技术资料

The physiologic roles of angiopoietin-like proteins (Angptls) in the hematopoietic system remain unknown. Here we show that hematopoietic stem cells (HSCs) in Angptl3-null mice are decreased in number and quiescence. HSCs transplanted into Angptl3-null recipient mice exhibited impaired repopulation. Bone marrow sinusoidal endothelial cells express high levels of Angptl3 and are adjacent to HSCs. Importantly,bone marrow stromal cells or endothelium deficient in Angptl3 have a significantly decreased ability to support the expansion of repopulating HSCs. Angptl3 represses the expression of the transcription factor Ikaros,whose unregulated overexpression diminishes the repopulation activity of HSCs. Angptl3,as an extrinsic factor,thus supports the stemness of HSCs in the bone marrow niche. View Publication

Cell-based therapies against HIV/AIDS have been gaining increased interest. Natural killer (NK) cells are a key component of the innate immune system with the ability to kill diverse tumor cells and virus-infected cells. While NK cells have been shown to play an important role in the control of HIV-1 replication,their functional activities are often compromised in HIV-1-infected individuals. We have previously demonstrated the derivation of NK cells from human embryonic stem cells (hESCs) with the ability to potently kill multiple types of tumor cells both in vitro and in vivo. We now demonstrate the derivation of functional NK cells from human induced pluripotent stem cells (iPSCs). More importantly,both hESC- and iPSC-derived NK cells are able to inhibit HIV-1 NL4-3 infection of CEM-GFP cells. Additional studies using HIV-1-infected human primary CD4(+) T cells illustrated that hESC- and iPSC-derived NK cells suppress HIV-1 infection by at least three distinct cellular mechanisms: killing of infected targets through direct lysis,antibody-dependent cellular cytotoxicity,and production of chemokines and cytokines. Our results establish the potential to utilize hESC- and iPSC-derived NK cells to better understand anti-HIV-1 immunity and provide a novel cellular immunotherapeutic approach to treat HIV/AIDS. View Publication

CD4(+)Vβ5(+) peripheral T cells in C57BL/6 mice respond to encounter with a peripherally expressed endogenous superantigen by undergoing either deletion or TCR revision. In this latter process,cells lose surface Vβ5 expression and undergo RAG-dependent rearrangement of endogenous TCRβ genes,driving surface expression of novel TCRs. Although postrevision CD4(+)Vβ5(-)TCRβ(+) T cells accumulate with age in Vβ5 transgenic mice and bear a diverse TCR Vβ repertoire,it is unknown whether they respond to homeostatic and antigenic stimuli and thus may benefit the host. We demonstrate in this study that postrevision cells are functional. These cells have a high rate of steady-state homeostatic proliferation in situ,and they undergo extensive MHC class II-dependent lymphopenia-induced proliferation. Importantly,postrevision cells do not proliferate in response to the tolerizing superantigen,implicating TCR revision as a mechanism of tolerance induction and demonstrating that TCR-dependent activation of postrevision cells is not driven by the transgene-encoded receptor. Postrevision cells proliferate extensively to commensal bacterial Ags and can generate I-A(b)-restricted responses to Ag by producing IFN-γ following Listeria monocytogenes challenge. These data show that rescued postrevision T cells are responsive to homeostatic signals and recognize self- and foreign peptides in the context of self-MHC and are thus useful to the host. View Publication

Recent findings indicate that NK cells are involved in cardiac repair following myocardial infarction. The aim of this study is to investigate the role NK cells in infarct angiogenesis and cardiac remodeling. In normal C57BL/6 mice,myelomonocytic inflammatory cells invaded infarcted heart within 24 h followed by a lymphoid/NK cell infiltrate by day 6,accompanied by substantial expression of IL-2,TNF-α,and CCL2. In contrast,NOD SCID mice had virtually no lymphoid cells infiltrating the heart and did not upregulate IL-2 levels. In vitro and in vivo,IL-2-activated NK cells promoted TNF-α-stimulated endothelial cell proliferation,enhanced angiogenesis and reduced fibrosis within the infarcted myocardium. Adoptive transfer of IL-2-activated NK cells to NOD SCID mice improved post-myocardial infarction angiogenesis. RNA silencing technology and neutralizing Abs demonstrated that this process involved α4β7 integrin/VCAM-1 and killer cell lectin-like receptor 1/N-cadherin-specific binding. In this study,we show that IL-2-activated NK cells reduce myocardial collagen deposition along with an increase in neovascularization following acute cardiac ischemia through specific interaction with endothelial cells. These data define a potential role of activated NK cells in cardiac angiogenesis and open new perspectives for the treatment of ischemic diseases. View Publication

