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Cellular decisions of self-renewal or differentiation arise from integration and reciprocal titration of numerous regulatory networks. Notch and Wnt/β-catenin signalling often intersect in stem and progenitor cells and regulate each other transcriptionally. The biological outcome of signalling through each pathway often depends on the context and timing as cells progress through stages of differentiation. Here,we show that membrane-bound Notch physically associates with unphosphorylated (active) β-catenin in stem and colon cancer cells and negatively regulates post-translational accumulation of active β-catenin protein. Notch-dependent regulation of β-catenin protein did not require ligand-dependent membrane cleavage of Notch or the glycogen synthase kinase-3β-dependent activity of the β-catenin destruction complex. It did,however,require the endocytic adaptor protein Numb and lysosomal activity. This study reveals a previously unrecognized function of Notch in negatively titrating active β-catenin protein levels in stem and progenitor cells. View Publication

Pluripotent stem cells exist in naive and primed states,epitomized by mouse embryonic stem cells (ESCs) and the developmentally more advanced epiblast stem cells (EpiSCs; ref. 1). In the naive state of ESCs,the genome has an unusual open conformation and possesses a minimum of repressive epigenetic marks. In contrast,EpiSCs have activated the epigenetic machinery that supports differentiation towards the embryonic cell types. The transition from naive to primed pluripotency therefore represents a pivotal event in cellular differentiation. But the signals that control this fundamental differentiation step remain unclear. We show here that paracrine and autocrine Wnt signals are essential self-renewal factors for ESCs,and are required to inhibit their differentiation into EpiSCs. Moreover,we find that Wnt proteins in combination with the cytokine LIF are sufficient to support ESC self-renewal in the absence of any undefined factors,and support the derivation of new ESC lines,including ones from non-permissive mouse strains. Our results not only demonstrate that Wnt signals regulate the naive-to-primed pluripotency transition,but also identify Wnt as an essential and limiting ESC self-renewal factor. View Publication

Human pluripotent stem cells (PSCs),which include human embryonic stem cells (ESCs) as well as induced pluripotent stem cells (iPSCs),represent an important source of cellular therapies in regenerative medicine and the study of early human development. As such,it is becoming increasingly important to develop methods for the large-scale banking of human PSC lines. There are several well-established methods for the propagation of human PSCs. The key to development of a good manufacturing practice (GMP) bank is to determine a manufacturing method that is amenable to large-scale production using materials that are fully documented. We have developed several banks of hESCs using animal feeder cells,animal-based matrices,or animal-free matrices. Protocols for growing hESCs on mouse embryonic fibroblasts (MEFs) are well established and are very helpful for producing research grade banks of cells. As most human ESCs cultured by research laboratories have been exposed to xenogeneic reagents,it is not imperative that all materials used in the production of a master cell bank be animal-free in origin. Nevertheless,as the field develops,it will no doubt become increasingly important to produce a bank of cells for clinical use without xenogeneic reagents,particularly nonhuman feeder cells which might harbor viruses with potential risk to human health or cell product integrity. Thus,even for cell lines previously exposed to xenogeneic reagents,it is important to minimize any subsequent exposure of the cell lines to additional adventitious agents. We have specifically described procedures for the growth of hESCs on Matrigel,an animal-matrix,and CELLstart,an animal-free matrix,and these can be used to produce hESCs as part of a clinical manufacturing process. View Publication

