Hematopoietic Stem Cell Cycling Research Articles

Mitochondrial Activity Determines Hematopoietic Stem Cell Fate Decisions

A tight control of hematopoietic stem cell (HSC) quiescence, self-renewal and differentiation is crucial for lifelong blood production. The mechanisms behind this control are still poorly understood. Here we show that mitochondrial activity determines HSC fate decisions. A low mitochondrial membrane potential (Δψm) predicts long-term multi-lineage blood reconstitution capability, as we show for freshly isolated and in vitro-cultured HSCs. However, as in vivo both quiescent and cycling HSCs have comparable Δψm distributions, a low Δψm is not per se related to quiescence but is also found in dividing cells. Indeed, using divisional tracking, we demonstrate that daughter HSCs with a low Δψm maintain stemness, whereas daughter cells with high Δψm have undergone differentiation.Strikingly, lowering the Δψm by chemical uncoupling of the electron transport chain leads to HSC self-renewal under culture conditions that normally induce rapid differentiation. Taken together, these data show that mitochondrial activity and fate choice are causally related in HSCs, and provides a novel method for identifying HSC potential after in vitro culture. DisclosuresNo relevant conflicts of interest to declare.

Regulation of Hematopoietic Stem Cells by Interferons and by DNA Methylation: Implications for Bone Marrow Failure

Bone marrow failure (BMF), the inability to regenerate the differentiated cells of the blood, has a number of genetic and environmental etiologies, such as mutation of telomere-associated protein genes and immune-related aplastic anemia. Recently, mutations in DNA methyltransferase 3A (DNMT3A) have been found to be associated with approximately 15% of cases of primary myelofibrosis (MF), which can be a cause of BMF. The role of DNMT3A more broadly in hematopoiesis, and specifically in BMF, is currently poorly understood. DNMT3A is one of two de novo DNA methylation enzymes important in developmental fate choice. We showed that Dnmt3a is critical for normal murine hematopoiesis, as hematopoietic stem cells (HSCs) from Dnmt3a knockout (KO) mice displayed greatly diminished differentiation potential while their self-renewal ability was markedly increased1, in effect, leading to failure of blood regeneration or BMF. Combined with loss of Dnmt3b, HSCs exhibited a profound differentiation block, mediated in part by an increase of stabilized b-catenin. While we did not initially observe bone marrow pathology or malignancy development in mice transplanted with Dnmt3a KO HSCs, when we aged a large cohort of mice, all mice succumbed to hematologic disease within about 400 days. Roughly one-third of mice developed frank leukemia (acute lymphocytic leukemia or acute myeloid leukemia), one-third developed MDS, and the remainder developed primary myelofibrosis or chronic myelomonocytic leukemia. The pathological characteristics of the mice broadly mirror those of patients, suggesting the Dnmt3a KO mice can serve as a model for human DNMT3A-mutation associated disease. Strikingly, bone marrow of mice with different disease types exhibit distinct DNA methylation features. These will findings and the implications for disease development will be discussed. We are currently investigating the factors that drive different outcomes in the mice, including stressors such as exposure to interferons. We have hypothesized that HSC proliferation accelerates the Dnnmt3a-associated disease phenotypes. We have previously shown that interferons directly impinge on HSCs in the context of infections. Interferons activate HSCs to divide, generating differentiated progeny and cycling HSCs. Repeated interferon stimulation may permanently impair HSC function and bias stem cell output. When combined with loss of Dnmt3a, interferons may promote BMF. We will discuss broadly how external factors such as aging and infection may collaborate with specific genetic determinants to affect long-term hematopoiesis and malignancy development. Reference: Challen GA, Sun D, Jeong M, et al. Dnmt3a is essential for hematopoietic stem cell differentiation. Nat Genet 2012; 44: 23-31 Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Altered HSC Metabolism in Response to Stress Leads to De Novo dna Damage and Cellular Attrition

Hematopoietic stem cells (HSCs) reside in a quiescent state, which is thought to preserve their genomic stability during aging. HSCs are forced to exit this so-called dormant state and enter into cycle in response to stress stimuli such as infections or severe bleeding. This situation may provoke high levels of proliferative stress in HSCs and a subsequent decline in stem cell function. We recently found that de novo DNA damage can be precipitated in HSCs in vivo by enforcing cell cycle progression using agonists that mimic physiologic stress, such as interferons, G-CSF, TPO or serial bleeding, (Walter et al., Blood, 122, 21:799). The Fanconi anemia (FA) DNA repair pathway is an important route via which this replication damage is resolved in HSCs in vivo. In FA deficient mice, DNA damage repair was impaired, provoking HSC depletion and severe aplastic anemia (p<0.01) upon serial treatment with the synthetic double-stranded RNA mimetic polyI:polyC (pI:pC). Here, we sought to identify the mechanistic basis of the stress-induced DNA damage acquisition and concomitant HSC attrition in vivo.Activated HSCs exhibited elevated mitochondrial membrane potential, indicative of increased energy production via oxidative phosphorylation (>2-fold increase, p<0.01). Next, to determine whether there was an associated increase in intracellular reactive oxygen species (ROS) production, we made use of genetically encoded fluorescent biosensors to detect the status of specific redox couples within different HSC compartments in vivo. Activated HSCs demonstrated increased levels of oxidized mitochondrial glutathione (2.3-fold increase, p<0.01) and cytoplasmic hydrogen peroxide (1.6-fold increase, p<0.05) compared to dormant HSC controls. These enhanced ROS levels directly correlated with elevated 8-Oxo-dG lesions on the DNA of HSCs that had been activated into cycle in vivo(>1.3-fold increase, p<0.05). Finally, retroviral over-expression of ROS-detoxifying enzymes completely rescued gH2AX foci formation in cycling HSCs, demonstrating a direct functional link between stress-induced DNA damage and altered redox biology.We next performed live cell video imaging on individual WT and Fanca-/- LT-HSCs in vitro in order to track cell fate decisions upon exit from quiescence. In the first division upon exit from quiescence, Fanca-/- HSCs were frequently observed to undergo abnormal mitoses while this was not evident in WT HSCs. At this time point, we observed elevated DNA damage in Fanca-/- HSCs as measured by gH2AX, 53BP1 and RAD51 foci, as well as increased ROS-induced 8-Oxo-dG lesions (>5-fold increase, p<0.01). HSCs from Fanca-/- mice demonstrated a significantly higher rate of replication-dependent cell death following the first division (24% vs. 6%, p<0.05%) suggesting that apoptosis is the major route via which HSCs are lost in response to stress-induced DNA damage.Taken together, these data strongly implicate stress-induced exit from dormancy as a cause of physiologic DNA damage in HSCs in vivo. Under stress conditions, the increased energy demand of cycling stem cells leads to elevated levels of ROS in mitochondria and cytoplasm, which is a direct source of DNA damage. If unresolved by the FA-dependent DNA damage response, this DNA damage accumulates in the cell and provokes apoptotic cell death. This recapitulates the highly penetrant bone marrow failure syndrome in FA patients and suggests that their HSCs are lost due to an aberrant response to HSC activation, most likely as a consequence of infection or other physiologic stress. These data provide a novel link between stress hematopoiesis, ROS, DNA damage and HSC loss and may have important clinical implications in the study of age-related hematopoietic defects in both FA and non-FA patients. Moreover, these data provide the first evidence that FA knockout mouse models can be utilized to accurately recapitulate the etiology of bone marrow failure through the progressive application of stress-induced alterations in HSC function that mimic usual physiologic stressors such as infection. DisclosuresNo relevant conflicts of interest to declare.

