技术资料

OBJECTIVE: The molecular mechanisms that maintain human pluripotent stem (PS) cells are not completely understood. Here we sought to identify new candidate PS cell regulators to facilitate future improvements in their generation,expansion,and differentiation. MATERIALS AND METHODS: We used bioinformatic analyses of multiple serial-analysis-of-gene-expression libraries (generated from human PS cells and their differentiated derivatives),together with small interfering RNA (siRNA) screening to identify candidate pluripotency regulators. Validation of candidate regulators involved promoter analyses,Affymetrix profiling,real-time PCR,and immunoprecipitation. RESULTS: Promoter analysis of genes differentially expressed across multiple serial-analysis-of-gene-expression libraries identified E2F motifs in the promoters of many PS cell-specific genes (e.g.,POU5F1,NANOG,SOX2,FOXD3). siRNA analyses identified two retinoblastoma binding proteins (RBBP4,RBBP9) as required for maintenance of multiple human PS cell types. Both RBBPs were bound to RB in human PS cells,and E2F motifs were present in the promoters of genes whose expression was altered by decreasing RBBP4 and RBBP9 expression. Affymetrix and real-time PCR studies of siRNA-treated human PS cells showed that reduced RBBP4 or RBBP9 expression concomitantly decreased expression of POU5F1,NANOG,SOX2,and/or FOXD3 plus certain cell cycle genes (e.g.,CCNA2,CCNB1),while increasing expression of genes involved in organogenesis (particularly neurogenesis). CONCLUSIONS: These results reveal new candidate positive regulators of human PS cells,providing evidence of their ability to regulate expression of pluripotency,cell cycle,and differentiation genes in human PS cells. These data provide valuable new leads for further elucidating mechanisms of human pluripotency. 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

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

Direct cell-cell contact between haematopoietic progenitor cells (HPCs) and their cellular microenvironment is essential to maintain 'stemness'. In cancer biology,focal adhesion (FA) proteins are involved in survival signal transduction in a wide variety of human tumours. To define the role of FA proteins in the haematopoietic microenvironment of myelodysplastic syndromes (MDS),CD73-positive mesenchymal stromal cells (MSCs) were immunostained for paxillin,pFAK [Y(397)],and HSP90α/β and p130CAS,and analysed for reactivity,intensity and cellular localisation. Immunofluorescence microscopy allowed us to identify qualitative and quantitative differences,and subcellular localisation analysis revealed that in pathological MSCs,paxillin,pFAK [Y(397)],and HSP90α/β formed nuclear molecular complexes. Increased expression of paxillin,pFAK [Y(397)],and HSP90α/β and enhanced nuclear co-localisation of these proteins correlated with a consistent proliferative advantage in MSCs from patients with refractory anaemia with excess blasts (RAEB) and negatively impacted clonogenicity of HPCs. These results suggest that signalling via FA proteins could be implicated in HPC-MSC interactions. Further,because FAK is an HSP90α/β client protein,these results suggest the utility of HSP90α/β inhibition as a target for adjuvant therapy for myelodysplasia. View Publication

Pluripotency,the ability of a cell to differentiate and give rise to all embryonic lineages,defines a small number of mammalian cell types such as embryonic stem (ES) cells. While it has been generally held that pluripotency is the product of a transcriptional regulatory network that activates and maintains the expression of key stem cell genes,accumulating evidence is pointing to a critical role for epigenetic processes in establishing and safeguarding the pluripotency of ES cells,as well as maintaining the identity of differentiated cell types. In order to better understand the role of epigenetic mechanisms in pluripotency,we have examined the dynamics of chromatin modifications genome-wide in human ES cells (hESCs) undergoing differentiation into a mesendodermal lineage. We found that chromatin modifications at promoters remain largely invariant during differentiation,except at a small number of promoters where a dynamic switch between acetylation and methylation at H3K27 marks the transition between activation and silencing of gene expression,suggesting a hierarchy in cell fate commitment over most differentially expressed genes. We also mapped over 50 000 potential enhancers,and observed much greater dynamics in chromatin modifications,especially H3K4me1 and H3K27ac,which correlate with expression of their potential target genes. Further analysis of these enhancers revealed potentially key transcriptional regulators of pluripotency and a chromatin signature indicative of a poised state that may confer developmental competence in hESCs. Our results provide new evidence supporting the role of chromatin modifications in defining enhancers and pluripotency. View Publication

