技术资料

Platelet-derived growth factor receptors (PDGFR) α and β have been suggested as potential targets for treatment of rhabdomyosarcoma,the most common soft tissue sarcoma in children. This study identifies biologic activities linked to PDGF signaling in rhabdomyosarcoma models and human sample collections. Analysis of gene expression profiles of 101 primary human rhabdomyosarcomas revealed elevated PDGF-C and -D expression in all subtypes,with PDGF-D as the solely overexpressed PDGFRβ ligand. By immunohistochemistry,PDGF-CC,PDGF-DD,and PDGFRα were found in tumor cells,whereas PDGFRβ was primarily detected in vascular stroma. These results are concordant with the biologic processes and pathways identified by data mining. While PDGF-CC/PDGFRα signaling associated with genes involved in the reactivation of developmental programs,PDGF-DD/PDGFRβ signaling related to wound healing and leukocyte differentiation. Clinicopathologic correlations further identified associations between PDGFRβ in vascular stroma and the alveolar subtype and with presence of metastases. Functional validation of our findings was carried out in molecularly distinct model systems,where therapeutic targeting reduced tumor burden in a PDGFR-dependent manner with effects on cell proliferation,vessel density,and macrophage infiltration. The PDGFR-selective inhibitor CP-673,451 regulated cell proliferation through mechanisms involving reduced phosphorylation of GSK-3α and GSK-3β. Additional tissue culture studies showed a PDGFR-dependent regulation of rhabdosphere formation/cancer cell stemness,differentiation,senescence,and apoptosis. In summary,the study shows a clinically relevant distinction in PDGF signaling in human rhabdomyosarcoma and also suggests continued exploration of the influence of stromal PDGFRs on sarcoma progression. 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

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

Aldehyde dehydrogenases (ALDHs),enzymes responsible for detoxification and retinoic acid biosynthesis,are considered a potent functional stem cell marker of normal and malignant cells in many tissues. To date,however,there are no available data on ALDH distributions and functions in oral mucosa. This study aims to clarify the levels and types of ALDH expression using immunohistochemistry with accompanying mRNA expression as well as an ALDEFLUOR assay,and to assess phenotypic and histological changes after manipulation of the ALDH activity of oral keratinocytes to increase the potency of a tissue-engineered oral mucosa by a specific ALDH inhibitor,diethylaminobenzaldehyde (DEAB),together with small interfering RNA of ALDH1A3 and ALDH3A1. Results showed the mRNA and cytoplasmic protein expression of ALDH1A3 and ALDH3A1 to be mostly localized in the upper suprabasal layer although no ALDH1A1 immunoreaction was detected throughout the epithelium. Oral keratinocytes with high ALDH activity exhibited a profile of differentiating cells. By pharmacological inhibition,the phenotypic analysis revealed the proliferating cell-population shifting to a more quiescent state compared with untreated cells. Furthermore,a well-structured epithelial layer showing a normal differentiation pattern and a decrease in Ki-67 immunopositive basal cells was developed by DEAB incubation,suggesting a slower turnover rate efficient to maintain undifferentiated cells. Histological findings of a regenerated oral epithelium by ALDH1A3 siRNA were similar to those when treated with DEAB while ALDH3A1 siRNA eradicated the epithelial regenerative capacity. These observations suggest the effects of phenotypic and morphological alterations by DEAB on oral keratinocytes are mainly consequent to the inhibition of ALDH1A3 activity. View Publication

A broad range of hematopoietic stem cells and progenitors reside within a fraction of umbilical cord blood (UCB) that exhibits low light scatter properties (SSC(lo)) and high expression of aldehyde dehydrogenase (ALDH(br)). Many SSC(lo) ALDH(br) cells coexpress CD34; however,other cells express either ALDH or CD34. To investigate the developmental potential of these cell subsets,purified ALDH(br) CD34+,ALDH(neg) CD34+,and ALDH(br) CD34(neg) UCB cells were characterized within a variety of in vivo and in vitro assays. Primitive progenitors capable of multilineage development were monitored in long- and short-term repopulation assays performed on nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice,and in primary and secondary long-term culture assays. These progenitors were highly enriched within the ALDH(br) CD34+ fraction. This cell fraction also enriched short-term myeloid progenitors that were detected in vitro. By comparison,ALDH(neg) CD34+ cells contained few primitive progenitors and had diminished short-term myeloid potential but exhibited enhanced short-term natural killer (NK) cell development in vitro. The ALDH(br) CD34(neg) cells were not efficiently supported by any of the assays used. These studies suggested that in particular the expression of ALDH delineated distinct CD34+ stem cell and progenitor compartments. The differential expression of ALDH may provide a means to explore normal and malignant processes associated with myeloid and lymphoid development. View Publication

