技术资料

Embryonic stem (ES) and induced-pluripotent stem (iPS) cells can be grown indefinitely under appropriate conditions whilst retaining the ability to differentiate to cells representative of the three primary germ layers. Such cells have the potential to revolutionize medicine by offering treatment options for a wide range of diseases and disorders as well as providing a model system for elucidating mechanisms involved in development and disease. In recent years,evidence for the function of E-cadherin in regulating pluripotent and self-renewal signaling pathways in ES and iPS cells has emerged. In this review,we discuss the function of E-cadherin and its interacting partners in the context of development and disease. We then describe relevant literature highlighting the function of E-cadherin in establishing and maintaining pluripotent and self-renewal properties of ES and iPS cells. In addition,we present experimental data demonstrating that exposure of human ES cells to the E-cadherin neutralizing antibody SHE78.7 allows culture of these cells in the absence of FGF2-supplemented medium. View Publication

BACKGROUND Anagrelide is a cytoreductive agent used to lower platelet counts in essential thrombocythemia. Although the drug has been known to selectively inhibit megakaryopoiesis for many years,the molecular mechanism accounting for this activity is still unclear. OBJECTIVES AND METHODS To address this issue we have compared the global gene expression profiles of human hematopoietic cells treated ex-vivo with and without anagrelide while growing under megakaryocyte differentiation conditions,using high-density oligonucleotide microarrays. Gene expression data were validated by the quantitative polymerase chain reaction and mined to identify functional subsets and regulatory pathways. RESULTS We identified 328 annotated genes differentially regulated by anagrelide,including many genes associated with platelet functions and with the control of gene transcription. Prominent among the latter was TRIB3,whose expression increased in the presence of anagrelide. Pathway analysis revealed that anagrelide up-regulated genes that are under the control of the transcription factor ATF4,a known TRIB3 inducer. Notably,immunoblot analysis demonstrated that anagrelide induced the phosphorylation of eIF2α,which is an upstream regulator of ATF4,and increased ATF4 protein levels. Furthermore,salubrinal,an inhibitor of eIF2α dephosphorylation,increased the expression of ATF4-regulated genes and blocked megakaryocyte growth. CONCLUSIONS These findings link signaling through eIF2α/ATF4 to the anti-megakaryopoietic activity of anagrelide and identify new potential modulators of megakaryopoiesis. View Publication

Cell-matrix interactions are paramount for the successful repair and regeneration of damaged and diseased tissue. Since many tissues have an anisotropic architecture,it has been proposed that aligned extracellular matrix (ECM) structures in particular could guide and support the differentiation of resident mesenchymal stem and progenitor cells (MSCs). We therefore created aligned collagen type I structures using a microfluidic set-up with the aim to assess their impact on MSC growth and differentiation. In addition,we refined our aligned collagen matrices by incorporating the glycosaminoglycan (GAG) heparin to demonstrate the versatility of the applied methodology to study multiple ECM components in a single system. Our reconstituted,aligned ECM structures maintained and allowed multilineage (osteogenic/adipogenic/chondrogenic) differentiation of MSCs. Most noticeable was the observation that during osteogenesis,aligned collagen substrates choreographed ordered matrix mineralization. Likewise,myotube assembly of C2C12 cells was profoundly influenced by aligned topographic features resulting in enhanced myotube organization and length. Our results shed light on the regulation of MSCs through directional ECM structures and demonstrate the versatility of these cell culture platforms for guiding the morphogenesis of tissue types with anisotropic structures. View Publication

A first step in hematopoiesis is the specification of the lymphoid and myeloid lineages from multipotent progenitor cells in the bone marrow. Using a conditional ablation strategy in adult mice,we show that this differentiation step requires Patched (Ptch),the cell surface-bound receptor for Hedgehog (Hh). In the absence of Ptch,the development of T- and B-lymphoid lineages is blocked at the level of the common lymphoid progenitor in the bone marrow. Consequently,the generation of peripheral T and B cells is abrogated. Cells of the myeloid lineage develop normally in Ptch mutant mice. Finally,adoptive transfer experiments identified the stromal cell compartment as a critical Ptch-dependent inducer of lymphoid versus myeloid lineage commitment. Our data show that Ptch acts as a master switch for proper diversification of hematopoietic stem cells in the adult organism. View Publication

