Loss Of Hematopoietic Stem Cells Research Articles

To be or not to be a stem cell: dissection of cellular and molecular components of haematopoietic stem cell niches

Recent studies have identified multiple cell types that regulate haematopoietic stem cells (HSCs); however, proof that a specific cell type produces a specific factor important for HSC function and maintenance is largely lacking. Ding et al (2012) reported recently that conditional deletion of stem cell factor (SCF) in Leptin receptor ( Lepr ) expressing perivascular cells or endothelial and haematopoietic cells resulted in significant reductions in number but less profound reduction in function of HSCs. Although the long‐term fate of HSCs in these models is largely unexplored and an underlying mechanism for reduction in HSCs not yet reported, these findings further implicate the vascular niche in the functional maintenance of HSCs in vivo and also raise intriguing questions for future studies in this field.

The Transcriptional Coactivator Cbp Regulates Self-Renewal and Differentiation in Adult Hematopoietic Stem Cells

The transcriptional coactivator Cbp plays an important role in a wide range of cellular processes, including proliferation, differentiation, and apoptosis. Although studies have shown its requirement for hematopoietic stem cell (HSC) development, its role in adult HSC maintenance, as well as the cellular and molecular mechanisms underlying Cbp function, is not clear. Here, we demonstrate a gradual loss of phenotypic HSCs and differentiation defects following conditional ablation of Cbp during adult homeostasis. In addition, Cbp-deficient HSCs reconstituted hematopoiesis with lower efficiency than their wild-type counterparts, and this response was readily exhausted under replicative stress. This phenotype relates to an alteration in cellular fate decisions for HSCs, with Cbp loss leading to an increase in differentiation, quiescence, and apoptosis. Genome-wide analyses of Cbp occupancy and differential gene expression upon Cbp deletion identified HSC-specific genes regulated by Cbp, providing a molecular basis for the phenotype. Finally, Cbp binding significantly overlapped at genes combinatorially bound by 7 major hematopoietic transcriptional regulators, linking Cbp to a critical HSC transcriptional regulatory network. Our data demonstrate that Cbp plays a role in adult HSC homeostasis by maintaining the balance between different HSC fate decisions, and our findings identify a putative HSC-specific transcriptional network coordinated by Cbp.

Retroviral Ectopic Expression of a Signaling-Defective Thrombopoietin Receptor (Mpl) Induces a Systemic Loss of Hematopoietic Stem Cells in Mice,

Abstract 4175MPL signaling, induced by the binding of Thrombopoietin (THPO), regulates megakaryopoiesis and platelet development, and is essential for hematopoietic stem cell (HSC) maintenance. Murine lineage negative, Sca-1 and c-kit positive bone marrow (BM) cells (LSK cells) and human CD34 positive cells expressing MPL have long term BM reconstitution capacities. In human patients, deficiency of MPL due to inactivating mutations in the MPL gene causes severe thrombocytopenia and progressive aplastic anemia, which is lethal if not treated by BM transplantation (BMT). This rare genetic disorder is termed congenital amegakaryocytic thrombocytopenia (CAMT).With the final aim of developing gene therapy to treat CAMT, we explored the effects of ectopic Mpl expression in wild-type C57Bl/6 mice. The enforced overexpression of Mpl induced a rapid expansion of all three hematological cell lineages, resembling chronic myeloproliferative disease (CMPD) 2 months after BMT (n=25 mice). Surprisingly, mice subsequently developed life threatening pancytopenia with normal Thpo levels despite low platelet counts (167+/−52 × 103/μl). The BM showed reduced LSK cell numbers (0.01+/−0.01 % versus controls 0.05+/−0.015%, p=0.03). Non-transduced, co-transplanted BM cells could not rescue the disturbed hematopoiesis. Based on these observations, we hypothesized that the ectopic overexpression of Mpl in hematopoietic cells induces a dominant-negative effect, dysregulating the Thpo-Mpl balance.To test our hypothesis, we ectopically expressed a signaling-defective truncated dominant-negative Mpl (dnMpl) receptor, lacking the complete intracellular domain, in a BMT model in wild-type C57Bl/6 mice (n=23). Lineage negative cells were transduced with gammaretroviral vectors expressing dnMpl from the SFFV promoter in the LTRs and transplanted into lethally irradiated mice. We observed long term engraftment with 57.6+/−1.9% dnMpl positive mononuclear cells in the peripheral blood (controls 76.7+/−7.8%) after 19 weeks and severe thrombocytopenia (174 +/− 32 x103/μl) in the dnMpl expressing animals (controls 951+/−233×103/μl). After 23 weeks, we detected 15–34% dnMpl positive BM cells (controls 15–99%). To address the question of whether the BM of dnMpl treated mice contained fewer HSCs compared to control transplanted mice, we analyzed the LSK cell number and saw a ∼4.6-fold reduction (0.02+/− 0.005% versus control mice 0.07+/−0.012%; p=0.0016). As Thpo/Mpl signaling is known to induce HSC quiescence, we speculated that the loss of these signals induces HSC cycling. Cell cycle analysis of the LSK cells of dnMpl treated mice showed a transition into G1/S/G2, indicating a loss of quiescent HSCs (G0 3.5 +/−1.6% vs controls 29.8 +/−10.5%). This effect was even more pronounced in CD34 negative LSK cells (5.4+/−4.0% vs. 44.5+/−8.5%). Secondary transplantation of dnMpl BM showed severely reduced repopulation capacity with 5 out of 6 secondary recipients dying due to graft failure.In selected animals we transplanted another batch of BM after 16 weeks without preconditioning of the mice. Long term repopulation of the second graft was seen in 71–79% of dnMpl treated mice (controls 0.2–1.1%) and BM cells further engrafted in secondary recipients.To address changes in HSC gene expression, we sorted LSK cells from dnMpl BM (pool of 4–5 dnMpl mice for each sample) and also separated dnMpl positive and negative cells. Our controls were LSK cells from control transplanted mice (one per sample). RNA was analyzed on an Affymetrix Mouse Genome 430 2.0 array in triplicates. Unsupervised clustering and principle component analysis revealed that dnMpl positive and negative samples clustered, suggesting a systemic effect on HSCs. We tested more than 4000 gene sets from the MySigDB Database (Broad Institute, Cambridge, MA) for enrichment in either of the phenotypes and found a profound downregulation of gene sets containing “stemness” genes in the LSK cells of dnMpl treated mice, irrespective of cellular dnMpl expression.In summary, we show that ectopic expression of dnMpl in a subset of BM cells disturbs Thpo/Mpl-signaling inducing thrombocytopenia and a systemic loss of HSCs. Besides underlining the strict requirement for regulated Mpl expression in gene therapy for CAMT, these data create new hypotheses for the pathogenesis of aplastic anemia and the development of non-cytotoxic conditioning regimens in BMT. Disclosures:No relevant conflicts of interest to declare.

