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The role of autophagy,a lysosomal degradation pathway which prevents cellular damage,in the maintenance of adult mouse hematopoietic stem cells (HSCs) remains unknown. Although normal HSCs sustain life-long hematopoiesis,malignant transformation of HSCs leads to leukemia. Therefore,mechanisms protecting HSCs from cellular damage are essential to prevent hematopoietic malignancies. In this study,we crippled autophagy in HSCs by conditionally deleting the essential autophagy gene Atg7 in the hematopoietic system. This resulted in the loss of normal HSC functions,a severe myeloproliferation,and death of the mice within weeks. The hematopoietic stem and progenitor cell compartment displayed an accumulation of mitochondria and reactive oxygen species,as well as increased proliferation and DNA damage. HSCs within the Lin(-)Sca-1(+)c-Kit(+) (LSK) compartment were significantly reduced. Although the overall LSK compartment was expanded,Atg7-deficient LSK cells failed to reconstitute the hematopoietic system of lethally irradiated mice. Consistent with loss of HSC functions,the production of both lymphoid and myeloid progenitors was impaired in the absence of Atg7. Collectively,these data show that Atg7 is an essential regulator of adult HSC maintenance. View Publication

BACKGROUND: Cancer stem cells (CSCs) are purported to be epithelial tumour cells expressing CD44(+)CD24(lo) that exhibit aldehyde dehydrogenase activity (Aldefluor(+)). We hypothesised that if CSCs are responsible for tumour dissemination,disseminated cells in the bone marrow (BM) would be positive for putative breast CSC markers. Therefore,we assessed the presence of Aldefluor(+) epithelial (CD326(+)CD45(dim)) cells for the presence of the CD44(+)CD24(lo) phenotype in BM of patients with primary breast cancer (PBC). METHODS: BM aspirates were collected at the time of surgery from 66 patients with PBC. Thirty patients received neoadjuvant chemotherapy (NACT) prior to aspiration. BM was analysed for Aldefluor(+) epithelial cells with or without CD44(+)CD24(lo) expression by flow cytometry. BM aspirates from three healthy donors (HD) were subjected to identical processing and analyses and served as controls. RESULTS: Patients with triple-receptor-negative (TN) tumours had a significantly higher median percentage of CD44(+)CD24(lo) CSC within Aldefluor(+) epithelial cell population than patients with other immunohistochemical subtypes (P=0.018). Patients with TN tumours or with pN2 or higher pathologic nodal status were more likely to have a proportion of CD44(+)CD24(lo) CSC within Aldefluor(+) epithelial cell population above the highest level of HD. Furthermore,patients who received NACT were more likely to have percentages of Aldefluor(+) epithelial cells than the highest level of HD (P=0.004). CONCLUSION: The percentage of CD44(+)CD24(lo) CSC in the BM is higher in PBC patients with high risk tumour features. The selection or enrichment of Aldefluor(+) epithelial cells by NACT may represent an opportunity to target these cells with novel therapies. View Publication

Patients with Fanconi anemia (FA) have a high risk of developing acute myeloid leukemia (AML). In this study,we attempted to identify cell-surface markers for leukemia-initiating cells in FA-AML patients. We found that the IL-3 receptor-α (IL-3Rα) is a promising candidate as an leukemia-initiating cell-specific antigen for FA-AML. Whereas IL-3Rα expression is undetectable on normal CD34(+)CD38(-) HSCs,it is overexpressed on CD34(+)CD38(-) cells from FA patients with AML. We examined the leukemia-initiating cell activity of IL-3Rα-positive FA-AML cells in a humanized" FA xenotransplant model in which we separated AML cells into IL-3Rα-positive and IL-3Rα-negative CD34 fractions and transplanted them into irradiated recipient mice. In all 3 FA-AML samples View Publication

