Scientific Resources

Hyperactive signaling of the MAP kinase pathway resulting from the constitutively active B-Raf(V600E) mutated enzyme has been observed in a number of human tumors,including melanomas. Herein we report the discovery and biological evaluation of GSK2118436,a selective inhibitor of Raf kinases with potent in vitro activity in oncogenic B-Raf-driven melanoma and colorectal carcinoma cells and robust in vivo antitumor and pharmacodynamic activity in mouse models of B-Raf(V600E) human melanoma. GSK2118436 was identified as a development candidate,and early clinical results have shown significant activity in patients with B-Raf mutant melanoma. View Publication

Nuclear factor,erythroid 2-like 2 (Nrf2) is a master transcription factor for cellular defense against endogenous and exogenous stresses by regulating expression of many antioxidant and detoxification genes. Here,we show that Nrf2 acts as a key pluripotency gene and a regulator of proteasome activity in human embryonic stem cells (hESCs). Nrf2 expression is highly enriched in hESCs and dramatically decreases upon differentiation. Nrf2 inhibition impairs both the self-renewal ability of hESCs and re-establishment of pluripotency during cellular reprogramming. Nrf2 activation can delay differentiation. During early hESC differentiation,Nrf2 closely colocalizes with OCT4 and NANOG. As an underlying mechanism,our data show that Nrf2 regulates proteasome activity in hESCs partially through proteasome maturation protein (POMP),a proteasome chaperone,which in turn controls the proliferation of self-renewing hESCs,three germ layer differentiation and cellular reprogramming. Even modest proteasome inhibition skews the balance of early differentiation toward mesendoderm at the expense of an ectodermal fate by decreasing the protein level of cyclin D1 and delaying the degradation of OCT4 and NANOG proteins. Taken together,our findings suggest a new potential link between environmental stress and stemness with Nrf2 and the proteasome coordinately positioned as key mediators. View Publication

Native porcine nucleus pulposus (NP) tissue harbors a number of notochordal cells (NCs). Whether the native NP matrix supports the homeostasis of notochordal cells is poorly understood. We hypothesized the NP matrix alone may contain sufficient regulatory factors and can serve as stimuli to generate notochordal cells (NCs) from human pluripotent stem cells. NCs are a promising cell sources for cell-based therapy to treat some types of intervertebral disc (IVD) degeneration. One major limitation of this emerging technique is the lack of available NCs as a potential therapeutic cell source. Human pluripotent stem cells derived from reprogramming or somatic cell nuclear transfer technique may yield stable and unlimited source for therapeutic use. We devised a new method to use porcine NP matrix to direct notochordal differentiation of human induced pluripotent stem cells (hiPSCs). The results showed that hiPSCs successfully differentiated into NC-like cells under the influence of devitalized porcine NP matrix. The NC-like cells expressed typical notochordal marker genes including brachyury (T),cytokeratin-8 (CK-8) and cytokeratin-18 (CK-18),and they displayed the ability to generate NP-like tissue in vitro,which was rich in aggrecan and collagen type II. These findings demonstrated the proof of concept for using native NP matrix to direct notochordal differentiation of hiPSCs. It provides a foundation for further understanding the biology of NCs,and eventually towards regenerative therapies for disc degeneration. View Publication

Background: Human induced pluripotent stem cells,which can be differentiated into hepatocyte-like cells,could provide a source for liver regeneration and bio-artificial liver devices. However,the functionality of hepatocyte-like cells is significantly lower than that of primary hepatocytes. Aims: To investigate whether serum from patients undergoing hepatectomy might promote differentiation from human induced pluripotent stem cells to hepatocyte-like cells. Methods: Serum from patients undergoing hepatectomy (acquired pre-hepatectomy and 3. hours,1 day and 3 days post-hepatectomy) was used to replace foetal bovine serum when differentiating human induced pluripotent stem cells into hepatocyte-like cells. Properties of hepatocyte-like cells were assessed and compared with cells cultured in foetal bovine serum. Results: The differentiation efficiency and functionality of hepatocyte-like cells cultured in human serum 3. hours and 1 day post-hepatectomy were superior to those cultured in foetal bovine serum and human serum pre-hepatectomy. Human serum 3 days post-hepatectomy had an equal effect to that of human serum pre-hepatectomy. Some cytochrome P450 isozyme transcript levels of hepatocyte-like cells cultured in human serum were higher than those cultured in foetal bovine serum. Conclusion: Human serum,particularly that acquired relatively soon after hepatectomy,can enhance the differentiation efficiency and functionality of hepatocyte-like cells derived from human induced pluripotent stem cells. textcopyright 2014 Editrice Gastroenterologica Italiana S.r.l. View Publication