BACKGROUND: Induced pluripotent stem (iPS) cells have the capability to undergo self-renewal and differentiation into all somatic cell types. Since they can be produced through somatic cell reprogramming,which uses a defined set of transcription factors,iPS cells represent important sources of patient-specific cells for clinical applications. However,before these cells can be used in therapeutic designs,it is essential to understand their genetic stability. METHODOLOGY/PRINCIPAL FINDINGS: Here,we describe DNA damage responses in human iPS cells. We observe hypersensitivity to DNA damaging agents resulting in rapid induction of apoptosis after γ-irradiation. Expression of pluripotency factors does not appear to be diminished after irradiation in iPS cells. Following irradiation,iPS cells activate checkpoint signaling,evidenced by phosphorylation of ATM,NBS1,CHEK2,and TP53,localization of ATM to the double strand breaks (DSB),and localization of TP53 to the nucleus of NANOG-positive cells. We demonstrate that iPS cells temporary arrest cell cycle progression in the G(2) phase of the cell cycle,displaying a lack of the G(1)/S cell cycle arrest similar to human embryonic stem (ES) cells. Furthermore,both cell types remove DSB within six hours of γ-irradiation,form RAD51 foci and exhibit sister chromatid exchanges suggesting homologous recombination repair. Finally,we report elevated expression of genes involved in DNA damage signaling,checkpoint function,and repair of various types of DNA lesions in ES and iPS cells relative to their differentiated counterparts. CONCLUSIONS/SIGNIFICANCE: High degrees of similarity in DNA damage responses between ES and iPS cells were found. Even though reprogramming did not alter checkpoint signaling following DNA damage,dramatic changes in cell cycle structure,including a high percentage of cells in the S phase,increased radiosensitivity and loss of DNA damage-induced G(1)/S cell cycle arrest,were observed in stem cells generated by induced pluripotency. View Publication

Conventionally,researchers remove spontaneously differentiated areas in human pluripotent stem cell (hPSC) colonies by using a finely drawn glass pipette or a commercially available syringe needle. However,when extreme differentiation occurs,it is inefficient to purify the remaining undifferentiated cells,as these undifferentiated areas are too small to be isolated completely with the mechanical method. Antibodies can be utilized to purify the rare undifferentiated cells; however,this type of purification cannot be used in xeno-free culture systems. To avoid the loss of valuable hPSCs,we developed a novel method to isolate undifferentiated hPSCs from extremely differentiated colonies that could be easily adapted to xeno-free culture conditions. This protocol involves dissecting away differentiated areas,dissociating the remaining colony into clumps,seeding small clumps into new dishes,and picking undifferentiated colonies for expansion. Using this method,we routinely achieve completely undifferentiated colonies in one passage without the use of antibody-based purification. View Publication

Infection with human immunodeficiency virus (HIV)-1 induces a progressive deterioration of the immune system that ultimately leads to acquired immune deficiency syndrome (AIDS). Murine models indicate that the common γ-chain (γ(c))-sharing cytokine interleukin (IL)-21 and its receptor (IL-21R) play a crucial role in maintaining polyfunctional T cell responses during chronic viral infections. Therefore,we analyzed the ability of this cytokine to modulate the properties of human CD8 T cells in comparison with other γ(c)-sharing cytokines (IL-2,IL-7,and IL-15). CD8 T cells from healthy volunteers were stimulated in vitro via T cell receptor signals to mimic the heightened status of immune activation of HIV-infected patients. The administration of IL-21 upregulated cytotoxic effector function and the expression of the costimulatory molecule CD28. Notably,this outcome was not accompanied by increased cellular proliferation or activation. Moreover,IL-21 promoted antiviral activity while not inducing HIV-1 replication in vitro. Thus,IL-21 may be a favorable molecule for immunotherapy and a suitable vaccine adjuvant in HIV-infected individuals. View Publication

Neuronal precursor cells (NPCs) are temporally regulated and have the ability to proliferate and differentiate into mature neurons,oligodendrocytes,and astrocytes in the presence of growth factors (GFs). In the present study,the role of the Jak pathway in brain development was investigated in NPCs derived from neurosphere cultures using Jak2 and Jak3 small interfering RNAs and specific inhibitors. Jak2 inhibition profoundly decreased NPC proliferation,preventing further differentiation into neurons and glial cells. However,Jak3 inhibition induced neuronal differentiation accompanied by neurite growth. This phenomenon was due to the Jak3 inhibition-mediated induction of neurogenin (Ngn)2 and NeuroD in NPCs. Jak3 inhibition induced NPCs to differentiate into scattered neurons and increased the expression of Tuj1,microtubule associated protein 2 (MAP2),Olig2,and neuroglial protein (NG)2,but decreased glial fibrillary acidic protein (GFAP) expression,with predominant neurogenesis/polydendrogenesis compared with astrogliogenesis. Therefore,Jak2 may be important for NPC proliferation and maintenance,whereas knocking-down of Jak3 signaling is essential for NPC differentiation into neurons and oligodendrocytes but does not lead to astrocyte differentiation. These results suggest that NPC proliferation and differentiation are differentially regulated by the Jak pathway. View Publication

