技术资料

To better understand endogenous parameters that influence pluripotent cell differentiation we used human embryonic stem cells (hESCs) as a model system. We demonstrate that differentiation trajectories in aggregate (embryoid body [EB])-induced differentiation,a common approach to mimic some of the spatial and temporal aspects of in vivo development,are affected by three factors: input hESC composition,input hESC colony size,and EB size. Using a microcontact printing approach,size-specified hESC colonies were formed by plating single-cell suspensions onto micropatterned (MP) extracellular matrix islands. Subsequently,size-controlled EBs were formed by transferring entire colonies into suspension culture enabling the independent investigation of colony and aggregate size effects on differentiation induction. Gene and protein expression analysis of MP-hESC populations revealed that the ratio of Gata6 (endoderm-associated marker) to Pax6 (neural-associated marker) expression increased with decreasing colony size. Moreover,upon forming EBs from these MP-hESCs,we observed that differentiation trajectories were affected by both colony and EB size-influenced parameters. In MP-EBs generated from endoderm-biased (high Gata6/Pax6) input hESCs,higher mesoderm and cardiac induction was observed at larger EB sizes. Conversely,neural-biased (low Gata6/Pax6) input hESCs generated MP-EBs that exhibited higher cardiac induction in smaller EBs. Our analysis demonstrates that heterogeneity in hESC colony and aggregate size,typical in most differentiation strategies,produces subsets of appropriate conditions for differentiation into specific cell types. Moreover,our findings suggest that the local microenvironment modulates endogenous parameters that can be used to influence pluripotent cell differentiation trajectories. View Publication

Our recent studies have shown that immune cell-produced complement provides costimulatory and survival signals to naive CD4(+) T cells. Whether these signals are similarly required during effector cell expansion and what molecular pathways link locally produced complement to T-cell survival were not clarified. To address this,we stimulated monoclonal and polyclonal T cells in vitro and in vivo with antigen-presenting cells (APCs) deficient in the complement regulatory protein,decay accelerating factor (DAF),and/or the complement component C3. We found that T-cell expansion induced by DAF-deficient APCs was augmented with diminished T-cell apoptosis,whereas T-cell expansion induced by C3(-/-) APCs was reduced because of enhanced T-cell apoptosis. These effects were traced to locally produced C5a,which through binding to T cell-expressed C5aR,enhanced expression of Bcl-2 and prevented Fas up-regulation. The results show that C5aR signal transduction in T cells is important to allow optimal T-cell expansion,as well as to maintain naive cell viability,and does so by suppressing programmed cell death. View Publication

Experimental autoimmune encephalomyelitis (EAE) models,in animals,many characteristics of multiple sclerosis,for which there is no adequate therapy. We investigated whether lithium,an inhibitor of glycogen synthase kinase-3 (GSK3),can ameliorate EAE in mice. Pretreatment with lithium markedly suppressed the clinical symptoms of EAE induced in mice by myelin oligodendrocyte glycoprotein peptide (MOG35-55) immunization and greatly reduced demyelination,microglia activation,and leukocyte infiltration in the spinal cord. Lithium administered postimmunization,after disease onset,reduced disease severity and facilitated partial recovery. Conversely,in knock-in mice expressing constitutively active GSK3,EAE developed more rapidly and was more severe. In vivo lithium therapy suppressed MOG35-55-reactive effector T cell differentiation,greatly reducing in vitro MOG35-55- stimulated proliferation of mononuclear cells from draining lymph nodes and spleens,and MOG35-55-induced IFN-gamma,IL-6,and IL-17 production by splenocytes isolated from MOG35-55-immunized mice. In relapsing/remitting EAE induced with proteolipid protein peptide139-151,lithium administered after the first clinical episode maintained long-term (90 days after immunization) protection,and after lithium withdrawal the disease rapidly relapsed. These results demonstrate that lithium suppresses EAE and identify GSK3 as a new target for inhibition that may be useful for therapeutic intervention of multiple sclerosis and other autoimmune and inflammatory diseases afflicting the CNS. View Publication