Human embryonic stem (hES) cells have a potential use for the repair and regeneration of injured tissues. However,teratoma formation can be a major obstacle for hES-mediated cell therapy. Therefore,tracking the fate and function of transplanted hES cells with noninvasive imaging could be valuable for a better understanding of the biology and physiology of teratoma formation. In this study,hES cells were stably transduced with a double fusion reporter gene consisting of firefly luciferase and enhanced green fluorescent protein. Following bioluminescence imaging and histology,we demonstrated that engraftment of hES cells was followed by dramatically increasing signaling and led to teratoma formation confirmed by histology. Studies of the angiogenic processes within teratomas revealed that their vasculatures were derived from both differentiated hES cells and host. Moreover,FACS analysis showed that teratoma cells derived from hES cells expressed high levels of CD56 and SSEA-4,and the subcultured SSEA-4(+) cells showed a similar cell surface marker expression pattern when compared to undifferentiated hES cells. We report here for the first time that SSEA-4(+) cells derived from teratoma exhibited multipotency,retained their differentiation ability in vivo as confirmed by their differentiation into representative three germ layers. View Publication

Human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) are promising can- didate cell sources for regenerative medicine. However,despite the common ability of hiPSCs and hESCs to dif- ferentiate into all 3 germ layers,their functional equivalence at the single cell level remains to be demonstrated. Moreover,single cell heterogeneity amongst stem cell populations may underlie important cell fate decisions. Here,we used single cell analysis to resolve the gene expression profiles of 362 hiPSCs and hESCs for an array of 42 genes that characterize the pluripotent and differentiated states. Comparison between single hESCs and single hiPSCs revealed markedly more heterogeneity in gene expression levels in the hiPSCs,suggesting that hiPSCs occupy an alternate,less stable pluripotent state. hiPSCs also displayed slower growth kinetics and impaired directed differentiation as compared with hESCs. Our results suggest that caution should be exer- cised before assuming that hiPSCs occupy a pluripotent state equivalent to that of hESCs,particularly when producing differentiated cells for regenerative medicine aims. View Publication

Previous studies have indicated that retinoic acid (RA) may be therapeutic for endometrial cancer. However,the downstream target genes and pathways triggered by ligand-activated RA receptor α (RARα) in endometrial cancer cells are largely unknown. In this study,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,flow cytometry,and immunoblotting assays were used to assess the roles of RA and the RA agonist (AM580) in the growth of endometrial cancer cells. Illumina-based microarray expression profiling of endometrial Ishikawa cells incubated with and without AM580 for 1,3,and 6 h was performed. We found that both RA and AM580 markedly inhibited endometrial cancer cell proliferation,while knockdown of RARα could block AM580 inhibition. Knockdown of RARα significantly increased proliferating cell nuclear antigen and BCL2 protein levels. Incubation of Ishikawa cells with or without AM580 followed by microarray expression profiling showed that 12 768 genes out of 47 296 gene probes were differentially expressed with significant P values. We found that 90 genes were the most regulated genes with the most significant P value (Ptextless0.0001) using F-test. We selected four highly regulated genes with diverse functions,namely G0S2,TNFAIP2,SMAD3,and NRIP1. Real-time PCR verified that AM580 highly regulated these genes,whereas chromatin immunoprecipitation-PCR assay demonstrated that ligand-activated RARα interacted with the promoter of these genes in intact endometrial cancer cells. AM580 also significantly altered 18 pathways including those related to cell growth,differentiation,and apoptosis. In conclusion,AM580 treatment of Ishikawa cells causes the differential expression of a number of RARα target genes and activation of signaling pathways. These pathways could,therefore,mediate the carcinogenesis of human endometrial cancer. View Publication