The development and emergence of the hematopoietic stem cell involves a series of tightly regulated molecular events that are not well characterized. The hematopoietically expressed homeobox (Hhex) gene,a member of the homeobox gene family,is an essential regulator of embryogenesis and hematopoietic progenitor development. To investigate the role of Hhex in hematopoiesis we adapted a murine embryonic stem (ES) cell coculture system,in which ES cells can differentiate into CD41(+) and CD45(+) hematopoietic progenitors in vitro. Our results show that in addition to delayed hemangioblast development,Hhex(-/-) ES-derived progeny accumulate as CD41(+) and CD41(+)c-kit(+) cells,or the earliest definitive hematopoietic progenitors. In addition,Hhex(-/-) ES-derived progeny display a significantly reduced ability to develop into mature CD45(+) hematopoietic cells. The observed reduction in hematopoietic maturation was accompanied by reduced proliferation,because Hhex(-/-) CD41(+)CD45(-)c-kit(+) hematopoietic progenitors accumulated in the G(2) phase of the cell cycle. Thus,Hhex is a critical regulator of hematopoietic development and is necessary for the maturation and proliferation of the earliest definitive hematopoietic progenitors. View Publication

The molecular basis for the unique proliferative and self-renewal properties that hierarchically distinguish human stem cells from progenitors and terminally differentiated cells remains largely unknown. We report a role for the Bcl-2 family member myeloid cell leukemia-1 (Mcl-1) as an indispensable regulator of self-renewal in human stem cells and show that a functional dependence on Mcl-1 defines the human stem cell hierarchy. In vivo pharmacologic targeting of the Bcl-2 family members in human hematopoietic stem cells (HSCs) and human leukemic stem cells reduced stem cell regenerative and self-renewal function. Subsequent protein expression studies showed that,among the Bcl-2 family members,only Mcl-1 was up-regulated exclusively in the human HSC fraction on in vivo regeneration of hematopoiesis. Short hairpin RNA-knockdown of Mcl-1 in human cord blood cells did not affect survival in the HSC or hematopoietic progenitor cell fractions in vitro but specifically reduced the in vivo self-renewal function of human HSCs. Moreover,knockdown of Mcl-1 in ontogenetically primitive human pluripotent stem cells resulted in almost complete ablation of stem cell self-renewal function. Our findings show that Mcl-1 is an essential regulator of stem cell self-renewal in humans and therefore represents an axis for therapeutic interventions. View Publication

BACKGROUND: Mesenchymal stromal cells (MSC) have been proven to have potent immunosuppressive action and hence have been proposed for the treatment of severe Graft Versus Host Disease. However,in most models,MSC were added at the same time of lymphocyte stimulation,which is quite different from what occurs in vivo. AIMS: To investigate how the timing of lymphocyte activation and the exposure to activation-related cytokines (licensing) can influence the immunosuppressive action of Wharton's jelly stromal cells (WJSC). METHODS: WJSC,licensed or not with activation-related cytokines,were added lymphocytes the same time or 24 hours after their stimulation with phytohaemoagglutinin. Proliferation of lymphocytes and cytokines production was measured after three days co-culture. RESULTS: Lymphocytes stimulated in the presence of WJSC displayed a dramatic decrease in proliferation and production of cytokines,in spite of normal expression of activation markers. The suppression was weakened when targeted lymphocytes were seperated by a membrane and partially rescued by the addition of exogenous l-tryptophan,suggesting a major role for indoleamine 2,3-dioxigenase with a probable paracrine effect. Licensing of WJSC increased the immunosuppressive effect,in both contact and non-contact settings. The timing of WJSC licensing was crucial for the immunosuppressive action. Lymphocytes pre-stimulated alone for 24 h,and added afterwards to non-licensed WJSC,showed normal or even increased proliferation. On the other hand,their proliferation was strongly inhibited by licensed WJSC. CONCLUSIONS: WJSC have a potent immunosuppressive function best realized with direct contact,and increased by licensing signals before and during lymphocyte stimulation. Our results could contribute to the set up of new WJSC-based therapies for severe autoimmuno disorders. View Publication

Current EC differentiation protocols are inefficient,and the phenotypes of the differentiated ECs are only briefly stable,which significantly inhibits their utility for basic science research. Here,a remarkably more efficient hiPSC-EC differentiation protocol that incorporates a three-dimensional (3D) fibrin scaffold is presented. With this protocol,up to 45% of the differentiated hiPSCs assumed an EC phenotype,and after purification,greater than 95% of the cells displayed the EC phenotype (based on CD31 expression). The hiPSC-ECs continued to display EC characteristics for 4 weeks invitro. Gene and protein expression levels of CD31,CD144 and von Willebrand factor-8 (vWF-8) were significantly up-regulated in differentiated hiPSC-ECs. hiPSC-ECs also have biological function to up-take Dil-conjugated acetylated LDL (Dil-ac-LDL) and form tubular structures on Matrigel. Collectively,these data demonstrate that a 3D differentiation protocol can efficiently generate ECs from hiPSCs and,furthermore,the differentiated hiPSC-ECs are functional and can maintain EC fate up to 4 weeks invitro. ?? 2014 Elsevier Ltd. View Publication

