Abstracts — Day 1

Normal and Neoplastic Stem Cells
Irving Weissman, MD, Stanford University

Following embryonic development, most of our tissues and organs are continuously regenerated from tissue/organ specific stem cells. The principal property that distinguishes such stem cells from their daughter cells is self-renewal; when stem cells divide they give rise to stem cells (by self-renewal) and progenitors (by differentiation). In most tissues, only the primitive stem cells self-renew. Stem cell isolation and transplantation is the basis for regenerative medicine. Self-renewal is dangerous, and therefore strictly regulated. Poorly regulated self renewal can lead to the genesis of cancer stem cells, the only self-renewing cells in the cancerous tumor. The Weissman lab has followed the progression from hematopoietic stem cells to myelogenous leukemias. They have found that the developing cancer clones progress at the stage of hematopoietic stem cells, until they become fully malignant. At this point, the ‘leukemia’ stem cell moves to a stage of a downstream oligolineage or multilineage progenitor that has evaded programmed cell death and programmed cell removal, while acquiring or keeping self-renewal. While there are many ways to defeat programmed cell death and senescence, there appears to be one dominant method to avoid programmed cell removal — the expression of the cell surface ‘don’t eat me’ protein, CD47, the ligand for macrophage SIRP-alpha. All cancers tested express CD47 to overcome expression of ‘eat me’ signals such as calreticulin and asialogylycoproteins. Antibodies that block the CD47–SIRP-alpha interaction enable phagocytosis and killing of the tumor cells in vitro and in vivo.
 

Myeloproliferative Neoplasm Development Remodels the Osteoblastic Bone Marrow Niche and Promotes Myelofibrosis
Emmanuelle Passegué, PhD, The Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, University of California, San Francisco

Hematopoietic stem cell (HSC) function is influenced by the activity of bone marrow (BM) niche cells, including mesenchymal stem cells (MSC) and their osteoblastic lineage cell (OBC) derivatives. We show that the development of myeloproliferative neoplasms (MPN), such as chronic myelogenous leukemia (CML), causes a severe remodeling of the BM microenvironment and negatively affects the ability of OBCs to support HSC function. This remodeling is a direct consequence of leukemic myeloid cells stimulating MSCs to overproduce functionally altered OBC derivatives, which accumulate in the BM cavity as inflammatory myelofibrotic cells. These remodeled OBCs, in turn, show profound molecular and functional deregulations, and favor myeloid differentiation at the expense of HSC maintenance. These changes preferentially affect the activity of normal HSCs with minimal effect on the leukemic stem cell (LSC) activity of transformed HSCs. Taken together, our results describe a novel self-reinforcing mechanism wherein MPN development remodels the osteoblastic BM niche and creates a leukemic niche that promote myelofibrosis and impaired maintenance of normal hematopoiesis. Targeting this vicious interplay could represent a novel avenue to treat myeloid malignancies and restore normal blood function.
 

Niche Targeting of Leukemia Stem Cells
Catriona Jamieson, MD, PhD, University of California, San Diego

Human leukemia stem cells (LSC) evolve from progenitors as a result of acquisition of subversion of stem cell properties, such as self-renewal, survival and dormancy. This process of malignant progenitor reprogramming occurs in inflammatory niches, in part, through alternative GSK3beta and BCL2 family gene splice isoform expression that promotes LSC maintenance. Lentivirally enforced expression of BCR-ABL and JAK2 promotes TNF and STAT-mediated activation of adenosine deaminase RNA associated (ADAR1), which contributes to RNA editing and alternative splicing. While shRNA knockdown of ADAR1 prevents LSC self-renewal in blast crisis CML humanized mouse models, overexpression leads to myeloid progenitor expansion and GSK3beta missplicing. Deregulation of GSK3beta prevents phosphorylation and degradation of both beta-catenin and GLI sonic hedgehog pathway transcriptional activators resulting in increased LSC self-renewal, survival and dormancy. Recent pre-clinical and clinical studies demonstrate that sonic hedgehog inhibition abrogates LSC dormancy and survival thereby reducing maintenance in the niche. Furthermore, whole transcriptome RNA sequencing reveals that these LSC upregulate pro-survival BCL2 isoforms and downregulate pro-apoptotic genes in the marrow niche, which can be abrogated by a novel panBCL2 inhibitor, sabutoclax. This panBCL2 inhibitor targets quiescent LSC in the marrow and prolongs survival of serially transplanted recipients while sparing normal hematopoietic stem cells thereby providing the impetus for scale up and production for potential LSC targeted clinical trials that will obviate relapse.
 