BACKGROUND: Primitive brain tumors are the leading cause of cancer-related death in children. Tumor cells with stem-like properties (TSCs),thought to account for tumorigenesis and therapeutic resistance,have been isolated from high-grade gliomas in adults. Whether TSCs are a common component of pediatric brain tumors and are of clinical relevance remains to be determined. METHODOLOGY/PRINCIPAL FINDINGS: Tumor cells with self-renewal properties were isolated with cell biology techniques from a majority of 55 pediatric brain tumors samples,regardless of their histopathologies and grades of malignancy (57% of embryonal tumors,57% of low-grade gliomas and neuro-glial tumors,70% of ependymomas,91% of high-grade gliomas). Most high-grade glioma-derived oncospheres (10/12) sustained long-term self-renewal akin to neural stem cells (textgreater7 self-renewals),whereas cells with limited renewing abilities akin to neural progenitors dominated in all other tumors. Regardless of tumor entities,the young age group was associated with self-renewal properties akin to neural stem cells (P = 0.05,chi-square test). Survival analysis of the cohort showed an association between isolation of cells with long-term self-renewal abilities and a higher patient mortality rate (P = 0.013,log-rank test). Sampling of low- and high-grade glioma cultures showed that self-renewing cells forming oncospheres shared a molecular profile comprising embryonic and neural stem cell markers. Further characterization performed on subsets of high-grade gliomas and one low-grade glioma culture showed combination of this profile with mesenchymal markers,the radio-chemoresistance of the cells and the formation of aggressive tumors after intracerebral grafting. CONCLUSIONS/SIGNIFICANCE: In brain tumors affecting adult patients,TSCs have been isolated only from high-grade gliomas. In contrast,our data show that tumor cells with stem cell-like or progenitor-like properties can be isolated from a wide range of histological sub-types and grades of pediatric brain tumors. They suggest that cellular mechanisms fueling tumor development differ between adult and pediatric brain tumors. View Publication

Reprogramming blood cells to induced pluripotent stem cells (iPSCs) provides a novel tool for modeling blood diseases in vitro. However,the well-known limitations of current reprogramming technologies include low efficiency,slow kinetics,and transgene integration and residual expression. In the present study,we have demonstrated that iPSCs free of transgene and vector sequences could be generated from human BM and CB mononuclear cells using non-integrating episomal vectors. The reprogramming described here is up to 100 times more efficient,occurs 1-3 weeks faster compared with the reprogramming of fibroblasts,and does not require isolation of progenitors or multiple rounds of transfection. Blood-derived iPSC lines lacked rearrangements of IGH and TCR,indicating that their origin is non-B- or non-T-lymphoid cells. When cocultured on OP9,blood-derived iPSCs could be differentiated back to the blood cells,albeit with lower efficiency compared to fibroblast-derived iPSCs. We also generated transgene-free iPSCs from the BM of a patient with chronic myeloid leukemia (CML). CML iPSCs showed a unique complex chromosomal translocation identified in marrow sample while displaying typical embryonic stem cell phenotype and pluripotent differentiation potential. This approach provides an opportunity to explore banked normal and diseased CB and BM samples without the limitations associated with virus-based methods. View Publication

We analyzed in B-chronic lymphocytic leukemia (B-CLL) whole blood assays the activity of therapeutic mAbs alemtuzumab,rituximab,and type II glycoengineered anti-CD20 mAb GA101. Whole blood samples were treated with Abs,and death of CD19(+) B-CLL was measured by flow cytometry. Alemtuzumab efficiently lysed B-CLL targets with maximal lysis at 1-4 h (62%). In contrast,rituximab induced a more limited cell death (21%) that was maximal only at 24 h. GA101 killed B-CLL targets to a similar extent but more rapidly than rituximab,with 19.2 and 23.5% cell death at 4 and 24 h,respectively,compared with 7.9 and 21.4% for rituximab. Lysis by both rituximab and GA101 correlated directly with CD20 expression levels (r(2) = 0.88 and 0.85,respectively). Interestingly,lysis by all three Abs at high concentrations was mostly complement dependent,because it was blocked by the anti-C5 Ab eculizumab by 90% in the case of alemtuzumab and rituximab and by 64% in the case of GA101. Although GA101 caused homotypic adhesion,it induced only limited (3%) direct cell death of purified B-CLL cells. Both rituximab and GA101 showed the same efficiency in phagocytosis assays,but phagocytosis was not significant in whole blood due to excess Igs. Finally,GA101 at 1-100 μg/ml induced 2- to 3-fold more efficient NK cell degranulation than rituximab in isolated B-CLL or normal PBMCs. GA101,but not rituximab,also mediated significant NK cell degranulation in whole blood samples. Thus,complement and Ab-dependent cellular cytotoxicity are believed to be the major effector mechanisms of GA101 in whole blood assays. View Publication

