SUMMARY Adipose tissue (AT) has previously been identified as an extra-medullary reservoir for normal hematopoietic stem cells (HSCs) and may promote tumor development. Here, we show a subpopulation of leukemic stem cells (LSCs) can utilize gonadal adipose tissue (GAT) as a niche to support their metabolism and evade chemotherapy. In a mouse model of blast crisis CML, adipose-resident LSCs exhibit a pro-inflammatory phenotype and induce lipolysis in GAT. GAT lipolysis fuels fatty acid oxidation in LSCs, especially within a subpopulation expressing the fatty acid transporter CD36. CD36+ LSCs have unique metabolic properties, are strikingly enriched in AT, and are protected from chemotherapy by the GAT microenvironment. CD36 also marks a fraction of human blast crisis CML and AML cells with similar biological properties. These findings suggest striking interplay between leukemic cells and adipose tissue to create a unique microenvironment that supports the metabolic demands and survival of a distinct LSC subpopulation.
A fundamental tenet of scientific research is that published results are open to independent validation and refutation. Minimum data standards aid data providers, users, and publishers by providing a specification of what is required to unambiguously interpret experimental findings. Here, we present the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard, stating the minimum information required to report flow cytometry (FCM) experiments. We brought together a crossdisciplinary international collaborative group of bioinformaticians, computational statisticians, software developers, instrument manufacturers, and clinical and basic research scientists to develop the standard. The standard was subsequently vetted by the International Society for Advancement of Cytometry (ISAC) Data Standards Task Force, Standards Committee, membership, and Council. The MIFlowCyt standard includes recommendations about descriptions of the specimens and reagents included in the FCM experiment, the configuration of the instrument used to perform the assays, and the data processing approaches used to interpret the primary output data. MIFlowCyt has been adopted as a standard by ISAC, representing the FCM scientific community including scientists as well as software and hardware manufacturers. Adoption of MIFlowCyt by the scientific and publishing communities will facilitate third-party understanding and reuse of FCM data. ' 2008 International Society for Advancement of Cytometry Key termsimmunology; fluorescence-activated cell sorting; knowledge representation FLOW cytometry (FCM) systems have been available to investigators for over 30 years, and the field continues to advance at a rapid rate. FCM has been responsible for major progress in basic and clinical research by enabling the phenotypic and functional characterization of individual cells in a high-throughput manner. Advances in the technology now allow for automated, multiparametric analyses of thousands of samples per day (1). Each data set can consist of multidimensional descriptions of millions of individual cells, producing data similar in size and complexity to gene expression microarrays. Like the microarray field, the ability to collect FCM data is outpacing the computational means for data handling and analysis. Furthermore, the lack of reporting standardization limits collaboration, independent validation/refutation, and meta-analysis, and thus minimizes the value of the wealth
IntroductionEach day, the hematopoietic system of the adult mouse produces billions of mature blood cells. The multistep process that underlies the production of these cells is controlled by complex mechanisms that enable changing physiologic demands to be met without overwhelming the system. It is now clear that a small population of cells known as hematopoietic stem cells (HSCs) are ultimately responsible for maintaining the lifelong output of new blood cells. 1 Nevertheless, a molecular understanding of what constitutes an HSC and how its key functions are maintained are still poorly understood.The existence of hematopoietic cells with the individual potential to produce large numbers of multiple blood cell types in vivo for prolonged periods was first suggested by retrospective clonal tracking experiments that used unique chromosomal, 2 and later, retroviral marking approaches. 3,4 The relatively short lifespan of many mature blood cell types and the contrasting longevity of some of the multilineage clones identified in these experiments implied an origin of the clones from undifferentiated cells with an extensive ability to divide and maintain a derivative population of similarly undifferentiated cells. Serial transplantation experiments demonstrated that such cell divisions did occur and thus provided the first definitive evidence of HSC self-renewal. 3,4 These findings prompted a search for functional end points that would allow HSCs to be specifically quantified independent of the presence of other cell types when assayed in limiting dilution transplantation strategies using suitably irradiated congenic hosts. 5,6 A sustained output of at least 1% of all the circulating white blood cells (WBCs) for at least 4 months is now widely assumed to be suitable for this purpose. 7 Interestingly, most analyses of individual pluripotent hematopoietic cells proliferating either in vivo or in vitro have generally found the self-renewal activity actually displayed to be highly variable. 3,4,8,9 How such a variable behavior is related to the molecular state of the initial cells is not well defined and remains a subject of intense interest and investigation. 10 Variations in external cues may be one contributing parameter, at least in vivo, because it is known that self-renewal responses can be directly and rapidly modulated in this way in vitro. 11,12 In addition, there is some evidence of predetermined heterogeneity in HSC self-renewal potential. This is exemplified by the differences in regenerative activity of HSCs from fetal and adult sources 13,14 and the finding of a consistent association of short-term and long-term multilineage WBC outputs with distinct phenotypes of adult bone marrow (BM) cells (eg, according to their expression of CD49b and CD34). 15,16 Taken together, these results suggest that some hematopoietic cells can remain pluripotent for several divisions even though they are already destined to undergo terminal differentiation within a finite period. This is the basis of the concept of durable versu...