Nitric oxide-induced murine hematopoietic stem cell fate involves multiple signaling proteins, gene expression, and redox modulation.

There are a growing number of reports showing the influence of redox modulation in cellular signaling. Although the regulation of hematopoiesis by reactive oxygen species (ROS) and reactive nitrogen species (RNS) has been described, their direct participation in the differentiation of hematopoietic stem cells (HSCs) remains unclear. In this work, the direct role of nitric oxide (NO(•)), a RNS, in the modulation of hematopoiesis was investigated using two sources of NO(•) , one produced by endothelial cells stimulated with carbachol in vitro and another using the NO(•)-donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) in vivo. Two main NO(•) effects were observed: proliferation of HSCs-especially of the short-term HSCs-and its commitment and terminal differentiation to the myeloid lineage. NO(•)-induced proliferation was characterized by the increase in the number of cycling HSCs and hematopoietic progenitor cells positive to BrdU and Ki-67, upregulation of Notch-1, Cx43, PECAM-1, CaR, ERK1/2, Akt, p38, PKC, and c-Myc. NO(•)-induced HSCs differentiation was characterized by the increase in granulocytic-macrophage progenitors, granulocyte-macrophage colony forming units, mature myeloid cells, upregulation of PU.1, and C/EBPα genes concomitantly to the downregulation of GATA-3 and Ikz-3 genes, activation of Stat5 and downregulation of the other analyzed proteins mentioned above. Also, redox status modulation differed between proliferation and differentiation responses, which is likely associated with the transition of the proliferative to differentiation status. Our findings provide evidence of the role of NO(•) in inducing HSCs proliferation and myeloid differentiation involving multiple signaling.

Abstract 4775: PI3K-p110β is required for leukemic transformation and HSC depletion in the absence of Pten

Abstract The tumor suppressor PTEN, which antagonizes PI3K signaling, plays critical roles in cell cycle, cell migration and stem cell self-renewal. PTEN is frequently mutated or inactivated in T-cell acute lymphoblastic leukemia (T-ALL) and acute myelogenous leukemia (AML). In mice, HSC-specific deletion of Pten leads to myeloproliferative phenotype, transplantable leukemia, and increased cycling of hematopoietic stem cells (HSCs), leading to their depletion. Rapamycin, an mTOR inhibitor, rescues this phenotype. Thus, it is likely that PI3K itself plays an important role in Pten-deficient HSCs and leukemic cells, but the specific contributions of the PI3K isoforms are poorly understood. We demonstrate that genetic ablation or pharmacological inhibition of PI3K-p110β prevents myeloid neoplasia and prolongs survival. Importantly, p110β ablation rescues HSCs in the bone marrow (BM), and reinstates long-term hematopoietic reconstitution. Moreover, Pten deficiency leads to aberrant expression of Cxcl12 in the BM and spleen, enabling the generation of an alternative splenic niche. Inactivation of p110β rescues retention of HSCs, and perturbs the splenic niche by interfering with the Cxcl12-CXCR4 axis. This suggests that selective inhibition of p110β offers an alternative therapeutic strategy in PTEN deficient leukemias while restoring untransformed HSCs to their proper niche. Citation Format: Haluk Yuzugullu, Lukas Baitsch, Allison Steiner, Linda K. Clayton, Kira Gritsman, Jean J. Zhao. PI3K-p110β is required for leukemic transformation and HSC depletion in the absence of Pten. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4775. doi:10.1158/1538-7445.AM2014-4775

Pleiotrophin mediates hematopoietic regeneration via activation of RAS.

Hematopoietic stem cells (HSCs) are highly susceptible to ionizing radiation-mediated death via induction of ROS, DNA double-strand breaks, and apoptotic pathways. The development of therapeutics capable of mitigating ionizing radiation-induced hematopoietic toxicity could benefit both victims of acute radiation sickness and patients undergoing hematopoietic cell transplantation. Unfortunately, therapies capable of accelerating hematopoietic reconstitution following lethal radiation exposure have remained elusive. Here, we found that systemic administration of pleiotrophin (PTN), a protein that is secreted by BM-derived endothelial cells, substantially increased the survival of mice following radiation exposure and after myeloablative BM transplantation. In both models, PTN increased survival by accelerating the recovery of BM hematopoietic stem and progenitor cells in vivo. PTN treatment promoted HSC regeneration via activation of the RAS pathway in mice that expressed protein tyrosine phosphatase receptor-zeta (PTPRZ), whereas PTN treatment did not induce RAS signaling in PTPRZ-deficient mice, suggesting that PTN-mediated activation of RAS was dependent upon signaling through PTPRZ. PTN strongly inhibited HSC cycling following irradiation, whereas RAS inhibition abrogated PTN-mediated induction of HSC quiescence, blocked PTN-mediated recovery of hematopoietic stem and progenitor cells, and abolished PTN-mediated survival of irradiated mice. These studies demonstrate the therapeutic potential of PTN to improve survival after myeloablation and suggest that PTN-mediated hematopoietic regeneration occurs in a RAS-dependent manner.

