技术资料

In this study,we have investigated the hypothesis that previously reported beneficial effect of peripheral blood mononuclear cells cultured under angiogenic conditions on cardiovascular function following ischemia is not limited to EPCs but also to monocytes contained therein. We first purified and analyzed the phenotype and secretome of human and murine blood monocytes cultured under angiogenic conditions (named MDs for monocyte derivatives) and tested their effect in a mouse model of myocardial infarction (MI). FACS analysis of MDs shows that these cells express mature endothelial cell markers and that their proliferative capacity is virtually absent,consistent with their end-differentiated monocytic ontogeny. MDs secreted significant levels of HGF,IGF-1,MCP-1,and sTNFR-1 relative to their monocyte precursors. MDs were unable to form vascular networks in vitro when cultured on matrix coated flasks. Treatment of murine HL-1 cardiomyocyte cell line with MD-conditioned medium reduced their death induced by TNF-alpha,staurosporine,and oxidative stress,and this effect was dependent upon MD-derived sTNFR-1,HGF,and IGF-1. We further demonstrate that MD secretome promoted endothelial cell proliferation and capacity to form vessels in vitro and this was dependent upon MD-derived MCP-1,HGF,and IGF-1. Echocardiography analysis showed that MD myocardial implantation improved left ventricle fractional shortening of mouse hearts following MI and was associated with reduced myocardial fibrosis and enhancement of angiogenesis. Transplanted MDs and their secretome participate in preserving functional myocardium after ischemic insult and attenuate pathological remodeling. 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

PURPOSE: To examine the role of cancer stem cells (CSC) in mediating metastasis in inflammatory breast cancer (IBC) and the association of these cells with patient outcome in this aggressive type of breast cancer. EXPERIMENTAL DESIGN: CSCs were isolated from SUM149 and MARY-X,an IBC cell line and primary xenograft,by virtue of increased aldehyde dehydrogenase (ALDH) activity as assessed by the ALDEFLUOR assay. Invasion and metastasis of CSC populations were assessed by in vitro and mouse xenograft assays. Expression of ALDH1 was determined on a retrospective series of 109 IBC patients and this was correlated with histoclinical data. All statistical tests were two sided. Log-rank tests using Kaplan-Meier analysis were used to determine the correlation of ALDH1 expression with development of metastasis and patient outcome. RESULTS: Both in vitro and xenograft assays showed that invasion and metastasis in IBC are mediated by a cellular component that displays ALDH activity. Furthermore,expression of ALDH1 in IBC was an independent predictive factor for early metastasis and decreased survival in this patient population. CONCLUSIONS: These results suggest that the metastatic,aggressive behavior of IBC may be mediated by a CSC component that displays ALDH enzymatic activity. ALDH1 expression represents the first independent prognostic marker to predict metastasis and poor patient outcome in IBC. The results illustrate how stem cell research can translate into clinical practice in the IBC field. 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

Both extrinsic and intrinsic mechanisms tightly govern hematopoietic stem cell (HSC) decisions of self-renewal and differentiation. However,transcription factors that can selectively regulate HSC self-renewal division after stress remain to be identified. Slug is an evolutionarily conserved zinc-finger transcription factor that is highly expressed in primitive hematopoietic cells and is critical for the radioprotection of these key cells. We studied the effect of Slug in the regulation of HSCs in Slug-deficient mice under normal and stress conditions using serial functional assays. Here,we show that Slug deficiency does not disturb hematopoiesis or alter HSC homeostasis and differentiation in bone marrow but increases the numbers of primitive hematopoietic cells in the extramedullary spleen site. Deletion of Slug enhances HSC repopulating potential but not its homing and differentiation ability. Furthermore,Slug deficiency increases HSC proliferation and repopulating potential in vivo after myelosuppression and accelerates HSC expansion during in vitro culture. Therefore,we propose that Slug is essential for controlling the transition of HSCs from relative quiescence under steady-state condition to rapid proliferation under stress conditions. Our data suggest that inhibition of Slug in HSCs may present a novel strategy for accelerating hematopoietic recovery,thus providing therapeutic benefits for patients after clinical myelosuppressive treatment. View Publication

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by defined factors. However,the low efficiency and slow kinetics of the reprogramming process have hampered progress with this technology. Here we report that a natural compound,vitamin C (Vc),enhances iPSC generation from both mouse and human somatic cells. Vc acts at least in part by alleviating cell senescence,a recently identified roadblock for reprogramming. In addition,Vc accelerates gene expression changes and promotes the transition of pre-iPSC colonies to a fully reprogrammed state. Our results therefore highlight a straightforward method for improving the speed and efficiency of iPSC generation and provide additional insights into the mechanistic basis of the reprogramming process. View Publication