Human endothelial cells (ECs) and pericytes are of great interest for research on vascular development and disease,as well as for future therapy. This protocol describes the efficient generation of ECs and pericytes from human pluripotent stem cells (hPSCs) under defined conditions. Essential steps for hPSC culture,differentiation,isolation and functional characterization of ECs and pericytes are described. Substantial numbers of both cell types can be derived in only 2-3 weeks: this involves differentiation (10 d),isolation (1 d) and 4 or 10 d of expansion of ECs and pericytes,respectively. We also describe two assays for functional evaluation of hPSC-derived ECs: (i) primary vascular plexus formation upon coculture with hPSC-derived pericytes and (ii) incorporation in the vasculature of zebrafish xenografts in vivo. These assays can be used to test the quality and drug sensitivity of hPSC-derived ECs and model vascular diseases with patient-derived hPSCs. View Publication

Recent methodological advances have improved the ease and efficiency of generating human induced pluripotent stem cells (hiPSCs),but this now typically results in a greater number of hiPSC clones being derived than can be wholly characterized. It is therefore imperative that methods are developed which facilitate rapid selection of hiPSC clones most suited for the downstream research aims. Here we describe a combination of procedures enabling the simultaneous screening of multiple clones to determine their genomic integrity as well as their cardiac differentiation potential within two weeks of the putative reprogrammed colonies initially appearing. By coupling splinkerette-PCR with Ion Torrent sequencing,we could ascertain the number and map the proviral integration sites in lentiviral-reprogrammed hiPSCs. In parallel,we developed an effective cardiac differentiation protocol that generated functional cardiomyocytes within 10 days without requiring line-specific optimization for any of the six independent human pluripotent stem cell lines tested. Finally,to demonstrate the scalable potential of these procedures,we picked 20 nascent iPSC clones and performed these independent assays concurrently. Before the clones required passaging,we were able to identify clones with a single integrated copy of the reprogramming vector and robust cardiac differentiation potential for further analysis. View Publication

Aims Hemophilia A (HA) is a severe,congenital bleeding disorder caused by the deficiency of clotting factor VIII (FVIII). For years,traditional laboratory animals have been used to study HA and its therapies,although animal models may not entirely mirror the human pathophysiology. Human induced pluripotent stem cells (iPSCs) can undergo unlimited self-renewal and differentiate into all cell types. This study aims to generate hemophilia A (HA) patient-specific iPSCs that differentiate into disease-affected hepatocyte cells. These hepatocytes are potentially useful for in vitro disease modeling and provide an applicable cell source for autologous cell therapy after genetic correction. Main methods In this study,we mainly generated iPSCs from urine collected from HA patients with integration-free episomal vectors PEP4-EO2S-ET2K containing human genes OCT4,SOX2,SV40LT and KLF4,and differentiated these iPSCs into hepatocyte-like cells. We further identified the genetic phenotype of the FVIII genes and the FVIII activity in the patient-specific iPSC derived hepatic cells. Key findings HA patient-specific iPSCs (HA-iPSCs) exhibited typical pluripotent properties evident by immunostaining,in vitro assays and in vivo assays. Importantly,we showed that HA-iPSCs could differentiate into functional hepatocyte-like cells and the HA-iPSC-derived hepatocytes failed to produce FVIII,but otherwise functioned normally,recapitulating the phenotype of HA disease in vitro. Significance HA-iPSCs,particular those generated from the urine using a non-viral approach,provide an efficient way for modeling HA in vitro. Furthermore,HA-iPSCs and their derivatives serve as an invaluable cell source that can be used for gene and cell therapy in regenerative medicine. textcopyright 2014 Elsevier Inc. View Publication

Recently,induced pluripotent stem cells (iPSCs) were established as promising cell sources for revolutionary regenerative therapies. The initial culture system used for iPSC generation needed fetal calf serum in the culture medium and mouse embryonic fibroblast as a feeder layer,both of which could possibly transfer unknown exogenous antigens and pathogens into the iPSC population. Therefore,the development of culture systems designed to minimize such potential risks has become increasingly vital for future applications of iPSCs for clinical use. On another front,although donor cell types for generating iPSCs are wide-ranging,T cells have attracted attention as unique cell sources for iPSCs generation because T cell-derived iPSCs (TiPSCs) have a unique monoclonal T cell receptor genomic rearrangement that enables their differentiation into antigen-specific T cells,which can be applied to novel immunotherapies. In the present study,we generated transgene-free human TiPSCs using a combination of activated human T cells and Sendai virus under defined culture conditions. These TiPSCs expressed pluripotent markers by quantitative PCR and immunostaining,had a normal karyotype,and were capable of differentiating into cells from all three germ layers. This method of TiPSCs generation is more suitable for the therapeutic application of iPSC technology because it lowers the risks associated with the presence of undefined,animal-derived feeder cells and serum. Therefore this work will lead to establishment of safer iPSCs and extended clinical application. View Publication