Niche and Signaling Regulation of the State and Fate of Stem Cells
Linheng Li, PhD, Stowers Institute for Medical Research

Hematopoietic stem cells (HSCs) are maintained in balance between quiescent state and proliferating state. While proliferating HSCs are critical for supporting the routine blood production, quiescent HSCs are essential for long-term maintenance and also can be roused to replenish lost active HSCs. How the different states of HSCs are regulated is a fundamental question. The underlying signaling to regulate the quiescence and activation in different niches remains largely unknown. To address this question, we have analyzed the expression profile of Wnt receptors, Frizzleds, in HSCs. We found that noncanonical Wnt signaling — via receptor Frizzled8 (Fz8) and co-receptor Flamingo — presents in and functionally maintains quiescent HSCs in the endosteal (an inner bone surface) zone. Recently, we detected another noncanonical Wnt receptor, Frizzled5 (Fz5), is expressed in metabolically active (indicated by Mitotracker) HSCs and also in Nestin-GFP+ mesenchymal stem cells (MSCs) in the perivascular zone of central marrow. Fz5 is not expressed in H2B-GFP label-retaining quiescent HSCs, or in endosteal cells, or in sinusoidal cells. Using an Mx1-Cre:Fz5 knockout mouse model, we found a 60% decrease of HSCs isolated from central marrow, but no change in HSCs isolated from endosteum. Functionally, hematopoietic reconstitution was not affected in the primary transplantation, but was substantially decreased (by 80%) in the secondary transplantation compared to the control. This indicates that Fz5 maintains HSCs in the perivascular zone. We propose that noncanonical Wnt signaling maintains quiescent and active HSCs residing, respectively, in the endosteal and perivascular zones. In these zones, Fz8 and Fz5 are differentially expressed and mediate noncanonical Wnt signaling to maintain HSCs in the endosteal niche and to regulate active HSCs in the perivascular niche.
 

Niche Initiated Oncogenesis
David T. Scadden, MD, Massachusetts General Hospital; Harvard University

The microenvironment in which tumor cells reside is a recognized modulator of tumor cell behavior. Whether that environment can participate in the induction of cancer is less clear and difficult to investigate, in part, because of our limited understanding of the specific cell types comprising ‘stroma.’ The hematopoietic system has been informative in exploring multiple aspects of tissue homeostasis and malignant transformation. Among these, is defining heterologous cells in the microenvironment that can serve a regulatory role including the regulation of hematopoietic stem cells. Specific mesenchymal cells in bone have been shown to serve as niche components for stem cells in the bone marrow stroma. By genetically modifying subsets of osteolineage cells, we induced perturbation of stem cell function and caused disordered hematopoiesis. The resulting myelodysplasia was microenvironment dependent and resulted in the emergence of a frank leukemia with distinctive secondary genetic abnormalities.These genetic abnormalities did not include the gene deletion we induced in the microenvironement. The multi-step process of oncogenesis may then include an initiating step in heterologous cells that comprise ‘stroma.’ To test whether a dependence on stroma was retained, we transplanted the leukemic cells and found that the leukemia could only engraft in recipients who had the genetically altered osteolineage cells. Therefore the interaction between the microenvironmental cells and the hematopoietic cells was capable of initiating malignancy and appeared to be necessary for its maintenance. The dependence on interaction between cell types offers the potential for intervention at the points of cell-cell interaction in treatment and prevention strategies.
 

