技术资料

Induced pluripotent stem cell technology has attracted enormous interest for potential application in regenerative medicine. Here,we report that a specific glycogen synthase kinase 3 (GSK-3) inhibitor,CHIR99021,can induce the reprogramming of mouse embryonic fibroblasts transduced by only two factors,Oct4 and Klf4. When combined with Parnate (also named tranylcypromine),an inhibitor of lysine-specific demethylase 1,CHIR99021 can cause the reprogramming of human primary keratinocyte transduced with the two factors,Oct4 and Klf4. To our knowledge,this is the first time that human iPS cells have been generated from somatic cells without exogenous Sox2 expression. Our studies suggest that the GSK-3 inhibitor might have a general application to replace transcription factors in both mouse and human reprogramming. View Publication

The active form of vitamin D,1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)),has potent immunomodulatory properties that have promoted its potential use in the prevention and treatment of infectious disease and autoimmune conditions. A variety of immune cells,including macrophages,dendritic cells,and activated T cells express the intracellular vitamin D receptor and are responsive to 1,25(OH)(2)D(3.) Despite this,how 1,25(OH)(2)D(3) regulates adaptive immunity remains unclear and may involve both direct and indirect effects on the proliferation and function of T cells. To further clarify this issue,we have assessed the effects of 1,25(OH)(2)D(3) on human CD4(+)CD25(-) T cells. We observed that stimulation of CD4(+)CD25(-) T cells in the presence of 1,25(OH)(2)D(3) inhibited production of proinflammatory cytokines including IFN- gamma,IL-17,and IL-21 but did not substantially affect T cell division. In contrast to its inhibitory effects on inflammatory cytokines,1,25(OH)(2)D(3) stimulated expression of high levels of CTLA-4 as well as FoxP3,the latter requiring the presence of IL-2. T cells treated with 1,25(OH)(2)D(3) could suppress proliferation of normally responsive T cells,indicating that they possessed characteristics of adaptive regulatory T cells. Our results suggest that 1,25(OH)(2)D(3) and IL-2 have direct synergistic effects on activated T cells,acting as potent anti-inflammatory agents and physiologic inducers of adaptive regulatory T cells. View Publication

Chondrogenesis of mesenchymal stem cells (MSCs) is typically induced when they are condensed into a single aggregate and exposed to transforming growth factor-beta (TGF-beta). Hypoxia,like aggregation and TGF-beta delivery,may be crucial for complete chondrogenesis. However,the pellet dimensions and associated self-induced oxygen gradients of current chondrogenic methods may limit the effectiveness of in vitro differentiation and subsequent therapeutic uses. Here we describe the use of embryoid body-forming technology to produce microscopic aggregates of human bone marrow MSCs (BM-MSCs) for chondrogenesis. The use of micropellets reduces the formation of gradients within the aggregates,resulting in a more homogeneous and controlled microenvironment. These micropellet cultures (approximately 170 cells/micropellet) as well as conventional pellet cultures (approximately 2 x 10(5) cells/pellet) were chondrogenically induced under 20% and 2% oxygen environments for 14 days. Compared to conventional pellets under both environments,micropellets differentiated under 2% O(2) showed significantly increased sulfated glycosaminoglycan (sGAG) production and more homogeneous distribution of proteoglycans and collagen II. Aggrecan and collagen II gene expressions were increased in pellet cultures differentiated under 2% O(2) relative to 20% O(2) pellets but 2% O(2) micropellets showed even greater increases in these genes,as well as increased SOX9. These results suggest a more advanced stage of chondrogenesis in the micropellets accompanied by more homogeneous differentiation. Thus,we present a new method for enhancing MSC chondrogenesis that reveals a unique relationship between oxygen tension and aggregate size. The inherent advantages of chondrogenic micropellets over a single macroscopic aggregate should allow for easy integration with a variety of cartilage engineering strategies. View Publication

The epithelium of the mammary gland exists in a highly dynamic state,undergoing dramatic morphogenetic changes during puberty,pregnancy,lactation,and regression. The recent identification of stem and progenitor populations in mouse and human mammary tissue has provided evidence that the mammary epithelium is organized in a hierarchical manner. Characterization of these normal epithelial subtypes is an important step toward understanding which cells are predisposed to oncogenesis. This review summarizes progress in the field toward defining constituent cells and key molecular regulators of the mammary epithelial hierarchy. Potential relationships between normal epithelial populations and breast tumor subtypes are discussed,with implications for understanding the cellular etiology underpinning breast tumor heterogeneity. View Publication