Nonmyelinating Schwann Cells Maintain Hematopoietic Stem Cell Hibernation in the Bone Marrow Niche

Hematopoietic stem cells (HSCs) reside and self-renew in the bone marrow (BM) niche. Overall, the signaling that regulates stem cell dormancy in the HSC niche remains controversial. Here, we demonstrate that TGF-β type II receptor-deficient HSCs show low-level Smad activation and impaired long-term repopulating activity, underlining the critical role of TGF-β/Smad signaling in HSC maintenance. TGF-β is produced as a latent form by a variety of cells, so we searched for those that express activator molecules for latent TGF-β. Nonmyelinating Schwann cells in BM proved responsible for activation. These glial cells ensheathed autonomic nerves, expressed HSC niche factor genes, and were in contact with a substantial proportion of HSCs. Autonomic nerve denervation reduced the number of these active TGF-β-producing cells and led to rapid loss of HSCs from BM. We propose that glial cells are components of a BM niche and maintain HSC hibernation by regulating activation of latent TGF-β.

Lentiviral gene transfer regenerates hematopoietic stem cells in a mouse model for Mpl-deficient aplastic anemia

AbstractThpo/Mpl signaling plays an important role in the maintenance of hematopoietic stem cells (HSCs) in addition to its role in megakaryopoiesis. Patients with inactivating mutations in Mpl develop thrombocytopenia and aplastic anemia because of progressive loss of HSCs. Yet, it is unknown whether this loss of HSCs is an irreversible process. In this study, we used the Mpl knockout (Mpl−/−) mouse model and expressed Mpl from newly developed lentiviral vectors specifically in the physiologic Mpl target populations, namely, HSCs and megakaryocytes. After validating lineage-specific expression in vivo using lentiviral eGFP reporter vectors, we performed bone marrow transplantation of transduced Mpl−/− bone marrow cells into Mpl−/− mice. We show that restoration of Mpl expression from transcriptionally targeted vectors prevents lethal adverse reactions of ectopic Mpl expression, replenishes the HSC pool, restores stem cell properties, and corrects platelet production. In some mice, megakaryocyte counts were atypically high, accompanied by bone neo-formation and marrow fibrosis. Gene-corrected Mpl−/− cells had increased long-term repopulating potential, with a marked increase in lineage−Sca1+cKit+ cells and early progenitor populations in reconstituted mice. Transcriptome analysis of lineage−Sca1+cKit+ cells in Mpl-corrected mice showed functional adjustment of genes involved in HSC self-renewal.