Our ability to guide differentiation of human pluripotent stem cells (hPSCs) toward desired lineages efficiently and reproducibly in xeno-free conditions is the key to advancing hPSC technology from the laboratory to clinical use. Here we report an engineered biomimetic substrate functionalized with both peptide ligands for α5β1 and α6β1 integrins to support efficient early mesodermal differentiation of human embryonic stem cells (hESCs) when cultured in a differentiation medium containing BMP4. In contrast,mesodermal differentiation is not induced on substrates functionalized with either ligand alone even though the culture medium is identical. Mesodermal differentiation was characterized by immunofluorescent staining,flow cytometric analysis,and RT-PCR analysis of early mesodermal markers Brachyury,Mixl1,and Wnt3. The early mesodermal progenitors derived on the substrate functionalized with both integrin ligands have the normal developmental potential to further differentiate along the hemato-endothelial and cardiac lineages. Immobilized ligands for α5β1 and α6β1 integrins both are permissive,necessary,and sufficient insoluble ligands in this engineered system to support early mesodermal differentiation of hESCs. This synthetic substrate,in conjunction with defined soluble factors,constructs a well-controlled and xeno-free early mesodermal differentiation niche that offers advantages over the previously reported niche constructed with the Matrigel-coated substrate. View Publication

Aberrant histone deacetylase (HDAC) activity is frequent in human leukemias. However,while classical,NAD(+)-independent HDACs are an established therapeutic target,the relevance of NAD(+)-dependent HDACs (sirtuins) in leukemia treatment remains unclear. Here,we assessed the antileukemic activity of sirtuin inhibitors and of the NAD(+)-lowering drug FK866,alone and in combination with traditional HDAC inhibitors. Primary leukemia cells,leukemia cell lines,healthy leukocytes and hematopoietic progenitors were treated with sirtuin inhibitors (sirtinol,cambinol,EX527) and with FK866,with or without addition of the HDAC inhibitors valproic acid,sodium butyrate,and vorinostat. Cell death was quantified by propidium iodide cell staining and subsequent flow-cytometry. Apoptosis induction was monitored by cell staining with FITC-Annexin-V/propidium iodide or with TMRE followed by flow-cytometric analysis,and by measuring caspase3/7 activity. Intracellular Bax was detected by flow-cytometry and western blotting. Cellular NAD(+) levels were measured by enzymatic cycling assays. Bax was overexpressed by retroviral transduction. Bax and SIRT1 were silenced by RNA-interference. Sirtuin inhibitors and FK866 synergistically enhanced HDAC inhibitor activity in leukemia cells,but not in healthy leukocytes and hematopoietic progenitors. In leukemia cells,HDAC inhibitors were found to induce upregulation of Bax,a pro-apoptotic Bcl2 family-member whose translocation to mitochondria is normally prevented by SIRT1. As a result,leukemia cells become sensitized to sirtuin inhibitor-induced apoptosis. In conclusion,NAD(+)-independent HDACs and sirtuins cooperate in leukemia cells to avoid apoptosis. Combining sirtuin with HDAC inhibitors results in synergistic antileukemic activity that could be therapeutically exploited. View Publication

Elucidating the in vitro differentiation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells is important for understanding both normal and pathological hematopoietic development in vivo. For this purpose,a robust and simple hematopoietic differentiation system that can faithfully trace in vivo hematopoiesis is necessary. In this study,we established a novel serum-free monolayer culture that can trace the in vivo hematopoietic pathway from ES/iPS cells to functional definitive blood cells via mesodermal progenitors. Stepwise tuning of exogenous cytokine cocktails induced the hematopoietic mesodermal progenitors via primitive streak cells. These progenitors were then differentiated into various cell lineages depending on the hematopoietic cytokines present. Moreover,single cell deposition assay revealed that common bipotential hemoangiogenic progenitors were induced in our culture. Our system provides a new,robust,and simple method for investigating the mechanisms of mesodermal and hematopoietic differentiation. View Publication