Multiple myeloma is characterized by the clonal expansion of neoplastic plasma cells within the bone marrow,elevated serum immunoglobulin,and osteolytic bone disease. The disease is highly responsive to a wide variety of anticancer treatments including conventional cytotoxic chemotherapy,corticosteroids,radiation therapy,and a growing number of agents with novel mechanisms of action. However,few if any patients are cured with these modalities and relapse remains a critical issue. A better understanding of clonogenic multiple myeloma cells is essential to ultimately improving long-term outcomes,but the nature of the cells responsible for myeloma regrowth and disease relapse is unclear. We review evidence that functional heterogeneity exists in multiple myeloma and discuss potential strategies and clinical implications of the stem-cell model of cancer in this disease. View Publication

Neuropilin-1 (Nrp1) is a multifunctional protein,identified principally as a receptor for the class 3 semaphorins and members of the vascular endothelial growth factor (VEGF) family,but it is capable of other interactions. It is a marker of regulatory T cells (Tr),which often carry Nrp1 and latency-associated peptide (LAP)-TGF-beta1 (the latent form). The signaling TGF-beta1 receptors bind only active TGF-beta1,and we hypothesized that Nrp1 binds the latent form. Indeed,we found that Nrp1 is a high-affinity receptor for latent and active TGF-beta1. Free LAP,LAP-TGF-beta1,and active TGF-beta1 all competed with VEGF165 for binding to Nrp1. LAP has a basic,arginine-rich C-terminal motif similar to VEGF and peptides that bind to the b1 domain of Nrp1. A C-terminal LAP peptide (QSSRHRR) bound to Nrp1 and inhibited the binding of VEGF and LAP-TGF-beta1. We also analyzed the effects of Nrp1/LAP-TGF-beta1 coexpression on T cell function. Compared with Nrp1(-) cells,sorted Nrp1+ T cells had a much greater capacity to capture LAP-TGF-beta1. Sorted Nrp1(-) T cells captured soluble Nrp1-Fc,and this increased their ability to capture LAP-TGF-beta1. Conventional CD4+CD25(-)Nrp1(-) T cells coated with Nrp1-Fc/LAP-TGF-beta1 acquired strong Tr activity. Moreover,LAP-TGF-beta was activated by Nrp1-Fc and also by a peptide of the b2 domain of Nrp1 (RKFK; similar to a thrombospondin-1 peptide). Breast cancer cells,which express Nrp1,also captured and activated LAP-TGF-beta1 in a Nrp1-dependent manner. Thus,Nrp1 is a receptor for TGF-beta1,activates its latent form,and is relevant to Tr activity and tumor biology. View Publication

Anaplastic thyroid cancers (ATC) are aggressive tumors,which exhibit cell cycle misregulations leading to uncontrolled cellular proliferation and genomic instability. They fail to respond to chemotherapeutic agents and radiation therapy,and most patients die within a few months of diagnosis. In the present study,we evaluated the in vitro effects on ATC cells of VX-680,an inhibitor of the Aurora serine/threonine kinases involved in the regulation of multiple aspects of chromosome segregation and cytokinesis. The effects of VX-680 on proliferation,apoptosis,soft agar colony formation,cell cycle,and ploidy were tested on the ATC-derived cell lines CAL-62,8305C,8505C,and BHT-101. Treatment of the different ATC cells with VX-680 inhibited proliferation in a time- and dose-dependent manner,with the IC50 between 25 and 150 nM. The VX-680 significantly impaired the ability of the different cell lines to form colonies in soft agar. Analysis of caspase-3 activity showed that VX-680 induced apoptosis in the different cell lines. CAL-62 cells exposed for 12 h to VX-680 showed an accumulation of cells with textgreater or =4N DNA content. Time-lapse analysis demonstrated that VX-680-treated CAL-62 cells exit metaphase without dividing. Moreover,histone H3 phosphorylation was abrogated following VX-680 treatment. In conclusion,our data demonstrated that VX-680 is effective in reducing cell growth of different ATC-derived cell lines and warrant further investigation to exploit its potential therapeutic value for ATC treatment. View Publication

