技术资料

Although activated inflammatory monocytes (IMCs) and inflammatory dendritic cells (IDCs) are potent T cell suppressors,nonactivated IMCs and IDCs promote T cell activation and Th1/Th17 cell differentiation. In this study,we investigated how to reduce the proinflammatory properties of IMCs and IDCs and further convert them into immune regulatory dendritic cells (DCs). We found that IL-4 and retinoic acid (RA) cotreatment of GM-CSF-differentiated IDCs synergistically induced the expression of aldehyde dehydrogenase family 1,subfamily A2,a rate-limiting enzyme for RA synthesis in DCs. IL-4 plus RA-treated IDCs upregulated CD103 expression and markedly reduced the production of proinflammatory cytokines upon activation. IL-4 plus RA-treated IDCs strongly induced CD4�?�Foxp3�?� regulatory T cell differentiation and suppressed Th1 and Th17 differentiation. Mechanistically,the transcription factors Stat6 and RA receptor $$ play important roles in aldehyde dehydrogenase family 1,subfamily A2,induction. In addition,IL-4 and RA signaling pathways interact closely to enhance the regulatory function of treated DCs. Adoptive transfer of IL-4 plus RA-treated DCs significantly increased regulatory T cell frequency in vivo. Direct treatment with IL-4 and RA also markedly suppressed actively induced experimental autoimmune encephalomyelitis. Our data demonstrate the synergistic effect of IL-4 and RA in inducing a regulatory phenotype in IDCs,providing a potential treatment strategy for autoimmune diseases. View Publication

Cancer stem cells (CSCs) are a small subset of cancer cells with indefinite potential for self-renewal and the capacity to drive tumorigenesis. Brefeldin A (BFA) is an antibiotic that is known to block protein transport and induce endoplasmic reticulum (ER) stress in eukaryotic cells,but its effects on colorectal CSCs are unknown. We investigated the inhibitory effect of BFA on human colorectal cancer Colo 205 cells. We found that BFA effectively reduced the survival of suspension Colo 205 cells (IC₅₀ = ˜15 ng/mL) by inducing apoptosis,and inhibited the clonogenic activity of Colo 205 CSCs in tumorsphere formation assay and soft agar colony formation assay in the same nanogram per milliliter range. We also discovered that at such low concentrations,BFA effectively induced endoplasmic reticulum (ER) stress response as indicated by the increased mRNA expression of ER stress-related genes,such as glucose-regulated protein 78 (GRP78),X-box binding protein 1 (XBP1),and C/EBP homologous protein (CHOP). Finally,we found that BFA reduced the activity of matrix metallopeptidase 9 (MMP-9). These findings suggest that BFA can effectively suppress the progression of colorectal cancer during the tumorigenesis and metastasis stages. These results may lead to the development of novel therapies for the treatment of colorectal cancer. View Publication

Although BMP9 is highly capable of promoting osteogenic differentiation of mesenchymal stem cell (MSCs),the molecular mechanism involved remains to be fully elucidated. Here,we explore the possible involvement and detail role of JNKs (c-Jun N-terminal kinases) in BMP9-induced osteogenic differentiation of MSCs. It was found that BMP9 stimulated the activation of JNKs in MSCs. BMP9-induced osteogenic differentiation of MSCs was dramatically inhibited by JNKs inhibitor SP600125. Moreover,BMP9-activated Smads signaling was decreased by SP600125 treatment in MSCs. The effects of inhibitor are reproduced with adenoviruses expressing siRNA targeted JNKs. Taken together,our results revealed that JNKs was activated in BMP9-induced osteogenic differentiation of MSCs. What is most noteworthy,however,is that inhibition of JNKs activity resulted in reduction of BMP9-induced osteogenic differentiation of MSCs,implying that activation of JNKs is essential for BMP9 osteoinductive activity. View Publication

We have examined a number of reagents for their ability to modulate activity of the Hh signaling pathway during embryonic development of Xenopus. In particular we have focused on regulation of events occurring during tailbud stages and later. Two inducible protein reagents based on the Gli1 and Gli3 transcription factors were generated and the activity of these proteins was compared to the Hh signaling pathway inhibitor,cyclopamine,and the activators,Smoothened agonist (SAG) and purmorphamine (PMA). Effectiveness of reagents was assayed using both molecular biological techniques and biological readouts. We found that the small molecule modulators of the Hh pathway were highly specific and effective and produced results generally superior to the more conventional protein reagents for examination of later stage developmental processes. View Publication