Inflammatory cytokines,particularly interferon-γ (IFN-γ),are markedly elevated in the peripheral blood of patients with immune checkpoint inhibitor-induced myocarditis (ICI-M). Endomyocardial biopsies from these patients also show GBP-associated inflammasome overexpression. While both factors are implicated in ICI-M pathophysiology,their interplay and cellular targets remain poorly characterized. Our aim was to elucidate how ICI-M-associated cytokines affect the viability and inflammatory responses of endothelial cells (ECs) and cardiomyocytes (CMs) using human induced pluripotent stem cell (hiPSC)-derived models. ECs and CMs were differentiated from the same hiPSC line derived from a healthy donor. Cells were exposed either to IFN-γ alone or to an inflammatory cytokine cocktail (CCL5,GZMB,IL-1β,IL-2,IL-6,IFN-γ,TNF-α). We assessed large-scale transcriptomic changes via microarray and evaluated inflammatory,apoptotic,and cell death pathways at cellular and molecular levels. hiPSC-ECs were highly sensitive to cytokine exposure,displaying significant mortality and marked transcriptomic changes in immunity- and inflammation-related pathways. In contrast,hiPSC-CM showed limited transcriptional changes and reduced susceptibility to cytokine-induced death. In both cell types,cytokine treatment upregulated key components of the inflammasome pathway,including regulators (GBP5,GBP6,P2X7,NLRC5),a core component (AIM2),and the effector GSDMD. Increased GBP5 expression and CASP-1 cleavage mirrored the findings found elsewhere in endomyocardial biopsies from ICI-M patients. This hiPSC-based model reveals a distinct cellular sensitivity to ICI-M-related inflammation,with endothelial cells showing heightened vulnerability. These results reposition endothelial dysfunction,rather than cardiomyocyte injury alone,as a central mechanism in ICI-induced myocarditis. Modulating endothelial inflammasome activation,particularly via AIM2 inhibition,could offer a novel strategy to mitigate cardiac toxicity while preserving antitumor efficacy. View Publication

Nanog,Oct4,and Sox2 are the core regulators of mouse (m)ESC pluripotency. Although their basic importance in human (h)ESCs has been demonstrated,the mechanistic functions are not well defined. Here,we identify general and cell-line-specific requirements for NANOG,OCT4,and SOX2 in hESCs. We show that OCT4 regulates,and interacts with,the BMP4 pathway to specify four developmental fates. High levels of OCT4 enable self-renewal in the absence of BMP4 but specify mesendoderm in the presence of BMP4. Low levels of OCT4 induce embryonic ectoderm differentiation in the absence of BMP4 but specify extraembryonic lineages in the presence of BMP4. NANOG represses embryonic ectoderm differentiation but has little effect on other lineages,whereas SOX2 and SOX3 are redundant and repress mesendoderm differentiation. Thus,instead of being panrepressors of differentiation,each factor controls specific cell fates. Our study revises the view of how self-renewal is orchestrated in hESCs. View Publication

Recent studies have suggested that human pluripotent stem cells (hPSCs) depend primarily on glycolysis and only increase oxidative metabolism during differentiation. Here,we demonstrate that both glycolytic and oxidative metabolism can support hPSC growth and that the metabolic phenotype of hPSCs is largely driven by nutrient availability. We comprehensively characterized hPSC metabolism by using 13C/2H stable isotope tracing and flux analysis to define the metabolic pathways supporting hPSC bioenergetics and biosynthesis. Although glycolytic flux consistently supported hPSC growth,chemically defined media strongly influenced the state of mitochondrial respiration and fatty acid metabolism. Lipid deficiency dramatically reprogramed pathways associated with fatty acid biosynthesis and NADPH regeneration,altering the mitochondrial function of cells and driving flux through the oxidative pentose phosphate pathway. Lipid supplementation mitigates this metabolic reprogramming and increases oxidative metabolism. These results demonstrate that self-renewing hPSCs can present distinct metabolic states and highlight the importance of medium nutrients on mitochondrial function and development. Zhang et al. apply metabolic flux analysis to comprehensively characterize the metabolism of human pluripotent stem cells cultured in different media. Cells maintained in chemically defined media significantly upregulate lipid biosynthesis and redox pathways to compensate for medium lipid deficiency while downregulating oxidative mitochondrial metabolism. View Publication