Hematopoietic stem cells undergo an IFNγ-dependent, MyD88-independent transition from dormancy to activity during acute bacterial infection (153.8)

Abstract How hematopoietic stem cells (HSCs) respond to and recover from immunologic stress is not well-understood. We demonstrate that infection with the intracellular bacteria, Ehrlichia muris, induces a transient expansion of bone marrow Lin-negative Sca-1+ cKit+ (LSK) progenitor cells. As inflammation can modulate the expression of surface markers used to phenotype stem and progenitor cells, we addressed whether the phenotypic changes correlated with functional changes in the progenitor cell population. The expansion of LSK cells was associated with a loss of dormant, long-term repopulating HSCs, reduced engraftment, and a bias towards myeloid lineage differentiation. The infection-induced changes required IFNγ signaling, and were accompanied by an expansion of more differentiated multipotent progenitor cells. IFNγ was required for the infection-induced loss of functionally-defined dormant HSCs in label retaining-cell assays. In competitive reconstitution experiments, LSK cells purified from infected IFNγR-deficient mice preferentially engrafted, relative to LSK cells purified from infected wild type mice. Furthermore, the infection-induced changes in progenitor cells were independent of intrinsic MyD88-signaling. We propose that IFNγ elicited during acute infection, provides a signal to dormant HSCs to undergo a rapid transition from dormancy to activity, ostensibly, to provide the host with additional or better-armed innate cells for host defense.

Hematopoietic Defects In ACD/TPP1-Deficient Mice Reveal An Essential Role for the Shelterin Complex In Blood-Forming Stem Cell Homeostasis

AbstractAbstract 882Bone marrow failure syndromes can be caused by loss-of-function mutations in telomerase components. Besides the enzymatic activity of telomerase, telomere maintenance requires the protective function of the shelterin protein complex. This complex consists of six proteins that bind to the telomere and prevent an inappropriate DNA damage response from being elicited by exposed telomeric DNA. Within this complex, TPP1, encoded by the gene Acd, plays a central role in complex organization as it links elements that bind the double-stranded portion of the telomere to those that bind the single-stranded overhang. We report an essential function for the shelterin complex and specifically for TPP1 in the maintenance of hematopoietic stem cells (HSCs). Utilizing adrenocortical dysplasia (acd), a spontaneous autosomal recessive mouse mutation causing profoundly hypomorphic Acd expression, we identified phenotypic and functional abnormalities in fetal hematopoietic progenitors. acd mutant Fetal liver HSCs were larger, more granular, and expressed higher levels of the cell surface protein Sca-1, as compared to wild-type counterparts. BrdU incorporation analysis showed an accumulation of Acd-deficient progenitors in the G2M phase of the cell cycle, consistent with G2M arrest. Absolute quantification of fetal liver HSCs revealed a significant reduction in stem cell numbers in acd mutants as compared to control littermates. p53 deficiency rescued the HSC depletion phenotype, but induced a paradoxical exacerbation of the increased progenitor cell size and Sca-1 expression. These findings suggest that p53-dependent and p53-independent elements contribute to the acd HSC phenotype. In competitive repopulation assays, acd HSCs failed to provide tri-lineage hematopoietic reconstitution in lethally irradiated recipients, indicating a profound functional defect. To further study the effects of Acd deficiency in adult HSCs, we generated an Acd allele in which exons 3–8 are flanked by loxP sites, allowing conditional inactivation following Mx1-Cre induction via poly(I:C) administration. Acd inactivation resulted in rapid HSC depletion within 2 weeks after initiation of poly(I:C) injections. To assess whether Acd exerted exclusively cell-autonomous effects in HSCs, we transplanted Acdf/–Mx1-Cre+ HSCs into wild-type recipients. Poly(I:C)-mediated Acd inactivation in these bone marrow chimeric mice resulted in rapid HSC loss and failure of tri-lineage hematopoiesis in animals reconstituted with Acd-deficient bone marrow. These data indicate an exquisite cell-autonomous requirement for Acd/TPP1 in adult mouse HSCs. In view of the extremely rapid HSC loss after Acd inactivation, these observations cannot be explained by telomere shortening. Together, our findings represent the first report of an essential role for Acd/TPP1 and the shelterin complex in HSC maintenance and hematopoietic homeostasis, independently of telomerase function. Disclosures:No relevant conflicts of interest to declare.

Expansion of Human Cord Blood Hematopoietic Stem Cells for Transplantation

A recent Science paper reported a purine derivative that expands human cord blood hematopoietic stem cells in culture (Boitano et al., 2010) by antagonizing the aryl hydrocarbon receptor. Major problems need to be overcome before ex vivo HSC expansion can be used clinically.