BACKGROUND/AIMS [corrected] Embryonic stem cells (ES cells) have the capacity to propagate indefinitely,maintain pluripotency,and differentiate into any cell type under defined conditions. As a result,they are considered to be the best model system for research into early embryonic development. AICA ribonucleotide (AICAR) is an activator of AMP-activated protein kinase (AMPK) that is thought to affect ES cell function,but its role in ES cell fate decision is unclear. METHODS In this study,we performed microarray analysis to investigate AICAR downstream targets and further understand its effect on ES cells. RESULTS Our microarray data demonstrated that AICAR can significantly up-regulate pluripotency-associated genes and down-regulate differentiation-associated transcription factors. Although AICAR cannot maintain ES cell identity without LIF,it can antagonize the action of RA-induced differentiation. Using those differentially expressed genes identified,we performed gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis with the Database for Annotation,Visualization and Integrated Discovery (DAVID) online system. AICAR was not only shown to influence the AMPK pathway,but also act on other signaling pathways such as BMP,MAPK and TGF-β,to maintain the stemness of J1 ES cells. Furthermore,AICAR modulated ES cell epigenetic modification by altering the expression of epigenetic-associated proteins,including Dnmt3a,Dnmt3b,Smarca2,Mbd3,and Arid1a,or through regulating the transcription of long intervening non-coding RNA (lincRNA). CONCLUSION Taken together,our work suggests that AICAR is capable of maintaining ES cell self-renewal and pluripotency,which could be useful in future medical treatment. View Publication

BACKGROUND To investigate the expression status of PIWIL2 and piR-932 in breast cancer stem cells and the role they could play in tumor cell growth and metastasis through Latexin. METHODS CD44(+)/CD24(-) tumor cells (CSC) from clinical specimens were sorted using flow cytometry. PIWIL2 expression status was detected in CSC cells by microarray analysis and 1086 breast cancer specimens by Western blot and immunohistochemistry staining. piR-932 expression was also detected in CSC cells by piRNA microarray assay. The relationship between the PIWIL2 protein and clinico-pathological parameters and prognosis was subsequently determined. RESULTS CSC cells are more likely to generate new tumors in mice and cell microspheres that are deficient in NOD/SCID compared to the control group. PIWIL2 protein was expressed higher in CSC cells compared to the control cells. In total,334 (30.76%) of the 1086 breast cases showed high PIWIL2 expression. PIWIL2 was observed to be related to age,tumor size,histological type,tumor stage,and lymph node metastasis (all P textless 0.05). Furthermore,we have found that one of the Piwi-interacting RNAs (piRNAs) called piR-932 expressed significantly higher in the breast cancer cells that were induced to EMT,and it could form immune complexes through immunoprecipitation with PIWIL2; in PIWIL2+ breast cancer stem cells,Latexin expression significantly reduced because of its promoter region CpG island methylation. CONCLUSIONS These results suggest that the combination of piR-932 and PIWIL2 may be a positive regulator in the process of breast cancer stem cells through promoting the methylation of Latexin,and they both could be the potential targets for blocking the metastasis of breast cancer. View Publication

Normal stem cells and cancer stem cells (CSCs) share similar properties,in that both have the capacity to self-renew and differentiate into multiple cell types. In both the normal stem cell and cancer stem cell fields,there has been a great need for a universal marker that can effectively identify and isolate these rare populations of cells in order to characterize them and use this information for research and therapeutic purposes. Currently,it would appear that certain isoenzymes of the aldehyde dehydrogenase (ALDH) superfamily may be able to fulfill this role as a marker for both normal and cancer stem cells. ALDH has been identified as an important enzyme in the protection of normal hematopoietic stem cells,and is now also widely used as a marker to identify and isolate various types of normal stem cells and CSCs. In addition,emerging evidence suggests that ALDH1 is not only a marker for stem cells,but may also play important functional roles related to self-protection,differentiation,and expansion. This comprehensive review discusses the role that ALDH plays in normal stem cells and CSCs,with focus on ALDH1 and ALDH3A1. Discrepancies in the functional themes between cell types and future perspectives for therapeutic applications will also be discussed. View Publication

MicroRNA miR-125b has been implicated in several kinds of leukemia. The chromosomal translocation t(2;11)(p21;q23) found in patients with myelodysplasia and acute myeloid leukemia leads to an overexpression of miR-125b of up to 90-fold normal. Moreover,miR-125b is also up-regulated in patients with B-cell acute lymphoblastic leukemia carrying the t(11;14)(q24;q32) translocation. To decipher the presumed oncogenic mechanism of miR-125b,we used transplantation experiments in mice. All mice transplanted with fetal liver cells ectopically expressing miR-125b showed an increase in white blood cell count,in particular in neutrophils and monocytes,associated with a macrocytic anemia. Among these mice,half died of B-cell acute lymphoblastic leukemia,T-cell acute lymphoblastic leukemia,or a myeloproliferative neoplasm,suggesting an important role for miR-125b in early hematopoiesis. Furthermore,coexpression of miR-125b and the BCR-ABL fusion gene in transplanted cells accelerated the development of leukemia in mice,compared with control mice expressing only BCR-ABL,suggesting that miR-125b confers a proliferative advantage to the leukemic cells. Thus,we show that overexpression of miR-125b is sufficient both to shorten the latency of BCR-ABL-induced leukemia and to independently induce leukemia in a mouse model. View Publication