Development requires coordinated interactions between the epiblast,which generates the embryo proper; the trophectoderm,which generates the placenta; and the hypoblast,which forms both the anterior signalling centre and the yolk sac. These interactions remain poorly understood in human embryogenesis because mechanistic studies have only recently become possible. Here we examine signalling interactions post-implantation using human embryos and stem cell models of the epiblast and hypoblast. We find anterior hypoblast specification is NODAL dependent,as in the mouse. However,while BMP inhibits anterior signalling centre specification in the mouse,it is essential for its maintenance in human. We also find contrasting requirements for BMP in the naive pre-implantation epiblast of mouse and human embryos. Finally,we show that NOTCH signalling is important for human epiblast survival. Our findings of conserved and species-specific factors that drive these early stages of embryonic development highlight the strengths of comparative species studies. Weatherbee,Weberling,Gantner et al. find contrasting requirements for BMP in the anterior signalling centre and pre-implantation epiblast between mice and humans. They further find that NOTCH may be indispensable for human epiblast survival. View Publication

NK cells are key regulators of innate defense against mouse CMV (MCMV). Like NK cells,NKT cells also produce high levels of IFN-γ rapidly after MCMV infection. However,whether similar mechanisms govern activation of these two cell types,as well as the significance of NKT cells for host resistance,remain unknown. In this article,we show that,although both NKT and NK cells are activated via cytokines,their particular cytokine requirements differ significantly in vitro and in vivo. IL-12 is required for NKT cell activation in vitro but is not sufficient,whereas NK cells have the capacity to be activated more promiscuously in response to individual cytokines from innate cells. In line with these results,GM-CSF-derived dendritic cells activated only NK cells upon MCMV infection,consistent with their virtual lack of IL-12 production,whereas Flt3 ligand-derived dendritic cells produced IL-12 and activated both NK and NKT cells. In vivo,NKT cell activation was abolished in IL-12(-/-) mice infected with MCMV,whereas NK cells were still activated. In turn,splenic NK cell activation was more IL-18 dependent. The differential requirements for IL-12 and IL-18 correlated with the levels of cytokine receptor expression by NK and NKT cells. Finally,mice lacking NKT cells showed reduced control of MCMV,and depleting NK cells further enhanced viral replication. Taken together,our results show that NKT and NK cells have differing requirements for cytokine-mediated activation,and both can contribute nonredundantly to MCMV defense,revealing that these two innate lymphocyte subsets function together to fine-tune antiviral responses. View Publication

In most human somatic cells,the lack of telomerase activity results in progressive telomere shortening during each cell division. Eventually,DNA damage responses triggered by critically short telomeres induce an irreversible cell cycle arrest termed replicative senescence. However,the cellular responses of human pluripotent stem cells to telomere uncapping remain unknown. We generated telomerase knockout human embryonic stem (ES) cells through gene targeting. Telomerase inactivation in ES cells results in progressive telomere shortening. Telomere DNA damage in ES cells and neural progenitor cells induces rapid apoptosis when telomeres are uncapped,in contrast to fibroblast cells that enter a state of replicative senescence. Significantly,telomerase inactivation limits the proliferation capacity of human ES cells without affecting their pluripotency. By targeting telomerase activity,we can functionally separate the two unique properties of human pluripotent stem cells,namely unlimited self-renewal and pluripotency. We show that the potential of ES cells to form teratomas in vivo is dictated by their telomere length. By controlling telomere length of ES cells through telomerase inactivation,we can inhibit teratoma formation and potentially improve the safety of cell therapies involving terminally differentiated cells as well as specific progenitor cells that do not require sustained cellular proliferation in vivo,and thus sustained telomerase activity. This article is protected by copyright. All rights reserved. View Publication

Hematopoietic stem cells (HSC) are tightly regulated through,as yet,undefined mechanisms that balance self-renewal and differentiation. We have identified a role for the transcriptional coactivators CREB-binding protein (CBP) and p300 in such HSC fate decisions. A full dose of CBP,but not p300,is crucial for HSC self-renewal. Conversely,p300,but not CBP,is essential for proper hematopoietic differentiation. Furthermore,in chimeric mice,hematologic malignancies emerged from both CBP(-/-) and p300(-/-) cell populations. Thus,CBP and p300 play essential but distinct roles in maintaining normal hematopoiesis,and,in mice,both are required for preventing hematologic tumorigenesis. View Publication