技术资料

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

CD200 is a transmembrane protein broadly expressed on a variety of cell types,which delivers immunoregulatory signals through binding to receptors (CD200Rs) expressed on monocytes/myeloid cells and T lymphocytes. Signals delivered through the CD200:CD200R axis have been shown to play an important role in the regulation of anti-tumor immunity,and overexpression of CD200 has been reported in a number of malignancies,including CLL,as well as on cancer stem cells. We investigated the effect of CD200 blockade in vitro on a generation of CTL responses against a poorly immunogenic CD200+ lymphoma cell line and fresh cells obtained from CLL patients using anti-CD200 mAb and CD200-specific siRNAs. Suppression of functional expression of CD200 augmented killing of the CD200+ cells,as well as production of the inflammatory cytokines IFN-gamma and TNF-alpha by effector PBMCs. Killing was mediated by CD8+ cytotoxic T cells,and CD4+ T cells play an important role in CD200-mediated suppression of CTL responses. Our data suggest that CD200 blockade may represent a novel approach to clinical treatment of CLL. 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

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

Natural killer cells are lymphocytes specialized to participate in host defense through their innate ability to mediate cytotoxicity by secreting the contents of preformed secretory lysosomes (lytic granules) directly onto a target cell. This form of directed secretion requires the formation of an immunological synapse and occurs stepwise with actin reorganization preceding microtubule-organizing center (MTOC) polarization to the synapse. Because MTOC polarization to the synapse is required for polarization of lytic granules,we attempted to define their interrelationship. We found that compared with the time required for MTOC polarization,lytic granules converged to the MTOC rapidly. The MTOC-directed movement of lytic granules was independent of actin and microtubule reorganization,dependent on dynein motor function,occurred before MTOC polarization,and did not require a commitment to cytotoxicity. This defines a novel paradigm for rapid MTOC-directed transport as a prerequisite for directed secretion,one that may prepare,but not commit cells for precision secretory function. 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

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

Raman microspectroscopy is an attractive approach for chemical imaging of biological specimens,including live cells,without the need for chemi-selective stains. Using a microspectrometer,near-infrared Raman spectra throughout the range 663 cm(-1) to 1220 cm(-1) were obtained from colonies of CA1 human embryonic stem cells (hESCs) and CA1 cells that had been stimulated to differentiate for 3 weeks by 10% fetal bovine serum on gelatin. Distributions and intensities of spectral bands attributed to proteins varied significantly between undifferentiated and differentiated cells. Importantly,compared to proteins and lipids,the band intensities of nucleic acids were dominant in undifferentiated cells with a dominance-reversal in differentiated cells. Thus,we could identify intensity ratios of particular protein-related bands (e.g.,757 cm(-1) tryptophan) to nucleic acid bands (784 cm(-1) DNA/RNA composite) that were effective in discriminating between spectra of undifferentiated and differentiated cells. We observed no discernible negative effects due to the laser exposure in terms of morphology,proliferation,or pluripotency of the stem cells. We conclude that Raman microscopy and complementary data processing procedures provide a rapid,noninvasive approach that can distinguish hESCs from differentiated cells. This is the first report to identify specific Raman markers for the differentiation status of hESCs. View Publication

The use of induced pluripotent stem cells (iPSCs) is an exciting frontier in the study and treatment of human diseases through the generation of specific cell types. Here we show the derivation of iPSCs from human nonmobilized peripheral blood (PB) and bone marrow (BM) mononuclear cells (MNCs) by retroviral transduction of OCT3/4,SOX2,KLF4,and c-MYC. The PB- and BM-derived iPSCs were quite similar to human embryonic stem cells with regard to morphology,expression of surface antigens and pluripotency-associated transcription factors,global gene expression profiles,and differentiation potential in vitro and in vivo. Infected PB and BM MNCs gave rise to iPSCs in the presence of several cytokines,although transduction efficiencies were not high. We found that 5 × 10(5) PB MNCs,which corresponds to less than 1 mL of PB,was enough for the generation of several iPSC colonies. Generation of iPSCs from MNCs of nonmobilized PB,with its relative efficiency and ease of harvesting,could enable the therapeutic use of patient-specific pluripotent stem cells. View Publication