More recently,reprogramming of somatic cells to an embryonic stem cell-like state presents a milestone in the realm of stem cells,making it possible to derive all cell types from any patients bearing specific genetic mutations. With the development of induced pluripotent stem (iPS) cells,we are now able to use the derivatives of iPS cells to study the mechanisms of disease and to perform drug screening and toxicology testing. In addition,differentiated iPS cells are now close to be used in clinical practice. Here we review the progress of iPS technique and the possible application in the area of Parkinson's disease treatment. View Publication

Although a large proportion of patients with polycythemia vera (PV) harbor a valine-to-phenylalanine mutation at amino acid 617 (V617F) in the JAK2 signaling molecule,the stage of hematopoiesis at which the mutation arises is unknown. Here we isolated and characterized hematopoietic stem cells (HSC) and myeloid progenitors from 16 PV patient samples and 14 normal individuals,testing whether the JAK2 mutation could be found at the level of stem or progenitor cells and whether the JAK2 V617F-positive cells had altered differentiation potential. In all PV samples analyzed,there were increased numbers of cells with a HSC phenotype (CD34+CD38-CD90+Lin-) compared with normal samples. Hematopoietic progenitor assays demonstrated that the differentiation potential of PV was already skewed toward the erythroid lineage at the HSC level. The JAK2 V617F mutation was detectable within HSC and their progeny in PV. Moreover,the aberrant erythroid potential of PV HSC was potently inhibited with a JAK2 inhibitor,AG490. View Publication

De novo DNA methylation is an essential aspect of the epigenetic reprogramming that takes place during early development,yet factors responsible for its instatement at particular genomic loci are poorly defined. Here,we demonstrate that the KRAB-ZFP-mediated recruitment of KAP1 to DNA in embryonic stem cells (ESCs) induces cytosine methylation. This process is preceded by H3K9 trimethylation,and genome-wide analyses reveal that it spreads over short distances from KAP1-binding sites so as to involve nearby CpG islands. In sharp contrast,in differentiated cells,KRAB/KAP1-induced heterochromatin formation does not lead to DNA methylation. Correspondingly,the methylation status of CpG islands in the adult mouse liver correlates with their proximity to KAP1-binding sites in ESCs,not in hepatocytes. Therefore,KRAB-ZFPs and their cofactor KAP1 are in part responsible for the establishment during early embryogenesis of site-specific DNA methylation patterns that are maintained through development View Publication

Human amniotic fluid cells (AFCs) are routinely obtained for prenatal diagnostics procedures. Recently,it has been illustrated that these cells may also serve as a valuable model system to study developmental processes and for application in regenerative therapies. Cellular reprogramming is a means of assigning greater value to primary AFCs by inducing self-renewal and pluripotency and,thus,bypassing senescence. Here,we report the generation and characterization of human amniotic fluid-derived induced pluripotent stem cells (AFiPSCs) and demonstrate their ability to differentiate into the trophoblast lineage after stimulation with BMP2/BMP4. We further carried out comparative transcriptome analyses of primary human AFCs,AFiPSCs,fibroblast-derived iPSCs (FiPSCs) and embryonic stem cells (ESCs). This revealed that the expression of key senescence-associated genes are down-regulated upon the induction of pluripotency in primary AFCs (AFiPSCs). By defining distinct and overlapping gene expression patterns and deriving the LARGE (Lost,Acquired and Retained Gene Expression) Principle of Cellular Reprogramming,we could further highlight that AFiPSCs,FiPSCs and ESCs share a core self-renewal gene regulatory network driven by OCT4,SOX2 and NANOG. Nevertheless,these cell types are marked by distinct gene expression signatures. For example,expression of the transcription factors,SIX6,EGR2,PKNOX2,HOXD4,HOXD10,DLX5 and RAXL1,known to regulate developmental processes,are retained in AFiPSCs and FiPSCs. Surprisingly,expression of the self-renewal-associated gene PRDM14 or the developmental processes-regulating genes WNT3A and GSC are restricted to ESCs. Implications of this,with respect to the stability of the undifferentiated state and long-term differentiation potential of iPSCs,warrant further studies. View Publication