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In mouse,a subset of dendritic cells (DCs) known as CD8alpha+ DCs has emerged as an important player in the regulation of T cell responses and a promising target in vaccination strategies. However,translation into clinical protocols has been hampered by the failure to identify CD8alpha+ DCs in humans. Here,we characterize a population of human DCs that expresses DNGR-1 (CLEC9A) and high levels of BDCA3 and resembles mouse CD8alpha+ DCs in phenotype and function. We describe the presence of such cells in the spleens of humans and humanized mice and report on a protocol to generate them in vitro. Like mouse CD8alpha+ DCs,human DNGR-1+ BDCA3hi DCs express Necl2,CD207,BATF3,IRF8,and TLR3,but not CD11b,IRF4,TLR7,or (unlike CD8alpha+ DCs) TLR9. DNGR-1+ BDCA3hi DCs respond to poly I:C and agonists of TLR8,but not of TLR7,and produce interleukin (IL)-12 when given innate and T cell-derived signals. Notably,DNGR-1+ BDCA3+ DCs from in vitro cultures efficiently internalize material from dead cells and can cross-present exogenous antigens to CD8+ T cells upon treatment with poly I:C. The characterization of human DNGR-1+ BDCA3hi DCs and the ability to grow them in vitro opens the door for exploiting this subset in immunotherapy. View Publication

During disease progression in myelodysplastic syndromes (MDS),clonal blasts gain a more aggressive nature,whereas nonclonal immune cells become less efficient via an unknown mechanism. Using MDS cell lines and patient samples,we showed that the expression of an immunoinhibitory molecule,B7-H1 (CD274),was induced by interferon-gamma (IFNgamma) and tumor necrosis factor-alpha (TNFalpha) on MDS blasts. This induction was associated with the activation of nuclear factor-kappaB (NF-kappaB) and nearly completely blocked by an NF-kappaB inhibitor,pyrrolidine dithiocarbamate (PDTC). B7-H1(+) MDS blasts had greater intrinsic proliferative capacity than B7-H1(-) MDS blasts when examined in various assays. Furthermore,B7-H1(+) blasts suppressed T-cell proliferation and induced T-cell apoptosis in allogeneic cocultures. When fresh bone marrow samples from patients were examined,blasts from high-risk MDS patients expressed B7-H1 molecules more often compared with those from low-risk MDS patients. Moreover,MDS T cells often overexpressed programmed cell death 1 (PD-1) molecules that transmit an inhibitory signal from B7-H1 molecules. Taken together,these findings provide new insight into MDS pathophysiology. IFNgamma and TNFalpha activate NF-kappaB that in turn induces B7-H1 expression on MDS blasts. B7-H1(+) MDS blasts have an intrinsic proliferative advantage and induce T-cell suppression,which may be associated with disease progression in MDS. View Publication

The endosteal niche is critical for the maintenance of hematopoietic stem cells (HSCs). However,it consists of a heterogeneous population in terms of differentiation stage and function. In this study,we characterized endosteal cell populations and examined their ability to maintain HSCs. Bone marrow endosteal cells were subdivided into immature mesenchymal cell-enriched ALCAM(-)Sca-1(+) cells,osteoblast-enriched ALCAM(+)Sca-1(-),and ALCAM(-)Sca-1(-) cells. We found that all 3 fractions maintained long-term reconstitution (LTR) activity of HSCs in an in vitro culture. In particular,ALCAM(+)Sca-1(-) cells significantly enhanced the LTR activity of HSCs by the up-regulation of homing- and cell adhesion-related genes in HSCs. Microarray analysis showed that ALCAM(-)Sca-1(+) fraction highly expressed cytokine-related genes,whereas the ALCAM(+)Sca-1(-) fraction expressed multiple cell adhesion molecules,such as cadherins,at a greater level than the other fractions,indicating that the interaction between HSCs and osteoblasts via cell adhesion molecules enhanced the LTR activity of HSCs. Furthermore,we found an osteoblastic marker(low/-) subpopulation in ALCAM(+)Sca-1(-) fraction that expressed cytokines,such as Angpt1 and Thpo,and stem cell marker genes. Altogether,these data suggest that multiple subsets of osteoblasts and mesenchymal progenitor cells constitute the endosteal niche and regulate HSCs in adult bone marrow. View Publication