BACKGROUND Evaluation of cystic fibrosis transmembrane conductance regulator (CFTR) functional activity to assess new therapies and define diagnosis of cystic fibrosis (CF) is cumbersome. It is known that leukocytes express detectable levels of CFTR but the molecule has not been characterized in these cells. In this study we aim at setting up and validating a blood test to evaluate CFTR expression and function in leukocytes. DESCRIPTION Western blot,PCR,immunofluorescence and cell membrane depolarization analysis by single-cell fluorescence imaging,using the potential-sensitive DiSBAC(2)(3) probe were utilized. Expression of PKA phosphorylated,cell membrane-localized CFTR was detected in non-CF monocytes,being undetectable or present in truncated form in monocytes derived from CF patients presenting with nonsense mutations. CFTR agonist administration induced membrane depolarization in monocytes isolated from non-CF donors (31 subjects) and,to a lesser extent,obligate CFTR heterozygous carriers (HTZ: 15 subjects),but it failed in monocytes from CF patients (44 subjects). We propose an index,which values in CF patients are significantly (ptextless0.001) lower than in the other two groups. Nasal Potential Difference,measured in selected subjects had concordant results with monocytes assay (Kappa statistic 0.93,95%CI: 0.80-1.00). RESULTS AND SIGNIFICANCE CFTR is detectable and is functional in human monocytes. We also showed that CFTR-associated activity can be evaluated in 5 ml of peripheral blood and devise an index potentially applicable for diagnostic purposes and both basic and translational research: from drug development to evaluation of functional outcomes in clinical trials. View Publication

Glioblastoma multiforme (GBM) is the most common and aggressive brain cancer,and despite treatment advances,patient prognosis remains poor. During routine animal studies,we serendipitously observed that fenbendazole,a benzimidazole antihelminthic used to treat pinworm infection,inhibited brain tumor engraftment. Subsequent in vitro and in vivo experiments with benzimidazoles identified mebendazole as the more promising drug for GBM therapy. In GBM cell lines,mebendazole displayed cytotoxicity,with half-maximal inhibitory concentrations ranging from 0.1 to 0.3 µM. Mebendazole disrupted microtubule formation in GBM cells,and in vitro activity was correlated with reduced tubulin polymerization. Subsequently,we showed that mebendazole significantly extended mean survival up to 63% in syngeneic and xenograft orthotopic mouse glioma models. Mebendazole has been approved by the US Food and Drug Administration for parasitic infections,has a long track-record of safe human use,and was effective in our animal models with doses documented as safe in humans. Our findings indicate that mebendazole is a possible novel anti-brain tumor therapeutic that could be further tested in clinical trials. View Publication

Identification of new techniques to express proteins into mammal cells is of particular interest for both research and medical purposes. The present study describes the use of engineered vesicles to deliver exogenous proteins into human cells. We show that overexpression of the spike glycoprotein of the vesicular stomatitis virus (VSV-G) in human cells induces the release of fusogenic vesicles named gesicles. Biochemical and functional studies revealed that gesicles incorporated proteins from producer cells and could deliver them to recipient cells. This protein-transduction method allows the direct transport of cytoplasmic,nuclear or surface proteins in target cells. This was demonstrated by showing that the TetR transactivator and the receptor for the murine leukemia virus (MLV) envelope [murine cationic amino acid transporter-1 (mCAT-1)] were efficiently delivered by gesicles in various cell types. We further shows that gesicle-mediated transfer of mCAT-1 confers to human fibroblasts a robust permissiveness to ecotropic vectors,allowing the generation of human-induced pluripotent stem cells in level 2 biosafety facilities. This highlights the great potential of mCAT-1 gesicles to increase the safety of experiments using retro/lentivectors. Besides this,gesicles is a versatile tool highly valuable for the nongenetic delivery of functions such as transcription factors or genome engineering agents. View Publication