BACKGROUND: Malignant melanoma is an exceptionally aggressive,drug-resistant and heterogeneous cancer. Recently it has been shown that melanoma cells with high clonogenic and tumourigenic abilities are common,but markers distinguishing such cells from cells lacking these abilities have not been identified. There is therefore no definite evidence that an exclusive cell subpopulation,i.e. cancer stem cells (CSC),exists in malignant melanoma. Rather,it is suggested that multiple cell populations are implicated in initiation and progression of the disease,making it of importance to identify subpopulations with elevated aggressive properties. METHODS AND FINDINGS: In several other cancer forms,Aldehyde Dehydrogenase (ALDH),which plays a role in stem cell biology and resistance,is a valuable functional marker for identification of cells that show enhanced aggressiveness and drug-resistance. Furthermore,the presence of ALDH(+) cells is linked to poor clinical prognosis in these cancers. By analyzing cell cultures,xenografts and patient biopsies,we showed that aggressive melanoma harboured a large,distinguishable ALDH(+) subpopulation. In vivo,ALDH(+) cells gave rise to ALDH(-) cells,while the opposite conversion was rare,indicating a higher abilities of ALDH(+) cells to reestablish tumour heterogeneity with respect to the ALDH phenotype. However,both ALDH(+) and ALDH(-) cells demonstrated similarly high abilities for clone formation in vitro and tumour initiation in vivo. Furthermore,both subpopulations showed similar sensitivity to the anti-melanoma drugs,dacarbazine and lexatumumab. CONCLUSIONS: These findings suggest that ALDH does not distinguish tumour-initiating and/or therapy-resistant cells,implying that the ALDH phenotype is not associated with more-aggressive subpopulations in malignant melanoma,and arguing against ALDH as a universal" marker. Besides� View Publication

Follicular helper T (T(FH)) cells,defined by expression of the surface markers CXCR5 and programmed death receptor-1 (PD-1) and synthesis of IL-21,require upregulation of the transcriptional repressor Bcl6 for their development and function in B cell maturation in germinal centers. We have explored the role of B cells and the cytokines IL-6 and IL-21 in the in vivo regulation of Bcl6 expression and T(FH) cell development. We found that T(FH) cells are characterized by a Bcl6-dependent downregulation of P-selectin glycoprotein ligand 1 (PSGL1,a CCL19- and CCL21-binding protein),indicating that,like CXCR5 and PD-1 upregulation,modulation of PSGL1 expression is part of the T(FH) cell program of differentiation. B cells were neither required for initial upregulation of Bcl6 nor PSGL1 downregulation,suggesting these events preceded T-B cell interactions,although they were required for full development of the T(FH) cell phenotype,including CXCR5 and PD-1 upregulation,and IL-21 synthesis. Bcl6 upregulation and T(FH) cell differentiation were independent of IL-6 and IL-21,revealing that either cytokine is not absolutely required for development of Bcl6(+) T(FH) cells in vivo. These data increase our understanding of Bcl6 regulation in T(FH) cells and their differentiation in vivo and identifies a new surface marker that may be functionally relevant in this subset. View Publication

Human embryonic stem cell (hESC)-based assay systems and genetically modified hESCs are very useful tools for screening drugs that regulate stemness and differentiation and for studying the molecular mechanisms involved in hESC fate determination. For these types of studies,feeder cell-dependent cultures of hESCs are often problematic because the physiology of the feeder cells is perturbed by the drug treatments or genetic modifications,which potentially obscures research outcomes. In this study,we evaluated three commonly used feeder-free culture conditions to determine whether they supported the undifferentiated growth of hESCs and to determine whether the hESCs grown in these conditions displayed gene expression patterns that were similar to the expression patterns of feeder cell-dependent hESCs. Our results demonstrate that hESCs grown in the three feeder-free conditions expressed undifferentiation marker genes as strongly as hESCs that were grown in the feeder-dependent cultures. Furthermore,genome-wide gene expression profiles indicated that the gene expression patterns of hESCs that were grown under feeder-free or feeder-dependent culture conditions were highly similar. These results indicate that the feeder-free culture conditions support the undifferentiated growth of hESCs as effectively as the feeder-dependent culture conditions. Therefore,feeder-free culture conditions are potentially suitable for drug screening and for the genetic manipulation of hESCs in basic research. View Publication