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p400/mDomino is an ATP-dependent chromatin-remodeling protein that catalyzes the deposition of histone variant H2A.Z into nucleosomes to regulate gene expression. We previously showed that p400/mDomino is essential for embryonic development and primitive hematopoiesis. Here we generated a conditional knock-out mouse for the p400/mDomino gene and investigated the role of p400/mDomino in adult bone marrow hematopoiesis and in the cell-cycle progression of embryonic fibroblasts. The Mx1-Cre- mediated deletion of p400/mDomino resulted in an acute loss of nucleated cells in the bone marrow,including committed myeloid and erythroid cells as well as hematopoietic progenitor and stem cells. A hematopoietic colony assay revealed a drastic reduction in colony-forming activity after the deletion of p400/mDomino. Moreover,the loss of p400/mDomino in mouse embryonic fibroblasts (MEFs) resulted in strong growth inhibition. Cell-cycle analysis revealed that the mDomino-deficient MEFs exhibited a pleiotropic cell-cycle defect at the S and G(2)/M phases,and polyploid and multi-nucleated cells with micronuclei emerged. DNA microarray analysis revealed that the p400/mDomino deletion from MEFs caused the impaired expression of many cell-cycle-regulatory genes,including G(2)/M-specific genes targeted by the transcription factors FoxM1 and c-Myc. These results indicate that p400/mDomino plays a key role in cellular proliferation by controlling the expression of cell-cycle-regulatory genes. View Publication

In up to one-third of patients with acute myeloid leukemia,a C-terminal frame-shift mutation results in abnormal and abundant cytoplasmic accumulation of the usually nucleoli-bound protein nucleophosmin (NPM),and this is thought to function in cancer pathogenesis. Here,we demonstrate a gain-of-function role for cytoplasmic NPM in the inhibition of caspase signaling. The NPM mutant specifically inhibits the activities of the cell-death proteases,caspase-6 and -8,through direct interaction with their cleaved,active forms,but not the immature procaspases. The cytoplasmic NPM mutant not only affords protection from death ligand-induced cell death but also suppresses caspase-6/-8-mediated myeloid differentiation. Our data hence provide a potential explanation for the myeloid-specific involvement of cytoplasmic NPM in the leukemogenesis of a large subset of acute myeloid leukemia. View Publication

The p53 tumor suppressor limits proliferation in response to cellular stress through several mechanisms. Here,we test whether the recently described ability of p53 to limit stem cell self-renewal suppresses tumorigenesis in acute myeloid leukemia (AML),an aggressive cancer in which p53 mutations are associated with drug resistance and adverse outcome. Our approach combined mosaic mouse models,Cre-lox technology,and in vivo RNAi to disable p53 and simultaneously activate endogenous Kras(G12D)-a common AML lesion that promotes proliferation but not self-renewal. We show that p53 inactivation strongly cooperates with oncogenic Kras(G12D) to induce aggressive AML,while both lesions on their own induce T-cell malignancies with long latency. This synergy is based on a pivotal role of p53 in limiting aberrant self-renewal of myeloid progenitor cells,such that loss of p53 counters the deleterious effects of oncogenic Kras on these cells and enables them to self-renew indefinitely. Consequently,myeloid progenitor cells expressing oncogenic Kras and lacking p53 become leukemia-initiating cells,resembling cancer stem cells capable of maintaining AML in vivo. Our results establish an efficient new strategy for interrogating oncogene cooperation,and provide strong evidence that the ability of p53 to limit aberrant self-renewal contributes to its tumor suppressor activity. View Publication

Changes in progenitor cell biology remain at the forefront of many theories of biologic aging,but there are limited studies evaluating this in humans. Aging has been associated with a progressive depletion of circulating progenitor cells,but age-related bone marrow-resident progenitor cell depletion has not been systematically determined in humans. Patients undergoing total hip replacement were consented,and bone marrow and peripheral progenitor cells were enumerated based on aldehyde dehydrogenase activity and CD34 and CD133 expression. Circulating progenitors demonstrated an age-dependent decline. In contrast,marrow-resident progenitor cell content demonstrated no age association with any progenitor cell subtype. In humans,aging is associated with depletion of circulating,but not marrow-resident,progenitors. This finding has impact on the mechanism(s) responsible for age-related changes in circulating stem cells and important implications for the use of autologous marrow for the treatment of age-related diseases. View Publication