Prep1 (pKnox1) regulates mouse embryonic HSC cycling and self-renewal affecting the Stat1-Sca1 IFN-dependent pathway.

A hypomorphic Prep1 mutation results in embryonic lethality at late gestation with a pleiotropic embryonic phenotype that includes defects in all hematopoietic lineages. Reduced functionality of the hematopoietic stem cells (HSCs) compartment might be responsible for the hematopoietic phenotype observed at mid-gestation. In this paper we demonstrate that Prep1 regulates the number of HSCs in fetal livers (FLs), their clonogenic potential and their ability to de novo generate the hematopoietic system in ablated hosts. Furthermore, we show that Prep1 controls the self-renewal ability of the FL HSC compartment as demonstrated by serial transplantation experiments. The premature exhaustion of Prep1 mutant HSCs correlates with the reduced quiescent stem cell pool thus suggesting that Prep1 regulates the self-renewal ability by controlling the quiescence/proliferation balance. Finally, we show that in FL HSCs Prep1 absence induces the interferon signaling pathway leading to premature cycling and exhaustion of fetal HSCs.

A Feeder-Free Differentiation System Identifies Autonomously Proliferating B Cell Precursors in Human Bone Marrow

The peripheral B cell compartment is maintained by homeostatic proliferation and through replenishment by bone marrow precursors. Because hematopoietic stem cells cycle at a slow rate, replenishment must involve replication of precursor B cells. To study proliferation of early human B cell progenitors, we established a feeder cell-free in vitro system allowing the development of B cells from CD34(+) hematopoietic stem cells up to the stage of immature IgM(+) B cells. We found that pro-B and pre-B cells generated in vitro can proliferate autonomously and persist up to 7 wk in culture in the absence of signals induced by exogenously added cytokines. Nevertheless, addition of IL-7 enhanced pre-B cell expansion and inhibited maturation into IgM(+) B cells. The B cell precursor subsets replicating in vitro were highly similar to the bone marrow B cell precursors cycling in vivo. The autonomous proliferation of B cell precursor subsets in vitro and their long-term persistence implies that proliferation during pro-B and pre-B cell stages plays an important role in the homeostasis of the peripheral B cell compartment. Our in vitro culture can be used to study defects in B cell development or in reconstitution of the B cell pool after depletion and chemotherapy.

The TIF1β-HP1 System Maintains Transcriptional Integrity of Hematopoietic Stem Cells

SummaryTIF1β is a transcriptional corepressor that recruits repressive chromatin modifiers to target genes. Its biological function and physiological targets in somatic stem cells remain largely unknown. Here, we show that TIF1β is essential for the maintenance of hematopoietic stem cells (HSCs). Deletion of Tif1b in mice induced active cycling and apoptosis of HSCs and promoted egression of HSCs from the bone marrow, leading to rapid depletion of HSCs. Strikingly, Tif1b-deficient HSCs showed a strong trend of ectopic expression of nonhematopoietic genes. Levels of heterochromatin protein 1 (HP1α, β and γ) proteins, which form a complex with TIF1β, were significantly reduced in the absence of TIF1β and depletion of HP1 recapitulated a part of the phenotypes of Tif1b-deficient HSCs. These results demonstrate that the TIF1β-HP1 system functions as a critical repressive machinery that targets genes not normally activated in the hematopoietic compartment, thereby maintaining the transcriptional signature specific to HSCs.

Developing a systems-based understanding of hematopoietic stem cell cycle control.

To maintain hematologic homeostasis, hematopoietic stem cells (HSCs) undergo multiple rounds of cell division throughout their lives. Under steady-state conditions, adult HSCs are relatively quiescent and reside primarily in hypoxic bone marrow niches. In response to physiologic stimuli, normal HSCs either reenter the cell division cycle or remain in quiescence. A large body of work has focused on understanding the mechanistic underpinnings balancing differentiation against self-renewal programs in cycling HSCs. Numerous reports from genetically engineered mouse models harboring mutations in key pathways governing proliferation control, DNA damage responses, and metabolic regulation indicate the critical roles these processes play in determining HSC self-renewing versus blood-lineage-reconstituting divisions. In this chapter, we integrate these findings and highlight the cellular networks that control HSC function and fitness by regulating HSC cycling.

Transient Activation Of Inflammatory Molecules By Epigenetic Modifiers Likely Plays a Role In Expansion Of Transplantable Human Cord Blood Stem/Progenitor Cells

