphologically distinct from human PSC progenitors on either substratum but still meet standard in vitro criteria for pluripotency. They form well-differentiated teratomas in immune-compromised mice that secrete human chorionic gonadotropin (hCG) into the host mouse and include small areas of trophoblast-like cells. The cells have a distinct transcriptome profile from the human PSCs from which they were derived (including higher expression of NANOG,LEFTY1,and LEFTY2). In nonconditioned medium lacking FGF2,the colonies spontaneously differentiated along multiple lineages,including trophoblast. They responded to PD173074 in the absence of both FGF2 and BMP4 by conversion to trophoblast,and especially syncytiotrophoblast,whereas an A83-01/PD173074 combination favored increased expression of HLA-G,a marker of extravillous trophoblast. Together,these data suggest that the cell lines exhibit totipotent potential and that BMP4 can prime human PSCs to a self-renewing alternative state permissive for trophoblast development. The results may have implications for regulation of lineage decisions in the early embryo. View Publication

Blood flow promotes emergence of definitive hematopoietic stem cells (HSCs) in the developing embryo,yet the signals generated by hemodynamic forces that influence hematopoietic potential remain poorly defined. Here we show that fluid shear stress endows long-term multilineage engraftment potential upon early hematopoietic tissues at embryonic day 9.5,an embryonic stage not previously described to harbor HSCs. Effects on hematopoiesis are mediated in part by a cascade downstream of wall shear stress that involves calcium efflux and stimulation of the prostaglandin E2 (PGE2)-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling axis. Blockade of the PGE2-cAMP-PKA pathway in the aorta-gonad-mesonephros (AGM) abolished enhancement in hematopoietic activity. Furthermore,Ncx1 heartbeat mutants,as well as static cultures of AGM,exhibit lower levels of expression of prostaglandin synthases and reduced phosphorylation of the cAMP response element-binding protein (CREB). Similar to flow-exposed cultures,transient treatment of AGM with the synthetic analogue 16,16-dimethyl-PGE2 stimulates more robust engraftment of adult recipients and greater lymphoid reconstitution. These data provide one mechanism by which biomechanical forces induced by blood flow modulate hematopoietic potential. View Publication

Non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations are responsive to EGFR-tyrosine kinase inhibitor (EGFR-TKI). However,NSCLC patients with secondary somatic EGFR mutations are resistant to EGFR-TKI treatment. In this study,we investigated the effect of TG101348 (a JAK2 inhibitor) on the tumor growth of erlotinib-resistant NSCLC cells. Cell proliferation,apoptosis,gene expression and tumor growth were evaluated by diphenyltetrazolium bromide (MTT) assay,flow cytometry,terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining,Western Blot and a xenograft mouse model,respectively. Results showed that erlotinib had a stronger impact on the induction of apoptosis in erlotinib-sensitive PC-9 cells but had a weaker effect on erlotinib-resistant H1975 and H1650 cells than TG101348. TG101348 significantly enhanced the cytotoxicity of erlotinib to erlotinib-resistant NSCLC cells,stimulated erlotinib-induced apoptosis and downregulated the expressions of EGFR,p-EGFR,p-STAT3,Bcl-xL and survivin in erlotinib-resistant NSCLC cells. Moreover,the combined treatment of TG101348 and erlotinib induced apoptosis,inhibited the activation of p-EGFR and p-STAT3,and inhibited tumor growth of erlotinib-resistant NSCLC cells in vivo. Our results indicate that TG101348 is a potential adjuvant for NSCLC patients during erlotinib treatment. View Publication

Cholangiocytes are the target of a heterogeneous group of liver diseases known as the cholangiopathies. An evolving understanding of the mechanisms driving biliary development provides the theoretical underpinnings for rational development of induced pluripotent stem cell (iPSC)-derived cholangiocytes (iDCs). Therefore,the aims of this study were to develop an approach to generate iDCs and to fully characterize the cells in vitro and in vivo. Human iPSC lines were generated by forced expression of the Yamanaka pluripotency factors. We then pursued a stepwise differentiation strategy toward iDCs,using precise temporal exposure to key biliary morphogens,and we characterized the cells,using a variety of morphologic,molecular,cell biologic,functional,and in vivo approaches. Morphology shows a stepwise phenotypic change toward an epithelial monolayer. Molecular analysis during differentiation shows appropriate enrichment in markers of iPSC,definitive endoderm,hepatic specification,hepatic progenitors,and ultimately cholangiocytes. Immunostaining,western blotting,and flow cytometry demonstrate enrichment of multiple functionally relevant biliary proteins. RNA sequencing reveals that the transcriptome moves progressively toward that of human cholangiocytes. iDCs generate intracellular calcium signaling in response to ATP,form intact primary cilia,and self-assemble into duct-like structures in three-dimensional culture. In vivo,the cells engraft within mouse liver,following retrograde intrabiliary infusion. In summary,we have developed a novel approach to generate mature cholangiocytes from iPSCs. In addition to providing a model of biliary differentiation,iDCs represent a platform for in vitro disease modeling,pharmacologic testing,and individualized,cell-based,regenerative therapies for the cholangiopathies. View Publication