Histone deacetylases (HDAC) are key enzymes in the epigenetic control of gene expression. Recently,inhibitors of class I and class II HDAC have been successfully employed for the treatment of different inflammatory diseases such as rheumatoid arthritis,colitis,airway inflammation and asthma. So far,little is known so far about a similar therapeutic effect of inhibitors specifically directed against sirtuins,the class III HDAC. In this study,we investigated the expression and localization of endogenous sirtuins in primary human dermal microvascular endothelial cells (HDMEC),a cell type playing a key role in the development and maintenance of skin inflammation. We then examined the biological activity of sirtinol,a specific sirtuin inhibitor,in HDMEC response to pro-inflammatory cytokines. We found that,even though sirtinol treatment alone affected only long-term cell proliferation,it diminishes HDMEC inflammatory responses to tumor necrosis factor (TNF)α and interleukin (IL)-1β. In fact,sirtinol significantly reduced membrane expression of adhesion molecules in TNFã- or IL-1β-stimulated cells,as well as the amount of CXCL10 and CCL2 released by HDMEC following TNFα treatment. Notably,sirtinol drastically decreased monocyte adhesion on activated HDMEC. Using selective inhibitors for Sirt1 and Sirt2,we showed a predominant involvement of Sirt1 inhibition in the modulation of adhesion molecule expression and monocyte adhesion on activated HDMEC. Finally,we demonstrated the in vivo expression of Sirt1 in the dermal vessels of normal and psoriatic skin. Altogether,these findings indicated that sirtuins may represent a promising therapeutic target for the treatment of inflammatory skin diseases characterized by a prominent microvessel involvement. View Publication

The ability to stimulate mammalian cells with light has significantly changed our understanding of electrically excitable tissues in health and disease,paving the way toward various novel therapeutic applications. Here,we demonstrate the potential of optogenetic control in cardiac cells using a hybrid experimental/computational technique. Experimentally,we introduced channelrhodopsin-2 into undifferentiated human embryonic stem cells via a lentiviral vector,and sorted and expanded the genetically engineered cells. Via directed differentiation,we created channelrhodopsin-expressing cardiomyocytes,which we subjected to optical stimulation. To quantify the impact of photostimulation,we assessed electrical,biochemical,and mechanical signals using patch-clamping,multielectrode array recordings,and video microscopy. Computationally,we introduced channelrhodopsin-2 into a classic autorhythmic cardiac cell model via an additional photocurrent governed by a light-sensitive gating variable. Upon optical stimulation,the channel opens and allows sodium ions to enter the cell,inducing a fast upstroke of the transmembrane potential. We calibrated the channelrhodopsin-expressing cell model using single action potential readings for different photostimulation amplitudes,pulse widths,and frequencies. To illustrate the potential of the proposed approach,we virtually injected channelrhodopsin-expressing cells into different locations of a human heart,and explored its activation sequences upon optical stimulation. Our experimentally calibrated computational toolbox allows us to virtually probe landscapes of process parameters,and identify optimal photostimulation sequences toward pacing hearts with light. ?? 2011 Biophysical Society. View Publication

Recurrent chromosomal translocations involving the mixed lineage leukaemia (MLL) gene initiate aggressive forms of leukaemia,which are often refractory to conventional therapies. Many MLL-fusion partners are members of the super elongation complex (SEC),a critical regulator of transcriptional elongation,suggesting that aberrant control of this process has an important role in leukaemia induction. Here we use a global proteomic strategy to demonstrate that MLL fusions,as part of SEC and the polymerase-associated factor complex (PAFc),are associated with the BET family of acetyl-lysine recognizing,chromatin 'adaptor' proteins. These data provided the basis for therapeutic intervention in MLL-fusion leukaemia,via the displacement of the BET family of proteins from chromatin. We show that a novel small molecule inhibitor of the BET family,GSK1210151A (I-BET151),has profound efficacy against human and murine MLL-fusion leukaemic cell lines,through the induction of early cell cycle arrest and apoptosis. I-BET151 treatment in two human leukaemia cell lines with different MLL fusions alters the expression of a common set of genes whose function may account for these phenotypic changes. The mode of action of I-BET151 is,at least in part,due to the inhibition of transcription at key genes (BCL2,C-MYC and CDK6) through the displacement of BRD3/4,PAFc and SEC components from chromatin. In vivo studies indicate that I-BET151 has significant therapeutic value,providing survival benefit in two distinct mouse models of murine MLL-AF9 and human MLL-AF4 leukaemia. Finally,the efficacy of I-BET151 against human leukaemia stem cells is demonstrated,providing further evidence of its potent therapeutic potential. These findings establish the displacement of BET proteins from chromatin as a promising epigenetic therapy for these aggressive leukaemias. View Publication