Methylation of cytosine is a DNA modification associated with gene repression. Recently,a novel cytosine modification,5-hydroxymethylcytosine (5-hmC) has been discovered. Here we examine 5-hmC distribution during mammalian development and in cellular systems,and show that the developmental dynamics of 5-hmC are different from those of 5-methylcytosine (5-mC); in particular 5-hmC is enriched in embryonic contexts compared to adult tissues. A detectable 5-hmC signal appears in pre-implantation development starting at the zygote stage,where the paternal genome is subjected to a genome-wide hydroxylation of 5-mC,which precisely coincides with the loss of the 5-mC signal in the paternal pronucleus. Levels of 5-hmC are high in cells of the inner cell mass in blastocysts,and the modification colocalises with nestin-expressing cell populations in mouse post-implantation embryos. Compared to other adult mammalian organs,5-hmC is strongly enriched in bone marrow and brain,wherein high 5-hmC content is a feature of both neuronal progenitors and post-mitotic neurons. We show that high levels of 5-hmC are not only present in mouse and human embryonic stem cells (ESCs) and lost during differentiation,as has been reported previously,but also reappear during the generation of induced pluripotent stem cells; thus 5-hmC enrichment correlates with a pluripotent cell state. Our findings suggest that apart from the cells of neuronal lineages,high levels of genomic 5-hmC are an epigenetic feature of embryonic cell populations and cellular pluri- and multi-lineage potency. To our knowledge,5-hmC represents the first epigenetic modification of DNA discovered whose enrichment is so cell-type specific. View Publication

RATIONALE: Human embryonic stem cells can form cardiomyocytes when cultured under differentiation conditions. Although the initiating step of mesoderm formation is well characterized,the subsequent steps that promote for cardiac lineages are poorly understood and limit the yield of cardiomyocytes. OBJECTIVE: Our aim was to develop a human embryonic stem cell-based high-content screening assay to discover small molecules that drive cardiogenic differentiation after mesoderm is established to improve our understanding of the biology involved. Screening of libraries of small-molecule pathway modulators was predicted to provide insight into the cellular proteins and signaling pathways that control stem cell cardiogenesis. METHODS AND RESULTS: Approximately 550 known pathway modulators were screened in a high-content screening assay,with hits being called out by the appearance of a red fluorescent protein driven by the promoter of the cardiac-specific MYH6 gene. One potent small molecule was identified that inhibits transduction of the canonical Wnt response within the cell,which demonstrated that Wnt inhibition alone was sufficient to generate cardiomyocytes from human embryonic stem cell-derived mesoderm cells. Transcriptional profiling of inhibitor-treated compared with vehicle-treated samples further indicated that inhibition of Wnt does not induce other mesoderm lineages. Notably,several other Wnt inhibitors were very efficient in inducing cardiogenesis,including a molecule that prevents Wnts from being secreted by the cell,which confirmed that Wnt inhibition was the relevant biological activity. CONCLUSIONS: Pharmacological inhibition of Wnt signaling is sufficient to drive human mesoderm cells to form cardiomyocytes; this could yield novel tools for the benefit of pharmaceutical and clinical applications. View Publication

A precise identification of adult human hemangioblast is still lacking. To identify circulating precursors having the developmental potential of the hemangioblast,we established a new ex vivo long-term culture model supporting the differentiation of both hematopoietic and endothelial cell lineages. We identified from peripheral blood a population lacking the expression of CD34,lineage markers,CD45 and CD133 (CD34⁻Lin⁻CD45⁻CD133⁻ cells),endowed with the ability to differentiate after a 6-week culture into both hematopoietic and endothelial lineages. The bilineage potential of CD34⁻Lin⁻CD45⁻CD133⁻ cells was determined at the single-cell level in vitro and was confirmed by transplantation into NOD/SCID mice. In vivo,CD34⁻Lin⁻CD45⁻CD133⁻ cells showed the ability to reconstitute hematopoietic tissue and to generate functional endothelial cells that contribute to new vessel formation during tumor angiogenesis. Molecular characterization of CD34⁻Lin⁻D45⁻CD133⁻ cells unveiled a stem cell profile compatible with both hematopoietic and endothelial potentials,characterized by the expression of c-Kit and CXCR4 as well as EphB4,EphB2,and ephrinB2. Further molecular and functional characterization of CD34⁻Lin⁻CD45⁻CD133⁻ cells will help dissect their physiologic role in blood and blood vessel maintenance and repair in adult life. View Publication