3D suspension culture is generally considered a promising method to achieve efficient expansion and controlled differentiation of human pluripotent stem cells (hPSCs). In this work,we focused on developing an integrated culture platform for expansion and neural commitment of hPSCs into neural precursors using 3D suspension conditions and chemically-defined culture media. We evaluated different inoculation methodologies for hPSC expansion as 3D aggregates and characterized the resulting cultures in terms of aggregate size distribution. It was demonstrated that upon single-cell inoculation,after four days of culture,3D aggregates were composed of homogenous populations of hPSC and were characterized by an average diameter of 139 ± 26 μm,which was determined to be the optimal size to initiate neural commitment. Temporal analysis revealed that upon neural specification it is possible to maximize the percentage of neural precursor cells expressing the neural markers Sox1 and Pax6 after nine days of culture. These results highlight our ability to define a robust method for production of hPSC-derived neural precursors that minimizes processing steps and that constitutes a promising alternative to the traditional planar adherent culture system due to a high potential for scaling-up. View Publication

The application of stem-cell-based therapies in regenerative medicine is hindered by the tumorigenic potential of residual human pluripotent stem cells. Previously,we identified a human pluripotent stem-cell-specific lectin probe,called rBC2LCN,by comprehensive glycome analysis using high-density lectin microarrays. Here we developed a recombinant lectin-toxin fusion protein of rBC2LCN with a catalytic domain of Pseudomonas aeruginosa exotoxin A,termed rBC2LCN-PE23,which could be expressed as a soluble form from the cytoplasm of Escherichia coli and purified to homogeneity by one-step affinity chromatography. rBC2LCN-PE23 bound to human pluripotent stem cells,followed by its internalization,allowing intracellular delivery of a cargo of cytotoxic protein. The addition of rBC2LCN-PE23 to the culture medium was sufficient to completely eliminate human pluripotent stem cells. Thus,rBC2LCN-PE23 has the potential to contribute to the safety of stem-cell-based therapies. View Publication

Environmental influences and insults by reproductive toxicant exposure can lead to impaired spermatogenesis or infertility. Understanding how toxicants disrupt spermatogenesis is critical for determining how environmental factors contribute to impaired fertility. While current animal models are available,understanding of the reproductive toxic effects on human fertility requires a more robust model system. We recently demonstrated that human pluripotent stem cells can differentiate into spermatogonial stem cells/spermatogonia,primary and secondary spermatocytes,and haploid spermatids; a model that mimics many aspects of human spermatogenesis. Here,using this model system,we examine the effects of 2-bromopropane (2-BP) and 1,2,dibromo-3-chloropropane (DBCP) on in vitro human spermatogenesis. 2-BP and DBCP are non-endocrine disrupting toxicants that are known to impact male fertility. We show that acute treatment with either 2-BP or DBCP induces a reduction in germ cell viability through apoptosis. 2-BP and DBCP affect viability of different cell populations as 2-BP primarily reduces spermatocyte viability,whereas DBCP exerts a much greater effect on spermatogonia. Acute treatment with 2-BP or DBCP also reduces the percentage of haploid spermatids. Both 2-BP and DBCP induce reactive oxygen species (ROS) formation leading to an oxidized cellular environment. Taken together,these results suggest that acute exposure with 2-BP or DBCP causes human germ cell death in vitro by inducing ROS formation. This system represents a unique platform for assessing human reproductive toxicity potential of various environmental toxicants in a rapid,efficient,and unbiased format. View Publication

Eosinophils are associated with type 2 immune responses to allergens and helminths. They release various proinflammatory mediators and toxic proteins on activation and are therefore considered proinflammatory effector cells. Eosinophilia is promoted by the cytokines interleukin (IL)-3,IL-5,and granulocyte macrophage-colony-stimulating factor (GM-CSF) and can result from enhanced de novo production or reduced apoptosis. In this study,we show that only IL-5 induces differentiation of eosinophils from bone marrow precursors,whereas IL-5,GM-CSF,and to a lesser extent IL-3 promote survival of mature eosinophils. The receptors for these cytokines use the common β chain,which serves as the main signaling unit linked to signal transducer and activator of transcription 5,p38 mitogen-activated protein kinase,and nuclear factor (NF)-κB pathways. Inhibition of NF-κB induced apoptosis of in vitro cultured eosinophils. Selective deletion of IκBα in vivo resulted in enhanced expression of Bcl-xL and reduced apoptosis during helminth infection. Retroviral overexpression of Bcl-xL promoted survival,whereas pharmacologic inhibition of Bcl-xL in murine or human eosinophils induced rapid apoptosis. These results suggest that therapeutic strategies targeting Bcl-xL in eosinophils could improve health conditions in allergic inflammatory diseases. View Publication