Contribution of the Reprogrammed Vascular Niche to Stem Cell Self-Renewal and Organ Regeneration
Jason M. Butler, PhD, Bisen Ding, PhD, Daylon James, PhD, Daniel Nolan, PhD, Michael Ginsberg, PhD, Sina Rabbany, PhD, and Shahin Rafii, MD, Weill Cornell Medical College, Ansary Stem Cell Institute; Howard Hughes Medical Institute, New York

Organ specific endothelial cells (ECs) are not just passive conduits to deliver oxygen and nutrients, but also establish an instructive vascular niche, which by elaboration of specific paracrine trophogens, (known as angiocrine factors), directly balance the rate of stem cell self-renewal and differentiation. Activation of Akt-mTOR pathway in the sinusoidal ECs (SECs) stimulates expression of angiocrine factors, including Notch-ligands, Wnts, FGFs and TGF modulators, that induce expansion of authentic hematopoietic stem cells (Cell Stem Cell, 3:251-64. 2010). While MAPkinase induces expression of angiocrine factors, that support differentiation of the stem cells into lineage committed progenitors.
 
After partial hepatectomy, SECs within the liver stimulated regeneration by angiocrine expression of Wnt2 and HGF (Nature, 468(7321):310-5, 2010). Pulmonary capillary ECs (PCECs), by deploying MMP14 and release of EGF ligands, sustain lung regeneration. Notably, transplantation of SECs or PCECs into mice restores organ regeneration (Cell, 47(3):539-53, 2011). These data establish the remarkable tissuespecific vascular heterogeneity in orchestrating organ regeneration.
 
To translate these findings to the clinical setting, we have differentiated human and mouse embryonic stem and iPSC cells into induced vascular endothelial cells (iVECs) (Cell, 151, 559-75, 2012). However, iVECs are unstable and have limited expansion potential. To circumvent this hurdle, we have developed new strategies by transcriptional reprogramming of amniotic cells into vascular ECs (rAC-VECs) (Cell, 151, 559-75, 2012). rACVECs phenocopy the specialized tissue-specific function of ECs, supporting long-term expansion of repopulating cells (Developmental Cell, In Press), such as hematopoietic stem cells in xenobiotic-free conditions. Given that rACVECs can be HLA-typed, cryopreserved, and publicly banked, these cells could establish an inventory for generating abundant tissue-specific vascular niche cells for promoting angiocrine-dependent organ regeneration.
 

Niche-Secreted Factors Regulate Stem Cell Behavior
Robert Oostendorp, PhD, Technical University of Munich

Somatic stem cells are critical to maintain highly regenerative tissues such as the skin, the gastro-intestinal mucosa and the blood system. Although it has long been known that hematopoietic stem cells (HSC) are located in the bone marrow, the surrounding micro-environment — the so-called niche — is thought to control the balance between HSC self-renewal and differentiation and may control HSC dormancy and proliferation. The molecular mechanisms involved in this regulation are, however, largely unknown. Most investigators, including our group, have started dissecting these mechanisms using in vitro models of the niche: stromal cell lines. The knowledge gathered in this manner is now validated in vivo. Our data shows that factors secreted by the niche, such as Sfrp1 and Ptn play a critical role in maintaining HSC self-renewal and the balance between myeloid and lymphoid regenerative potential. One of the main HSC pathways affected is the Wnt signaling pathway, as well as associated Smad-signaling modulators. In particular, the niche seems to have a critical role in maintaining the balance between canonical and non-canonical Wnt signaling in HSC. Disturbances in secretion of niche factors may thus dysregulate HSC pathways, facilitating disturbances in self-renewal and differentiation. In ultimate cases, this may lead to malignant transformation. Thus, the knowledge generated will aid in developing new tools for early detection of malignant transformation, as well as help improve therapies for eradicating malignant disease.
 