The leukemic stem cells in patients with chronic myeloid leukemia (CML) are well known to be clinically resistant to conventional chemotherapy and may also be relatively resistant to BCR-ABL-targeted drugs. Here we show that the lesser effect of imatinib mesylate (IM) on the 3-week output of cells produced in vitro from lin À CD34 þ CD38 À CML (stem) cells compared with cultures initiated with the CD38 þ subset of lin À CD34 þ cells is markedly enhanced (410-fold) when conditions of reduced growth factor stimulation are used. Quantitative analysis of genes expressed in these different CML subsets revealed a differentiation-associated decrease in IL-3 and G-CSF transcripts, a much more profound decrease in expression of BCR-ABL than predicted by changes in BCR expression, decreasing expression of ABCB1/MDR and ABCG2 and increasing expression of OCT1. p210 BCR-ABL and kinase activity were also higher in the lin À CD34 þ CD38 À cells and formal evidence that increasing BCR-ABL expression decreases IM sensitivity was obtained from experiments with a cell line model. Nevertheless, within the entire CD34 þ subset of CML cells, BCR-ABL expression was not strongly affected by changes in cell cycle status. Taken together, these results provide the first evidence of multiple mechanisms of innate IM resistance in primitive and quiescent CML cells. Leukemia (2007) 21, 926-935.
Objective-Strategies to expand hematopoietic stem cells (HSCs) ex vivo are of key interest. The objective of this study was to resolve if ability of HOXB4, previously documented to induce a significant expansion of HSCs in culture, may extend to other HOX genes and also to further analyze the HOX sequence requirements to achieve this effect.Methods-To investigate the ability of Nucleoporin98-Homeobox fusion genes to stimulate HSC self-renewal, we evaluated their presence in 10-to 20-day cultures of transduced mouse bone marrow cells. Stem cell recovery was measured by limiting-dilution assay for long-term competitive repopulating cells (CRU Assay).Results-These experiments revealed remarkable expansions of Nucleoporin98-Homeoboxtransduced HSCs (1000-fold to 10,000-fold over input) in contrast to the expected decline of HSCs in control cultures. Nevertheless, the Nucleoporin98-Homeobox-expanded HSCs displayed no proliferative senescence and retained normal lympho-myeloid differentiation activity and a controlled pool size in vivo. Analysis of proviral integration patterns showed the cells regenerated in vivo were highly polyclonal, indicating they had derived from a large proportion of the initially targeted HSCs. Importantly, these effects were preserved when all HOX sequences flanking the homeodomain were removed, thus defining the homeodomain as a key and independent element in the fusion.Conclusion-These findings create new possibilities for investigating HSCs biochemically and genetically and for achieving clinically significant expansion of human HSCs.The self-renewal function of hematopoietic stem cells (HSCs) is essential to their ability to sustain lifelong hematopoiesis and to regenerate the hematopoietic system after myeloablative treatments. This property is the basis of an increasing range of applications of HSC transplants to treat various malignant and genetic disorders [1,2]. Further improvements in the safety and therapeutic utility of HSC transplants can be readily envisaged if robust methods for largescale ex vivo expansion of HSCs were available. For example, the low absolute numbers of HSCs in most cord blood samples [3] [4] restrict the clinical utility of these products. A method for significantly expanding HSCs could not only address these insufficiencies but also broaden the exploitation of reduced conditioning regimens and associated therapeutic benefits.One approach that has allowed some HSC expansion in vitro to be achieved has focused on the identification of optimized combinations and concentrations of externally acting growth factors and related molecules [5][6][7][8][9][10][11][12]. A complementary approach has been to identify intrinsic regulators such as transcription factors [13] and key mediators of signaling pathways [14,15] that can be manipulated to activate or promote HSC self-renewal divisions. A striking example of the latter strategy is the use of retrovirally engineered overexpression of the homeobox transcription factor HOXB4 to stimulate expansions of HSC numb...
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