Aldehyde dehydrogenase (ALDH) is an enzyme that is expressed in the liver and is required for the conversion of retinol (vitamin A) to retinoic acids. ALDH is also highly enriched in hematopoietic stem cells (HSCs) and is considered a selectable marker of human HSCs, although its contribution to stem cell fate remains unknown. In this study, we demonstrate that ALDH is a key regulator of HSC differentiation. Inhibition of ALDH with diethylaminobenzaldehyde (DEAB) delayed the differentiation of human HSCs that otherwise occurred in response to cytokines. Moreover, short-term culture with DEAB caused a 3.4-fold expansion in the most primitive assayable human cells, the nonobese diabetic͞severe combined immunodeficiency mouse repopulating cells, compared with day 0 CD34 ؉ CD38 ؊ lin ؊ cells. The effects of DEAB on HSC differentiation could be reversed by the coadministration of the retinoic acid receptor agonist, all-trans-retinoic acid, suggesting that the ability of ALDH to generate retinoic acids is important in determining HSC fate. DEAB treatment also caused a decrease in retinoic acid receptor-mediated signaling within human HSCs, suggesting directly that inhibition of ALDH promotes HSC self-renewal via reduction of retinoic acid activity. Modulation of ALDH activity and retinoid signaling is a previously unrecognized and effective strategy to amplify human HSCs.retinoic acid ͉ self-renewal ͉ diethylaminobenzaldehyde ͉ long-term repopulating cells H ematopoietic stem cells (HSCs) possess the unique capacity to self-renew and give rise to all mature lymphohematopoietic progeny throughout the lifetime of an individual (1, 2). Several molecular pathways that regulate HSC self-renewal have now been identified, including Notch (3), HOXB4 (4), Wnt (5), and bone morphogenetic protein signaling pathways (6). The osteoblastic niche for HSCs within the bone marrow (BM) has also been characterized (7,8). Despite these advances in understanding HSC biology, clinical methods to amplify human HSCs have yet to be realized, and characterization of the pathways that regulate HSC self-renewal continues to evolve.Two decades ago, Colvin et al. (9,10) demonstrated that the intracellular enzyme, aldehyde dehydrogenase (ALDH), protected BM progenitors from the cytotoxic effects of cyclophosphamide by deactivation of its metabolite, 4-hydroxycyclophosphamide (9, 10). Several isoforms of ALDH have been identified, with ALDH1 being the primary isoform expressed within human hematopoietic progenitors (11,12). Recent studies have shown that human and murine hematopoietic progenitors can be isolated by using a fluorescently labeled dye specific for ALDH activity (13-16) and cord blood (CB) ALDH br lin Ϫ cells are enriched for nonobese diabetic͞severe combined immunodeficiency (NOD͞SCID) mouse repopulating cells [SCID-repopulating cells (SRCs)] (15, 16). Although these data demonstrate that ALDH is a selectable marker for human stem͞progenitor cells, the HSC-specific function of ALDH remains unknown. In the liver, ALDH1 contributes prima...

Hematopoietic stem cell (HSC) self-renewal is regulated by both intrinsic and extrinsic signals. Although some of the pathways that regulate HSC self-renewal have been uncovered, it remains largely unknown whether these pathways can be triggered by deliverable growth factors to induce HSC growth or regeneration. Here we show that pleiotrophin, a neurite outgrowth factor with no known function in hematopoiesis, efficiently promotes HSC expansion in vitro and HSC regeneration in vivo. Treatment of mouse bone marrow HSCs with pleiotrophin caused a marked increase in long-term repopulating HSC counts in culture, as measured in competitive repopulating assays. Treatment of human cord blood CD34+CDCD38−Lin− cells with pleiotrophin also substantially increased severe combined immunodeficient (SCID)-repopulating cell counts in culture, compared to input and cytokine-treated cultures. Systemic administration of pleiotrophin to irradiated mice caused a pronounced expansion of bone marrow stem and progenitor cells in vivo, indicating that pleiotrophin is a regenerative growth factor for HSCs. Mechanistically, pleiotrophin activated phosphoinositide 3-kinase (PI3K) signaling in HSCs; antagonism of PI3K or Notch signaling inhibited pleiotrophin-mediated expansion of HSCs in culture. We identify the secreted growth factor pleiotrophin as a new regulator of both HSC expansion and regeneration

BackgroundThe capacity to assess environmental inputs to biological phenotypes is limited by methods that can accurately and quantitatively measure these contributions. One such example can be seen in the context of exposure to ionizing radiation.Methods and FindingsWe have made use of gene expression analysis of peripheral blood (PB) mononuclear cells to develop expression profiles that accurately reflect prior radiation exposure. We demonstrate that expression profiles can be developed that not only predict radiation exposure in mice but also distinguish the level of radiation exposure, ranging from 50 cGy to 1,000 cGy. Likewise, a molecular signature of radiation response developed solely from irradiated human patient samples can predict and distinguish irradiated human PB samples from nonirradiated samples with an accuracy of 90%, sensitivity of 85%, and specificity of 94%. We further demonstrate that a radiation profile developed in the mouse can correctly distinguish PB samples from irradiated and nonirradiated human patients with an accuracy of 77%, sensitivity of 82%, and specificity of 75%. Taken together, these data demonstrate that molecular profiles can be generated that are highly predictive of different levels of radiation exposure in mice and humans.ConclusionsWe suggest that this approach, with additional refinement, could provide a method to assess the effects of various environmental inputs into biological phenotypes as well as providing a more practical application of a rapid molecular screening test for the diagnosis of radiation exposure.

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BackgroundPrevious work has demonstrated the potential for peripheral blood (PB) gene expression profiling for the detection of disease or environmental exposures.Methods and FindingsWe have sought to determine the impact of several variables on the PB gene expression profile of an environmental exposure, ionizing radiation, and to determine the specificity of the PB signature of radiation versus other genotoxic stresses. Neither genotype differences nor the time of PB sampling caused any lessening of the accuracy of PB signatures to predict radiation exposure, but sex difference did influence the accuracy of the prediction of radiation exposure at the lowest level (50 cGy). A PB signature of sepsis was also generated and both the PB signature of radiation and the PB signature of sepsis were found to be 100% specific at distinguishing irradiated from septic animals. We also identified human PB signatures of radiation exposure and chemotherapy treatment which distinguished irradiated patients and chemotherapy-treated individuals within a heterogeneous population with accuracies of 90% and 81%, respectively.ConclusionsWe conclude that PB gene expression profiles can be identified in mice and humans that are accurate in predicting medical conditions, are specific to each condition and remain highly accurate over time.