The Ah Receptor in Stem Cell Cycling, Regulation and Quiescence
Thomas A. Gasiewicz, PhD, University of Rochester Medical Center

Processes that regulate quiescence, self-renewal, and senescence of hematopoietic stem cells (HSCs) are not well understood. Due in part to the ability of xenobiotic ligands to have persistent effects on the immune system in experimental animals, there has been much work to define a physiological role of the aryl hydrocarbon receptor (AhR) and relationships to human disease. Persistent AhR activation by dioxin, a potent agonist, results in altered numbers and function of HSCs in mice. HSCs from AhR null-allele (KO) mice are hyperproliferative and have altered cell cycle. In addition, aging KO mice show characteristics consistent with premature bone marrow senescence and are prone to hematopoietic disease development. Furthermore, the Ahr gene appears to be regulated under conditions that control HSC proliferation. These data, and others, present a compelling argument for a function of the AhR in HSC regulation. We propose that the increased proliferation of HSCs lacking AhR expression or activity is a result of loss of quiescence, and as such, AhR normally acts as a negative regulator to curb excessive or unnecessary proliferation. Similarly, prolonged and/or inappropriate stimulation of AhR activity may compromise the ability of HSCs to sense environmental signals that allow these cells to balance quiescence, proliferation, migration, and differentiation. These data also support a hypothesis that deregulation of AhR function has an important role in the etiology and/or progression of certain hematopoietic diseases, many of which are associated with aging.
 
Supported by NIH Grants ES01247, ES07026, and ES04862.

Stress Induced Activation of Hematopoietic Stem Cells In Vivo
Stefanie Thamm, Raphael Lutz, Andrea Kuck, Stephan Wurzer, Marieke A.G. Essers, PhD, HI-STEM and DKFZ

Tissue stem cells are responsible for the maintenance and repair of most organs and tissues. In the hierarchical organized blood system, dormant hematopoietic stem cells (HSCs) with lifelong self-renewal capacity are at the top of the hierarchy, giving rise to active HSCs that typically control the blood cell production during healthy homeostasis. However, under stress conditions such as during virus infections or after blood loss, where large amounts of mature blood cells are lost, feedback signals are thought to signal back to the dormant HSCs, leading to their activation, and thus production of new mature blood cells. The molecular and cellular mechanisms, including which cytokines are part of these feedback loops, remain largely unexplored.
 
Our work has recently demonstrated that, very surprisingly, the cytokine IFNα, which is produced by virally infected immune cells to block the infection of more mature blood cells, is able to activate the entire HSC pool including dormant HSCs. Activated HSCs start to proliferate in vivo and up-regulate stem cell antigen 1 (Sca-1). In order to explain this surprising effect of IFNα on HSCs, we are currently exploring whether during infections IFNα might be part of a feedback loop leading to the activation of HSCs. Using a reporter mouse to monitor IFNα production in the bone marrow, several forms of bone marrow stress were tested. Interestingly, injection of mice with lipopolysaccharide (LPS) lead to increased IFNα production, followed by a TLR4-dependent activation of quiescent HSCs. Similar to IFNα, LPS induced activation is accompanied by and dependent on up-regulation of Sca-1 on the surface of HSCs. However, though IFNα has a direct effect on HSCs, both in vivo and in vitro experiments show that LPS has an indirect effect on HSCs. We are currently unraveling the mechanism underlying the indirect activation of HSCs in response to LPS and the potential role the bone marrow niche plays in this process. These data will further increase our knowledge on the mechanism of activation of HSCs under stress conditions.
 

Aldehyde Dehydrogenases in Normal and Malignant Hematopoietic Stem Cells
Clayton Smith, MD, University of Colorado, Denver

Hematopoietic stem cells (HSCs) sustain hematopoiesis in a highly controlled fashion through a dynamic balance of self-renewal, differentiation, apoptosis and other processes. Perturbation of these processes can lead to marrow failure, myelodysplasia (MDS), chronic or acute leukemia and other diseases. Control of these processes occurs through a complex interplay between HSCs and their microenvironment, as well as through increasingly well-characterized cellular processes. We have found that the Aldehyde Dehydrogenase (ALDH) gene family, which consists of at least 19 members, may also play an important role in regulating HSC cell fate decisions by metabolizing reactive oxygen species (ROS) and reactive aldehydes (RAld). Data from our group and others suggest that the intracellular ROS and RAld can provide fine level control over a variety of normal cellular process, as well as cause protein and DNA damage leading to cellular dysfunction. As an example, we have found that loss of 2 ALDH family isoforms in HSCs, ALDH1A1 and ALDH3A1, leads to increases in intracellular ROS and reactive aldehydes and widespread perturbations in cell signaling, gene expression and cell cycle progression, as well as a predisposition to leukemia formation. Approximately 1/3 of human leukemias also fail to express ALDH1A1 and ALDH3A1 and are exquisitely sensitive to toxic ALDH substrates. These observations set the stage for future studies designed to understand better the role of ROS and RAld in normal and malignant stem cells and to develop new therapies for AML, MDS and other disorders.
 