The clinical success of stem cell therapy for myocardial repair hinges on a better understanding of cardiac fate mechanisms. We have identified small molecules involved in cardiac fate by screening a chemical library for activators of the signature gene Nkx2.5,using a luciferase knockin bacterial artificial chromosome (BAC) in mouse P19CL6 pluripotent stem cells. We describe a family of sulfonyl-hydrazone (Shz) small molecules that can trigger cardiac mRNA and protein expression in a variety of embryonic and adult stem/progenitor cells,including human mobilized peripheral blood mononuclear cells (M-PBMCs). Small-molecule-enhanced M-PBMCs engrafted into the rat heart in proximity to an experimental injury improved cardiac function better than control cells. Recovery of cardiac function correlated with persistence of viable human cells,expressing human-specific cardiac mRNAs and proteins. Shz small molecules are promising starting points for drugs to promote myocardial repair/regeneration by activating cardiac differentiation in M-PBMCs. View Publication

BCR/ABL kinase-positive chronic myelogenous leukemia (CML) cells display genomic instability leading to point mutations in various genes including bcr/abl and p53,eventually causing resistance to imatinib and malignant progression of the disease. Mismatch repair (MMR) is responsible for detecting misincorporated nucleotides,resulting in excision repair before point mutations occur and/or induction of apoptosis to avoid propagation of cells carrying excessive DNA lesions. To assess MMR activity in CML,we used an in vivo assay using the plasmid substrate containing enhanced green fluorescent protein (EGFP) gene corrupted by T:G mismatch in the start codon; therefore,MMR restores EGFP expression. The efficacy of MMR was reduced approximately 2-fold in BCR/ABL-positive cell lines and CD34(+) CML cells compared with normal counterparts. MMR was also challenged by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG),which generates O(6)-methylguanine and O(4)-methylthymine recognized by MMR system. Impaired MMR activity in leukemia cells was associated with better survival,accumulation of p53 but not of p73,and lack of activation of caspase 3 after MNNG treatment. In contrast,parental cells displayed accumulation of p53,p73,and activation of caspase 3,resulting in cell death. Ouabain-resistance test detecting mutations in the Na(+)/K(+) ATPase was used to investigate the effect of BCR/ABL kinase-mediated inhibition of MMR on mutagenesis. BCR/ABL-positive cells surviving the treatment with MNNG displayed approximately 15-fold higher mutation frequency than parental counterparts and predominantly G:C--textgreaterA:T and A:T--textgreaterG:C mutator phenotype typical for MNNG-induced unrepaired lesions. In conclusion,these results suggest that BCR/ABL kinase abrogates MMR activity to inhibit apoptosis and induce mutator phenotype. View Publication

Expansion of the CGG.CCG-repeat tract in the 5' UTR of the FMR1 gene to textgreater200 repeats leads to heterochromatinization of the promoter and gene silencing. This results in Fragile X syndrome (FXS),the most common heritable form of mental retardation. The mechanism of gene silencing is unknown. We report here that a Class III histone deacetylase,SIRT1,plays an important role in this silencing process and show that the inhibition of this enzyme produces significant gene reactivation. This contrasts with the much smaller effect of inhibitors like trichostatin A (TSA) that inhibit Class I,II and IV histone deacetylases. Reactivation of silenced FMR1 alleles was accompanied by an increase in histone H3 lysine 9 acetylation as well as an increase in the amount of histone H4 that is acetylated at lysine 16 (H4K16) by the histone acetyltransferase,hMOF. DNA methylation,on the other hand,is unaffected. We also demonstrate that deacetylation of H4K16 is a key downstream consequence of DNA methylation. However,since DNA methylation inhibitors require DNA replication in order to be effective,SIRT1 inhibitors may be more useful for FMR1 gene reactivation in post-mitotic cells like neurons where the effect of the gene silencing is most obvious. View Publication