There is increasing evidence to suggest that embryonic stem cells (ESCs) are capable of differentiating into hepatocytes in vitro. In this study,we used a combination of cytokines and sodium butyrate in a novel three-step procedure to efficiently direct the differentiation of mouse ESCs into hepatocytes. Mouse ESCs were first differentiated into definitive endoderm cells by 3 days of treatment with Activin A. The definitive endoderm cells were then differentiated into hepatocytes by the addition of acidic fibroblast growth factor (aFGF) and sodium butyrate to the culture medium for 5 days. After 10 days of further in vitro maturation,the morphological and phenotypic markers of hepatocytes were characterized using immunohistochemistry,immunoblotting,and reverse transcription-polymerase chain reaction (RT-PCR). Furthermore,the cells were tested for functions associated with mature hepatocytes,including glycogen storage and indocyanine green uptake and release,and the ratio of hepatic differentiation was determined by counting the percentage of albumin-positive cells. In the presence of medium containing cytokines and sodium butyrate,numerous epithelial cells resembling hepatocytes were observed,and approximately 74% of the cells expressed the hepatic marker,albumin,after 18 days in culture. RT-PCR analysis and immunohistochemistry showed that these cells expressed adult liver cell markers,and had the abilities of glycogen storage and indocyanine green uptake and release. We have developed an efficient method for directing the differentiation of mouse ESCs into cells that exhibit the characteristics of mature hepatocytes. This technique will be useful for research into the molecular mechanisms underlying liver development,and could provide a source of hepatocytes for transplantation therapy and drug screening. View Publication

BACKGROUND: Differentiation of human embryonic stem cells into endothelial cells (hESC-ECs) has the potential to provide an unlimited source of cells for novel transplantation therapies of ischemic diseases by supporting angiogenesis and vasculogenesis. However,the endothelial differentiation efficiency of the conventional embryoid body (EB) method is low while the 2-dimensional method of co-culturing with mouse embryonic fibroblasts (MEFs) require animal product,both of which can limit the future clinical application of hESC-ECs. Moreover,to fully understand the beneficial effects of stem cell therapy,investigators must be able to track the functional biology and physiology of transplanted cells in living subjects over time. METHODOLOGY: In this study,we developed an extracellular matrix (ECM) culture system for increasing endothelial differentiation and free from contaminating animal cells. We investigated the transcriptional changes that occur during endothelial differentiation of hESCs using whole genome microarray,and compared to human umbilical vein endothelial cells (HUVECs). We also showed functional vascular formation by hESC-ECs in a mouse dorsal window model. Moreover,our study is the first so far to transplant hESC-ECs in a myocardial infarction model and monitor cell fate using molecular imaging methods. CONCLUSION: Taken together,we report a more efficient method for derivation of hESC-ECs that express appropriate patterns of endothelial genes,form functional vessels in vivo,and improve cardiac function. These studies suggest that hESC-ECs may provide a novel therapy for ischemic heart disease in the future. View Publication

The molecular mechanisms governing breast tumor cellular self-renewal contribute to breast cancer progression and therapeutic resistance. The ErbB2 oncogene is overexpressed in approximately 30% of human breast cancers. c-Jun,the first cellular proto-oncogene,is overexpressed in human breast cancer. However,the role of endogenous c-Jun in mammary tumor progression is unknown. Herein,transgenic mice expressing the mammary gland-targeted ErbB2 oncogene were crossed with c-jun(f/f) transgenic mice to determine the role of endogenous c-Jun in mammary tumor invasion and stem cell function. The excision of c-jun by Cre recombinase reduced cellular migration,invasion,and mammosphere formation of ErbB2-induced mammary tumors. Proteomic analysis identified a subset of secreted proteins (stem cell factor (SCF) and CCL5) induced by ErbB2 expression that were dependent upon endogenous c-Jun expression. SCF and CCL5 were identified as transcriptionally induced by c-Jun. CCL5 rescued the c-Jun-deficient breast tumor cellular invasion phenotype. SCF rescued the c-Jun-deficient mammosphere production. Endogenous c-Jun thus contributes to ErbB2-induced mammary tumor cell invasion and self-renewal. View Publication

Hematopoietic stem cells (HSCs) are enriched for aldehyde dehydrogenase (ALDH) activity and ALDH is a selectable marker for human HSCs. However,the function of ALDH in HSC biology is not well understood. We sought to determine the function of ALDH in regulating HSC fate. Pharmacologic inhibition of ALDH with diethylaminobenzaldehyde (DEAB) impeded the differentiation of murine CD34(-)c-kit(+)Sca-1(+)lineage(-) (34(-)KSL) HSCs in culture and facilitated a ninefold expansion of cells capable of radioprotecting lethally irradiated mice compared to input 34(-)KSL cells. Treatment of bone marrow (BM) 34(-)KSL cells with DEAB caused a fourfold increase in 4-week competitive repopulating units,verifying the amplification of short-term HSCs (ST-HSCs) in response to ALDH inhibition. Targeted siRNA of ALDH1a1 in BM HSCs caused a comparable expansion of radioprotective progenitor cells in culture compared to DEAB treatment,confirming that ALDH1a1 was the target of DEAB inhibition. The addition of all trans retinoic acid blocked DEAB-mediated expansion of ST-HSCs in culture,suggesting that ALDH1a1 regulates HSC differentiation via augmentation of retinoid signaling. Pharmacologic inhibition of ALDH has therapeutic potential as a means to amplify ST-HSCs for transplantation purposes. View Publication