Recent studies indicate that inflammatory molecules, including interferon, possess stimulatory effects which trigger excessive proliferation leading to hematopoietic stem cell (HSC) exhaustion and bone marrow (BM) failure. We hypothesized that transient activation of inflammatory molecules by chromatin modifying agents (CMA) stimulates proliferation of transplantable HSC in culture. We previously demonstrated that sequential in vitro addition of a hypomethylating drug, 5-aza-2'-deoxycytidine (5azaD) followed by addition of a histone deacetylase (HDAC) inhibitor, trichostatin A (TSA) during human umbilical cord blood CD34+ cell culture results in expansion of serially transplantable long term HSC (Araki et al. Blood 2007, Exp Hematol 2009). However treatment with valproic acid (VPA), a HDAC inhibitor, only expands short term HSC, which are not serially transplantable (Araki et al. Abst. 827, Blood 2010). This failure of VPA to expand long-term repopulating HSC is not however due to poor BM homing (homing efficiency VPA-expanded cells 1.68 ± 0.35%, n = 10 NOD/SCID mice, 5azaD/TSA-expanded cells 0.39 ± 0.056%, n = 7, p = 0.008). Molecular studies indicate that expression of genes implicated in HSC self-renewal (HoxB4, Bmi1, Ezh2, and GATA2) are equally increased in 5azaD/TSA or VPA expanded CD34+ cells (n = 3, control vs. 5azaD/TSA or control vs. VPA p < 0.05). Similarly, increased HoxB4, Ezh2 and PU.1 protein levels are observed in VPA or 5azaD/TSA expanded cells compared to controls. Our global microarray data (Affymetrix chip HG U133 plus 2.0, n = 3) reveals a set of 113 differentially expressed genes preferentially detectable in 5azaD/TSA expanded cells associated with transplantable HSC expansion in culture and 278 HSC maintenance genes differentially expressed in CD34+ cells expanded in VPA or 5azaD/TSA, but not in control. Differential expression of genes representative of HSC expansion or maintenance gene sets were validated by real time qPCR. Intriguingly, functional pathway analyses of the 113 HSC expansion genes indicate the importance of inflammation signaling pathways (genes such as S100A8, Alox5). Furthermore, global genomic DNA methylation analysis of uncultured vs expanded, enriched CD34+ cells by LINE-1 methylation assay indicates transient hypomethylation of 5azaD/TSA-expanded cells at day 3 (55.4 ± 2.2 vs pre–treatment levels 77.3 ± 1.9) which later returned towards baseline (69.7 ± 2.1), corresponding with recovery of DNMT1 expression. Notably, control cultures or CD34+ cells expanded in VPA do not display changes in LINE-1 methylation (78.5 ± 2.2 and 77.2 ± 2 respectively, n = 2, mean % ± SE of 4 CpG sites). Interestingly, ELISA assays of conditioned medium from 5azaD/TSA-expanded cells reveals that levels of the inflammatory mediator leukotriene B4 (LTB4; synthesized from arachidonic acid by the Alox5 gene products, 5-lipooxygenase and LTA4 hydrolase) are 426.87 ± 101.7 pg/ml, significantly greater than that of control cultures (109.5 ± 11.6 pg/ml; n = 3, p = 0.03). VPA-treated cultures had slightly higher levels than controls (346.8 ± 138.8 pg/ml; p = 0.16). Similarly 5azaD/TSA IL-1β levels were significantly greater than controls (10.1 ± 0.8 pg/ml vs 6.0 ± 0.6 pg/ml, n= 2, triplicate wells, p = 0.05). IL-1β levels from VPA cultures were 10.1 ± 4.5 pg/ml (p = n.s.). Notably, expansion of CD34+CD90+ cells in the presence of 5azaD/TSA is reduced by 47.6% by addition of dexamethasone, an anti-inflammatory agent, without affecting total cell numbers. Furthermore, demethylation of CpG sites of the promoters of inflammatory/stress response genes correspond with higher expression of these genes in CMA-expanded cultures (e.g., S100A8 and Cyp11A1; pre treatment: 76.42% ± 4.91% and 80.73 % ± 4.16%, post 5azaD/TSA treatment 54.5% ± 3.84% and 54.29% ± 3.72% respectively; n = 2, mean ± SE of 3 to 11 CpG sites methylation). S100A8 is a known agonist of toll-like receptor 4, which plays a role in the maintenance and proliferation of endothelial progenitor cells, in keeping with the influence of systemic infection on HSC cycling and of interferon on HSC proliferation. Our studies, for the first time, identify inflammatory molecules regulated by epigenetic mechanisms that are potentially important in governing HSC fate choices. Further studies should determine the mechanisms by which CMAs influence HSC expansion or maintenance through inflammatory and other stress response pathway genes. Disclosures:No relevant conflicts of interest to declare.

Hemopoietic Stem Cells In Polycythemia Vera and Essential Thrombocythemia Have Increased TNF-Alpha That Decreases After Pegylated Interferon-a Therapy, But Not Hydroxyurea, and Correlates With Suppression Of PV and ET Clones

The mechanism whereby suppression of normal hematopoietic stem cells (HSC) by polycythemia vera (PV) and essential thrombocytopenia (ET) clones occurs remains an enigma, yet PV and ET clones dominate mature myelopoiesis even when increased numbers of circulating clonal cells are normalized by hydroxyurea or other myelosuppressive therapies. Interferon-a (Infa) is the only treatment that not only decreases JAK2V617F allelic burden and ameliorates marrow cytogenetic abnormalities, but may also reverse clonal hematopoiesis (E. Liu, Blood, 2003). The mechanism of this response is not certain, and some data suggest this reversal may be due to: a) increased cycling of normal HSC (see K. King abstract at this ASH mtg), b) possible immune response against the malignant clone, or c) direct suppression of the malignant clone by an as-yet-unknown mechanism. As TNFa is a cytokine known to suppress normal hematopoiesis, we have examined its role in possible reversal to polyclonal hematopoiesis in patients treated in a Phase III trial for PV and ET with either pegylated interferon-α (pegInfa) or hydroxyurea.We found that plasma TNFa levels were not markedly elevated in PV and ET patients (range = 1-10 pg/ml, mean = 4 pg/ml, normal = <5 pg/ml) and did not change before or after therapy with pegInfa or hydroxyurea. However, immunohistochemical staining of PV and ET marrows showed a marked increase in TNFa protein expression in PV and ET marrow cells compared to normal controls. We found that TNFa mRNA, measured by QT-PCR, of early and late myeloid progenitors isolated from marrow was mainly confined to early hematopoietic precursors. Furthermore, sorted PV & ET CD34+ cells had increased TNFa transcripts when compared to normal HSC cells (17 fold, p>0.0005). Patients who responded to therapy with pegInfa had a greater decrease in TNFa transcripts in CD34+ cells compared to patients treated with hydroxyurea (p value =0.0008) (see Figure). In pegInfa-treated patients, the decrement in TNFa mRNA corresponded to the decline in JAK2V617F level (Spearman's Rho = 0.4254; p-value = 0.0097). Additionally, 2 female PV patients who converted from clonal to polyclonal hematopoiesis (detected by X-chromosome allelic usage ratio) also had marked decline of CD34+ TNFa mRNA. Furthermore, 1 PV patient, who had a dramatic decline of JAK2V617F and TNFa mRNA on pegInfa, which was stopped because of side-effects and then switched to hydroxyurea, reverted to increased CD34+ TNFa mRNA and an increased JAK2V617F allelic burden. Then, because of an incomplete response to hydroxyurea, this patient was started on low-dose pegInfa and had marked corresponding changes of both JAK2V617F and TNFa. In 2 pegInfa-treated PV and 1 untreated PV patients, 9 JAK2V617F homozygous BFU-E, 35 JAK2V617F heterozygous BFU-E, and 64 wild-type JAK2 BFU-E colonies were analyzed for TNFa transcripts. Unexpectedly, at the time of harvest of BFU-E colonies (14 days after culture, cells were largely late normoblasts), the JAK2V617F-positive colonies had undetectable TNFa transcripts, unlike wild-type JAK2 in PV BFU-E and BFU-E colonies from normal healthy controls.These data indicate that intracellular and possibly paracrine TNFa is strongly associated with the suppression of normal hematopoiesis in PV and ET. We demonstrated that this increase in HSC TNFa is markedly reduced in those patients successfully treated with pegInfa, and this decrease is not seen when increased numbers of circulating clonal cells are normalized by hydroxyurea. These data provide a foundation for the addition of TNFa inhibitors as possible combination therapies for PV & ET.This work was supported by 1P01CA108671-O1A2 (NCI) Myeloproliferative Disorders (MPD) Consortium (PI Ron Hoffman) project#1 (PI Prchal) and Leukemia&Lymphoma Society.First and second authors contributed equally [Display omitted] Disclosures:Swierczek:University of Utah: No financial compensation, No financial compensation Patents & Royalties. Prchal:University of Utah : No financial compensation, No financial compensation Patents & Royalties.