Osteoclasts are resident cells of the bone that are primarily involved in the physiological and pathological remodeling of this tissue. Mature osteoclasts are multinucleated giant cells that are generated from the fusion of circulating precursors originating from the monocyte/macrophage lineage. During inflammatory bone conditions in vivo,de novo osteoclastogenesis is observed but it is currently unknown whether,besides increased osteoclast differentiation from undifferentiated precursors,other cell types can generate a multinucleated giant cell phenotype with bone resorbing activity. In this study,an animal model of calvaria-induced aseptic osteolysis was used to analyze possible bone resorption capabilities of dendritic cells (DCs). We determined by FACS analysis and confocal microscopy that injected GFP-labeled immature DCs were readily recruited to the site of osteolysis. Upon recruitment,the cathepsin K-positive DCs were observed in bone-resorbing pits. Additionally,chromosomal painting identified nuclei from female DCs,previously injected into a male recipient,among the nuclei of giant cells at sites of osteolysis. Finally,osteolysis was also observed upon recruitment of CD11c-GFP conventional DCs in Csf1r(-/-) mice,which exhibit a severe depletion of resident osteoclasts and tissue macrophages. Altogether,our analysis indicates that DCs may have an important role in bone resorption associated with various inflammatory diseases. View Publication

CD44+/CD24-/low tumor cells or aldehyde dehydrogenase 1 (ALDH1) positive tumor cells are considered cancer stem cells (CSCs) that possess the properties of self-renewal and tumorigenicity. However,their clinical value and significance in breast cancer remain controversial. A meta-analysis based on published studies was performed with the aim of obtaining an accurate evaluation of the association between the presence of CSCs in clinical samples and clinical outcome. A total of 12 eligible studies with 898 cases and 1,853 controls were included. CSC positive breast cancers,in particular those positive for ALDH1,were significantly associated with high histological grade,estrogen receptor (ER) negativity,progesterone receptor (PR) negativity,and human epidermal growth factor receptor type 2 (HER2) positivity. However,the presence of cancer stem cells was not associated with tumor size or nodal status. ALDH1 positive (RR = 2.83,95% CI: 2.16-3.67,P textless 0.001) and CD44+/CD24-/low tumor cells (RR = 2.32,95% CI: 1.51-3.60,P textless 0.001) were significantly associated with poor overall survival (OS). The stem cell markers are prognostic factors in breast cancer. Larger clinical studies are required to further evaluate the role of these markers in clinical practice. View Publication

Bone Morphogenetic Protein (BMP) signaling pathways are involved in differentiation of stem cells into diverse cell types,and thus BMPs can be used as main guidance molecules for in vitro differentiation of human stem cells. View Publication

Cancer stem cells (CSCs) are a subpopulation of tumor cells with preferential tumor-initiating capacity and have been purported to be resistant to chemotherapy. It has been shown that breast CSC are,on average,enriched in patient tumors after combination neoadjuvant chemotherapy including docetaxel,doxorubicin,and cyclophosphamide (CPA). Here,we investigate the resistance of breast CSC to CPA alone in a xenograft model. CPA treatment led to a 48% reduction in tumor volume during a 2-week period. Cells bearing the CD44(+) CD24(-) phenotype were reduced by 90% (2.5% to 0.24%) in CPA-treated tumors,whereas cells with aldehyde dehydrogenase activity were reduced by 64% (4.7% to 1.7%). A subsequent functional analysis showed that CPA-treated tumors were impaired in their ability to form tumors,indicating loss of functional tumor-initiating activity. These results are consistent with a CSC phenotype that is sensitive to CPA and indicate that some patient CSC may not display the expected resistance to therapy. Deciphering the mechanism for this difference may lead to therapies to counteract resistance. View Publication

Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of the blastocyst. Because of their ability to differentiate into a variety of cell types,human embryonic stem cells (hESCs) provide an unlimited source of cells for clinical medicine and have begun to be used in clinical trials. Presently,although several hundred hESC lines are available in the word,only few have been widely used in basic and applied research. More and more hESC lines with differing genetic backgrounds are required for establishing a bank of hESCs. Here,we report the first Canadian hESC lines to be generated from cryopreserved embryos and we discuss how we navigated through the Canadian regulatory process. The cryopreserved human zygotes used in this study were cultured to the blastocyst stage,and used to isolate ICM via microsurgery. Unlike previous microsurgery methods,which use specialized glass or steel needles,our method conveniently uses syringe needles for the isolation of ICM and subsequent hESC lines. ICM were cultured on MEF feeders in medium containing FBS or serum replacer (SR). Resulting outgrowths were isolated,cut into several cell clumps,and transferred onto fresh feeders. After more than 30 passages,the two hESC lines established using this method exhibited normal morphology,karyotype,and growth rate. Moreover,they stained positively for a variety of pluripotency markers and could be differentiated both in vitro and in vivo. Both cell lines could be maintained under a variety of culture conditions,including xeno-free conditions we have previously described. We suggest that this microsurgical approach may be conducive to deriving xeno-free hESC lines when outgrown on xeno-free human foreskin fibroblast feeders. View Publication