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by ectopic expression of four transcription factors. However,the efficiency of human iPS cell generation is extremely low and therefore elucidating the mechanisms underlying cellular reprogramming is of prime importance. We demonstrate that 8-Bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) improves the reprogramming efficiency of human neonatal foreskin fibroblast (HFF1) cells transduced with the four transcription factors by 2-fold. The combination of 8-Br-cAMP and VPA synergistically increases the efficiency to 6.5-fold. The effect of 8-Br-cAMP or VPA may in part be due to the up-regulation of cytokine-related and inflammatory pathways. Remarkably,the synergistic effect of 8-Br-cAMP and VPA on cellular reprogramming may be due to the transient decrease of p53 protein during the early stages of reprogramming. However,it could also be due to additional differentially regulated genes and pathways such as the up-regulation of cytokine-related,inflammatory pathways and self-renewal supporting gene,namely cyclin-encoding CCND2,and the associated genes CCNA1 and CCNE1. Conversely,we also see the down-regulation of the p53 (CCNB2,GTSE1,SERPINE1) and cell cycle (PLK1,CCNB2) pathways. Our data demonstrates that a cyclic AMP analog,8-Br-cAMP,enhances the efficiency of cellular reprogramming. In addition,8-Br-cAMP and VPA have a synergistic effect on cellular reprogramming,which may be in part due to the transient down-regulation of the p53 signaling pathway during the early stages of reprogramming. View Publication

OBJECTIVE: To explore the effects of anti-CD44 monoclonal antibody-IM7 on the in vitro adhesion and migration of chronic myeloid leukemia stem cell (CML-LSC) and its mechanism. METHODS: CD34(+)CD38(-)CD123(+) leukemic stem cells (LSC) from 20 newly-diagnosed chronic myeloid leukemia (CML) patients BM cells and CD34(+)CD38(-) hematopoietic stem cells (HSC) from 20 full-term newborn cord blood cells were isolated with EasySep(TM) magnet beads. The CD44 expression of the LSC and HSC was detected by flow cytometry (FCM),and the adhesion and migration ability of the LSC and HSC pre- and post-incubated with IM7 in vitro by MTT assay and transendothelial migration assay,respectively. RESULTS: (1) After incubated with IM7,the LSC and HSC CD44 expression rates were (86.60 ± 2.10)% vs. (25.40 ± 1.70)% (P textless 0.05),respectively. (2) The adhesive ability of the LSC to endothelial cells was decreased markedly after incubated with IM7,the OD value (A(570)) changing from pre-incubation of (0.62 ± 0.11) to post-incubation of (0.34 ± 0.07),while there was little change of A(570) in the HSC group. (3) The migration ability of the LSC group was inhibited evidently after incubated with IM7,the inhibition rate being 46% ∼ 63%,while little change of that in HSC group was detected. (4) The adhesive ability of the LSC group to marrow stromal cells was decreased markedly after incubated with IM7,while little change was found in that of HSC group. CONCLUSION: The anti-CD44 monoclonal antibody-IM7 can effectively inhibit the adhesion and migration abilities of the LSC in vitro,which might provide a theoretical evidence for targeting therapy. View Publication

Cancer-associated IDH mutations are characterized by neomorphic enzyme activity and resultant 2-hydroxyglutarate (2HG) production. Mutational and epigenetic profiling of a large acute myeloid leukemia (AML) patient cohort revealed that IDH1/2-mutant AMLs display global DNA hypermethylation and a specific hypermethylation signature. Furthermore,expression of 2HG-producing IDH alleles in cells induced global DNA hypermethylation. In the AML cohort,IDH1/2 mutations were mutually exclusive with mutations in the α-ketoglutarate-dependent enzyme TET2,and TET2 loss-of-function mutations were associated with similar epigenetic defects as IDH1/2 mutants. Consistent with these genetic and epigenetic data,expression of IDH mutants impaired TET2 catalytic function in cells. Finally,either expression of mutant IDH1/2 or Tet2 depletion impaired hematopoietic differentiation and increased stem/progenitor cell marker expression,suggesting a shared proleukemogenic effect. View Publication

Osteoblasts play a crucial role in the hematopoietic stem cell (HSC) niche; however,an overall increase in their number does not necessarily promote hematopoiesis. Because the activity of osteoblasts and osteoclasts is coordinately regulated,we hypothesized that active bone-resorbing osteoclasts would participate in HSC niche maintenance. Mice treated with bisphosphonates exhibited a decrease in proportion and absolute number of Lin(-)cKit(+)Sca1(+) Flk2(-) (LKS Flk2(-)) and long-term culture-initiating cells in bone marrow (BM). In competitive transplantation assays,the engraftment of treated BM cells was inferior to that of controls,confirming a decrease in HSC numbers. Accordingly,bisphosphonates abolished the HSC increment produced by parathyroid hormone. In contrast,the number of colony-forming-unit cells in BM was increased. Because a larger fraction of LKS in the BM of treated mice was found in the S/M phase of the cell cycle,osteoclast impairment makes a proportion of HSCs enter the cell cycle and differentiate. To prove that HSC impairment was a consequence of niche manipulation,a group of mice was treated with bisphosphonates and then subjected to BM transplantation from untreated donors. Treated recipient mice experienced a delayed hematopoietic recovery compared with untreated controls. Our findings demonstrate that osteoclast function is fundamental in the HSC niche. View Publication