Loss Of p53 Rescues The Defective Function Of Foxo3-/- Hematopoietic Stem Cells But Enhances Their Predisposition To Malignancy

Leukemic stem cells (LSCs) share many of the same properties of normal hematopoietic stem cells (HSCs) including their highly quiescent state, capacity to self-renew, low levels of reactive oxygen species (ROS) and enhanced DNA repair program. These properties make the efficient and specific eradication of these cells challenging. Foxo3 and p53 are two transcription factors essential for the modulation of HSC quiescence and self-renewal. While Foxo3 is inhibited by signaling from several oncoproteins but crucial for the maintenance of the LSCs in both chronic and acute myeloid leukemia (CML and AML respectively), mutations of p53 although rare, are associated with poor prognosis in advanced stages of these diseases. In vivo ROS-mediated activation of p53 is known to lead to loss of quiescence, alterations of cell cycle and exhaustion of the Foxo3-/- HSC pool. Seeking to understand the contribution of p53 to Foxo3-/- HSC cycling defects, we crossed p53+/- and Foxo3+/- mice. To our surprise we found the bone marrow (BM) frequency of both p53+/-Foxo3-/- and p53-/-Foxo3-/- LSK (Lin-Sca1+cKit+) and long-term-HSC (LT-HSC, LSK Flk2-CD34-) populations greatly increased as compared to their Foxo3-/- counterparts (n=5 mice per genotype; p<0.05). Using Ki67 and DAPI staining we found that loss of one or both alleles of p53 gradually rescued the cell cycle defect of Foxo3-/- HSC and increased the frequency of LSK cells in Go by 2-fold. Loss of p53 also rescued the impaired capacity of Foxo3-/- LSK cells to competitively repopulate multilineage blood over 16 weeks, as shown by the higher frequency of p53+/-Foxo3-/- and p53-/-Foxo3-/- donor-derived cells in the peripheral blood of recipient animals (∼47% recipients of double-mutant cells versus 20% in Foxo3-/- recipients, n=5 per group). Furthermore, loss of p53 significantly improved the compromised self-renewal of Foxo3 mutant HSC in serial BM transplantations. In our quest to identify mechanisms whereby p53 depletion improves Foxo3-/- HSC function, we noticed that the DNA damage accumulated in Foxo3-/- HSC at the steady-state was remarkably ameliorated by removal of one or both alleles of p53 from Foxo3-/- HSCs, as measured by flow cytometry levels of phospho-H2AX (gamma-H2AX) and DNA breaks by comet assay (n=3, p<0.05). Unexpectedly, ROS levels were also significantly reduced by 30% in p53+/-Foxo3-/- in comparison to Foxo3-/- LSK cells, while ROS levels in p53+/- LSK cells were similar to that in WT cells. Consistent with these results, the expression of several anti-oxidant enzymes including Sod1, Sod2, Catalase, Gpx1, Sesn1 and Sesn2 (n≥2), was highly upregulated while a number of genes implicated in mitochondrial generation of ROS were significantly deregulated as a result of loss of one or both alleles of p53. These combined findings suggest that a switch from anti-oxidant to pro-oxidant activity of p53 contributes to Foxo3-/- HSC defects. Despite their apparent normal stem cell function, p53+/-Foxo3-/- HSC were highly altered in their gene expression profile. Interestingly, Gene Set Enrichment Analysis (GSEA) of the microarray analysis (Illumina bead chip mouse-Ref8) of WT, p53+/-, Foxo3-/-, and p53+/-Foxo3-/- LSK cells showed that a cluster of genes associated with fatty acid metabolism was highly enriched in p53+/-Foxo3-/- HSCs (ES=0.746; p<0.01). In addition, from 3976 genes exclusively deregulated in p53+/-Foxo3-/- LSK cells, 201 (out of 1051) overlapped with genes downregulated, while 9 (out of 14) overlapped with genes exclusively upregulated in a LSC-gene signature. To evaluate whether this pre-leukemic profile was associated with increased susceptibility to malignancy, we compared the potential and timeline of BCR-ABL-transformed p53+/-Foxo3-/- HSC as compared to controls in establishing CML in mice. We found a shorter time to the onset of the disease and decreased survival of the recipients of p53+/-Foxo3-/- transformed HSCs (n=4 per group, p<0.05) as compared to WT and Foxo3-/- controls. We propose that the p53+/-Foxo3-/- double-mutant HSCs are enriched for preleukemic stem cells based on their quiescence and self-renewal capacity, low ROS, robust DNA repair, susceptibility to transformation and aberrant gene expression profile. These findings raise the possibility that the coordinated Foxo3 and p53 regulation of ROS wires together the stem cell program. Disclosures:No relevant conflicts of interest to declare.

The murine long-term multi-lineage renewal marrow stem cell is a cycling cell

Prevailing wisdom holds that hematopoietic stem cells (HSCs) are predominantly quiescent. Although HSC cycle status has long been the subject of scrutiny, virtually all marrow stem cell research has been based on studies of highly purified HSCs. Here we explored the cell cycle status of marrow stem cells in un-separated whole bone marrow (WBM). We show that a large number of long-term multi-lineage engraftable stem cells within WBM are in S/G2/M phase. Using bromodeoxyuridine, we show rapid transit through the cell cycle of a previously defined relatively dormant purified stem cell, the long-term HSC (LT-HSC; Lineage(-)/c-kit(+)/Sca-1(+)/Flk-2(-)). Actively cycling marrow stem cells have continually changing phenotype with cell cycle transit, likely rendering them difficult to purify to homogeneity. Indeed, as WBM contains actively cycling stem cells, and highly purified stem cells engraft predominantly while quiescent, it follows that the population of cycling marrow stem cells within WBM are lost during purification. Our studies indicate that both the discarded lineage-positive and lineage-negative marrow cells in a stem cell separation contain cycling stem cells. We propose that future work should encompass this larger population of cycling stem cells that is poorly represented in current studies solely focused on purified stem cell populations.