EBV infects most of the human population and is associated with a number of human diseases including cancers. Moreover,evasion of the immune system and chronic infection is an essential step for EBV-associated diseases. In this paper,we show that EBV can alter the regulation and expression of TLRs,the key effector molecules of the innate immune response. EBV infection of human primary B cells resulted in the inhibition of TLR9 functionality. Stimulation of TLR9 on primary B cells led to the production of IL-6,TNF-α,and IgG,which was inhibited in cells infected with EBV. The virus exerts its inhibitory function by decreasing TLR9 mRNA and protein levels. This event was observed at early time points after EBV infection of primary cells,as well as in an immortalized lymphoblastoid cell line. We determined that the EBV oncoprotein latent membrane protein 1 (LMP1) is a strong inhibitor of TLR9 transcription. Overexpression of LMP1 in B cells reduced TLR9 promoter activity,mRNA,and protein levels. LMP1 mutants altered in activating the NF-κB pathway prevented TLR9 promoter deregulation. Blocking the NF-κB pathway recovered TLR9 promoter activity. Mutating the NF-κB cis element on the TLR9 promoter restored luciferase transcription in the presence of LMP1. Finally,deletion of the LMP1 gene in the EBV genome abolished the ability of the virus to induce TLR9 downregulation. Our study describes a mechanism used by EBV to suppress the host immune response by deregulating the TLR9 transcript through LMP1-mediated NF-κB activation. View Publication

The longevity of organisms is maintained by stem cells. If an organism loses the ability to maintain a balance between quiescence and differentiation in the stem/progenitor cell compartment due to aging and/or stress,this may result in death or age-associated diseases,including cancer. Ewing sarcoma is the most lethal bone tumor in young patients and arises from primitive stem cells. Here,we demonstrated that endogenous Ewing sarcoma gene (Ews) is indispensable for stem cell quiescence,and that the ablation of Ews promotes the early onset of senescence in hematopoietic stem progenitor cells. The phenotypic and functional changes in Ews-deficient stem cells were accompanied by an increase in senescence-associated β-galactosidase staining and a marked induction of p16(INK4a) compared with wild-type counterparts. With its relevance to cancer and possibly aging,EWS is likely to play a significant role in maintaining the functional capacity of stem cells and may provide further insight into the complexity of Ewing sarcoma in the context of stem cells. View Publication

Information on natural killer (NK)-cell receptor-ligand interactions involved in the response to human cytomegalovirus (HCMV) is limited and essentially based on the study of infected fibroblasts. Experimental conditions were set up to characterize the NK response to HCMV-infected myeloid dendritic cells (DCs). Monocyte-derived DCs (moDCs) infected by the TB40/E HCMV strain down-regulated the expression of human leukocyte antigen class I molecules and specifically activated autologous NK-cell populations. NKG2D ligands appeared virtually undetectable in infected moDCs,reflecting the efficiency of immune evasion mechanisms,and explained the lack of antagonistic effects of NKG2D-specific monoclonal antibody. By contrast,DNAM-1 and DNAM-1 ligands (DNAM-1L)-specific monoclonal antibodies inhibited the NK response at 48 hours after infection,although the impact of HCMV-dependent down-regulation of DNAM-1L in infected moDCs was perceived at later stages. moDCs constitutively expressed ligands for NKp46 and NKp30 natural cytotoxicity receptors,which were partially reduced on HCMV infection; yet,only NKp46 appeared involved in the NK response. In contrast to previous reports in fibroblasts,human leukocyte antigen-E expression was not preserved in HCMV-infected moDCs,which triggered CD94/NKG2A(+) NK-cell activation. The results provide an insight on key receptor-ligand interactions involved in the NK-cell response against HCMV-infected moDCs,stressing the importance of the dynamics of viral immune evasion mechanisms. View Publication

Mesenchymal stem cells (MSCs) are fibroblast-like multipotent stem cells that can differentiate into cell types of mesenchymal origin. Because of their immune properties and differentiation,potential MSCs are discussed for the use in tissue regeneration and tolerance induction in transplant medicine. This cell type can easily be obtained from the umbilical cord tissue (UCMSC) without medical intervention. Standard culture conditions include fetal bovine serum (FBS) which may not be approved for clinical settings. Here,we analyzed the phenotypic and functional properties of UCMSC under xeno-free (XF,containing GMP-certified human serum) and serum-free (SF) culture conditions in comparison with standard UCMSC cultures. Phenotypically,UCMSC showed no differences in the expression of mesenchymal markers or differentiation capacity. Functionally,XF and SF-cultured UCMSC have comparable adipogenic,osteogenic,and endothelial differentiation potential. Interestingly,the UCMSC-mediated suppression of T cell proliferation in an allogeneic mixed lymphocyte reaction (MLR) is more effective in XF and SF media than in standard FBS-containing cultures. Regarding the mechanism of action of MLR suppression,transwell experiments revealed that in neither UCMSC culture a direct cell-cell contact is necessary for inhibiting T cell proliferation,and that the major effector molecule is prostaglandin E₂ (PGE₂). Taken together,GMP-compliant growth media qualify for long-term cultures of UCMSC which is important for a future clinical study design in regenerative and transplant medicine. View Publication