The number of colony forming unit-endothelial cells (CFU-EC) in human peripheral blood was found to be a biological marker for several vascular diseases. In this study,the heterogeneous composition of immune cells in the CFU-ECs was investigated. We confirmed that monocytes are essential for the formation of CFU-ECs. Also,however,CD4(+) T cells were found to be indispensable for the induction of CFU-EC colonies,mainly through cell-cell contact. By blocking or activating CD3 receptors on CD4(+) T cells or blocking MHC class II molecules on monocytes,it was shown that TCR-MHCII interactions are required for induction of CFU-EC colonies. Because the supernatant from preactivated T cells could also induce colony formation from purified monocytes,the T cell support turned out to be cytokine mediated. Gene expression analysis of the endothelial-like colonies formed by CD14(+) cells showed that colony formation is a proangiogenic differentiation and might reflect the ability of monocytes to facilitate vascularization. This in vitro study is the first to reveal the role of TCR-MHC class II interactions between T cells and monocytes and the subsequent inflammatory response as stimulus of monocytic properties that are associated with vascularization. View Publication

Anacardic acid (6-pentadecylsalicylic acid) is derived from traditional medicinal plants,such as cashew nuts,and has been linked to anticancer,anti-inflammatory,and radiosensitization activities through a mechanism that is not yet fully understood. Because of the role of nuclear factor-kappaB (NF-kappaB) activation in these cellular responses,we postulated that anacardic acid might interfere with this pathway. We found that this salicylic acid potentiated the apoptosis induced by cytokine and chemotherapeutic agents,which correlated with the down-regulation of various gene products that mediate proliferation (cyclin D1 and cyclooxygenase-2),survival (Bcl-2,Bcl-xL,cFLIP,cIAP-1,and survivin),invasion (matrix metalloproteinase-9 and intercellular adhesion molecule-1),and angiogenesis (vascular endothelial growth factor),all known to be regulated by the NF-kappaB. We found that anacardic acid inhibited both inducible and constitutive NF-kappaB activation; suppressed the activation of IkappaBalpha kinase that led to abrogation of phosphorylation and degradation of IkappaBalpha; inhibited acetylation and nuclear translocation of p65; and suppressed NF-kappaB-dependent reporter gene expression. Down-regulation of the p300 histone acetyltransferase gene by RNA interference abrogated the effect of anacardic acid on NF-kappaB suppression,suggesting the critical role of this enzyme. Overall,our results demonstrate a novel role for anacardic acid in potentially preventing or treating cancer through modulation of NF-kappaB signaling pathway. View Publication

Chronic graft-versus-host disease (cGVHD) is a common late complication of allogeneic stem cell transplantation (allo-SCT). Some cGVHD patients develop skin lesions,and the skin lesions in sclerodermatous cGVHD (s-cGVHD) patients resemble those in progressive systemic sclerosis (PSS),which is characterized by impaired production of circulating endothelial progenitor cells (EPCs). We investigated,retrospectively,whether low EPC production may promote the development of sclerodermatous lesions in cGVHD. Peripheral blood (PB) was obtained from 14 healthy volunteers and 27 allo-SCT patients. Five patients developed s-cGVHD. CD34(+) cells were purified by using the magnetic cell-sorting separation system,and the CD34(+)/CD133(+)/vascular endothelial growth factor (VEGF) receptor-2(+) EPCs were quantified. The endothelial cell colony-formation potential was evaluated. Serum VEGF and basic fibroblast growth factor (b-FGF) concentrations were measured by ELISA. The s-cGVHD patients had significantly lower median circulating EPCs frequencies than non-s-cGVHD patients or control (145 of 20 mL [interquartial range-IQR 107-193] versus 1083.5 [IQR 669.3-2151]; P = .0023,and versus 1530.5 [IQR 961.3-2158]; P = .0012,respectively). They also had impaired median endothelial-forming ability compared to non-s-cGVHD patients or controls (3.8 [IQR 1.0-4.3] versus 12.8 [IQR 8.8-28.8],and versus 26.4 [IQR 23.6-30.6],respectively; P = .0012). Their VEGF and b-FGF serum levels were also higher than in controls. In conclusion,s-cGVHD patients show findings consistent with those seen in PSS with impaired vasculogenesis that may limit blood perfusion and may contribute to the development of sclerodermatous lesions. View Publication