The development and emergence of the hematopoietic stem cell involves a series of tightly regulated molecular events that are not well characterized. The hematopoietically expressed homeobox (Hhex) gene,a member of the homeobox gene family,is an essential regulator of embryogenesis and hematopoietic progenitor development. To investigate the role of Hhex in hematopoiesis we adapted a murine embryonic stem (ES) cell coculture system,in which ES cells can differentiate into CD41(+) and CD45(+) hematopoietic progenitors in vitro. Our results show that in addition to delayed hemangioblast development,Hhex(-/-) ES-derived progeny accumulate as CD41(+) and CD41(+)c-kit(+) cells,or the earliest definitive hematopoietic progenitors. In addition,Hhex(-/-) ES-derived progeny display a significantly reduced ability to develop into mature CD45(+) hematopoietic cells. The observed reduction in hematopoietic maturation was accompanied by reduced proliferation,because Hhex(-/-) CD41(+)CD45(-)c-kit(+) hematopoietic progenitors accumulated in the G(2) phase of the cell cycle. Thus,Hhex is a critical regulator of hematopoietic development and is necessary for the maturation and proliferation of the earliest definitive hematopoietic progenitors. View Publication

The development of mature blood cells from hematopoietic stem cells requires coordinated activities of transcriptional networks. Transcriptional repressor growth factor independence 1 (Gfi-1) is required for the development of B cells,T cells,neutrophils,and for the maintenance of hematopoietic stem cell function. However,the mechanisms by which Gfi-1 regulates hematopoiesis and how Gfi-1 integrates into transcriptional networks remain unclear. Here,we provide evidence that Id2 is a transcriptional target of Gfi-1,and repression of Id2 by Gfi-1 is required for B-cell and myeloid development. Gfi-1 binds to 3 conserved regions in the Id2 promoter and represses Id2 promoter activity in transient reporter assays. Increased Id2 expression was observed in multipotent progenitors,myeloid progenitors,T-cell progenitors,and B-cell progenitors in Gfi-1(-/-) mice. Knockdown of Id2 expression or heterozygosity at the Id2 locus partially rescues the B-cell and myeloid development but not the T-cell development in Gfi-1(-/-) mice. These studies demonstrate a role of Id2 in mediating Gfi-1 functions in B-cell and myeloid development and provide a direct link between Gfi-1 and the B-cell transcriptional network by its ability to repress Id2 expression. View Publication

Human embryonic stem cells (hESCs) share an identical genome with lineage-committed cells,yet possess the remarkable properties of self-renewal and pluripotency. The diverse cellular properties in different cells have been attributed to their distinct epigenomes,but how much epigenomes differ remains unclear. Here,we report that epigenomic landscapes in hESCs and lineage-committed cells are drastically different. By comparing the chromatin-modification profiles and DNA methylomes in hESCs and primary fibroblasts,we find that nearly one-third of the genome differs in chromatin structure. Most changes arise from dramatic redistributions of repressive H3K9me3 and H3K27me3 marks,which form blocks that significantly expand in fibroblasts. A large number of potential regulatory sequences also exhibit a high degree of dynamics in chromatin modifications and DNA methylation. Additionally,we observe novel,context-dependent relationships between DNA methylation and chromatin modifications. Our results provide new insights into epigenetic mechanisms underlying properties of pluripotency and cell fate commitment. textcopyright 2010 Elsevier Inc. View Publication