Despite the fact that glucocorticoids and long acting beta agonists are effective treatments for asthma,their effects on human mast cells (MC) appear to be modest. Although MC are one of the major effector cells in the underlying inflammatory reactions associated with asthma,their regulation by these drugs is not yet fully understood and,in some cases,controversial. Using a human immortalized MC line (LAD2),we studied the effects of fluticasone propionate (FP) and salmeterol (SM),on the release of early and late phase mediators. LAD2 cells were pretreated with FP (100 nM),SM (1 µM),alone and in combination,at various incubation times and subsequently stimulated with agonists substance P,C3a and IgE/anti-IgE. Degranulation was measured by the release of β-hexosaminidase. Cytokine and chemokine expression were measured using quantitative PCR,ELISA and cytometric bead array (CBA) assays. The combination of FP and SM synergistically inhibited degranulation of MC stimulated with substance P (33% inhibition compared to control,n = 3,P>05). Degranulation was inhibited by FP alone,but not SM,when MC were stimulated with C3a (48% inhibition,n = 3,P>05). As previously reported,FP and SM did not inhibit degranulation when MC were stimulated with IgE/anti-IgE. FP and SM in combination inhibited substance P-induced release of tumor necrosis factor (TNF),CCL2,and CXCL8 (98%,99% and 92% inhibition,respectively,n = 4,P>05). Fluticasone and salmeterol synergistically inhibited mediator production by human MC stimulated with the neuropeptide substance P. This synergistic effect on mast cell signaling may be relevant to the therapeutic benefit of combination therapy in asthma. View Publication

Nanofibrous gelatin substrates are suited for long-term expansion of human pluripotent stem cells (hPSCs) under feeder- and serum-free culture conditions. A combinatorial library with different sets of processing parameters was established to assess the culture performance of hPSCs on nanofibrous substrates in terms of cell adhesion and growth rate,using Matrigel as control. Then,the optimal conditions were applied to long-term expansion of hPSCs with several cell lines,showing a maintained pluripotency over more than 20 passages without introducing any abnormal chromosome. In addition,this approach allowed us to avoid enzymatic disassociation and mechanic cutting during passages,thereby promoting a better hPSC culture and long-term expansion. ?? 2014 Elsevier Ltd. View Publication

Human pluripotent stem cell (hPSC)-derived endothelial lineage cells constitutes a promising source for therapeutic revascularization,but progress in this arena has been hampered by a lack of clinically-scalable differentiation protocols and inefficient formation of a functional vessel network integrating with the host circulation upon transplantation. Using a human embryonic stem cell reporter cell line,where green fluorescent protein expression is driven by an endothelial cell-specific VE-cadherin (VEC) promoter,we screened for textgreater 60 bioactive small molecules that would promote endothelial differentiation,and found that administration of BMP4 and a GSK-3β inhibitor in an early phase and treatment with VEGF-A and inhibition of the Notch signaling pathway in a later phase led to efficient differentiation of hPSCs to the endothelial lineage within six days. This sequential approach generated textgreater 50% conversion of hPSCs to endothelial cells (ECs),specifically VEC(+)CD31(+)CD34(+)CD14(-)KDR(high) endothelial progenitors (EPs) that exhibited higher angiogenic and clonogenic proliferation potential among endothelial lineage cells. Pharmaceutical inhibition or genetical knockdown of Notch signaling,in combination with VEGF-A treatment,resulted in efficient formation of EPs via KDR(+) mesodermal precursors and blockade of the conversion of EPs to mature ECs. The generated EPs successfully formed functional capillary vessels in vivo with anastomosis to the host vessels when transplanted into immunocompromised mice. Manipulation of this VEGF-A-Notch signaling circuit in our protocol leads to rapid large-scale production of the hPSC-derived EPs by 12- to 20-fold vs current methods,which may serve as an attractive cell population for regenerative vascularization with superior vessel forming capability compared to mature ECs. View Publication

Oxidative stress serves as an important regulator of both apoptosis and metabolic reprogramming in tumor cells. Chaetocin,a histone methyltransferase inhibitor,is known to induce ROS generation. As elevating basal ROS level sensitizes glioma cells to apoptosis,the ability of Chaetocin in regulating apoptotic and metabolic adaptive responses in glioma was investigated. Chaetocin induced glioma cell apoptosis in a ROS-dependent manner. Increased intracellular ROS induced (i) Yes-associated protein 1 (YAP1) expression independent of the canonical Hippo pathway as well as (ii) ATM and JNK activation. Increased interaction of YAP1 with p73 and p300 induced apoptosis in an ATM-dependent manner. Chaetocin induced JNK modulated several metabolic parameters like glucose uptake,lactate production,ATP generation,and activity of glycolytic enzymes hexokinase and pyruvate kinase. However,JNK had no effect on ATM or YAP1 expression. Coherent with the in vitro findings,Chaetocin reduced tumor burden in heterotypic xenograft glioma mouse model. Chaetocin-treated tumors exhibited heightened ROS,pATM,YAP1 and pJNK levels. Our study highlights the coordinated control of glioma cell proliferation and metabolism by ROS through (i) ATM-YAP1-driven apoptotic pathway and (ii) JNK-regulated metabolic adaptation. The elucidation of these newfound connections and the roles played by ROS to simultaneously shift metabolic program and induce apoptosis could provide insights toward the development of new anti-glioma strategies. View Publication