Niche Regulation for Hematopoietic Stem Cells
Toshio Suda, MD, PhD, School of Medicine, Keio University

Hematopoietic stem cells (HSCs) are sustained in a specific microenvironment known as the stem cell niche. Adult HSCs are kept quiescent during the cell cycle in the endosteal niche of the bone marrow (BM). The quiescent state is thought to be a characteristic property for the maintenance of HSCs. Normal HSCs maintain intracellular hypoxia, stabilize the hypoxia-inducible factor-1α (HIF-1α) protein and generate ATP by anaerobic metabolism. In HIF-1α-deficiency, HSCs become metabolically aerobic, lost cell cycle quiescence, and finally exhausted. An increased dose of HIF-1α protein in VHL mutated HSCs and their progenitors induced cell cycle quiescence and accumulation of HSCs in the BM. Restored glycolysis by pyruvate dehydrogenase kinases ameliorated cell cycle quiescence and stem cell capacity. Taken together, HSCs directly utilize the hypoxic microenvironment to maintain their cell cycle by HIF-1α-dependent metabolism. On the basis of the physiological nature of HSCs, I would like to discuss the abnormal HSCs and niches in hematological malignancies; multiple myeloma and chronic myelogenous leukemia (CML). Comparison between normal and abnormal HSCs and niches will be important to development of the new treatment for the cancer.
 

Apoptosis-Related Gene Expression Profiling of Hematopoietic Stem/Progenitor Cells after Radiation Exposure
Yoko Hirabayashi, MD, National Institute of Health Sciences

Because senescence is considered to be related to xenobiotic responses to ‘time’, a series of quantitative and qualitative studies was conducted, specifically focusing on the evaluation of hematopoietic stem/progenitor cells with or without radiation exposure followed by natural aging. As a result, the lineage negative, c-kit positive, stem cell antigen positive (LKS) fraction did not recover in 2Gy whole-body irradiated mice but remained at approximately 50-80% of those in age-matched nonirradiated controls until 18 months of age. Accordingly, the expression of genes, specifically those related to apoptosis, was elucidated by microarray data from mice one month after whole-body irradiation, and was quantitatively evaluated by real-time PCR analysis using bone-marrow cells and cells in the LKS fraction from 21-month-old mice with or without radiation exposure at 6 weeks of age, in comparison with 2-month-old non-irradiated control mice. In mice more than one year after radiation exposure, five out of eleven selected genes showed significant alterations of expression patterns. Among them, Ccnd1, Fyn, and Pik3r1 showed up-regulation of their expressions in the LKS fraction with radiation exposure compared with the fraction without radiation exposure. These findings may indicate a possible prolonged proliferation of cells in the LKS fraction after single-dose irradiation. Interestingly, the increased expression level of Ccnd1 was observed, specifically in mice with radiation exposure, in addition to the up-regulation of the gene in the LKS fraction of aged mice. These findings suggest that the aging phenotype may be enhanced by radiation exposure.
 