Modeling Human Hematopoietic Stem Cell Biology in the Mouse

Hematopoietic stem cells (HSCs) have the immense task of supplying an organism with enough blood to sustain a lifespan. Much of what is known about how this scant population of cells can meet the varying demand of producing more than 10(11) cells per day comes from studies conducted in an animal that is a fraction of our size and lives roughly 1/30th of our lifespan. The differences in longevity can be expected to impose different demands on a cell essential for existence. It is therefore unsurprising that while the mouse has proven invaluable in defining the organizing principals of how hematopoiesis is governed, mediators of cell localization as well as a range of experimental methods, the differences in cell cycling, DNA repair and specific molecular features of HSCs in humans are evident and important. Here, the utility and drawbacks of the mouse as an experimental model for human HSC biology are discussed.

TGFβ restores hematopoietic homeostasis after myelosuppressive chemotherapy

Myelosuppression is a life-threatening complication of antineoplastic therapy, but treatment is restricted to a few cytokines with unilineage hematopoietic activity. Although hematopoietic stem cells (HSCs) are predominantly quiescent during homeostasis, they are rapidly recruited into cell cycle by stresses, including myelosuppressive chemotherapy. Factors that induce HSCs to proliferate during stress have been characterized, but it is not known how HSC quiescence is then reestablished. In this study, we show that TGFβ signaling is transiently activated in hematopoietic stem and progenitor cells (HSPCs) during hematopoietic regeneration. Blockade of TGFβ signaling after chemotherapy accelerates hematopoietic reconstitution and delays the return of cycling HSCs to quiescence. In contrast, TGFβ blockade during homeostasis fails to induce cycling of HSPCs. We identified the cyclin-dependent kinase inhibitor Cdkn1c (p57) as a key downstream mediator of TGFβ during regeneration because the recovery of chimeric mice, incapable of expressing p57 in HSPCs, phenocopies blockade of TGFβ signaling after chemotherapy. This study demonstrates that context-dependent activation of TGFβ signaling is central to an unrecognized counterregulatory mechanism that promotes homeostasis once hematopoiesis has sufficiently recovered from myelosuppressive chemotherapy. These results open the door to new, potentially superior, approaches to promote multilineage hematopoietic recovery by blocking the TGFβ signaling that dampens regeneration.

Foxo3-Mediated Redox Regulation Is Required for DNA Damage Response in Hematopoietic Stem Cells.

Abstract 2295Stringent regulation of redox status is critical to the control of hematopoietic stem cell (HSC) quiescence and to the maintenance of HSC pool. However mechanisms by which oxidative stress controls HSC quiescence versus cycling remain unknown. Foxo3 transcription factor is required for the regulation of HSC quiescence and for the maintenance of hematopoietic and leukemic stem cell pool. Redox regulation is key to the Foxo3 control of HSC pool. ROS accumulation in Foxo3 null HSC mediates in vivo activation of p53, and increased p21 expression leading to an arrest in the G2/M phase of cell cycle associated with loss of quiescence. We hypothesized that ROS may regulate HSC quiescence versus cycling via control of DNA damage repair program. To address this question, we examined whether Foxo3 is involved in DNA damage response of HSC. We first evaluated by immunostaining phosphorylation of histone H2AX variant (γH2AX), a hallmark sensor of DNA strand break, in LSK (Lin−Sca-1+c-Kit+) cells freshly isolated from Foxo3−/− bone marrow. We found the number of cells with nuclear γH2AX foci significantly increased in Foxo3−/− LSK cells (n=100; >5 foci/nuclei) in comparison with wild type (WT)-LSK. We subsequently confirmed and quantified these data by flow cytometry analysis of γH2AX. Together these analyses showed that loss of Foxo3 leads to increased γH2AX levels in LSK cells at the steady state. We next evaluated the presence of DNA breaks, by submitting Foxo3−/− versus WT LSK FACS-sorted cells to single-cell gel electrophoresis (Comet Assay). These investigations confirmed that LSK cells from Foxo3−/− mice accumulate DNA breaks at the steady state, as the percentage of comet shape cells (4 fold) and comet length (3 fold) were all increased in Foxo3 mutant LSK. We then asked whether the increased ROS accumulation had any direct role in damaging DNA in Foxo3−/− LSK. Using a fluorescent probe specific for the most common oxidative DNA damage lesion, the 8-hydroxyguanine base (8-OxoG), we further showed that Foxo3−/− LSK cells exhibit oxidative DNA damage. To further investigate the potential function of ROS in the control of HSC DNA damage response, we treated Foxo3−/− and WT mice for 14 days with the ROS scavenger N-acetyl-cysteine (NAC; 100 mg/Kg/day) in vivo. NAC treatment reduced by four fold γH2AX in Foxo3−/− LSK cells to levels similar to that in WT-LSK cells. Similarly, comet assay analysis of FACS-sorted LSK cells from NAC-treated WT and Foxo3−/− mice showed a two fold reduction of DNA breaks. These results suggest that increase in ROS damage DNA and triggers DNA damage response in Foxo3−/− LSK cells at the steady state. Additionally, expression of a number of genes involved in DNA damage repair including Xrcc5 (Ku80) and Xrcc6 (Ku70) was highly downregulated in both long-term-HSC (LT-HSC, LSK-CD150+CD48−) and LSK populations as evidenced by Q-RT-PCR on the Fluidigm™ microfluidics array technology. Together these results strongly suggest that Foxo3-mediated redox regulation is required for protection of DNA from accumulating damage at the steady state in HSC. We further investigated whether ROS-mediated activation of p53 in Foxo3 null HSCs limits the extent of accumulation of DNA damage in HSC. To address this question we crossed p53+/−Foxo3+/− double heterozygous animals to generate p53-Foxo3 double knockout mice. Loss of p53 in Foxo3−/− mice led to significant rise in lymphocyte counts and decrease in neutrophil counts in comparison with Foxo3−/−, indicating a potential shift in lineage determination from HSC. To our surprise, loss of one allele of p53 in Foxo3-null mice significantly reduced gH2AX staining and DNA breaks, as analyzed respectively by flow cytometry and comet assay of sorted LSK cells. While the rescue of DNA damage in Foxo3−/− HSCs as result of loss of p53 was unexpected it is not clear whether it is related to the impact on the fate of HSC. The clarification of these questions in future studies will be important for understanding mechanisms that control the emergence of leukemic stem cells. Together these studies suggest that Foxo3 guards DNA from damage in HSC at the steady state. In addition they indicate an important function for ROS modulation in the in vivo regulation of DNA damage response in HSC. Altogether understanding mechanisms that control ROS modulation of DNA damage response are likely to advance our understanding of the regulation of normal hematopoietic and leukemic stem cell quiescence. Disclosures:No relevant conflicts of interest to declare.