C-C chemokine receptor type 5 (CCR5) is a major co-receptor for the entry of human immunodeficiency virus type-1 (HIV-1) into target cells. Human hematopoietic stem cells (hHSCs) with naturally occurring CCR5 deletions (Δ32) or artificially disrupted CCR5 have shown potential for curing acquired immunodeficiency syndrome (AIDS). However,Δ32 donors are scarce,heterologous bone marrow transplantation is not exempt of risks,and genetic engineering of autologous hHSCs is not trivial. Here,we have disrupted the CCR5 locus of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) using specific zinc finger nucleases (ZFNs) combined with homologous recombination. The modified hESCs and hiPSCs retained pluripotent characteristics and could be differentiated in vitro into CD34(+) cells that formed all types of hematopoietic colonies. Our results suggest the potential of using patient-specific hHSCs derived from ZFN-modified hiPSCs for treating AIDS. View Publication

Glioblastoma multiforme is the most common glioma variant in adults and is highly malignant. Tumors are thought to harbor a subpopulation of stem-like cancer cells,with the bulk resembling neural progenitor-like cells that are unable to fully differentiate. Although multiple pathways are known to be involved in glioma tumorigenesis,the role of Wnt signaling has been poorly described. Here,we show that Dishevelled 2 (Dvl2),a key component of the Wnt signaling pathway,is overexpressed in human gliomas. RNA interference-mediated depletion of Dvl2 blocked proliferation and promoted the differentiation of cultured human glioma cell lines and primary,patient-derived glioma cells. In addition,Dvl2 depletion inhibited tumor formation after intracranial injection of glioblastoma cells in immunodeficient mice. Inhibition of canonical Wnt/β-catenin signaling also blocked proliferation,but unlike Dvl2 depletion,did not induce differentiation. Finally,Wnt5a,a noncanonical Wnt ligand,was also required for glioma cell proliferation. The data therefore suggest that both canonical and noncanonical Wnt signaling pathways downstream of Dvl2 cooperate to maintain the proliferative capacity of human glioblastomas. View Publication

Genetic comparison between human embryonic stem cells and induced pluripotent stem cells has been hampered by genetic variation. To solve this problem,we have developed an isogenic system that allows direct comparison of induced pluripotent stem cells (hiPSCs) to their genetically matched human embryonic stem cells (hESCs). We show that hiPSCs have a highly similar transcriptome to hESCs. Global transcriptional profiling identified 102-154 genes (textgreater2 fold) that showed a difference between isogenic hiPSCs and hESCs. A stringent analysis identified NNAT as a key imprinted gene that was dysregulated in hiPSCs. Furthermore,a disproportionate number of X-chromosome localized genes were over-expressed in female hiPSCs. Our results indicate that despite a remarkably close transcriptome to hESCs,isogenic hiPSCs have alterations in imprinting and regulation of X-chromosome genes. View Publication

Low oxygen (O(2)) levels characterize the microenvironment of both stem cells and rapidly growing tumors. Moreover,hypoxia is associated with the maintenance of stem cell-like phenotypes and increased invasion,angiogenesis and metastasis in cancer patients. Metastatic cancers,such as breast cancer and melanoma,aberrantly express the embryonic morphogen Nodal,and the presence of this protein is correlated with metastatic disease. In this paper,we demonstrate that hypoxia induces Nodal expression in melanoma and breast cancer cells concomitant with increased cellular invasion and angiogenic phenotypes. Of note,Nodal expression remains up-regulated up to 48 h following reoxygenation. The oxygen-mediated regulation of Nodal expression occurs via a combinatorial mechanism. Within the first 24 h of exposure to low O(2),there is an increase in protein stability. This increase in stability is accompanied by an induction of transcription,mediated by the HIF-1α-dependent activation of Notch-responsive elements in the node-specific enhancer of the Nodal gene locus. Finally,Nodal expression is maintained upon reoxygenation by a canonical SMAD-dependent feed-forward mechanism. This work provides insight into the O(2)-mediated regulation of Nodal,a key stem cell-associated factor,and reveals that Nodal may be a target for the treatment and prevention of hypoxia-induced tumor progression. View Publication