Role of the polycomb group proteins in hematopoietic stem cells

Polycomb group (PcG) proteins play a role in the transcriptional repression of genes through histone modifications. Recent studies have clearly demonstrated that PcG proteins are required for the maintenance of embryonic as well as a broad range of adult stem cells, including hematopoietic stem cells (HSCs). PcG proteins maintain the self-renewal capacity of HSCs by repressing tumor suppressor genes and keep differentiation programs poised for activation in HSCs by repressing a cohort of hematopoietic developmental regulator genes via bivalent chromatin domains. Enforced expression of one of the PcG genes, Bmi1, augments the self-renewal capacity of HSCs. PcG proteins also maintain redox homeostasis to prevent premature loss of HSCs. These findings established PcG proteins as essential regulators of HSCs and underscored epigenetics as a new field of HSC research. In this review, we focus on the role of PcG proteins in the epigenetic regulation of the self-renewal capacity and multipotency of HSCs.

Cited2 Is an Essential Regulator of Adult Hematopoietic Stem Cells

SummaryThe regulatory pathways necessary for the maintenance of adult hematopoietic stem cells (HSCs) remain poorly defined. By using loss-of-function approaches, we report a selective and cell-autonomous requirement for the p300/CBP-binding transcriptional coactivator Cited2 in adult HSC maintenance. Conditional deletion of Cited2 in the adult mouse results in loss of HSCs causing multilineage bone marrow failure and increased lethality. In contrast, conditional ablation of Cited2 after lineage specification in lymphoid and myeloid lineages has no impact on the maintenance of these lineages. Additional deletion of Ink4a/Arf (encoding p16Ink4a and p19Arf) or Trp53 (encoding p53, a downstream target of p19Arf) in a Cited2-deficient background restores HSC functionality and rescues mice from bone marrow failure. Furthermore, we show that the critical role of Cited2 in primitive hematopoietic cells is conserved in humans. Taken together, our studies provide genetic evidence that Cited2 selectively maintains adult HSC functions, at least in part, via Ink4a/Arf and Trp53.

The Earliest Stages of Loss of Stem Cell Self-Renewal in-Vivo Is Linked to Upregulated Biosynthesis of “Quiet” Mitochondria and Is Influenced by CXCR4 Activation and STAT3 Gene Deletion.

Abstract 2546Poster Board II-523One attribute of all stem cells is self-renewal (SR). Progress has been made in identifying genes/proteins involved in stem cell SR mechanisms, especially in embryonic stem cells, but very little is known in adult stem cells such as hematopoietic stem cells (HSCs). Mitochondria (Mt) have recently been considered more than an ATP generator and mediator of apoptosis. They act as a kind of scaffold for integrating numerous signals to and from the nucleus and from the extracellular environment, and signals involved in differentiation and cell cycle. They also stabilize microtubules of the mitotic spindle and suppress rotation of the mitotic division plane, potentially influencing asymmetric/symmetric stem cell divisions. Populations of primitive hematopoietic cells with low Mt activity (i.e. Rho123low) are highly enriched in long-term repopulating (LTR)-HSC. Because Mt can suppress mitotic spindle rotation by stabilizing spindle microtubules, potentially influencing asymmetric divisions, we hypothesized that Mt biogenesis/function could be important for mechanisms of SR loss and early-stage differentiation of mouse HSC. We examined Mt mass and Mt membrane potential in primitive mouse CD34lo/neg LINnegSca-1posc-kitpos (CD34-LSK) cells, CD34+LSK cells, and related populations. We note that as the small CD34-LSK cells begin to express CD34, Mt mass significantly increases (2×), while Mt membrane potential does not change in these CD34+LSK cells. The CD34+LSK cells then increase in size without further increases in Mt mass, but with upregulated Mt membrane potential. Because CD34 expression in LSK cells is associated with loss of LTR-HSC (i.e. self-renewal) and retention of only short-term repopulating ability, we propose this previously uncharacterized population of LSK cells with increased numbers/mass of “quiet” Mt may be an important component during initial stages of loss of SR and asymmetric HSC division. We found that primitive LSK cells from mice constitutively expressing an SDF-1α/CXCL12 transgene (TG mice) have shifted ratios of these various mitochondrially distinct populations that may be related to CXCR4 signaling and its known influence on glycolysis and STAT3 activity, both of which can influence Mt metabolism. LSK cells in TG mouse total bone marrow significantly increased from .039+/_.01% to .085+/_.02% (n=6), but they were predominantly CD34+LSK cells. These CD34+LSK cells were composed of two distinct types of cells; small/high-Mt mass and large/high-Mt mass cells. The small LSK cells have lower Mt membrane potential while the large LSK cells had increased Mt membrane potential. Because CXCR4 can signal through STAT3, and because STAT3 can bind to Mt and influence their metabolic activities (i.e. membrane potential), we hypothesized that mice with a tissue-specific targeted gene deletion of STAT3 in hematopoietic cells might result in LSK cells that are defective in upregulating Mt membrane potential, which could shift the proportions of the three mitochondrially distinct populations of LSK cells toward the smaller, Mt mass-low, Mt membrane potential-low type of LSK cell. Bone marrow from targeted STAT3-deleted had LSK numbers increased 50%, but they were composed almost entirely of small, Mt mass-high, membrane potential-low, CD34+ type of LSK cells. This is consistent with our hypothesis and suggests that Mt membrane potential may be important for latter stages of CD34-LSK cell differentiation to more lineage-committed progenitor cells but may not be required for initial stages of SR loss. We propose a model where Mt biogenesis occurs before/during loss of SR in CD34-LSK cells. These Mt are metabolically quiescent (“quiet”), attach to spindle microtubules of dividing cells, and influence spindle orientation resulting in loss of SR (small, Mt mass high, Mt potential low, CD34+LSK cells). Before further differentiation, these cells upregulate Mt metabolism/membrane potential and increase in size to facilitate further expansion and differentiation (large, Mt mass high, Mt potential high, CD34+LSK cells). This model could help explain the observed phenotypes of SDF-1 α/CXCL12 transgenic mice and hematopoietic cell-targeted STAT3 deleted mice. This information may also be useful in the search for strategies to prevent loss of SR capacity of HSCs in culture and expansion of HSCs in-vitro. Disclosures:No relevant conflicts of interest to declare.