Induced pluripotent stem cells (iPSCs) offer immense potential for regenerative medicine and studies of disease and development. Somatic cell reprogramming involves epigenomic reconfiguration,conferring iPSCs with characteristics similar to embryonic stem (ES) cells. However,it remains unknown how complete the reestablishment of ES-cell-like DNA methylation patterns is throughout the genome. Here we report the first whole-genome profiles of DNA methylation at single-base resolution in five human iPSC lines,along with methylomes of ES cells,somatic cells,and differentiated iPSCs and ES cells. iPSCs show significant reprogramming variability,including somatic memory and aberrant reprogramming of DNA methylation. iPSCs share megabase-scale differentially methylated regions proximal to centromeres and telomeres that display incomplete reprogramming of non-CG methylation,and differences in CG methylation and histone modifications. Lastly,differentiation of iPSCs into trophoblast cells revealed that errors in reprogramming CG methylation are transmitted at a high frequency,providing an iPSC reprogramming signature that is maintained after differentiation. View Publication

Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis that in many cases is caused by mutations of the ELANE gene,which encodes neutrophil elastase (NE). Recent data suggest a model in which ELANE mutations result in NE protein misfolding,induction of endoplasmic reticulum (ER) stress,activation of the unfolded protein response (UPR),and ultimately a block in granulocytic differentiation. To test this model,we generated transgenic mice carrying a targeted mutation of Elane (G193X) reproducing a mutation found in SCN. The G193X Elane allele produces a truncated NE protein that is rapidly degraded. Granulocytic precursors from G193X Elane mice,though without significant basal UPR activation,are sensitive to chemical induction of ER stress. Basal and stress granulopoiesis after myeloablative therapy are normal in these mice. Moreover,inaction of protein kinase RNA-like ER kinase (Perk),one of the major sensors of ER stress,either alone or in combination with G193X Elane,had no effect on basal granulopoiesis. However,inhibition of the ER-associated degradation (ERAD) pathway using a proteosome inhibitor resulted in marked neutropenia in G193X Elane. The selective sensitivity of G913X Elane granulocytic cells to ER stress provides new and strong support for the UPR model of disease patho-genesis in SCN. View Publication

BACKGROUND: Cancer stem cells (CSCs) have been shown to be a small stem cell-like cell population which appears to drive tumorigenesis,tumor recurrence and metastasis. Thus,identification and characterization of CSCs may be critical to defining effective anticancer therapies. In prostate cancer (PCa),the CD44(+) cell population appears to have stem cell-like properties including being tumorigenic. The enzyme aldehyde dehydrogenase (ALDH) has been found to identify hematopoietic stem cells and our aim was to determine the utility of ALDH activity and CD44 in identifying PCa stem cell-like cells in PCa cell lines. MATERIALS AND METHODS: LNCaP cells and PC-3 cells were sorted based on their expression of CD44 and ALDH activity. The cell populations were investigated using colony-forming assays,invasion assays,sphere formation experiments in a non-adherent environment and 3-D Matrigel matrix culture to observe the in vitro stem-cell like properties. Different sorted cell populations were injected subcutaneously into NOD/SCID mice to determine the corresponding tumorigenic capacities. RESULTS: ALDH(hi) CD44(+) cells exhibit a higher proliferative,clonogenic and metastatic capacity in vitro and demonstrate higher tumorigenicity capacity in vivo than did ALDH(lo) CD44(-) cells. The tumors recapitulated the population of the original cell line. However,ALDHlo CD44(-) cells were able to develop tumors,albeit with longer latency periods. CONCLUSION: ALDH activity and CD44 do not appear to identify PCa stem cells; however,they do indicate increased tumorigenic and metastatic potential,indicating their potential importance for further exploration. View Publication