Umbilical cord blood (CB) represents a main source of circulating endothelial progenitor cells (cEPCs). In view of their clinical use,in either the autologous or allogeneic setting,cEPCs should likely be expanded from CB kept frozen in CB banks. In this study,we compared the expansion,functional features,senescence pattern over culture,and in vivo angiogenic potential of cEPCs isolated from fresh or cryopreserved CB (cryoCB). cEPCs could be isolated in only 59% of cryoCB compared to 94% for fresh CB,while CB units were matched in terms of initial volume,nucleated and CD34(+) cell number. Moreover,the number of endothelial colony-forming cells was significantly decreased when using cryoCB. Once cEPCs culture was established,the proliferation,migration,tube formation,and acetylated-LDL uptake potentials were similar in both groups. In addition,cEPCs derived from cryoCB displayed the same senescence status and telomeres length as that of cEPCs derived from fresh CB. Karyotypic aberrations were found in cells obtained from both fresh and cryoCB. In vivo,in a hind limb ischemia murine model,cEPCs from fresh and cryoCB were equally efficient to induce neovascularization. Thus,cEPCs isolated from cryoCB exhibited similar properties to those of fresh CB in vitro and in vivo. However,the low frequency of cEPCs colony formation after cryopreservation shed light on the need for specific freezing conditions adapted to cEPCs in view of their future clinical use. View Publication

Human mesenchymal stromal cells (hMSCs) have generated significant interest due to their potential use in clinical applications. hMSCs are present at low frequency in vivo,but after isolation can be expanded considerably,generating clinically useful numbers of cells. In this study,we demonstrate the use of a defined embryonic stem cell expansion medium,mTeSR (Stem Cell Technologies),for the expansion of bone-marrow-derived hMSCs. The hMSCs grow at comparable rates,demonstrate tri-lineage differentiation potential,and show similar surface marker profiles (CD29(+),CD44(+),CD49a(+),CD73(+),CD90(+),CD105(+),CD146(+),CD166(+),CD34(-),and CD45(-)) in both the fetal bovine serum (FBS)-supplemented medium and mTeSR. However,expression of early differentiation transcription factors runt-related transcription factor 2,sex-determining region Y box 9,and peroxisome proliferator-activated receptor gamma changed significantly. Both runt-related transcription factor 2 and sex-determining region Y box 9 were upregulated,whereas peroxisome proliferator-activated receptor gamma was downregulated in mTeSR compared with FBS. Although osteogenic and chondrogenic differentiation was comparable in cells grown in mTeSR compared to FBS,adipogenic differentiation was significantly decreased in mTeSR-expanded cells,both in terms of gene expression and absolute numbers of adipocytes. The removal of the FBS from the medium and the provision of a defined medium with disclosed composition make mTeSR a superior study platform for hMSC biology in a controlled environment. Further,this provides a key step toward generating a clinical-grade medium for expansion of hMSCs for clinical applications that rely on osteo- and chondroinduction of MSCs,such as bone repair and cartilage generation. View Publication

HOXA9-mediated up-regulation of miR-155 was noted during an array-based analysis of microRNA expression in Hoxa9(-/-)bone marrow (BM) cells. HOXA9 induction of miR-155 was confirmed in these samples,as well as in wild-type versus Hoxa9-deficient marrow,using northern analysis and qRT-PCR. Infection of wild-type BM with HOXA9 expressing or GFP(+) control virus further confirmed HOXA9-mediated regulation of miR-155. miR-155 expression paralleled Hoxa9 mRNA expression in fractionated BM progenitors,being highest in the stem cell enriched pools. HOXA9 capacity to induce myeloid colony formation was blunted in miR-155-deficient BM cells,indicating that miR-155 is a downstream mediator of HOXA9 function in blood cells. Pu.1,an important regulator of myelopoiesis,was identified as a putative down stream target for miR-155. Although miR-155 was shown to down-regulate the Pu.1 protein,HOXA9 did not appear to modulate Pu.1 expression in murine BM cells. View Publication