Abstracts — Day 3

Regulation of Leukemia Cell Dormancy by the Bone Marrow Niche
Dorothy A. Sipkins, MD, PhD, The University of Chicago

Current research in our laboratory is defining the features of the bone marrow microenvironment (BMM) that facilitate leukemic metastasis as well as the microenvironment changes that occur as leukemia progresses in the BM. We have developed unique in vivo confocal and multiphoton microscopy approaches to visualize leukemia – microenvironment cross-talk in real time in mouse xenograft models. Our techniques facilitate the study of cellular and molecular relationships within the three-dimensional, temporally dynamic BMM. They also allow us to rapidly assess responses to therapeutic interventions and monitor these responses over time. With this approach, we have demonstrated that leukemic cells hijack normal hematopoietic progenitor cell (HPC) niche signaling pathways to enter the BMM. The subsequent growth of leukemia within these niches leads to a molecular restructuring of the microenvironment that favors leukemic spread while suppressing normal HPCs. Conversely, normal HPC niche signals are also able to instruct the fate of leukemic cells. Recently, we have shown that osteopontin (OPN), an extracellular matrix molecule normally present in HPC niches, can function to anchor leukemic blasts in anatomic locations that support tumor dormancy. This interaction with extracellular OPN induces cell cycle exit in leukemic blasts, protecting them from cytotoxic chemotherapy. Through multiple mechanisms, dynamic interplay between leukemia and the hematopoietic niche can critically regulate leukemic growth in the BM; interrupting these cues should enhance our current disease treatments.
 

Methods to Analyze Homing of Stem Cells in Bone Marrow
Susie K. Nilsson, PhD, Commonwealth Scientific and Industrial Research Organisation (CSIRO)

The existence of a “niche” in which hemopoietic stem cells (HSC) reside within the bone marrow (BM) cavity was proposed more than 30 years ago. Recent data supports the theory that the interaction of HSC with cellular and extracellular components within the endosteal BM region is critical for HSC regulation. The tracking of immuno-fluorescent labelled prospectively isolated HSC to and within the BM cavity allows the assessment of the regulatory processes involved in trans-endothelial migration, trans-marrow migration and finally lodgement into the HSC niche. This is of interest, as the extra-cellular and cellular components involved in the regulation of HSC quiescence and differentiation are still not well understood. Homing of transplanted HSC is the first critical step in the interaction between HSC and the microenvironment of the BM. I will describe a method to analyze the homing efficiency and spatial distribution of HSC harvested from endosteal and central BM regions in a competitive homing assay. This allows direct comparison of different cell types into the same recipient, devoid of intraexperimental variability. Furthermore, I will present a method to functionally label BM vasculature in situ allowing the analysis of the relation between HSC, the endosteal surface and the vasculature BM components. Using this technique the modification of the microenvironment of the recipient or the surface protein expression of the donor HSC in transgenic animal models allows insight into components influencing the homing and engraftment process.
 

Modeling Exposure-Induced Leukemogenesis in the Mouse
Michael J. Thirman, MD, The University of Chicago

Mixed-lineage leukemia (MLL) fusion proteins confer a selective advantage to hematopoietic stem and progenitor cells. Gene array studies have demonstrated that progenitor cells which express MLL fusions acquire the genetic signatures of hematopoietic stem cells. To identify direct targets of MLL fusion proteins, we used an integrated whole genome ChIP-chip to identify target genes in both mouse and human hematopoietic cells. In addition to known targets of MLL fusion proteins such as HOXA9 and MEIS1, we found that EYA1 and SIX1, which comprise a heterodimeric transcription factor, are both upregulated by MLL fusion proteins. To characterize these leukemias in detail, we used two alternative strategies to develop mouse models of MLL-ELL leukemia. Using retroviral infection of bone marrow followed by transplantation, recipient mice all develop acute leukemia. In contrast, in a knock-in model of MLL-ELL generated by gene targeting, chimeric and heterozygous mice do not develop leukemia and exhibit no apparent hematopoietic phenotype, indicating that expression of the MLLELL fusion gene is insufficient by itself for the development of acute leukemia. However, treatment of the knock-in mice with ENU, exposure to ionizing radiation, or infection of newborn knock-in mice with the MOL4070LTR retrovirus, induces acute leukemia that recapitulates the phenotype observed in human AML in patients with (11;19)(q23;p13.1) translocations. Using the MLL-ELL mouse leukemia models, we have performed insertional mutagenesis to identify potential cooperating mutations that are required for the development of MLL-associated leukemia. Taken together, these data indicate that multiple genetic pathways cooperate with MLL fusion proteins to induce the development of leukemia. In view of the negligible background of leukemia in unexposed mice and the high incidence of AML following mutagenic exposures, MLL-ELL knock-in mice provide an ideal model for evaluating the leukemic potential of environmental exposures.
 