Disruption of Bis Leads to Deterioration of Mesenchymal Stromal Cells Predominantly Affecting Vascular Compartment of Hematopoietic Stem Cell Niche.

Abstract 3627Poster Board III-563The stem cell niche plays an important role in the microenvironmental regulation of hematopoietic stem cells (HSCs), but the integration of niche activity remains poorly understood. In this study explored the hematopoietic defect of mice disrupted with Bis/BAG-3/CAIR-1, a protein related to apoptosis and response to cellular stress and show that functional loss of bis leads to series of hematopoietic derangements due to perturbation of vascular stem cell niche. First, mice with targeted disruption of bis (bis−/−) exhibited severe hypocellularity in the bone marrows and spleen starting from 16 days after birth. Affected mice exhibited loss of primitive neonatal HSCs (CD34+Lin-Sca-1+c-kit+) and defect in early stage B-lymphopoiesis including common lymphoid progenitors (IL-17R+LSK), pre-B and pro-B cell populations, but not for mature recycling B-lymphocytes (IgD+B220+). However, this hematological defect of bis−/− mice was not reproduced when bis−/− bone marrow cells were transplanted into wild-type (WT) recipients, pointing to the microenvironmental origin of the phenotypes. Subsequent analysis of bis−/− mice bone marrow revealed a characteristic defect in the mesenchymal stromal component that included a quantitative loss of stromal cells (CD45-CD31-TER119-CD105+) in the bone marrows and rapid sensescence of stromal cells comprising colony forming unit-fibroblast (CFU-F) when re-plated in the ex-vivo culture. Moreover, mesenchymal stromal cells expressing CXCL-12 or IL-7 was lost in the affected bone marrows with lowered density of vascular development, together indicating a perturbation of peri-vascular stem cell niche in the bone marrow. In contrast, no abnormalities were observed in the growth and hematopoietic supporting activities of osteoblasts obtained from bis−/− mice. Collectively, these results indicate that Bis functions to mediate cellular regulation of the stem cell niche activities selectively on the vascular compartment without affecting osteoblastic niche, and suggest that Bis may serve as a molecule that can bridge the microenvironment niche and cellular stress/apoptotic signals during the in-vivo orchestration of hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Heterogenous Nuclear Ribonucleoprotein L (hnRNPL) Is Required for the Functional Integrity of Hematopoietic Stem Cells.