Human and mouse embryonic stem cells (ESCs) are derived from blastocyst-stage embryos but have very different biological properties,and molecular analyses suggest that the pluripotent state of human ESCs isolated so far corresponds to that of mouse-derived epiblast stem cells (EpiSCs). Here we rewire the identity of conventional human ESCs into a more immature state that extensively shares defining features with pluripotent mouse ESCs. This was achieved by ectopic induction of Oct4,Klf4,and Klf2 factors combined with LIF and inhibitors of glycogen synthase kinase 3beta (GSK3beta) and mitogen-activated protein kinase (ERK1/2) pathway. Forskolin,a protein kinase A pathway agonist which can induce Klf4 and Klf2 expression,transiently substitutes for the requirement for ectopic transgene expression. In contrast to conventional human ESCs,these epigenetically converted cells have growth properties,an X-chromosome activation state (XaXa),a gene expression profile,and a signaling pathway dependence that are highly similar to those of mouse ESCs. Finally,the same growth conditions allow the derivation of human induced pluripotent stem (iPS) cells with similar properties as mouse iPS cells. The generation of validated naïve" human ESCs will allow the molecular dissection of a previously undefined pluripotent state in humans and may open up new opportunities for patient-specific� View Publication

Current induction schemes directing hematopoietic differentiation of human embryonic stem cells (hESCs) are not well defined to mimic the sequential stages of hematopoietic development in vivo. Here,we report a 3-stage method to direct differentiation of hESCs toward hematopoietic progenitors in chemically defined mediums. In the first 2 stages,we efficiently generated T-positive primitive streak/mesendoderm cells and kinase domain receptor-positive (KDR(+)) platelet-derived growth factor receptor α-negative (PDGFRα(-)) hemato-vascular precursors sequentially. In the third stage,we found that cells in a spontaneous differentiation condition mainly formed erythroid colonies. Addition of all-trans retinoic acid (RA) greatly enhanced generation of hematopoietic progenitors in this stage while suppressing erythroid development. The RA-treated cells highly expressed definitive hematopoietic genes,formed large numbers of multilineage and myeloid colonies,and gave rise to greater than 45% CD45(+) hematopoietic cells. When hematopoietic progenitors were selected with CD34 and C-Kit,greater than 95% CD45(+) hematopoietic cells could be generated. In addition,we found that endogenous RA signaling at the second stage was required for vascular endothelial growth factor/basic fibroblast growth factor-induced hemato-vascular specification,whereas exogenously applied RA efficiently induced KDR(-)PDGFRα(+) paraxial mesoderm cells. Our study suggests that RA signaling plays diverse roles in human mesoderm and hematopoietic development. View Publication

Aldehyde dehydrogenase isoform 1 (ALDH1) has been proved useful for the identification of cancer stem cells. However,our knowledge of the expression and activity of ALDH1 in common epithelial cancers and their corresponding normal tissues is still largely absent. Therefore,we characterized ALDH1 expression in 24 types of normal tissues and a large collection of epithelial tumor specimens (six cancer types,n = 792) by immunohistochemical staining. Using the ALDEFUOR assay,ALDH1 activity was also examined in 16 primary tumor specimens and 43 established epithelial cancer cell lines. In addition,an ovarian cancer transgenic mouse model and 7 murine ovarian cancer cell lines were analyzed. We found that the expression levels and patterns of ALDH1 in epithelial cancers are remarkably distinct,and they correlate with their corresponding normal tissues. ALDH1 protein expression levels are positively correlated with ALDH1 enzymatic activity measured by ALDEFLUOR assay. Long-term in vitro culture doesn't significantly affect ALDH1 activity in epithelial tumor cells. Consistent with research on other cancers,we found that high ALDH1 expression is significantly associated with poor clinical outcomes in serous ovarian cancer patients (n = 439,p = 0.0036). Finally,ALDH(br) tumor cells exhibit cancer stem cell properties and are resistant to chemotherapy. As a novel cancer stem cell marker,ALDH1 can be used for tumors whose corresponding normal tissues express ALDH1 in relatively restricted or limited levels such as breast,lung,ovarian or colon cancer. View Publication