Stress-Mediated Activation of Dormant Hematopoietic Stem Cells In Vivo

Abstract SCI-41Maintenance of the blood system is dependent on dormant hematopoietic stem cells (HSCs), which are characterized by pluripotency and lifelong self-renewal capacity. In order to both maintain a supply of mature blood cells and not exhaust HSCs throughout the lifespan of the organism, most adult HSCs remain deeply quiescent during homeostasis, and only a limited number are cycling at any given time. The balance between self-renewal and differentiation of HSCs is controlled by external factors such as chemokines and cytokines, as well as by interactions of HSCs with their niche environment. The transcriptome of dormant CD34-CD150+CD48-LSK- HSCs significantly differs from that of active HSCs with the same phenotype, while the latter are highly similar to MPP1 progenitors which express CD34. One of the genes differentially expressed is the cylindromatosis (CYLD) gene, which encodes a negative regulator of the NF-κB signaling pathway. HSCs failing to express functional CYLD show various defects associated with a disturbed balance between dormant and active HSCs, suggesting a role for NF-κB signaling in establishing dormancy in HSCs. In addition, our studies have recently shown that the cytokine interferon-α (IFNα) very efficiently activates dormant HSCs in vivo. Within hours after treatment of mice with IFNα, HSCs exit G0 and enter an active cell cycle. In general, IFNα is produced in response to viral infections by cells of the immune system, and plays an important role in the antiviral host defense. We now questioned whether endogenous IFNα is also produced in response to other types of bone marrow stress and whether this affects the proliferation rate of HSCs. To monitor IFNα production in the bone marrow in vivo, we have generated MxCre ROSA-R26-EYFP mice and found that treatment with both the chemotherapeutic agent 5-FU as well as the endotoxin LPS leads to the production of IFNα in the vicinity of HSCs and progenitors. In addition, LPS treatment in vivo induced a strong increase in HSC cycling. Surprisingly, since mice lacking the IFNα receptor (Ifnar−/−) still respond to LPS, this effect is independent of IFNAR signaling. Strikingly, LPS-induced HSC activation correlated with increased expression of Sca-1, similar to what occurs upon IFNα treatment. Moreover, as for IFNα, the upregulation of SCA-1 is required for LPS-induced proliferation, since Sca-1−/− mice fail to respond to LPS stimulation. In summary, these data suggest that not only virus-inducible IFNα, but also infections by gram-negative-bacteria-produced LPS induce cycling of progenitors and otherwise dormant HSCs. Disclosures:No relevant conflicts of interest to declare.

Bone Marrow Arteriolar Niches Maintain Hematopoietic Stem Cell Quiescence

Abstract 638A prevailing idea suggests that quiescent hematopoietic stem cells (HSCs) reside near osteoblasts in the bone marrow (BM) whereas actively cycling HSCs are found near sinusoids. However, this idea is not been proven experimentally. Our recent studies have identified Nestin+ perivascular cells containing all BM mesenchymal stem cell activity as a candidate HSC niche cell (Nature 2010;466:829). To gain more insight in the 3-dimensional (3D) structure of the HSC niche, we have established a novel whole-mount immunofluorescence imaging technique in which the 3D spatial relationships between vascular structures and endogenous CD150+CD48−CD41−Lineage− HSCs in the sternal or femoral BM can be precisely determined. Our imaging analyses in both bone types revealed a prominent sinusoidal network interrupted by rare small caliber (∼10μm) arterioles whose identity was confirmed by staining with artery-specific Alexa Fluor 633 (Nat Methods 2012;9:273) or Sca-1 (Cell Stem Cell 2009;4:263). 3D analyses using Nes-GFP transgenic mice showed two distinct types of Nestin+ cells associated with distinct vascular structures: Nes-GFPbright cells exhibiting a pericyte-like morphology were tightly associated with arterioles (referred to as periarteriolar Nestin (Nesperi) cells) whereas more abundant Nes-GFPdim cells exhibiting a reticular shape (Nesretic cells) were largely associated with sinusoids. We sorted Nesperi and Nesretic cells based on GFP expression and cell size for further characterization. Nesperi cells were very rare (∼0.002% of BM) and expressed ∼3-fold and 10–50-fold higher levels of genes associated with HSC maintenance and retention (Cxcl12, Kitl, Angpt1, Vcam1) compared to Nesretic cells and Nes-GFPneg CD45−CD31−Ter119− stromal cells, respectively. These factors were down-regulated after G-CSF stimulation in Nesperi cells but not in Nesretic cells, which led us to hypothesize that Nesperi cells played an important role in HSC maintenance. We examined the spatial correlation between endogenous HSCs and Nesperi cells by whole-mount immunostaining and found that 38.6 ± 2.9% and 54.9 ± 6.8% of HSCs were localized within 20μm and 40μm distance from Nesperi cells, respectively. After G-CSF treatment, HSCs entered cell cycle (control/G-CSF; 86.8 ± 2.6%/52.2 ± 3.8% in G0, as defined by Ki67 and Hoechst 33342 staining, p=0.002) and expanded around Nesperi cells (control/G-CSF; 34.3 ± 1.0%/48.2 ± 1.6% in 0–20μm proximity, p<0.0001), suggesting that Nesperi cells may regulate cell cycle of HSCs by expression of HSC retention factors. Cell cycle quiescence is a hallmark of stem cells and protecting them from exogenous insults. We thus analyzed the cell cycle status and distribution of HSCs using Ki67 staining. Ki67+ cycling HSCs were distributed significantly further away from Nesperi cells than Ki67− non-cycling cells (Ki67+/Ki67−; 8.3 ± 8.3%/36.8 ± 4.7% in 0–20 μm proximity, p<0.05, 53.4 ± 7.0%/24.6 ± 7.0% > 80μm distance, p<0.05). We evaluated a genetic model (mice deficient in promyelocytic leukemia protein, Pml−/−) that leads to a loss of HSC quiescence in a cell autonomous manner (Nature 2008;453:1072). In Pml−/− BM, HSCs were distributed significantly further away from arterioles compared to wild-type mice (WT/Pml−/−; 34.2 ± 3.9%/9.4 ± 5.1% in 0–20μm proximity, p<0.05, 9.2 ± 4.7%/59.0 ± 12.7% >80μm distance, p<0.05). These data indicate that proximity to arterioles surrounded by Nesperi cells is critical for HSC behavior. Interestingly, we found that Nesperi cells themselves were more quiescent than Nesretic cells (88.7 ± 4.6%/64.2 ± 5.5% in G0, p=0.01). To test the relevance of this quiescent niche, we challenged mice with 5-fluorouracil (5FU). After 5FU administration, the number of Nesperi cells was largely preserved compared to Nesretic cells (63.4 ± 17.1%/13.8 ± 2.9% of untreated control, p=0.01). In addition, the vast majority of HSCs were closely associated with Nesperi cells on day 7 (Control/Day 7 5FU; 34.4 ± 1.0%/70.6 ± 4.0% in 0–20μm, p<0.0001) and returned to baseline levels on day 21 (Day 21 5FU; 36.0 ± 11.4% in 0–20μm), indicating that both HSCs and the niche cells are quiescent and thus resistant to myeloablation. These results indicate that bone marrow arterioles comprise a specialized microenvironment that promotes quiescence of both Nesperi niche cells and the HSCs. Understanding its regulation will have important implications in healthy and cancerous hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Histone Alterations Are Associated with Hematopoietic Stem Cell (HSC) Differentiation and Aging