Abstract 1486Poster Board I-509Hematopoietic differentiation has to be tightly regulated since uncontrolled or exaggerated development of blood cells may lead to the development of leukemia or autoimmune diseases. Many mechanisms exist to control hematopoiesis on a molecular level, including the regulation of transcription, which has been intensely studied. However, new evidence suggests the process of alternative splicing to be an important regulator of the maturation and activation of blood- and immune effector cells. One of the factors that has been identified as a potential regulator of the immune response and controls alternative splicing is “heterogenous nuclear ribonucleoprotein L” (hnRNP L). This factor affects among others the alternative splicing of the CD45 gene, which encodes the major tyrosine phosphatase expressed on all hematopoietic cells. To investigate the biological role of hnRNP L as a regulator of alternative splicing in hematopoiesis, we have generated conditional hnRNP L knockout (KO) mice carrying floxed alleles that can be deleted by co expression of Cre recombinase. Both the inducible MxCre transgene or Vav-Cre transgene, which is active in all hematopoietic cells were introduced into hnRNP Lfl/fl mice. We found that the conditional deletion of hnRNP L by the Vav Cre transgene led to early mortality before birth (at stage E17.5) and flow cytometric analysis of fetal liver of hnRNP Lfl/fl, Vav-Cre mice or bone marrow from pIpC induced hnRNP Lfl/fl Mx-Cre mice showed a deficit in erythrocyte maturation. In addition, fetal thymi from hnRNP Lfl/fl X Vav-Cre mice were severely reduced in cellularity and showed disturbed T cell maturation. Moreover, the deletion of hnRNP L results in reduced numbers of Lin−Sca1+ckit+ (LSK) cells, common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs), granulocyte-monocyte progenitors (GMPs) and megakaryocyte-erythrocyte progenitors (MEPs). Strikingly, while most of the progenitors and the short-term hematopoietic stem cells (HSCs) were affected by the deletion of hnRNP L, the population of long term HSCs was not reduced. We found a high percentage of Annexin V positive cells in the LSK population suggesting that the loss of progenitors and short term HSCs in hnRNP L deficient mice is due to an accelerated cell death. To test whether stem cells lacking hnRNP L were still functional, we sorted Lin−Sca1+ckit+ (LSK) cells and cultured them on either methylcellulose or the feeder cell lines OP9 and OP9-DL1. The co-culture with OP9 or OP9-DL1 cells demonstrated that hnRNP L−/− LSK cells had lost their potential to differentiate into B and T lymphocytes. Similarly, hnRNP L deficient LSK cells were unable to give rise to lymphoid, myeloid or erythroid colonies on methylcellulose. This suggests that hnRNP L is required to maintain not only the numbers of hematopoietic stem cells, but also their ability for multilineage differentiation. We conclude that the regulation of alternative splicing is an essential component of the regulatory network required to maintain hematopoietic differentiation and the functional integrity of hematopoietic stem cells. Disclosures:No relevant conflicts of interest to declare.

Inhibition of Notch Signaling in T Cells Prevents Immune-Mediated Bone Marrow Failure.

Abstract 180Aplastic anemia is a severe bone marrow disorder characterized by the loss of hematopoietic stem cells (HSC). HSC destruction is thought to be T cell-mediated in a majority of patients with aplastic anemia. Global immunosuppression and HSC transplantation can induce disease remission, but these treatments are not effective in all patients and can promote life-threatening complications. Thus, novel immunomodulatory approaches are needed in this disorder. Notch is a conserved cell-cell communication pathway that can regulate T cell differentiation and function with context-dependent effects. To study the role of Notch signaling in pathogenic T cells causing immune-mediated bone marrow failure, we inhibited canonical Notch signaling in mature T cells through conditional expression of the pan-Notch inhibitor DNMAML (ROSA-DNMAMLf × Cd4-Cre mice). We used two complementary mouse models of immune-mediated bone marrow failure that mimic features of aplastic anemia: administration of C57BL/6 (B6) T cells into sublethally irradiated (500 rads) minor histocompatibility antigen mismatched BALB/b recipients (Chen et al., J Immunol 2007; 178:4159), or infusion of B6 lymphocytes into unirradiated MHC-mismatched B6×DBA F1 recipients. In contrast to control B6 T cells which led to lethal bone marrow failure in virtually all recipients, DNMAML-expressing Notch-deprived T cells were profoundly deficient at inducing HSC loss in both disease models, leading to markedly improved long-term survival (>90%). Notch-deficient T cells showed a modest decrease in overall expansion within secondary lymphoid organs, but their accumulation in the target bone marrow was preserved. Upon restimulation with anti-CD3 and anti-CD28 antibodies, DNMAML T cells had decreased production of IL-2 and interferon gamma. Activated CD4+ and CD8+ DNMAML T cells had reduced interferon gamma, granzyme B, and perforin transcripts despite preserved induction of the master transcription factors Tb×21 (encoding T-bet) and Eomes. In vivo infusion of CFSE-labeled host-type target cells revealed a decreased cytotoxicity in DNMAML as compared to control B6 T cell recipients. These observations point to a novel spectrum and mechanism of Notch action in mature T cells. Since we have shown recently that canonical Notch signaling is dispensable for the maintenance of adult HSCs (Maillard et al., Cell Stem Cell 2008, 2:356), our findings suggest that Notch inhibition could represent a novel therapeutic modality to target the T cell response and reverse immune-mediated HSC destruction in aplastic anemia. Disclosures:Shan:American Society of Hematology: Research Funding. Zhang:University of Michigan Comprehensive Cancer Center: Research Funding; Damon Runyon Cancer Research Foundation: Research Funding. Maillard:Damon Runyon Cancer Research Foundation: Research Funding; American Society of Hematology: Research Funding; University of Michigan Comprehensive Cancer Center: Research Funding.