There is no licensed vaccine against the intracellular pathogen Francisella tularensis. The use of conventional mouse strains to screen protective vaccine antigens may be problematic,given the differences in the major histocompatibility complex (MHC) binding properties between murine and human antigen-presenting cells. We used engineered humanized mice that lack endogenous MHC class II alleles but that express a human HLA allele (HLA-DR4 transgenic [tg] mice) to identify potential subunit vaccine candidates. Specifically,we applied a biochemical and immunological screening approach with bioinformatics to select putative F. tularensis subsp. novicida T-cell-reactive antigens using humanized HLA-DR4 tg mice. Cell wall- and membrane-associated proteins were extracted with Triton X-114 detergent and were separated by fractionation with a Rotofor apparatus and whole-gel elution. A series of proteins were identified from fractions that stimulated antigen-specific gamma interferon (IFN-gamma) production,and these were further downselected by the use of bioinformatics and HLA-DR4 binding algorithms. We further examined the validity of this combinatorial approach with one of the identified proteins,a 19-kDa Francisella tularensis outer membrane protein (designated Francisella outer membrane protein B [FopB]; FTN0119). FopB was shown to be a T-cell antigen by a specific IFN-gamma recall assay with purified CD4(+) T cells from F. tularensis subsp. novicida DeltaiglC-primed HLA-DR4 tg mice and cells of a human B-cell line expressing HLA-DR4 (DRB1*0401) functioning as antigen-presenting cells. Intranasal immunization of HLA-DR4 tg mice with the single antigen FopB conferred significant protection against lethal pulmonary challenge with an F. tularensis subsp. holarctica live vaccine strain. These results demonstrate the value of combining functional biochemical and immunological screening with humanized HLA-DR4 tg mice to map HLA-DR4-restricted Francisella CD4(+) T-cell epitopes. View Publication

Although Th17 cells play critical roles in the pathogenesis of many inflammatory and autoimmune diseases,their prevalence among tumor-infiltrating lymphocytes (TILs) and function in human tumor immunity remains largely unknown. We have recently demonstrated high percentages of Th17 cells in TILs from ovarian cancer patients,but the mechanisms of accumulation of these Th17 cells in the tumor microenvironment are still unclear. In this study,we further showed elevated Th17 cell populations in the TILs obtained from melanoma and breast and colon cancers,suggesting that development of tumor-infiltrating CD4(+) Th17 cells may be a general feature in cancer patients. We then demonstrated that tumor microenvironmental RANTES and MCP-1 secreted by tumor cells and tumor-derived fibroblasts mediate the recruitment of Th17 cells. In addition to their recruitment,we found that tumor cells and tumor-derived fibroblasts produce a proinflammatory cytokine milieu as well as provide cell-cell contact engagement that facilitates the generation and expansion of Th17 cells. We also showed that inflammatory TLR and nucleotide oligomerization binding domain 2 signaling promote the attraction and generation of Th17 cells induced by tumor cells and tumor-derived fibroblasts. These results identify Th17 cells as an important component of human TILs,demonstrate mechanisms involved in the recruitment and regulation of Th17 cells in tumor microenvironments,and provide new insights relevant for the development of novel cancer immunotherapeutic approaches. View Publication

The Tal1 transcription factor is essential for the development of the hematopoietic system and plays a role during definitive erythropoiesis in the adult. Despite the importance of Tal1 in erythropoiesis,only a small number of erythroid differentiation target genes are known. A chromatin precipitation and cloning approach was established to uncover novel Tal1 target genes in erythropoiesis. The BirA tag/BirA ligase biotinylation system in combination with streptavidin chromatin precipitation (Strep-CP) was used to co-precipitate genomic DNA bound to Tal1. Tal1 was found to bind in the vicinity of 31 genes including the E2-ubiquitin conjugase UBE2H gene. Binding of Tal1 to UBE2H was confirmed by chromatin immunoprecipitation. UBE2H expression is increased during erythroid differentiation of hCD34(+) cells. Tal1 expression activated UBE2H expression,whereas Tal1 knock-down reduced UBE2H expression and ubiquitin transfer activity. This study identifies parts of the ubiquitinylation machinery as a cellular target downstream of the transcription factor Tal1 and provides novel insights into Tal1-regulated erythropoiesis. View Publication

Slow-onset adaptive changes that arise from sustained antidepressant treatment,such as enhanced adult hippocampal neurogenesis and increased trophic factor expression,play a key role in the behavioral effects of antidepressants. alpha(2)-Adrenoceptors contribute to the modulation of mood and are potential targets for the development of faster acting antidepressants. We investigated the influence of alpha(2)-adrenoceptors on adult hippocampal neurogenesis. Our results indicate that alpha(2)-adrenoceptor agonists,clonidine and guanabenz,decrease adult hippocampal neurogenesis through a selective effect on the proliferation,but not the survival or differentiation,of progenitors. These effects persist in dopamine beta-hydroxylase knock-out (Dbh(-/-)) mice lacking norepinephrine,supporting a role for alpha(2)-heteroceptors on progenitor cells,rather than alpha(2)-autoreceptors on noradrenergic neurons that inhibit norepinephrine release. Adult hippocampal progenitors in vitro express all the alpha(2)-adrenoceptor subtypes,and decreased neurosphere frequency and BrdU incorporation indicate direct effects of alpha(2)-adrenoceptor stimulation on progenitors. Furthermore,coadministration of the alpha(2)-adrenoceptor antagonist yohimbine with the antidepressant imipramine significantly accelerates effects on hippocampal progenitor proliferation,the morphological maturation of newborn neurons,and the increase in expression of brain derived neurotrophic factor and vascular endothelial growth factor implicated in the neurogenic and behavioral effects of antidepressants. Finally,short-duration (7 d) yohimbine and imipramine treatment results in robust behavioral responses in the novelty suppressed feeding test,which normally requires 3 weeks of treatment with classical antidepressants. Our results demonstrate that alpha(2)-adrenoceptors,expressed by progenitor cells,decrease adult hippocampal neurogenesis,while their blockade speeds up antidepressant action,highlighting their importance as targets for faster acting antidepressants. View Publication