Redox Proteomics to Measure Oxidative Stress
Dean P. Jones, PhD, Emory University

The redox proteome includes both reversible and irreversible covalent modifications of the translated proteome. Oxidative stress causes both types of modifications, with increased peroxide generation often causing reversible oxidation of Cys and Met and reactive aldehydes from radical processes causing irreversible modifications of multiple amino acids. Large-scale trials with radical scavengers failed to show benefit in humans, indicating that pathologic consequences of oxidative stress may be due to disruption of redox signaling and control via reversible effects on the redox proteome, rather than principally due to irreversible macromolecular damage, as earlier thought. Mass spectrometry based redox proteomics data show that the Cys redox proteome exists in a partially oxidized steady state in cells, reflecting an ongoing oxidation countered by thioredoxin- and GSH-dependent reduction systems. These redox-sensing Cys provide an evolved redox system to integrate and regulate cell functions. Aberrant activity or disruption of this redox system appears likely to underlie central mechanisms attributed to oxidative stress.
 

Long-Term Quantitative Single Cell Imaging: New Tools for Old Questions
Timm Schroeder, PhD, Helmholtz Center Munich

The hematopoietic system is highly complex and dynamic, and consists of large numbers of different cells expressing many molecules controlling their fates. Despite intensive investigation, many long-standing questions in hematopoiesis research remain unsolved. One major reason is that this research usually involves analyzing the fate of populations of cells — rather than individual cells — at very few time points of an experiment, and without knowing their individual identities. Real time tracking of individual cells in culture, tissues or whole organisms would be an extremely powerful approach to fully understand the developmental complexity of this stem cell driven regeneration.
 
We have therefore developed culture and imaging systems to follow the fate of individual cells over long periods of time. This new software is programmed to record and display the divisional history, position, properties, interaction, etc., of all individual cells in a culture over many generations. Our approaches also allow the continuous long term quantification of protein expression or activity in living cells. The novel quantitative data of single cell behavioral and molecular expression are used as the basis for the improved generation and falsification of models describing hematopoiesis. I will discuss how we use these approaches to try to find answers for long standing questions in hematopoiesis research.
 

Genome-Exposome Interactions in Leukemia Etiology
Martyn T. Smith, PhD, School of Public Health, University of California, Berkeley

Hematological malignancies most likely arise as a result of adverse geneenvironment interactions. For some forms of leukemia, genome-wide association studies (GWAS) have revealed some genetic variants of importance, but few are known for AML and MDS. Those that are known come from candidate gene studies and may not be reproducible. GWAS studies are needed of AML and MDS to identify susceptibility loci, which may also give clues as to the environmental causes of the disease. Such studies are underway. The established non-genetic causes of AML are tobacco smoking, ionizing radiation, cancer chemotherapy and occupational exposure to benzene or formaldehyde. Even though benzene is leukemogenic at relatively low occupational levels of exposure, it seems unlikely that it is a major cause of leukemia in the general population exposed to benzene in the ppb range. Other established non-genetic causes of AML can typically only explain about 20-30% of AML incidence, leaving ~70-80% unexplained. The question arises as to how to find the causes of the majority of de novo AML and MDS cases that remain unexplained. We propose that we should attempt to characterize the ‘exposome’ of human leukemia by using unbiased laboratory based methods to find the unknown ‘environmental’ factors that contribute to leukemia etiology. The exposome is defined as the totality of environmental exposures from conception onwards. It offers a conceptual leap towards understanding the environmental causes of human disease through comprehensive studies of the internal chemical environment using repeated blood and other biological samples taken during critical life stages (Rappaport and Smith, Science, 2010. 330:460-1). It augments the chemical-by-chemical approach to finding causes of disease, as it includes both endogenous and exogenous exposures.