Dual requirement for the ETS transcription factors Fli-1 and Erg in hematopoietic stem cells and the megakaryocyte lineage

Fli-1 and Erg are closely related members of the Ets family of transcription factors. Both genes are translocated in human cancers, including Ewing's sarcoma, leukemia, and in the case of Erg, more than half of all prostate cancers. Although evidence from mice and humans suggests that Fli-1 is required for megakaryopoiesis, and that Erg is required for normal adult hematopoietic stem cell (HSC) regulation, their precise physiological roles remain to be defined. To elucidate the relationship between Fli-1 and Erg in hematopoiesis, we conducted an analysis of mice carrying mutations in both genes. Our results demonstrate that there is a profound genetic interaction between Fli-1 and Erg. Double heterozygotes displayed phenotypes more dramatic than single heterozygotes: severe thrombocytopenia, with a significant deficit in megakaryocyte numbers and evidence of megakaryocyte dysmorphogenesis, and loss of HSCs accompanied by a reduction in the number of committed hematopoietic progenitor cells. These results illustrate an indispensable requirement for both Fli-1 and Erg in normal HSC and megakaryocyte homeostasis, and suggest these transcription factors may coregulate common target genes.

OsteoMacs maintain the endosteal hematopoietic stem cell niche and participate in mobilization

OsteoMacs are tissue macrophages that reside within bone lining tissues. OsteoMacs enhance osteoblast function in vitro and maintain osteoblast bone forming surface in vivo. Given the endosteum is a preferred niche for hematopoietic stem cells (HSC), we investigated whether OsteoMacs participate in HSC mobilization induced by granulocyte colony-stimulating factor (G-CSF). Immunohistochemistry (IHC) using the F4/80 antibody demonstrated that mobilization induced by G-CSF resulted in a sharp reduction in the number of F4/80+ OsteoMacs within the endosteal niche from days 2–6 post initiation of G-CSF administration. At day 2, clustering of F4/80+ cells in the central marrow and in particular around endothelial sinuses was evident. In contract, localization and maintenance of OsteoMacs within the periosteum was unaffected by G-CSF administration. Flow cytometry demonstrated increased numbers of F4/80+ macrophages in blood from days 1–6, indicating egress of macrophages from bone marrow to blood. Static and dynamic histomorphemtry and IHC for osteocalcin expression demonstrated a concomitant significant reduction in endosteal osteoblast number and function as well as osteoid. Notably, the kinetics of OsteoMac loss strikingly parallels, if not precedes, that of endosteal osteoblasts loss and mobilization of HSC to blood. In contrast, osteoclast numbers were not significantly altered by G-CSF administration and treatment with the bisphosphonate zoledronate increased HSC mobilization in response to G-CSF. F4/80+ macrophages repopulation of bone marrow and endosteal surfaces initiated at day 8, 2 days after cessation of G-CSF administration, and was quickly followed at day 10 by recovery of OsteoMac canopy covering cubiodal osteoblast-like cells. Prior experimentation has indicated that mobilization induced by G-CSF acts through an intermediary G-CSF receptor expressing myeloid population. Finally, we demonstrate a pivotal role of macrophages in maintaining endosteal HSC niches in transgenic MAFIA mice in which macrophages can be specifically deleted in vivo using AP20187. At day 5 following macrophages deletion, we observed a collapse in the number of osteoblasts lining the endosteum and robust mobilization of HSC in peripheral blood and spleen. Given macrophages can express the G-CSF receptor and the observations reported here, we propose that G-CSF induces HSC mobilization through OsteoMac-directed collapse of the endosteal HSC niches.

Identification of a Stroma-Mediated Wnt/β-Catenin Signal Promoting Self-Renewal of Hematopoietic Stem Cells in the Stem Cell Niche

With contrasting observations on the effects of beta-catenin on hematopoietic stem cells (HSCs), the precise role of Wnt/beta-catenin signals on HSC regulation remains unclear. Here, we show a distinct mode of Wnt/beta-catenin signal that can regulate HSCs in a stroma-dependent manner. Stabilization of beta-catenin in the bone marrow stromal cells promoted maintenance and self-renewal of HSCs in a contact-dependent manner, whereas direct stabilization in hematopoietic cells caused loss of HSCs. Interestingly, canonical Wnt receptors and beta-catenin accumulation were predominantly enriched in the stromal rather than the hematopoietic compartment of bone marrows. Moreover, the active form of beta-catenin accumulated selectively in the trabecular endosteum in "Wnt 3a-stimulated" or "irradiation-stressed," but not in "steady-state" marrows. Notably, notch ligands were induced in Wnt/beta-catenin activated bone marrow stroma and downstream notch signal activation was seen in the HSCs in contact with the activated stroma. Taken together, Wnt/beta-catenin activated stroma and their cross-talk with HSCs may function as a physiologically regulated microenvironmental cue for HSC self-renewal in the stem cell niche.