Lysine-specific demethylase 1 (LSD1) functions as a transcriptional coregulator by modulating histone methylation. Its role in neural stem cells has not been studied. We show here for the first time that LSD1 serves as a key regulator of neural stem cell proliferation. Inhibition of LSD1 activity or knockdown of LSD1 expression led to dramatically reduced neural stem cell proliferation. LSD1 is recruited by nuclear receptor TLX,an essential neural stem cell regulator,to the promoters of TLX target genes to repress the expression of these genes,which are known regulators of cell proliferation. The importance of LSD1 function in neural stem cells was further supported by the observation that intracranial viral transduction of the LSD1 small interfering RNA (siRNA) or intraperitoneal injection of the LSD1 inhibitors pargyline and tranylcypromine led to dramatically reduced neural progenitor proliferation in the hippocampal dentate gyri of wild-type adult mouse brains. However,knockout of TLX expression abolished the inhibitory effect of pargyline and tranylcypromine on neural progenitor proliferation,suggesting that TLX is critical for the LSD1 inhibitor effect. These findings revealed a novel role for LSD1 in neural stem cell proliferation and uncovered a mechanism for neural stem cell proliferation through recruitment of LSD1 to modulate TLX activity. View Publication

Transplantation studies have demonstrated the existence of mammary progenitor cells with the ability to self-renew and regenerate a functional mammary gland. Although these progenitors are the likely targets for oncogenic transformation,correlating progenitor populations with certain oncogenic stimuli has been difficult. Cyclin D1 is required for lobuloalveolar development during pregnancy and lactation as well as MMTV-ErbB2- but not MMTV-Wnt1-mediated tumorigenesis. Using a kinase-deficient cyclin D1 mouse,we identified two functional mammary progenitor cell populations,one of which is the target of MMTV-ErbB2. Moreover,cyclin D1 activity is required for the self-renewal and differentiation of mammary progenitors because its abrogation leads to a failure to maintain the mammary epithelial regenerative potential and also results in defects in luminal lineage differentiation. View Publication

BACKGROUND: Protein 4.2 deficiency caused by mutations in the EPB42 gene results in hereditary spherocytosis with characteristic alterations of CD47,CD44 and RhAG. We decided to investigate at which stage of erythropoiesis these hallmarks of protein 4.2 deficiency arise in a novel protein 4.2 patient and whether they cause disruption to the band 3 macrocomplex. DESIGN AND METHODS: We used immunoprecipitations and detergent extractability to assess the strength of protein associations within the band 3 macrocomplex and with the cytoskeleton in erythrocytes. Patient erythroblasts were cultured from peripheral blood mononuclear cells to study the effects of protein 4.2 deficiency during erythropoiesis. RESULTS: We report a patient with two novel mutations in EPB42 resulting in complete protein 4.2 deficiency. Immunoprecipitations revealed a weakened ankyrin-1-band 3 interaction in erythrocytes resulting in increased band 3 detergent extractability. CD44 abundance and its association with the cytoskeleton were increased. Erythroblast differentiation revealed that protein 4.2 and band 3 appear simultaneously and associate early in differentiation. Protein 4.2 deficiency results in lower CD47,higher CD44 expression and increased RhAG glycosylation starting from the basophilic stage. The normal downregulation of CD44 expression was not seen during protein 4.2(-) erythroblast differentiation. Knockdown of CD47 did not increase CD44 expression,arguing against a direct reciprocal relationship. CONCLUSIONS: We have established that the characteristic changes caused by protein 4.2 deficiency occur early during erythropoiesis. We postulate that weakening of the ankyrin-1-band 3 association during protein 4.2 deficiency is compensated,in part,by increased CD44-cytoskeleton binding. View Publication

Following activation through the TCR,CD4+ T cells can differentiate into three major subsets: Th1,Th2,and Th17 cells. IL-17-secreting Th17 cells play an important role in the pathogenesis of several autoimmune diseases and in immune responses to pathogens,but little is known about the regulation of apoptosis in Th17 cells. In this study,the sensitivity of in vitro-polarized Th1,Th2,and Th17 cells to Fas-mediated apoptosis was compared directly by different methods. The order of sensitivity of T cell subsets to Fas-mediated apoptosis is: Th1 textgreater Th17 textgreater Th2. The greater sensitivity of Th17 cells to Fas-mediated apoptosis compared with Th2 cells correlated with their higher expression of FasL and comparable expression of the antiapoptotic molecule FLIP. The decreased sensitivity of Th17 compared with Th1 cells correlated with the higher expression of FLIP by Th17 cells. Transgenic overexpression of FLIP in T cells protected all three subsets from Fas-mediated apoptosis. These findings provide new knowledge for understanding how survival of different subsets of T cells is regulated. View Publication