The 8p11 myeloproliferative syndrome (EMS),also referred to as stem cell leukemia/lymphoma,is a chronic myeloproliferative disorder that rapidly progresses into acute leukemia. Molecularly,EMS is characterized by fusion of various partner genes to the FGFR1 gene,resulting in constitutive activation of the tyrosine kinases in FGFR1. To date,no previous study has addressed the functional consequences of ectopic FGFR1 expression in the potentially most relevant cellular context,that of normal primary human hematopoietic cells. Herein,we report that expression of ZMYM2/FGFR1 (previously known as ZNF198/FGFR1) or BCR/FGFR1 in normal human CD34(+) cells from umbilical-cord blood leads to increased cellular proliferation and differentiation toward the erythroid lineage in vitro. In immunodeficient mice,expression of ZMYM2/FGFR1 or BCR/FGFR1 in human cells induces several features of human EMS,including expansion of several myeloid cell lineages and accumulation of blasts in bone marrow. Moreover,bone marrow fibrosis together with increased extramedullary hematopoiesis is observed. This study suggests that FGFR1 fusion oncogenes,by themselves,are capable of initiating an EMS-like disorder,and provides the first humanized model of a myeloproliferative disorder transforming into acute leukemia in mice. The established in vivo EMS model should provide a valuable tool for future studies of this disorder. View Publication

BACKGROUND: CBL missense mutations have recently been associated with juvenile myelomonocytic leukaemia (JMML),an aggressive myeloproliferative and myelodysplastic neoplasm of early childhood characterised by excessive macrophage/monocyte proliferation. CBL,an E3 ubiquitin ligase and a multi-adaptor protein,controls proliferative signalling networks by downregulating the growth factor receptor signalling cascades in various cell types. METHODS AND RESULTS: CBL mutations were screened in 65 patients with JMML. A homozygous mutation of CBL was found in leukaemic cells of 4/65 (6%) patients. In all cases,copy neutral loss of heterozygosity of the 11q23 chromosomal region,encompassing the CBL locus,was demonstrated. Three of these four patients displayed additional features suggestive of an underlying developmental condition. A heterozygous germline CBL p.Y371H substitution was found in each of them and was inherited from the father in one patient. The germline mutation represents the first hit,with somatic loss of heterozygosity being the second hit positively selected in JMML cells. The three patients display a variable combination of dysmorphic features,hyperpigmented skin lesions and microcephaly that enable a 'CBL syndrome' to be tentatively delineated. Learning difficulties and postnatal growth retardation may be part of the phenotype. CONCLUSION: A report of germline mutations of CBL in three patients with JMML is presented here,confirming the existence of an unreported inheritable condition associated with a predisposition to JMML. View Publication

Understanding the complex mechanisms that govern the fate decisions of human embryonic stem cells (hESCs) is fundamental to their use in cell replacement therapies. The progress of dissecting these mechanisms will be facilitated by the availability of robust high-throughput screening assays on hESCs. In this study,we report an image-based high-content assay for detecting compounds that affect hESC survival or pluripotency. Our assay was designed to detect changes in the phenotype of hESC colonies by quantifying multiple parameters,including the number of cells in a colony,colony area and shape,intensity of nuclear staining,and the percentage of cells in the colony that express a marker of pluripotency (TRA-1-60),as well as the number of colonies per well. We used this assay to screen 1040 compounds from two commercial compound libraries,and identified 17 that promoted differentiation,as well as 5 that promoted survival of hESCs. Among the novel small compounds we identified with activity on hESC are several steroids that promote hESC differentiation and the antihypertensive drug,pinacidil,which affects hESC survival. The analysis of overlapping targets of pinacidil and the other survival compounds revealed that activity of PRK2,ROCK,MNK1,RSK1,and MSK1 kinases may contribute to the survival of hESCs. View Publication