Pharmacological Manipulation of the RAR/RXR Signaling Pathway Maintains the Repopulating Capacity of Hematopoietic Stem Cells in Culture

The retinoid X receptor (RXR) contributes to the regulation of diverse biological pathways via its role as a heterodimeric partner of several nuclear receptors. However, RXR has no established role in the regulation of hematopoietic stem cell (HSC) fate. In this study, we sought to determine whether direct modulation of RXR signaling could impact human HSC self-renewal or differentiation. Treatment of human CD34(+)CD38(-)lin(-) cells with LG1506, a selective RXR modulator, inhibited the differentiation of HSCs in culture and maintained long-term repopulating HSCs in culture that were otherwise lost in response to cytokine treatment. Further studies revealed that LG1506 had a distinct mechanism of action in that it facilitated the recruitment of corepressors to the retinoic acid receptor (RAR)/RXR complex at target gene promoters, suggesting that this molecule was functioning as an inverse agonist in the context of this heterodimer. Interestingly, using combinatorial peptide phage display, we identified unique surfaces presented on RXR when occupied by LG1506 and demonstrated that other modulators that exhibited these properties functioned similarly at both a mechanistic and biological level. These data indicate that the RAR/RXR heterodimer is a critical regulator of human HSC differentiation, and pharmacological modulation of RXR signaling prevents the loss of human HSCs that otherwise occurs in short-term culture.

Disruption of the Osteoblast Niche by G-CSF Is Associated with Hematopoietic Stem Cell Quiescence and Loss of Long-Term Repopulating Activity: Role of Cdkn1a

The bone marrow microenvironment plays a key role in regulating hematopoietic stem cell (HSC) function. In particular, bone marrow stromal signals contribute to the maintenance of HSC quiescence, a property that is thought to be associated with long-term repopulating activity. We previously reported that G-CSF treatment disrupts the osteoblast niche by inducing osteoblast apoptosis and inhibiting osteoblast differentiation. In this altered bone marrow microenvironment, we also showed that the number of HSCs in the bone marrow after G-CSF treatment (as defined by CD34− Kit+ Sca+ lineage-cells or CD150+ CD48− CD41− lineage-[SLAM] cells) was unchanged and that the HSCs were more quiescent than HSCs from untreated mice. However, despite the quiescent phenotype, there was a marked loss of HSC long-term repopulating activity. To define mechanisms for this phenotype, we first asked whether G-CSF acts directly on HSCs to inhibit their long-term repopulating activity. Bone marrow chimeras containing wild type and G-CSFR−/− cells were established and treated with G-CSF. The contribution of G-CSFR−/− cells to hematopoiesis remained stable for at least 3 months after G-CSF treatment, demonstrating that the effects of G-CSF on HSC function are not direct. We next performed RNA expression profiling on sorted SLAM cells, a cell population highly enriched for HSCs. These data showed that expression of Cdkn1a (p21cip1/waf1) was increased in HSCs harvested from G-CSF treated mice. To define the contribution of Cdkn1a to HSC quiescence and loss of repopulating activity following treatment with G-CSF, Cdkn1a−/− mice (inbred on a C57BL/6 background) were studied. Wild-type or Cdkn1a−/− mice were treated with G-CSF for 7 days and pulse labeled with bromo-deoxyuridine (BrdU), and the percentage of SLAM cells that labeled with BrdU was determined. Consistent with our previous observations, treatment of wild-type mice with G-CSF resulted in a significant decrease in the percentage of BrdU+ SLAM cells in the bone marrow. In contrast, in Cdkn1a−/− mice, no change in the percentage of BrdU+ SLAM cells after G-CSF treatment was observed [10.08 ± 2.26% (untreated); 10.96 ± 2.80% (G-CSF treated); p = NS]. To assess HSC function, competitive repopulation assays were performed using untreated or G-CSF treated bone marrow from wild type or Cdkn1a−/− mice. Surprisingly, G-CSF had a similar deleterious effect on HSC repopulating activity in both wild type and Cdkn1a−/− mice. Collectively, these data show G-CSF treatment, possibly through disruption of the osteoblast niche, induces HSC quiescence and loss of long-term repopulating activity. HSC quiescence, but not loss of repopulating activity, is dependent upon Cdkn1a−/−. The mechanisms by which G-CSF treatment results in a loss of HSC function are under investigation.

Fbxw7 acts as a critical fail-safe against premature loss of hematopoietic stem cells and development of T-ALL

Common molecular machineries between hematopoietic stem cell (HSC) maintenance and leukemia prevention have been highlighted. The tumor suppressor Fbxw7 (F-box and WD-40 domain protein 7), a subunit of an SCF-type ubiquitin ligase complex, induces the degradation of positive regulators of the cell cycle. We demonstrate that inactivation of Fbxw7 in hematopoietic cells causes premature depletion of HSCs due to active cell cycling and p53-dependent apoptosis. Interestingly, Fbxw7 deletion also confers a selective advantage to cells with suppressed p53 function, eventually leading to development of T-cell acute lymphoblastic leukemia (T-ALL). Thus, Fbxw7 functions as a fail-safe mechanism against both premature HSC loss and leukemogenesis.