AbstractAbstract 1188Chromatin regulators, including both Trithorax-group (trxG) and Polycomb-group (PcG) families, have been reported to maintain hematopoietic stem cells (HSC) self-renewal and differentiation. However, their primary targets in HSC remain to be fully identified. Moreover, those histone modifiers have been recently found to control longevity in C.elegans, which leads us to further investigate their roles in HSC aging. HSCs increase in number, decrease in regeneration capacity, and exhibit a myeloid biased differentiation with age. In this study, we profiled global mRNA (RNA-seq) and chromatin changes (ChIP-seq) in highly purified young (4 month) and old (24 month) murine bone marrow-derived HSCs (SP-KSL-CD150+). One key challenge in this study is that mouse HSCs represent less than 0.01% of all bone marrow cells. Thus, we first developed and optimized a method for successful application of ChIP-seq to a limited number of cells (<20,000 cells). This method allowed us to generate the binding profiles for H3K4me3, H3K27me3 and H3K36me3 in young and old HSCs, differentiated granulocytes (Gr1+) and B (B220+) cells.In young HSCs, we determined ∼18,000 peaks for the active gene mark H3K4me3. For the repressive mark H3K27me3, we identified ∼6000 peaks across the genome, 2591 of which were present in the promoter region defined as the transcriptional start site (TSS) ± 100 bp. Strikingly, 70.2% (1820 out of 2591) of H3K27me3-enriched genes were also bound by H3K4me3. These so-called “Bivalent genes”, with both H3K4me3 and H3K27me3, are also found in embryonic stem cell (ESC) where they represent master regulators for lineage development. Here we found that these 1820 genes in HSC are prevalent in development, transcriptional regulation and RNA metabolism. They include many lineage-specific transcription factors, such as Cebpa, Ebf-1, Pax5, Gata3, Tbx21, Runx3 and Eomes. During HSC differentiation to granulocytes or B cells, HSC pluripotency regulators acquired the H3K27me3 repressive mark, while lineage regulators lost it in differentiated lineages. In addition, we observed with HSC differentiation alternative promoter usage on many epigenetic modifiers or transcription factors, such as Dnmt3a, Dnmt3b and Kdm6b (Jmjd3) and Runx3. The different isoforms may have different functions in hematopoiesis.Compared to HSC differentiation, HSC aging showed less extensive chromatin changes. Increased H3K4me3 binding at the TSS was identified for ∼300 genes, including Selp, Nupr1, Sdpr, Plscr2, Slamf1 (CD150) and Mt1. Interestingly, several genes in the Hoxb cluster showed increased H3K4me3 binding and higher expression with age. For H3K27me3, although PcG family member EZh2 expression decreases with HSC aging, we did not detect a global H3K27me3 decrease. On the contrary, H3K27me3 binding increased at ∼500 genes and decreased at ∼100 genes. Among them, the lymphoid transcription factors Pax5 and Ebf1 exhibited increased H3K27me3 binding. The enhanced repression of these lymphoid regulators may explain the myeloid-skewed differentiation that occurs with age. We also observed increased H3K27me3 on several Wnt ligands, including Wnt2a, Wnt8a and Wnt8b. As Wnt signaling is required for active HSC cycling, the increased H3K27me3 binding on these ligands may reinforce HSC quiescence. One well known target of the PcG family in aging is Cdkn2a (p16), which showed progressive loss of H3K27me3 repression with aging in neural stem cell (NSC). In contrast, our results revealed that p16 is repressed by H3K27me3 in both 4M and 24M HSC, and we did not observe its expression increase with aging. However, another cell cycle regulator p21 expression increases with aging, accompanied by a decrease of H3K27me3.In summary, we mapped chromatin state alterations with HSC differentiation and aging in this study. These findings will advance our fundamental understanding of HSC biology. Furthermore, chromatin regulators, such as Bmi1 and EZh2, have been implicated in leukemia transformation. This study provides a mechanistic link into how deregulation of these factors correlates with cancer progression. Disclosures:No relevant conflicts of interest to declare.